The Role of Intravenous Immunoglobulin Anti-A and Anti-B in Complement

Activation and Red Blood Cell Phagocytosis

By

Daniella Perri

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Laboratory Medicine and Pathobiology

University of Toronto

© Copyright by Daniella Perri 2009

ii

The role of IVIg Anti-A and Anti-B in Complement Activation and Red Blood Cell

Phagocytosis

Daniella Perri

Master of Science

Department of Laboratory Medicine & Pathobiology

University of Toronto

2009

Abstract

Intravenous immunoglobulin is a human blood derived product that is used to treat

immunodeficiencies and autoimmune disorders. An adverse side effect of IVIg therapy is

hemolysis. Patients who experience hemolysis are mainly blood group A or AB. Clinical

laboratory studies have demonstrated that IVIg contains ABO blood group antibodies, which

can bind complement proteins. This study hypothesizes that anti-A/B in IVIg will bind to

A/B antigens and activate complement in a dose dependant manner, which may lead to

enhanced RBC phagocytosis. This study observed that the quantity of ABO antigens does not

affect the in vitro binding of IVIg to RBCs. IVIg induced C3b deposition at high doses;

however, the amount of complement deposition was insufficient to enhance phagocytosis of

IVIg-sensitized RBCs by monocytic THP-1 cells in vitro. These studies emphasize that

hemolytic reactions involve many factors in conjunction with antibodies and complement

proteins.

iii

Acknowledgements

I would like to thank my supervisor, Dr. Greg Denomme, for supporting and guiding me

through the triumphs and tribulations of scientific research, experimental planning, and

manuscript writing.

I would also like to extend my gratitude to my committee members, Dr. J. Pendergrast, Dr. J.

Semple, and Dr. D. Branch, for their expertise and advice.

I would also like to thank the members of the Denomme and Semple lab who have supported

and encouraged me through the duration of this project. Also, many thanks go to Igor, Dipen,

Rukshana, and Mike for their technical support.

My sincerest gratitude goes towards the Canadian Blood Services employees who have

contributed to my project by either donating blood samples or providing technical support.

Your willingness and enthusiasm to contribute to my project was greatly appreciated.

Last, but not least, I would like to thank my parents, Marianne and Joe for providing me with

the support and determination I needed to reach my potential and beyond during my masters

program.

iv

Table of Contents

Abstract ii

Acknowledgements iii

Table of Contents iv

List of Tables viii

List of Figures ix

List of Appendices x

Abbreviations xi

1.0 Introduction 1

1.1 Blood Transfusion Reactions 2

1.1.1. Acute Immunologically Mediated Reactions 2

1.1.2 Delayed Immunological Reactions 3

1.1.3 Passive Antibodies in Transfusion Products 4

1.1.3.1 Anti-A and Anti-B in Transfusion Products 4

1.2 Transfusion of Blood Components: Intravenous Immunoglobulin (IVIg) 5

1.2.1 Biochemical Characteristics of IVIg 5

1.2.2 Clinical Use of IVIg 6

1.2.3 Mechanisms of Action of IVIg 6

1.2.3.1 IVIg Contains Anti-A/B That Can Cause Erythrophagocytosis 7

1.2.3.2 Fc-Dependant Mechanisms 7

1.2.3.3 Formation of Immune Complexes 9

1.2.3.4 Immunomodulatory Mechanisms 10

1.2.4 Hemolysis Associated with High Dose IVIg: In vivo Studies 10

1.3 The Classical Complement System 12

1.3.1 Complement Components and the Classical Complement Cascade 12

1.3.2 Complement Receptor 1 (CR1/CD35): C3b Receptor 13

1.3.2.1 Biochemical Properties 13

1.3.2.2 Genetic Polymorphisms of CR1 14

1.3.2.2.1 Structural Polymorphism 14

v

1.3.2.2.2 Expression Levels 15

1.3.2.2.3 Knops Antigens 15

1.3.3 Complement Receptor 2 (CR2) 16

1.3.4 Inhibition of the Classical Complement Pathway 16

1.3.5 Decay Accelerating Factor (DAF) 17

1.3.5.1 Cromer Blood Group System 19

1.4 Fc gamma receptor (FcγR)-mediated Immune Response 19

1.4.1 FcγR Biology 19

1.4.1.1 FcγRI (CD64) 20

1.4.1.2 FcγRII (CD32) 20

1.4.1.3 FcγRIII (CD16) 21

1.4.2 Macrophage Phagocytosis of Sensitized Red Blood Cells (RBCs) 21

1.4.2.1 Role of FcγRs and CRs 22

1.5 ABO Blood Group System 23

1.5.1 The ABO gene: Glycosyltransferases 23

1.5.1.1 The Structure of the ABO Glycosyltransferases 24

1.5.2 Differences Between Blood Groups A1 and A2 25

1.6 Lewis Blood Group System 25

1.6.1 Soluble ABO Substances 26

1.7 Rationale 27

1.7.1 Objectives 27

1.7.2 Hypothesis 27

2.0 Materials and Methods 28

2.1 Cell Line: THP-1 29

2.2 Blood Sample Donors 29

2.3 Intravenous Immunoglobulin (IVIg) 29

2.4 Effect of IVIg Dose on Anti-A/B Binding to RBCs 29

2.4.1 ABO Genotyping: DNA Extraction and Quantification 30

2.4.1.1 ABO PCR 31

2.4.1.2 Exon 6 and 7 Restriction Enzyme Digestion 31

2.4.2 Assessing the Level of Membrane-bound A-antigens 32

vi

2.4.3 Lewis Blood Group Serology Testing 32

2.5 Complement Activation with IVIg 33

2.5.1 Flow Cytometric Analysis of Complement Proteins 33

2.5.2 Absorption of Anti-A/B from IVIg 33

2.6 Inhibition of Rosette Formation by IVIg 34

2.7 RBC Phagocytosis Bioassay 34

2.7.1 Setting the Phagocytosis Assay Controls 35

2.7.2 Flow Cyometric Analysis of THP-1 Cell Viability 36

2.8 Statistical Analysis 36

3.0 Results 37

3.1 Characteristics of the Antibodies in IVIg: Clinical Laboratory Studies 38

3.1.1 ABO Antibodies in IVIg 38

3.1.2 Antibody Titres in IVIg 39

3.1.3 IVIg Anti-A/B and Complement Activation 40

3.2 Studies on IVIg with Whole Blood 40

3.2.1 The Effect of IVIg Concentration on Whole Blood 41

3.2.1.1 The Relationship Between Blood Group and IVIg Dose on 41

Anti-A/B Binding

3.2.1.2 The Effect of A-antigen quantity on IVIg 41

Anti-A binding

3.2.1.3 The Effect of Membrane-bound A/B Antigens on 44

Anti-A/B Binding

3.2.1.4 The Effect of Soluble A Substance on Anti-A Binding 46

3.2.1.5 The Effect of IVIg Dose on the Presence of Anti-A/B 47

in the Plasma

3.3 The Ability of IVIg anti-A/B to Activate the Classical Complement Cascade 48

3.4 The Interaction Between Red Blood Cells and Mononcytes 50

3.4.1 THP-1 Cell and IgG-sensitized RBC Interaction: Inhbition of 50

Rosette Formation

3.4.2 THP-1 Cell and RBC Interaction is Fcγ-receptor Dependent 53

3.4.3 IVIg-induced RBC Phagocytosis with and without 56

Complement Activation

4.0 Discussion 60

vii

4.1 Reviewing ABO Blood Group Antibodies and Hemolysis 61

4.2 Assessment of IVIg and Complement Activation: Clinical Studies 61

4.3 The Effect of IVIg Dose and ABO Genotype on Anti-A/B Binding 62

4.3.1 The Effect of IVIg Binding in the Presence of Soluble A Substance 62

4.4 Activation of the Classical Complement Cascade by IVIg 63

4.4.1 The Effect of Soluble A Substance on Complement Activation 63

4.4.2 Complement Activation on Group O RBCs 63

4.4.3 The Role of Complement Regulators on C3b Deposition 64

4.5 The Interaction Between Monocytes and RBCs is FcγR-dependant 64

4.6 Studies on Complement-mediated Synergism of IgG Sensitized RBC 65

Phagocytosis

4.6.1 The Effect of Receptor Expression on THP-1 cells on RBC 66

Phagocytosis

4.6.2 The Presence of Complement Regulators and Their Influence on 67

RBC Phagocytosis

4.7 Summary 67

4.8 Future Directions 69

5.0 References 72

6.0 Appendix 91

viii

List of Tables

Table 1. Molecular Basis of Cr Antigens 19

Table 2. ABO Antibodies in IVIg 39

Table 3. Antibodies Other than Anti-A/B in IVIg 39

Table 4. Anti-A Titres in Gamunex IVIg 40

Table 5. Complement Activation and Fixation by IVIg 40

Table 6. IVIg Anti-A/B Binding Associated with Blood Group, A Subclass 42

and Genotype

Table 7. Anti-A Binding in Association with Secretor Status 46

Table 8. Presence of anti-A/B in the Plasma After the Addition of IVIg 47

at the Concentrations Indicated

Table 9. The Effect of Serum Incubation on the Binding of IVIg anti-A/B 59

ix

List of Figures

Figure 1. Classical Complement Cascade 13

Figure 2. Synthesis of ABO and Lewis Antigens 24

Figure 3. The Effect of Secretor Status and Blood Group on Soluble A Substance 26

in the Plasma

Figure 4. Quantification of A-antigen by Flow Cytometric Analysis 45

Figure 5. Complement Activation by IVIg. 49

Figure 6. Images of Rosette Formation under an Inverted Microscope 51

Figure 7. Rosette Inhibition Assay 52

Figure 8. Histograms of Inhibition of Phagocytosis by Soluble IgG 54

Figure 9. Inhibition of FcγR-mediated phagocytosis 55

Figure 10. Experimental Controls for Phagocytosis Bioassay 57

Figure 11. IVIg-induced Complement-mediated RBC Phagocytosis 58

x

List of Appendices

Appendix A. Scores for Grading Agglutination by Direct Antiglobulin Tests 92

Appendix B. ABO Exon 6 and 7 Restriction Enzyme Digestion 93

Appendix C. Deposition of Complement Proteins 95

Appendix D. Binding of Anti-D 96

Appendix E. C3b Deposition and IgG Binding of RBCs Treated with Sucrose 97

Appendix F. Activation of Complement with Fresh Human Serum 98

Appendix G. Receptor Expression on THP-1 Cells 99

Appendix H. THP-1 Cell Viability 100

xi

Abbreviations

AABB American Association of Blood Banks

Ab antibody

ABO ABO blood group system

Ag antigen

AHTR acute haemolytic transfusion reaction

ATCC American type culture collection

BSA bovine serum albumin

CHO chinese hamster ovarian cells

CMFDA cell tracker green 5-chloromethylfluorescein diacetate

CR1 (CD35) complement receptor 1

CROM Cromer blood group system

DAF (CD55) decay acceleration factor

DAT direct antiglobulin test

DC dendritic cells

DHTR delayed hemolytic transfusion reaction

DMEM Dulbecco‘s modified eagle medium

DMSO dimethyl sulfoxide

dNTP deoxynucleotide triphosphate

DSTR delayed serological transfusion reaction

EDTA Ethylenediaminetetraacetic Acid

ELISA enzyme-linked immunosorbant assay

FACS fluorescent-activated cell sorter

FcγR-I,II,II Fc gamma receptor class I, II, III

FcRn neonatal Fc receptor

FITC Fluorescein Isothiocyanate

GPA glycophorin A

GPI glycosylphosphatidylinositol

GVHD graft versus host disease

Hb hemoglobin

HBV heptatitis B virus

HBsAg HBV surface antigen

HIV human immunodeficiency virus

HLA human leukocyte antigen

HTR hemolytic transfusion reaction

HR high responder

HSC hematopoetic stem cell

IC immune complexes

IgG,A,M immunoglobulin class G, A, M

IMDM Iscove‘s modified dulbecco‘s medium

ISBT International Society of Blood Transfusion

ITP immune thrombocytopenia purpura

ITAM tyrosine based activation motif

ITIM tyrosine based inhibition motif

IVIg intravenous immunoglobulin

xii

KD Kawasaki disease

KNOPS Knops blood group system

LE Lewis blood group system

LHR long homologous repeat

LR low responder

MHC I/II Major histocompatibility complex class I/II

moAb monoclonal antibody

mø monocyte/macrophage

MPS mononuclear phagocyte system

NK natural killer cells

Oh Bombay phenotype

PAF platelet activating factor

PBS phosphate buffered saline

PBMC peripheral blood mononuclear cells

PCR-RFLP polymerase chain reaction-restriction fragment length polymorphsism

PEG polyethyleneglycol

PID primary immunodeficiency

PIG-A phosphatidylinositol glycan anchor biosynthesis, class A

PKC protein kinase C

PMA phorbol-12-myristate-13-acetate

PMN polymorphonuclear cells

PNH paroxysmal nocturnal hemoglobinuria

PRP platelet rich plasma

PVP polyvinylpyrrolidone

RBC red blood cell

RCA regulator of complement activation gene

Rh Rhesus factor

SCID severe-combined immunodeficiency

SCR short consensus repeat

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM standard error of the mean

SIRS systemic inflammatory reaction syndrome

SLE systemic lupus erythematosis

SNP single nucleotide polymorphism

WNV west nile virus

1

Chapter 1

Introduction

2

1.1 Transfusion Reactions

There is a variety of transfusion reactions that can occur and they have been classified into a

number of categories. Two main categories are infectious and non-infectious related transfusion

reactions. The main viruses associated with infectious related reactions are hepatitis B virus

(HBV) and human immunodeficiency virus (HIV); however, zoonotic and bacterial

contaminations also pose hazardous risks to the blood supply used for transfusions. The main

reactions in the non-infectious category include acute and delayed hemolytic transfusion

reactions (HTRs). Acute transfusion reactions are further subdivided into intravascular and

extravascular. A major ABO blood group incompatible transfusion can cause acute intravascular

HTRs. These transfusion reactions involve A/B antigens on donor red cells and anti-A/B

antibodies in the recipient‘s plasma, and result in the activation of complement through to C9.

On the other hand, a minor ABO incompatible transfusion involves passively transfused donor

anti-A/B that bind to A/B antigens on recipient RBCs and can cause delayed extravascular

hemolysis, with or without the activation of complement through to C3b. Furthermore,

transfusion reactions can be categorized into immunologically- and non-immunologically-

mediated reactions.

1.1.1. Acute Immunologically-Mediated Reactions

Acute intravascular hemolytic transfusion reactions (AHTRs) are mainly associated with donor

red blood cell (RBC) antigen mismatched with an antibody produced by the recipient, with the

classical example being major ABO incompatibility. An international survey conducted in 2003,

showed that of 690 000 blood samples from 62 different hospitals, 1 in every 165 is mislabelled

or miscollected[1]. The recipient possesses natural immunoglobulins (Ig) against the donor`s

RBCs; natural IgM antibodies are able to activate the classical complement cascade.

Consequently, the RBCs (both the recipient`s and donor`s) suffer from complement-mediated

hemolysis. Activated complement can hemolyse recipient red cells in a process termed innocent

bystander. Regardless, this form of red cell destruction occurs intravascularly. As a result of

RBC lysis, intact hemoglobin (Hb) is released into the bloodstream. The Hb is bound by

haptoglobin and is broken down in the mononuclear phagocytic system (MPS), previously

termed reticuloendothelial system[2]. Consequently, there is an increase in bilirubin in the

plasma. In addition, free Hb bound to haptoglobin can be filtered through the kidney and when it

3

cannot be reabsorbed, it is excreted in the urine. Therefore, patients with AHTRs also present

with hemoglobinuria (ie. reddish urine) along with fever and changes in skin colour (ie.

jaundice)[2-5]. As little as 20 ml of blood can cause an acute HTR[4].

Immunoglobulin G (IgG) subclasses type 1 and 3 are able to activate the classical complement

cascade by binding to RBCs at 37oC[2;4]. Therefore, other blood group alloantibodies can cause

acute extravascular transfusion reactions[6]. Blood tests of recipients who suffered from IgG

associated extravascular transfusion reactions showed positive direct antiglobulin tests (DATs)

[with or without complement] and a less rapid decrease in hematocrit[7]. The symptoms can

appear within the first 15 minutes to 24 hours after the blood transfusion[2]. The incidence of a

transfusion reaction is approximately 1 in 70 000 units of blood[2].

1.1.2. Delayed Immunological Reactions

Delayed hemolytic transfusion reactions (DHTRs) are different from AHTR in many ways. One

important difference is that the reaction occurs 1-14 days after the blood transfusion[5]. The

blood that is assigned for transfusion is mistakenly labelled as compatible. The reason being that,

serologically, no antibodies directed against the donor`s RBCs are detected. This is due to the

fact that antibody, from previous sensitizations (ie. previous blood transfusion; pregnancy),

diminish and the titres (a measure of the amount of antibody) reach undetectable levels.[3-5]

When the ‗compatible‘ blood is transfused, the recipient‘s immune system engages in a rapid

secondary immune response, termed an anamnestic response. The amount of IgG rises rapidly

and exponentially, which leads to extravascualar hemolysis. IgG can activate the classical

complement cascade; however, not all complement proteins are used. The pathway ends after

C3b is deposited on the RBC membrane[5]. This cleaved complement protein opsonizes the

RBCs for destruction by the MPS. Also, it has been described that DHTR may be comparable to

anaphylaxis reactions engaging IgG antibodies, Fc gamma receptors (FcγRs), macrophages, and

platelet-activating factor (PAF)[5]. Extravascular hemolysis is associated with slower decreased

hematocrit and less rapid increase in bilirubin. As seen in AHTRs, DHTRs also present with

positive DATs and sometimes fever. A transfusion reaction with a positive DAT and a newly

developed alloantibody, however, without evidence of hemolysis, is described as a delayed

serological transfusion reaction (DSTR)[8].

4

1.1.3 Passive Antibodies in Transfusion Products

Passive antibodies are those that are transferred either naturally or artificially. Naturally-acquired

antibodies are those that are transferred from mother to fetus during pregnancy, namely IgG.

Artificially-acquired antibodies are those that are administered intravenously either from whole

blood transfusions or, blood component therapy. The passive antibodies in transfusion products

such as whole blood, platelets, or pooled Ig, include antibodies to minor blood group antigens (a

rare event) and anti-A/B. Under certain circumstances, the inadvertent transfusion of these

antibodies can have profound effects on red cell survival if they are directed to an antigen

expressed on the recipient‘s RBCs. As previously mentioned, there are two categories of ABO

incompatibilities. One is minor, in which hemolysis of the recipient‘s RBCs occurs due to the

presence of anti-A/B present in the small amount of plasma left over in the transfusion bag. The

second is major, in which the recipient‘s anti-A/B cause hemolysis of the donor‘s RBCs. What is

important to note is that the passive transfusion of antibodies (a minor ABO incompatibility) can

result in an acute extravascular transfusion reaction.

1.1.3.1 Anti-A and anti-B Antibodies in Transfusion Products

Patients who are group A, B or AB sometimes undergo platelet transfusion from donors that are

blood group O and are, therefore, at risk for ABO incompatible hemolytic reactions. The two

most important factors that can influence the hemolytic episode are the amount of plasma and

anti-A/B titre in the donated platelet unit[9]. Generally, the anti-A and anti-B titres in group O

donors are higher than the respective antibodies in group B and A donors. In a recent study, it

was found that approximately 60% of group O pooled platelet units can be considered as ―high

titre‖ units using the gel method for antibody detection when compared to the tube method[10];

the authors used a critical titre of 64[11]. The authors also reported that the antibody titre from

mixed (group O + group A or B) pooled platelet units was significantly lower than pooled group

O units, to which they hypothesized was the effect of ABO substances in the plasma that

neutralized the anti-A/B antibodies[10]. A case report by Sadani et al.[12] describes a Group A

patient who received 5 group O platelet units and subsequently suffered an AHTR. IgG anti-A

was eluted from the patient‘s RBCs and the last unit of platelets transfused, when tested,

contained an anti-A titre of 1280. In another study, a 2 year-old Group A patient was also

transfused with group O platelets and developed intravascular hemolysis (according to clinical,

5

serological tests) as a result of the platelet unit containing a high titre of anti-A. The antibody

titre in the platelet unit was 1024[13].

1.2 Transfusion of Blood Components: Intravenous Immunglobulin (IVIg)

In the early 20th

century, patients were only transfused with whole blood, as the technology

available then did not allow blood to be separated into its various components. The processing of

blood components for transfusion began shortly before World War II (WWII) allowing injured

soldiers to benefit from the transfusion of plasma[14]. In 1951, Edwin Cohn developed the first

cell separator, today known as a centrifuge, which could fractionate whole blood into red cells,

white cells, platelets, and plasma.[15] A newer technique involves a soft spin to separate RBCs

from the platelet-rich plasma, which is then subjected to a hard spin to separate the platelets[16].

The advent of separating techniques made it possible to advance blood component therapy.

Today, RBCs as well as plasma derivatives are commonly used for transfusion therapy. One such

derivative is Intravenous Imunoglobulin (IVIg).

1.2.1 Biochemical Characteristics of IVIg

IVIg is prepared by pooling human plasma from 10 000+ healthy donors and subjecting it to a

combination of cold ethanol fraction, caprylate precipitation and filtration, and anion-exchange

chromatography[17]. The first process isolates IgG molecules while the latter two are intended to

remove and inactivate viruses that may be present in the collected plasma. Glycine or, a

nonessential amino acid, is used as a protein stabilizer to maintain isotonicity. Proline may also

be used. The final product is stored at a pH level of 4.0-4.5. IVIg mainly (>98%) consists of

monomeric, polyspecific IgG[18;19], with trace amounts of IgA and IgM.[20] However, studies

have shown that IVIg contains small amounts of IgG dimers that behave as immune complexes

(IC)[21].

Stabilizers such as, sugar and sodium, as well as, osmolality and pH vary among IVIg products

from different manufacturers[22]. The large number of donors used for IVIg preparation enables

the drug to represent a broad range of natural antibodies that is present in healthy human serum.

IVIg is polyspecific to foreign bacteria, viruses and their toxins, as well as, cell membrane

proteins, receptors and autoantibodies[23]. Some adverse reactions include headache, anxiety,

6

flushing, wheezing, abdominal cramps, myalgias, arthralgia, dizziness, and rash. There is <0.1%

occurance of hemolysis.[17] It is a rare reaction, however, both moderate and severe hemolytic

episodes have been documented.

1.2.2 Clinical Use of IVIg

IVIg is used to treat patients with primary immune deficiencies (PID) such as, severe combined

immunodeficiency (SCID) and autoimmnune conditions such as idiopathic thrombocytopenia

purpura (ITP). IVIg is also given to patients following a bone marrow transplant (BMT) since

they are immunosuppressed and are therefore susceptible to infections. For treating PID, a 0.1-

0.6 g/Kg dose is administered every 3-4 weeks in order to achieve serum levels of 5 g/L[24]. In

clinical trials, IVIg has been shown to prevent and/or attenuate infections[17]. Some of the most

common adverse reactions PID patients experience include increased cough (1.7%), headache

(0.8%), fever (0.1%), and nausea (0.5%).[17]

Imbach et al.[25] was the first group to use high dose IVIg therapy to treat the autoimmune

disorder, ITP, in children. Today, patients are administered a total dose of 2 g/Kg either given

over 2 consecutive days at a 1 g/Kg dose or, over 5 consecutive days at a 0.4 g/Kg dose. Patients

mainly experience headaches (50%), as well as, fever (10%) and nausea (10%).[17] Adverse

reactions can be associated with administration rate. Generally, IVIg is infused at a rate of 0.01-

0.02 ml/Kg/min for the first 30 minutes. If well tolerated, it can gradually be increased to a

maximal rate of 0.14 ml/Kg/min. The rate should not exceed 0.08 ml/Kg/min for the first

infusion.

1.2.3 Mechanisms of Action of IVIg

The mechanisms of IVIg activity can be associated with IgG biology. IgG contains an Fc, as well

as, an F(ab‘)2 portion that can mediate various biological effects. The F(ab‘)2 region can bind to

various antigens including complement components, which can provide an anti-inflammatory

effect and surface proteins/receptors, which can disrupt cellular activities, leading to therapeutic

effects[20]. IVIg IgG can bind to complement components, C3b and C4b, in order to circumvent

complement-mediated lysis by preventing their deposition on targeted cells[23]. In the treatment

of toxic epidermal necrolysis (TEN), CD95-specific antibodies within IVIg bind to CD95 and

7

prevents the Fas-mediated pathway of apoptosis; thereby, inhibiting keratinocyte death.[26]

Additionally, anti-idiotypic antibodies within IVIg, bind to autoantibodies, therefore,

neutralizing them or, preventing their binding to autoantigens[23;27;28]. However, s

tudies have shown that the anti-idiotypic mechanism has failed to ameliorate murine ITP, both in

vitro and in vivo[29]. Other IgG within IVIg include those that target antigens of the ABO blood

group system. This can have detrimental effects, since these Igs have the capacity to behave as

hemolysins.

1.2.3.1 IVIg Contains Anti-A/B that can Cause Erythrophagocytosis

Multiple studies have shown that hemolysis, subsequent to the administration of high doses of

IVIg, has been associated with the presence of anti-A and anti-B antibodies found in the IVIg

preparation used[30-35]. These anti-A and anti-B antibodies can become saturated on RBCs

when incubated in levels of IVIg that correlate to high dose regiments[36]. To further support the

role of anti-A and anti-B antibodies within IVIg and their association with accelerated

extravascular hemolysis, Shomam-Kessary and Gershon have demonstrated that in the absence

of these isoagglutinins, phagocytosis of A+ and B+ RBCs is prevented[36]. Anti-A and anti-B

IgG also have the capacity to activate the classical complement pathway, which may augment

hemolysis[22;37]. The European Pharmacopeia has set a standard for anti-A and anti-B titres in

IVIg, being <32 in a 3% w/v solution[38;39]. However, the hemagglutination method of

antibody detection that various manufacturers employ may result in an underestimation of anti-A

and anti-B titres[35]. A recent collaborative study has shown that the use of IVIg reference

reagents (positive, negative, high anti-A/B titre controls), if used in parallel with IVIg lots, can

reduce the vaiability between tests when the direct hemagglutination test is used[40].

Furthermore, there are no published Health Canada recommendations for IVIg to have the titre

of anti-A/B below a certain threshold.

1.2.3.2 Fc-Dependant Mechanisms

Several models have been proposed for the Fc-dependant mechanisms of IVIg activity[20]. One

model suggests that IVIg competes with autoantibodies for binding sites on neonatal Fc receptors

(FcRn) therefore, causing enhanced clearance of autoantibodies[41]. Consequently, the half-life

of IgG is prolonged by binding to FcRn. Using a murine model for ITP, Hansen and

8

Balthasar[42] suggested that IVIg binds to and saturates FcRn, increasing the availability of

antiplatelet antibodies for clearance. However, inhibition of phagocytosis via activation of the

inhibitory receptor, FcγRIIb, is thought to be the major mechanism of action for amelioration of

ITP[43].

Another model suggests that IgGs within IVIg bind, with their Fc portion, to activating FcγRs,

therefore, competing for binding sites on FcγRs with autoantibody immune complexes. This

results in attenuation of autoantibody-mediated effects seen in autoimmune disorders, such as

ITP. Fehr et al.[44] was one of the first groups to show that FcγR blockade prolonged the

clearance of IgG-sensitizied RBCs, as well as, increased platelet counts in patients with ITP who

were treated with IVIg. Blockade of activating FcγRs may act in concert with the modulation of

expression of the low affinity, inhibitory receptor, FcγRIIb. Studies have shown that mice that

were genetically deficient for FcγRIIb, were not ameliorated of immune thrombocytopenia when

treated with IVIg; thus, demonstrating that the therapeutic effects of IVIg requires

FcγRIIb[43;45]. Conversely, Bazin et al.[46] demonstrated that FcγRIIb knock-out mice with

immune thrombocytomepnia respond effectively to IVIg treatment. Furthermore, IVIg has been

shown to cause upregulation of FcγRIIb expression[43]. A review on the therapeutic effects of

IVIg suggested that when IVIg is admininstered, IC form as either IgG dimers or soluble

antigen-Ig complexes that can stimulate FcγRIIb expression[47].

In addition to FcγR blockade, high dose IVIg may also enhance non-immune-mediated

phagocytosis as suggested by Newland et al.[48] The effects of IVIg on phagocytic function in

ITP patients was evaluated by measuring parameters for splenic and liver function. They

demonstrated that the clearance of heat-damaged RBCs was enhanced, while that of anti-D

sensitized RBCs was reduced; platelet counts increased in conjunction. They suggested that high

dose IVIg inhibits FcγR-mediated phagocytic function via FcγR blockade while, simultaneously,

enhancing non-immune-mediated phagocyte function. However, the increase in non-immune-

mediated phagocytic activity was not significantly correlated to increased platelet counts[48].

Newland et al. do suggest that high dose IVIg may form IgG aggregates which, when

internalized, can activate the non-immune-mediated response.

9

1.2.3.3 Formation of Immune Complexes

Numerous studies have shown that IVIg can indeed form IC. An earlier study on the anti-

complement effects of IVIg by D.H. Bing[49], showed that IVIg, when heated, caused a 49%

increase in complement activation. The heat aggregated IVIg had a 3000-fold increase in affinity

for C1q. Bing concluded that IVIg is suitable for immunodeficient patients since it has a weaker

ability to cause non-specific complement activation compared to heat aggregated IVIg, yet can

still bind to C1q to activate complement required for normal host defenses. In 1998, Shoham et

al.[21] demonstrated that IVIg in the presence of complement, caused an equivalent level of

erythrophagocytosis when compared to that of tetnas-anti-tetnas IC in the presence of

complement. In addition, this group also fractionated IVIg (Sandoglobin) and found that it

contained a portion of molecules that had a molecular weight similar to that of IC (≥300 kDa).

These high molecular weight moieties were considered to be IgG dimers. In a separate

experiment, Shoham et al. showed, in vitro, that these IgG dimers were able to bind to human

erythrocytes via CR1 in the presence of complement. In 1999, the same group showed that the

IC-like moieties in IVIg (Isiven) can bind RBCs in vivo and cause elevated erythrophagocytosis

as demonstrated by the decrease in hematocrit and hemoglobin measured from the patients in

their study[50].

The IC formed by IVIg in whole blood do not necessarily have to be IgG dimers. For example,

IVIg is known to contain antibodies to a wide variety of human plasma proteins. When infused,

IVIg can bind to these plasma proteins and form IC. The human body has mechanisms that can

remove IC when they form within the plasma. When IVIg is administered in high doses, it can

oversaturate the body‘s natural mechanisms to remove the IC. Consequently, formed IC remain

within the plasma. Using the reactivity of IVIg to human ferritin, Lamoureux et al.[51]

demonstrated that when IVIg is added to human serum auto-IC form. These can be precipitated

out of the serum with polyethyleneglycol (PEG) and tested for reactivity to plasma proteins. The

observation was that the PEG precipitate contained IC that were mostly IVIg IgG and had greater

reactivity to ferritin compared to PEG precipitates from IVIg alone. In a following study,

Lamoureux et al.[52] purified auto-IgG from IVIg using affinity chromatography, which made

up <3% of the total IVIg, yet had 78% of the anti-ferritin activity. The purified auto-IgG also

formed IC when added to human serum in therapeutic doses.

10

1.2.3.4 Immunomodulatory Mechanisms

IVIg has anti-inflammatory properties. In addition to the inhibition of phagocytosis, increased

IgG clearance, and complement activation interference[20], IVIg can also influence the activities

of cytokines, dendritic cells (DCs), T-cells and B-cells, which are important mediators in an

immune response. IVIg can modulate the production of cytokine antagonists[23] and pro-

inflammatory cytokines.[27] IVIg, in vitro, was able to stimulate the production of interleukin

(IL)-1 receptor antagonist (IL-1ra) from myeloid cells[53] and inhibit macrophage response to

interferon gamma (IFNγ) by suppressing the expression of IFNγR2 subunit via FcγRIII.[54] In

another investigation, IVIg inhibited the production of interleukin (IL)-6, which prevented

antibody secretion from plasma cells.[55] IVIg has also been shown to interfere with B-cell

differentiation in vitro.[56] Stohl et al.[56] demonstrated that early and late additions of IVIg to

cell cultures, inhibited the superantigen driven T-cell-dependant differentiation of B-cells by

examining plaque formation and cytolytic activity. Furthermore, Toyoda et al.[57] showed that

IVIg can significantly reduce B-cell numbers in peripheral blood mononuclear cell (PBMC)

cultures by inducing apoptosis, which requires intact IgG molecules.

IVIg also contains antibodies that bind to cell membrane proteins, such as CD4. Anti-CD4

antibodies isolated from IVIg prevented T-cell proliferation and HIV infection in vitro[58]. In

addition, antibodies to MHC class I proteins were also isolated and shown to bind to soluble and

membrane bound HLA class I molecules, as well as, inhibit MHC class I-mediated T-cell

cytotoxicity[59]. T-cell activity is controlled by DCs. DCs are antigen presenting cells (APCs)

that interact with naive T-cells and are key players in initiating the primary immune response.

Consequently, they pose excellent targets for IVIg to exert its immunosuppressive effects. IVIg

can inhibit the differentiation and maturation of DCs in vitro; IVIg abrogated the release of IL-12

from DCs. The release of co-stimulatory cytokines by DC is also prevented, therefore,

suppressing the activation and proliferation of alloreactive T-cells[58].

1.2.4 Hemolysis Associated with High Dose IVIg: in vivo Studies

In an early study by Thomas et al.[60], acute hemolysis was reported after the administration of

high dose IVIg therapy in a Caucasian male. He demonstrated characteristics of both

extravascular and intravascular hemolysis, with a drop in hemoglobin (15-8.7 g/L) and free

11

hemoglobin in his plasma and urine. The patient also had decreased haptoglobin levels in his

plasma, as well as, increased bilirubin levels. The patient‘s blood group was A1 and his RBCs

had bound anti-A1 IgG. Treatement for this patient required a single blood transfusion.

In 2000, Nakagawa et al.[61] reported a case of severe acute autoimmune hemolytic anemia

(AIHA) after the administration of IVIg. The patient was a 5 month old infant with Kawasaki

disease (KD), a condition in which arteries become inflamed and, if left untreated, can lead to

cardiovascular complications. Before IVIg treatment, the patient had no evidence of anemia. The

patient was treated with a total dose of 2g/Kg of IVIg; however, when the symptoms did not

improve, another total dose of 2g/Kg was administered. Sixteen days after the onset of the

disease, the infant was observed to have anemia; hematocrit decreased to 2.55 x 106 cells/µl,

hemoglobin level was 6.8 g/dL, tested positive for both direct and indirect Coombs tests, and the

patient‘s RBCs had bound anti-A IgG. The infant‘s blood type was A+. The authors believe the

cause for the anemia to be a result of the infant receiving a total dose of 4 g/Kg of IVIg.

In a recent case study analysis by Daw et al.,[62] it was demonstrated that hemolysis can occur

after the administration of high-cummulative-dose IVIg therapy. Hemolysis was characterized by

a drop in hemoglobin, in which the decrease ranged from 8-52 g/L. Patients received 100g or

more of IVIg consecutively for 2-4 days. Of the 16 patients studied, 15 were either blood group

A or B and each presented RBCs with a positive DAT score. Only one patient was positive for

complement deposition. In addition, when patient plasma was assessed, blood group antibodies

directed against the patients‘ RBCs were present.

Another study by Mollnes et al.[37] involving 7 patients, examined complement activation post

IVIg infusion. All patients were female and given 0.5 g/Kg of body weight of IVIg, infused for 3

hours. Blood samples were obtained, before and 30 minutes after treatement, and compared for

complement activation products and IgG levels. Both IgG and complement activation products

(C4bc, C4d, Bb, C3bc, C5a, and TCC [terminal complement complex]) were significantly

elevated in all patients‘ plasma. The authors conclude that IVIg can activate the classical

complement pathway and suggested that it may do so by diverting complement activation away

from targeted membranes.

12

Shoham-Kessary et al.[21] demonstrated that RBCs can bind IgG and complement (C3d) in vivo

after IVIg treatment. They further showed that the IgG and C3d bound to patient RBCs via CR1

as immune complexes and this was correlated to in vivo hemolysis. Hemolysis was measured by

the decrease in hematocrit and hemoglobin in the patients studied.

1.3 The Complement System

1.3.1 Complement Components and the Classical Complement Cascade

The classical and alternative complement systems are integral components to the innate immune

response. Like the alternative system, the classical complement system involves a protease

cascade that results in the proteolytic cleavage of complement component-3 (C3). However, the

classical pathway is activated by immunoglobulins (Igs) binding to target antigens (Ags)[63].

The antibody (Ab)-Ag complex activates a complement protein C1qrs, which initiates the

proteolytic cascade of C3[63]. (Figure 1) C3 is a 185 kDa protein that is made of two

polypeptide chains (α and β) held together by disulfide bonds and noncovalent forces.[63-65]

The α-chain is cleaved, by C3-convertase (C4b2a)—there is controversy in the terminiology for

the larger C2 cleaved product to be labeled as ‗C2b‘ form; however, many scientists still name

the larger fragment, C2a[15]—to produce C3a and C3b[63;65] and as a result, a thiolester bond

in C3b is revealed to mediate attachment to target surfaces[63;66;67]. Complement receptor 1

(CR1) or Factor H then binds to C3b, which allows Factor I, a serine protease, to cleave C3b

twice in the α-chain to produce iC3b and then a third time, to produce another two complement

products, C3d,g and C3c[65;68;69]. The end products of C3 regulation are C3d and C3e[65]. As

C3b is amplified, C3-convertases then bind to membrane-bound C3b to produce the C5-

convertase (C4b2a3b), which cleaves C5 to C5a and C5b. The C5b fragment associates with C6

and C7 and this complex binds to target membrane to assemble C8 and C9 into membrane attack

complexes (MACs). MACs are essentially pores embedded with the target membrane, which

disrupts the osmotic conditions of the cell causing lysis[70]. Alternatively, C3b can bind to its

receptor on phagocytic cells, which then remove C3b or C3b bearing IC from the circulation.

13

Figure 1. Classical Complement Cascade. This pathway shows

complement activation up to C3b deposition and subsequent breakdown

to C3d. The membrane attack complex (MAC) formation is not shown.

1.3.2 Complement Receptor 1 (CR1/CD35): C3b Receptor

1.3.2.1 Biochemical Properties

Characteristic of all complement receptors (CR1,2,3,4) are short consensus repeat (SCR)

units[71]. CR1, in particular, is a 200 kDa cellular surface protein that is composed of four long

homologous repeat (LHR) domains [A-D]. Each domain is comprised of 30 SCRs in which each

SCR contains 60 amino acids and 2 disulfide bonds for structural rigidity[63;72-75]. Conserved

amino acids, such as glycine, proline, tyrosine, phenyalanine, isoleucine, leucine and valine,

compose the hydrophobic core within CR1[71]. CR1 was discovered by Nelson Jr.[76] who

Ab/Ag complex

C4b2a (C3 convertase)

C3b

iC3b

C3d.g

C4, C2

C4a, C2b

C3

C3a

Factor I

Factor I

enzymes

C3c

C1qrs

C3e

14

identified the receptor as the immune adherence receptor. Nearly 20 years later, D.T.

Fearon[77;78] isolated the membrane receptor from human erythrocytes and identified it as the

C3b receptor. CR1 is located on a number of cell types including red blood cells (RBCs),

polymorphonuclear leukocytes, monocytes/macrophages, B-cells, T-cells, glomerular podocytes,

and splenic follicular dendritic cells[78-82]. On monocytes, for example, there are 30,000

CR1/cell with a Ka of 4 x 107[78]. However, the copy number can be upregulated via the

influence of chemoattractants[83] and/or by the influence of a HindIII restriction binding site

present within the CR1 gene[84].

1.3.2.2 Genetic Polymorphisms of CR1

The gene for CR1 is located on chromosome 1 q32.[71;72] Organization of the CR1 gene was

described in 1993 by Vik et al.[85] Recent genetic studies[86-91] have demonstrated that single

nucleotide polymorphisms (SNPs) in the promoter enhancer region, particularly in the first

intron, are important in gene regulation and disease[74]. Consequently, the complement system

has been implicated in inflammatory disorders, such as systemic inflammatory reaction

syndrome (SIRS), nephritis, and hyperacute graft rejection, complement-deficient disorders, such

as systemic lupus erythematosis[92;93], and autoimmune diseases[94]. Currently, there are three

well known polymorphisms of CR1. These include size polymorphisms, RBC expression levels,

and Knops (Kn) antigens (Ags)[95;96].

1.3.2.2.1 Structural Polymorphisms

There are four allotypic variants of CR1. Increasing in molecular weight are, CR1-3 (190 kDa),

CR1-1 (220 kDa), CR1-2 (250 kDa), and CR1-4 (280 kDa)[95;97]. The two most common

variants (named F [CR1-1] and S [CR1-2] or A and B, respectively) are the result of a SNP at

position bp159 (SCR 8), which substitutes a cytosine for a thymine; the amino acid arginine is

substituted with a cysteine[71;72;98;99]. The result is the S allotype containing 7 more

SCRs[100]. A recent study[72] demonstrated that homozygous BB CR1-2 binds C3b with higher

avidity than the BA and AA variants; the AA CR1-1 variant bound 80% less C3b. CR1-1 (for

simplicity, will be denoted as CR1) is the most common allotype compared with CR1-2.

Therefore, it is the receptor that is most frequently used for investigations involving CR1

complement mediated immune responses.

15

In 1988, Klickstein et al.[101] identified C3b binding sites within CR1 SCRs 8 and 9 with

identical binding sites in SCRs 15 and 16. To identify specific amino acids that are involved in

ligand binding, Krych et al.[73] carried out mutagenesis experiments in which they employed a

truncated form of CR1 and substituted amino acids in SCRs 1 and 2 with those in SCRs 8 and 9

that were known for C4b and C3b binding, respectively. There is 55% homology between SCRs

1 and 8 and 70% homology between SCRs 2 and 9[73]; thus, the differences in ligand binding

must be attributed to specific amino acids[71]. The conclusion was that amino acids in both

SCRs 1 and 2 are needed for C4b binding and one area in SCR 9, particularly residues

asparagine and lysine, is required for C3b binding[73]. It is the positive charge within the

binding site of CR1 that is important in binding the negatively charged α-chain of C3b[73;102].

1.3.2.2.2 Expression Levels

Expression level of CR1 is also genetically controlled. Between healthy individuals, the

expression levels can vary 10-fold[96;103]. The cis-acting regulatory element that regulates CR1

expression is identified by the HindIII restriction fragment length polymorphism (RFLP), which

was shown to be linked to the CR1 gene through its ability to control the amount of structural

allotypes that existed within three generations as was demonstrated by pedigree analysis[84].

Using SDS-PAGE analysis, those that had high CR1 expression levels displayed one band of 7.4

kb, those with low expression showed one band of 6.9 kb, and those individuals with

intermediate expression levels displayed both band sizes. This has implications on genotyping

and phenotyping for Knops Ags as they are associated with CR1 expression levels.

1.3.2.2.3 Knops Antigens (Kn Ags)

Knops is the 22nd

blood group system, as recognized by the International Society of Blood

Transfusion (ISBT).[97] Classically, Knops Abs are classified as high-titre, low-avidity (HTLA)

Abs[95;97;104;105]. The Ags have weak and variable reactions to the Abs[106] due to their

variable expression on CR1[95] and when in the heterozygous state, can result in a falsely

negative phenotype even when CR1 expression is normal[96]. Investigation of this blood group

system was initiated when the antiglobulin reactive Ab to a high frequency Ag in a Caucasian

woman did not react with RBCs from a blood bank technologist, named Helgeson. Her RBCs

were compatible with the Knops Ab serum and therefore, were considered to have the null

16

phenotype[107]; Kna-:McC(a-b-c-d-e-f-). Rao and Moulds[104;105] identified Knops (Kn

a),

McCoy (McCa), Swain-Langley (Sl

a), and Villien (Vil) Ags on CR1. The presumed allelic pairs

are Kna/Kn

b, McC

a/McC

b, Sl

a, Vil. The Helgeson phenotype is not associated with any particular

Kn genotype[95]. However, it is strongly correlated to a low copy number of CR1[104;108]. The

McC and Sl Ags were localized to LHR-D of CR1 in SCR 25.[96] At bp 4795 an adenosine (A)

is replaced with guanine (G) to yield an amino acid change of proline (P) to aspartic acid

(D)[104]. The amino acid change results in the allelic switch of McCa to McC

b. Similarly at bp

4828, there exists the same A to G displacement, but the amino acid change instead is an

arginine (R) to a glycine (G)[104]. This amino acid difference changes the allele for Sla to Sl

b.

The Kna/Kn

b allelic pair was also localized to LHR-D of CR1. Moulds et al.[106] demonstrated

that LHR-D with a valine (V) at position 1561 corresponded to the Kna(+) allele, whereas, LRH-

D with a methionine (M) instead resulted in the Knb(+) allele. Two experiments were done to

confirm that the Knops Ags existed on CR1. They showed that antisera that reacted with

Kn/McC, Kna, McC

a, Sl

a precipitated a 220 kDa protein that aligned with the common

polymorphic size variant of CR1 and that it was the same protein that was also precipitated by

monoclonal Abs to CR1[105;106]. In addition, Helgeson‘s RBCs bound anti-CR1 Abs and anti-

Kn/McC Abs very weakly, supporting the hypothesis of low CR1 copy number for the apparent

null phenotype.

1.3.3 Complement Receptor 2 (CR2)

CR2 is the complement receptor for iC3b, C3dg and C3d. It is a 140 kDa protein that binds to

C3d-coated RBCs creating rosettes in vitro. CR2 is present on monocytes, however, in low

quantities. When RBCs are opsonized with C3d, antibody-dependant cellular cytotoxicity

(ADCC) is enhanced by a subset of peripheral blood lymphocytes, K cells[109;110].

1.3.4 Inhibition of the Classical Complement Pathway

CR1 is known to inhibit the classical complement pathway. It has been demonstrated that CR1

decreases C3 consumption by C4b2a (C3 convertase) in a dose dependant manner[111]. The

mechanism of inhibition is that CR1 competes with C2 for a binding site on C4b. Consequently,

C3b is neither produced nor deposited on the target surface. Additionally, CR1 acts as a co-factor

to Factor I, which cleaves C3b to iC3b (Figure 1)[112]. Complement-mediated phagocytosis

17

requires protein kinase C (PKC)[113-116], which has been demonstrated through the inhibition

of phagocytosis using PKC inhibitors[117]. As mentioned previously, an Ab-Ag complex is

needed to activate the complement cascade. Abs that activate complement are natural Abs

(NAbs)[118-120] that are present in the sera of normal, non-immunized humans[121;122].

However, not all NAbs activate complement. It has been demonstrated that natural IgM activates

complement, whereas natural IgG inhibits complement fixation on the target cell membrane by

competing with IgM for binding sites on the target surface[123].

Conversely, Circolo et al.[124] demonstrated that IgG can activate complement when a high

density of haptens (Ag), in a patchy distribution, exist on the surface of RBCs. This allows for

aggregation of the Abs that activates complement. IgM-mediated complement activation was

significantly lower under the same conditions[124]. Therefore, IgG and Fc-gamma receptors

(FcγR) play a role in the complement-mediated immune response. In previous studies, CRs

played a significant role in the attachment of complement components, wheresas FcγRs were

responsible for inducing phagocytosis[125-127]. FcγR Abs did not affect CR binding to RBCs

coated with Ab-Ag-complement complex, but did result in a 5-fold decrease in

phagocytosis[126]. Furthermore, low concentrations of IgG enhanced ingestion and attachment

to RBCs 6-fold[125].

1.3.5 Decay Accelerating Factor (DAF) (CD55)

DAF is an intrinsic complement regulatory protein that acts to prevent the assembly of C3 and

C5 convertases, thereby protecting cells from complement-mediated lysis[128]. By inhibiting the

formation of C3 convertases, the amplification of C3b is avoided and therefore, insufficient

amount of this opsonin is deposited on the red cell membrane. Consequently, the cell

circumvents lysis. DAF is a glycosylphosphatidylinositol (GPI)-linked, single chain membrane

protein that has a molecular weight of 70 kDa. Using digestion experiments and SDS-PAGE

analysis, DAF was assigned to have one N-linked oligosaccharide which is modified as the

precursor protein passes through the Golgi; here, multiple O-linked oligosaccharides are added to

produce the mature form of DAF[129].

18

The GPI-link allows DAF to move laterally within the outer leaflet of the red cell membrane

making contact with many C3b molecules. In addition, the GPI link allows soluble DAF to

reincorporate into red cell membranes[129]. DAF is a 381 amino acid protein and has 4 SCR

units. Each unit is made of approximately 60 amino acids, four of which are cysteine residues.

After the SCR units, there is a serine and threonine-rich domain to which many O-linked

oligosaccharides are attached. Following this domain is the GPI, which is anchored into the red

cell membrane. However, if the GPI link is absent, DAF and all other GPI-anchored proteins, are

absent as well[130]. This cellular phenotype is observed in patients who suffer from Paroxysmal

Nocturnal Hemaglobinuria (PNH). It is an acquired clonal disorder of hematopoetic stem cells

(HSCs). Without DAF, PNH patients cannot regulate their complement system and so their red

cells are sensitive to complement-mediated lysis[130]. DAF can be reconstituted on PNH cells,

and it retains its function to reduce the cell‘s susceptibility to complement-mediated

destruction[131].

The DAF gene is a single copy gene and is located on chromosome 1q3.2. It is, therefore, a

member of the regulator of complement activation (RCA) gene cluster, which also includes

genes for C4 binding protein (C4BP), CR1, and C3dg receptor (CR2). On each RBC there is

approximately 5-15 x 103 DAF molecules. The presence or absence of DAF on RBCs is

associated with the PIG-A gene, which is responsible for the expression of the GPI link. The

PIG-A gene has been mapped to the X chromosome, which explains why a single mutation in an

HSC leads to DAF-deficiency in that cell and all of its progenitors[132]. However, a mutation in

the PIG-A gene is not sufficient to cause PNH. It has been hypothesized that PNH cells are

selected against by activated T-cells that target GPI[130]. Therefore, they are given the

opportunity to proliferate.

Nicholson-Weller et al.[133] isolated DAF from sheep and human erythrocytes by n-butanol

extraction. The functional role that DAF plays has been investigated by many groups. Medof et

al.[134] showed that DAF, incorporated into DAF-deficient sheep erythrocytes, inhibits

formation of C3 and C5 convertases. DAF rapidly dissociates C2a and Bb complement proteins

from their binding sites, inhibiting formation of C3 convertases. The exact mechanism of action

of DAF has yet to be delineated.

19

1.3.5.1 Cromer (Cr) Blood Group Sytem

Cromer is the 21st blood group system according to the Interanational Society of Blood

Transfusion (ISBT). The Cromer blood group system resides on the complement regulator, DAF.

In 1965, this antigen was discovered in a woman, named Mrs. Cromer, whose serum contained

antibodies that reacted to all other RBCs except her own. These antibodies were named anti-

Goa[135], until Stroup and McCreary[135] renamed it anti-Cr

a after discovering 4 additional

examples of the same antibody. The null phenotype for the Cr blood group system was

discovered in 1982 in a man named, Inab[136]. By 2002, the number of Cr antigens totaled to

11, divided into high (Cra, Tc

a, Dr

a, Es

a, IFC, WES

b, UMC, GUT1) and low (Tc

b, Tc

c, WES

a)

frequency variants. Three additional high frequency variants, named ZENA, CROV and CRAM,

were discovered in 2007 by Hue-Roye et al.[137] DAF consists of four SCRs to which the Cr

antigens have been mapped using DNA sequencing and mutant proteins expressed in Chinese

hamster ovarian (CHO) cells. The Tc, Es and WES antigen variants are located on SCR1; Dr

variants on SCR3; and, Cr, GUT, UMC variants located on SCR4[138-140]. The molecular basis

of these antigens can be seen in Table 1.

Table 1. Molecular Basis of Cr Antigens

Antigen Nucleotide

Substitution

Change in

Amino Acid

Cra+Cr

a- G679C

G155T

G155C

C596T

T239A

G261A

C262A

G245T

Ala193Pro

Arg18Leu

Arg18Pro

Ser165Leu

Ile46Asn

Trp54stop

Ser54stop

Arg48Leu

TcaTc

b

Tcc

Dra+Dr

a-

Esa+Es

a-

IFC (null)

WESbWES

a

UMC C749T Thr216Met

GUT1 G719A Arg206His

1.4 FcγR-mediated Immune Response

1.4.1 Fc-gamma receptor (FcγR) Biology

FcγRs are part of the immunoglobulin superfamily[141]. For every antibody class (IgA, E, M, D

and G) there is a specific FcR. For example, FcγRs bind to IgG.[142;143] In addition, FcγRs can

20

be either activating or inhibitory[144]. Those receptors that are activating have a tyrosine-based

activation motif (ITAM)[145] whereas, inhibitory receptors possess a tyrosine-based inhibition

motif (ITIM). There are 3 types of FcγRs, which include FcγR I, II, and III[144;146;147]. These

types differ in molecular weight, cellular distribution, and IgG affinity[148;149]. FcγRI (CD64)

is a high affinity receptor, whereas, FcγRII (CD32) and FcγRIII (CD16) are low affinity

receptors[141;150;151]. All FcγRs contain Ig-like domains and each is composed of 7 anti-

parallel β-strands connected by peptide loops[152]. High affinity receptors (FcγRI) have 3 Ig-

like domains while low affinity receptors (FcγRII, III) have only two[153]; these extracellular

domains are conserved between individuals[154]. FcγRs also possess a single membrane

domain—with the exception of FcγRIIIb, which is anchored to the membrane with a

glycosylphosphatidylinisitol (GPI) link—and a cytoplasmic domain of variable length according

to the specific receptor[155]. The genes that encode each FcγR class are located on chromosome

1[155]. Genetic polymorphisms of these receptors have been well characterized[146;147;156-

160].

1.4.1.1 FcγRI (CD64)

FcγRI is a 71 kDa, high affinity receptor that is mainly expressed on myeloid cells, including

human monocytes/macrophages and the cell lines U937, HL60[161] and THP-1[162]. Using

flow cytometry and binding assays, it was shown that monoclonal antibodies to FcγRI and II

bound to THP-1 cells, while those of FcγRIII failed to bind[162]. There are 35 000 FcγRI

molecules per THP-l cell. Expression of this receptor is upregulated when THP-l cells are treated

with interferon-gamma (IFNγ)[162;163]. FcγRI preferentially binds to monomeric IgG1 and IgG3

with an affinity of 5 x 108 M[164]. The third extracellular Ig-like domain, which is not present

on FcγRII and FcγRIII[146], is likely responsible for this high affinity binding.

1.4.1.2 FcγRII (CD32)

FcγRII is a 40 kDa, low affinity receptor[148] that has the widest cellular distribution with

receptor expression on monocytes/macrophages, neutrophils, eosinophils, platelets, and B-

cells[141;151;165;166]. FcγRII is expressed as 2 subtypes, IIb and IIa; IIa has 2 allelic forms

known as high responder (HR) and low responder (LR).[167-169]; this receptor preferentially

binds to aggregated IgG[143;166]. These variants correspond to the binding affinity, which is

21

dependant upon certain amino acids within the ligand binding site. Three nucleotide (nt)

substitutions, GATG (nt 192-3) and GA (nt 505), correspond to a change of glutamine (Gln)

to tryptophan (Trp) at position 27 and arginine (Arg) to histidine (His) at position 131[143;148];

these mutations occurred within the second Ig-like domain[148]. Phagocytic assays have

demonstrated that Arg131 is essential for IgG1 binding while the amino acid change at position

27 does not affect IgG binding affinity [141;149;170]; expression levels of receptor allotypic

variants did not affect binding as they were equal between HRs and LRs[143]. These allotypic

variants play an important role in the function of FcγRII. In particular, the A131H polymorphism

has been associated with diseases such as, systemic lupus erythematosis (SLE),

thrombocytopenia, and bacterial infections[160;171-173].

1.4.1.3 FcγRIII (CD16)

FcγRIII is a medium affinity[141] receptor that is expressed on macrophages,

polymorphonuclear cells (PMN)s, natural killer cells (NK), and monocytes.[141;174-176] In

human males, monocytes express 32,400 receptors, whereas in females, monocytes express

41,000 receptors[177]. FcγRIII is the most glycosylated of all the FcRs[146] and it is anchored

into the cell membrane through a glycosyl-phosphatidylinositol (GPI) linkage on

PMNs[178;179]. Like, FcγRII, the genes for FcγRIII are located on chromosome 1[154]. FcγRIII

also has 2 subclasses, IIIa and IIIb. FcγRIIIa has functional polymorphisms that govern the

binding affinity to IgG. The most common polymorphism is FcγRIIIa-158 valine (V) versus

phenylalanine (F)[180-182]. Possessing homozygosity for FcγRIIIa-158V (as well as FcγRIIa-

131H) has been significantly related to patients with immune thrombocytopenic purpura (ITP); it

has been shown that FcγRIIIa-V binds IgG1 and IgG3 more effectively than FcγRIIIa-F[183]. In

addition, FcγRIIIa-V has a higher affinity for IgG1 than for IgG3.

1.4.2 Macrophage Phagocytosis Activated by FcγRs

Particles within the body destined for destruction and elimination are opsonized with antibodies.

IgG opsonized particles then bind to macrophage FcγRs, which are activated in order to initiate

the phagocytic process. Both FcγRI and FcγRIIa possess ITAMs, which contain tyrosine residues

that become phosphorylated when aggregation of the FcγRs occurs[146]. Upon FcγR activation,

a variety of intracellular activities are initiated: tyrosine kinases are activated, phosphorylation of

22

phospholipase C (PLC) occurs, and intracellular calcium is released. Other biochemical

pathways are also initiated, which lead to the activation of PKC and the Ras pathway.

Using THP-1 cells, an in vitro assay demonstrated that activation of FcγRI and FcγRII activated

similar intracellular proteins, which included phosphorylation of Syk kinases, as well as PLCγ1,

PLCγ2, Vav, and GAP; CD45 inhibited these activities[184]. The authors conclude that

activation of both FcγRI and FcγRII are associated with the same intracellular signaling cascade.

Activation of PLC and PKC leads to the increase in intracellular calcium, while activation of the

Ras pathway leads to the activation of transcription factors and upregulation of required

genes[146]. Stimulation of FcγRs leads to macrophage membrane extension around the IgG

opsonized particle and subsequent phagocytosis. Once inside the macrophage, lysosomal

enzymes are secreted into the phagosome destroying the ingested particle. Complement proteins,

also on the IgG opsonized particles, bind to CR1, which aids in the phagocytic process[164].

1.4.2.1 Role of CRs and FcγRs in the Attachment and Ingestion Phases of Phagocytosis

Early studies have shown that phagocytosis can be separated into two distinct phases:

attachement and ingestion. Using an in vitro system, it was demonstrated that both binding and

ingestion are separate stages being characterized by certain requirements for each stage to occur.

CRs and FcγRs play essential roles in the interaction between sensitized RBCs and

monocyte/macrophages. Literature review has shown that each receptor has a unique role; CRs

are for attachment of immune complexes to macrophages, while FcγRs are responsible for

phagocytosis[125-127]. Mantovani et al.[126] has shown that when anti-mouse IgG F(ab‘)2

fragments are bound to RBCs sensitized with IgG and complement, ingestion of RBCs

significantly decreases, while attachment of RBCs is essentially unaffected. The F(ab‘)2

fragments do not inhibit binding to CR1 and therefore, RBC remain attached to the macrophages.

In an earlier investigation, Mantovani et al.[125] demonstrated that as the number of IgG

molecules on the surface of RBCs increase, so does phagocytosis, emphasizing the role of FcγRs

in mediating phagocytosis. In order for macrophages to interact with RBCs approximately 103-

104 IgG molecules are required. They also showed that with the addition of complement,

attachment significantly increased; however, phagocytosis increased then reached a plateau.

Furthermore, it was shown that antibodies to complement on the RBCs inhibited both the

23

attachment and ingestion of RBCs coated with IgG and complement, while that of IgG-coated

RBCs remained unaffected. Antibodies to IgG, however, prevented the binding and ingestion of

IgG-coated RBCs. All of these experiments elucidated the role of FcγRs in phagocytosis of

sensitized RBCs.

Both FcγRs and CRs can act synergistically to affect levels of phagocytosis. An in vitro study

conducted by Frank et al.[185] demonstrated that a heterodimer of IgG and C3 bound to RBCs

significantly increases phagocytosis when compared to that of RBCs opsonized with IgG or C3

alone.

1.5 ABO Blood Group System

The ABO blood group system is the first system recognized by the ISBT and is the most

important of all 29 blood groups. The ABO blood group antigens are inheritable, polymorphic

traits that are governed by a single gene, located on chromosome 9q34, and three alleles; A, B

and O. The classification of this blood group into distinct phenotypes and characterization of the

mechanism of inheritance have been described by Landsteiner[186;187] and Bernstein,

respectively[188]. Natural antibodies are produced against the antigen that is not present. This

serves as an important basis for the safety of transfusion medicine.

1.5.1 The ABO gene: Glycosyltransferases

The ABO gene encodes glycosyltransferases, which are enzymes that transfer specific sugar

moieties to a predetermined substrate, such as glycoproteins and glycolipids, producing the

respective blood group antigens (Figure 2). These substrates have been identified as chains 1-4.

Sugars are predominantly added to type 2 chains to produce RBC blood group antigens. The

blood group A gene encodes galactosaminyl transferase and adds N-acetylgalactosamine to type

2 chains; whereas, the B gene encodes a galactosyl transferase, which adds D-galactose to type 2

chains. The absence of A or B antigens corresponds to blood group O, or H. The H antigen is

produced by a fucosyl transferase, which adds L-fucose to type 2 chains. The H antigen serves as

the precursor for A and B antigens. In addition, the H gene (FUT 1) is independent of the ABO

gene locus[189]. A rare phenotype, discovered in 1952, that has been observed within the human

population is the Bombay (Oh) phenotype. These individuals do not possess any H antigen on

24

their RBC membranes; therefore, they also do not carry any A or B antigens. Consequently,

these individuals possess anti-A, -B, and –H antibodies.

Figure 2. Synthesis of ABO and Lewis antigens. Oligosaccharide precursor chains, Type 1 and

Type 2, are outlined in black. Links between carbohydrates are written beneath the bond (ie.

β1,4). Terminal carbohydrates determine the antigen. Figure modified from Blood Banking and

Transfusion Medicine, 2nd

ed.[112]

1.5.1.1 The Structure of the ABO glycosyltransferases

The glycosyltransferases encoded by the ABO gene exists as a membrane bound protein. It has 4

domains, which include the cytoplasmic tail, transmembrane domain, stem, and the catalytic

R

R

R

R

β1,4 β1,3 β1,4

R

R

R

R

β1,3 β1,4

β1,3

R

R

α1,2

α1,2

α1,3

A gene

B gene

H (FUT1)

gene

Se (FUT2)

gene

Le

(FUT3)

gene

Se (FUT2)

gene

H

H

A

A

B

B

Le(a)

Le(b)

Type 2 Sugar Chain:

RBC antigens

Type 1 Sugar Chain:

soluble antigens

= galactose

= N-acetylglucosamine

= fucose

= N-acetylgalactosamine

Key

B

C

Key

Precursors

25

activity domain. The ABO gene contains 7 exons, each of which governs a different domain of

the glycosyltransferase. The most important exons that are considered when studying ABO

subgroups are exons 6 and 7, which are responsible for the catalytic domain. Four critical single

base pair mutations exist that allow the A and B glycosyltransferases to differ from one another.

Specifically, these are C526G, G703A, C796A, and G803A. Additional mutations exist to

distinguish A1 individuals from A2 individuals and between O1, O

1v, and O

2/O

03 individuals.

These mutations change the efficiency and/or the sugar specificity of the glycosyltransferase. As

a result, different antigen subgroups are expressed and in different copy numbers. Researchers

use the exon sequences from A1 RBCs as a consensus sequence to which all other antigen

subgroups and different blood group antigen sequences are compared.

1.5.2 Differences Between Blood Groups A1 and A2

The differences between A1 and A2 RBCs are both quantitative and qualitative; however,

different investigating groups emphasize one over the other. One major distinction is that A1

RBCs possess more A epitopes on their membranes than A2 RBCs do (ie. 4 times as

many)[190;191]. Additionally, A1 RBCs contain a different sugar chain than A2 RBCs. The

precursor for the A antigen is an H epitope present on type 2 sugar chains. A2 RBCs possess a

type 3 sugar chain, which, essentially, is an H type 2 sugar chain with an internal αGalNAc (N-

acetylgalactosamine)[192;193]. The A1 transferase, and not the A2 transferase, is able to attach

an additional αGalNAc residue to type 3 chains; thus, creating a type 4 sugar chain. Based on

immunochemical analysis using the A1-specific monoclonal antibody (moAb), TH-1, it was

determined that the qualitative difference between A1 and A2 RBCs is the presence of A type 4

glycolipids[192;193].

1.6 Lewis Blood Group System

The Lewis blood group system is the 7th

system recognized by the ISBT. It is governed by the Le

gene (FUT3), which is located on chromosome 19. The gene codes for a fucosyltransferase that

covalently attaches a fucose residue in an α1-4 linkage to N-acetyl-D-glucosamine in type 1

sugar chains[194]. Type 1 chains are present in saliva and plasma. The gene has two alleles,

which create 3 genotypes: LeLe, Lele, lele. The le allele is considered to be silent. Lewis

antigens are directly inserted into RBC membranes through the glycolipid portion of the

26

chain.[194] The presence of Lewis antigens in the plasma/saliva and on RBCs is also influenced

by the secretor gene Se (FUT2).

1.6.1 Soluble ABO Substances

FUT2 encodes a fucosyltransferase that attaches fucose in an α1-2 linkage to the terminal

galactose on type 1 sugar chains; thus, creating type 1 H chains, which allows for the production

of soluble ABO antigens (Figure 2). In the presence of Se and Le alleles, 2 fucose residues are

attached to type 1 chains creating the Le(b) antigen. RBCs, therefore, react with anti-Le(b)

antibodies and are labeled as Le(a-b+) ABO secretors. In the absence of the Se allele, Le(a)

antigens are produced with the addition of a single fucose residue; hence, an Le(a+b-) ABO

nonsecretor. Furthermore, possessing the lele genotype, regardless of which FUT2 alleles are

present, will produce no Lewis antigens, Le(a-b-). RBCs that are Le(a-b-) do not possess Lewis

antigens on their membranes and, as a result, secretor status cannot be

determined by serological testing. A study was conducted in which the authors compared ABO

genotype to secretor status and it was found that A1 or A1B secretors have a greater amount of

soluble A substance in the plasma versus A2 or A2B nonsecretors (Figure 3).

Figure 3. The effect of secretor status and blood group on soluble A substance (SAS) in

the plasma. Fig 1. taken from Achermann et al.[195]

27

1.7 Rationale

Understanding the pathophysiology by which ABO antibodies cause hemolysis can be clinically

beneficial to patients receiving transfusion products. Although rare, IVIg is known to cause

hemolysis after high dose regiments. The hemolysis has mainly been observed in patients who

are either blood group A or AB (less commonly group B, seldom group O) and who received a

total dose of ≥ 2 g/Kg of IVIg. The effect of IVIg dose on hemolysis will be evaluated by

investigating the in vitro role of the anti-A and anti-B antibodies in IVIg to activate complement

C3 and to mediate or enhance RBC phagocytosis. Currently, the conditions by which IVIg anti-

A/B activate complement and cause RBC destruction is unknown. Many group A, AB, and B

patients receive IVIg without any incident or clinical evidence of hemolysis.

1.7.1 Hypothesis

IVIg Anti-A and anti-B binds to blood group antigens on RBCs and activates complement in a

dose-dependant manner. This IVIg-induced complement activation may enhance FcγR-mediated

phagocytosis of IVIg sensitized RBCs.

1.7.2 Objectives

The main objectives of this study were to: define the relationship between anti-A/B binding,

IVIg dose and blood group; determine the effect of IVIg dose on complement activation; and,

evaluate the effects of IVIg-induced complement activation on RBC destruction using an in vitro

bioassay and flow cytometric analysis.

28

Chapter 2

Materials and Methods

29

2.1 Cell Line: THP-1

THP-1 is a myeloid leukemic cell line derived from a boy with acute monocytic leukemia[196].

This cell line possesses FcγRI ans II and CR1. THP-1 cells (ATCC), frozen in liquid nitrogen,

were thawed in a 37oC water bath and washed once in culture media. On the fourth day the

culture was split and new media was added. Subsequently, cells were maintained in logarithmic

growth (2-5 x 105 cells/ml) by passage every 3 days. Cells were cultured in Gibco‘s RPM1 1640

medium (Invitrogen) containing 2mM L-glutamine supplemented with 10% fetal calf serum, 100

µg/ml streptomycin, 100 U/ml penicillin, and 5x105

mM mercaptoethanol and were maintained

at 37oC in a humidified atmosphere with 5% CO2. Cell viability and count were determined by

using trypan blue (Gibco, U.S.A.) exclusion and a hemocytometer, respectively. For the

phagocytosis experiments, THP-1 cell viability was determined by using a flow cytometric assay

(section 2.6.2) [Appendix H].

2.2 Donors

This study used blood samples from 54 randomly chosen, healthy volunteers. Blood samples

were obtained after written informed consent and in accordance with Review Board approved

protocol. Age, gender, or ethnicities were not considered in obtaining consent. Four additional

donors were obtained for use in the in vitro complement activation and phagocytic assays. Donor

samples were centrifiuged and plasma, RBCs, and buffy coat separated. Plasma and buffy coat

were stored at -80oC, while RBCs were frozen using the PVP method (protocol available from

(http://www.uni-ulm.de/%7Efwagner/RH/RB/2006) and stored in liquid nitrogen.

2.3 Intravenous Immunoglobulin (IVIg)

The IVIg preparation used in this study was manufactured by Talecris Biotherapeutics Inc. The

specific product was Gamunex (Lot. 26N6KX1), 10% Caprylate/Chromatography purified.

2.4 Effect of IVIg Dose on Anti-A/B Binding to RBCs in Whole Blood

To determine whether IVIg anti-A/B binding occurred in a dose-dependant manner, whole blood

was treated in vitro with varying concentrations of IVIg. Binding was assessed by direct

antiglobulin tests (DATs) and agglutination scores were recorded as described by the AABB

Techincal Manual (Appendix A)[197]. For each donor, two vials of whole blood were pooled

30

and 1 ml aliquots were transferred to four 10x75 plastic culture tubes. IVIg was added to give

final concentrations of 8, 20, 40, and 80 mg/ml, which was equivalent to 0.4, 1, 2, and 4 g of

IVIg infused per kilogram of body weight[198]. The tubes were capped and mixed gently by

inverting 6-8 times. For each sample, a 5% RBC suspension was made to perform the DAT. One

drop of 5% RBCs was added to a new 10x75 glass culture tube. Cells were washed with

phosphate buffered saline (PBS) 4 times, after which 2 drops of Novaclone Anti-IgG (murine

monoclonal blend; Lot. NIGG3806) was added and centrifuged on high for 15 s (seconds).

Aliquots of each group A sample were mixed with an anti-A1 lectin (Ortho, Dolichos Biflorus) to

detect the presence of the A1 antigen only. If the test was negative, the sample was considered A2

positive. Agglutination scores (determined according to the AABB technical manual [Appendix

A]) of each sample were compared to their ABO genotypes and Lewis phenotypes to evaluate

the effect of membrane-bound and soluble ABO antigens, respectively, on the level of IVIg-anti

A/B binding.

2.4.1 ABO Genotyping: DNA Extraction and Quantification

DNA extraction was performed by utilizing the Qiagen Kit (Mississauga, Ont, Can.). Blood

samples were centrifuged at 2600 g for 5 mins (minutes) at 4oC. Plasma and buffy coat (white

blood cells) were removed and stored in 1.5 ml microtubes at -20oC. Frozen buffy coat was

thawed at 37oC for approximately 10 min. Approximately 500 µL of nuclease-free water was

added and samples were microcentrifuged at 2000 rpm for 2 mins. The supernatant was removed

and washed again with nuclease-free water 2-3 times. After the last wash, 250 µL of nuclease-

free water was added and the microtubes were vortexed. In clean microtubes, 20 µL Qiagen

protease, 200 µL of washed buffy coat, and 200 µL of AL buffer were added; samples were

vortexed and incubated for 10 mins at 56oC. After incubation, 200 µL of ethanol was added and

the microtubes were vortexed.

The contents were transferred to QIAamp filter+collection tube and microcentrifuged at 8000

rpm for 1 min. The filters were transferred to another collected tube; 500 µL of AW1 wash

buffer was added and the samples were microcentrifuged again at 8000 rpm for 1 min. The filters

were transferred again to new collection tubes, where 500 µL of AW2 wash buffer was added.

The tubes were microcentrifuged at 13 000 rpm for 3 mins. The filters were transferred to clean

31

microtubes, 200 µL of 2mM Tris-HCl (pH=8.3) was added and the microtubes were incubated at

70oC for 1 hr. The samples were centrifuged one last time at 8000 rpm for 1 min. Extracted DNA

was stored at -20oC. DNA quatification was done using 1/20 dilution of stock DNA;

concentrations were read using Life Science UV/Vis Spectrophotometer.

2.4.1.1 ABO PCR

To determine the ABO genotype of the blood samples, PCR was performed according to the

procedure outlined by M. Olsson et al.[199] with slight modifications. Briefly, a master mix of

25.6 μL nuclease free water (Promega Lot. 25228701), 5 μL 10X PCR Buffer (Lot. JP9099), 3

μL 25mM magnesium chloride (MgCl2) solution (Lot. JP4985), 5 μL 2mM dNTP (Fermentas,

Lot. 00023887), 0.5 μL of exon 6 primers (mo46, mo57) and exon 7 primers (mo71, mo101)

[Invitrogen], and 0.4 μL Ampli-Taq Gold with GENEAMP (Applied Biosystems, Roche) was

prepared for ‗n‘ number of samples plus 1. The master mix was aliquoted into PCR tubes, each

containing 40 μL, of which either 10 μL of nuclease-free water (reagent control) or extracted

DNA [Section 2.3.1] (25μg/mL) was added. The PTC 200 DNA Engine SystemTM

Thermacycler

was utilized for thermocycling with different conditions for exon 6 and exon 7. The PCR for

exon 6 starts with an initial denaturation at 85oC for 8 mins, then 12 cycles of denaturation at

94oC for 30 s, annealing at 65

oC for 90 s and extension at 72

oC for 90 s, followed by 25 cycles of

denaturation at 94oC for 30 s, annealing at 61

oC for 90 s and extension at 72

oC for 90 s. The final

elongation was at 72oC for 10 mins. The PCR conditions for exon 7 is the same except that the

second round of denaturation, annealing and extension is repeated 27 times with the annealing

step occurring at 59oC for 45 s. PCR products were stored at 4

oC until separated by

electrophresis for 35 mins at 100 mV using 2% agarose gels. The remaining PCR products were

stored in a -20oC freezer until used for restriction enzyme digestion.

2.4.1.2 ABO exon 6 and 7 Restriction Enzyme Digestion

ABO exon restriction digestion was performed after ABO exon PCR. A master mix of nuclease-

free H2O, 10x restriction enzyme buffers (Fermentas) including, KpnI (exon 6) or Tango (exon

7), and restriction enzymes (Fermentas), KpnI (exon 6) or HpaII (exon 7), was prepared for ‗n‘

number of samples plus 1. Five µl was aliquoted to each PCR tube that contained 10 µl of either

nuclease-free H2O or PCR product at 50 ug/ml. The tubes were vortexed and spun to remove

32

bubbles. The digestion was performed in the Thermocycler for 2 hours at 37oC. The reaction was

stopped by adding 3 µl of 6x loading dye (Fermentas, Cat. No. R0611). Digested products were

separated by electrophoresis for 55 mins at 200 mV using a 12% polyacrylamide gel in 1x TBE

buffer (0.0089 M Tris, 0.089 M Boric Acid, 0.002 M EDTA).

2.4.2 Assessing the Level of Membrane-bound A-antigens

To correlate the amount of membrane bound A-antigen to genotype, blood samples were

analysed by flow cytometry. The protocol for A-antigen quantification was derived from a

previous protocol by Yazer et al.[200] Briefly, PVP frozen RBCs were thawed and washed 3

times with PBS, making a final 5% suspension of washed RBCs. In a 96-well, round bottom

microplate, 2.5 µL of 5% RBCs was added to 50 µL of PBS. RBCs were then fixed with 100 µL

of 1% glutaraldehyde for 10 mins under constant mixing to prevent agglutination of A-antigen

positive cells. The cells were centrifuged for 5 mins at 300 g and the supernatant discarded. Fifty

µL of PBS was added to each well along with 2 µL of FITC anti-A antibody (BD Pharmingen;

clone J606, Cat. No. 555578). Cells were incubated at room temperature for 20 mins.

Approximately 200 µL of stain buffer [SB] (BD Pharmingen cat. 554656) was added to the

wells, which were centrifuged at 300 g for 5 mins. The supernatant was discarded and the

samples were resuspended in 300 µL of SB. For each sample, 10 000 events were acquired

through a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lake, NJ) and analysed

with CellQuest Pro software. Group O RBCs served as the negative control. Detection of the

FITC-label was calibrated on the flow cytometer using a mouse IgG1 isotype control (AbD

Serotec, U.S.A.)

2.4.3 Lewis Blood Group Serology Test

Presence of soluble A antigens was assessed by determining the secretor status of only group A

blood samples. Secretor status is indirectly determined by evaluating the presence or absence of

Lewis antigens. Briefly, PVP frozen RBCs from each sample were thawed and washed 3 times

with warm PBS. After each wash, RBCs were centrifuged on high for 15 s. A 5% solution of

RBCs was prepared for testing. One drop of 5% RBCs was added to a 10x75 glass culture tube,

along with 1 drop of either anti-Le(a) or -Le(b) (Immucor Gamma) antibodies. Samples were

33

centrifuged on high for 15 s, after which, agglutination was scored according to the AABB

technical manual (Appendix A).

2.5 Complement Activation with IVIg

To determine the effect of IVIg dose on complement activation, an in vitro assay was employed.

Samples of each blood group (O, A, B, AB) were taken from 4 healthy donors. For each blood

group, whole blood was aliquoted to four 12x75 glass tubes. Stock IVIg at 100 mg/ml was added

to give final concentrations of 8, 20, 40, and 80 mg/ml, which correlated to 0.4, 1, 2, and 4 g/Kg

of body weight. Whole blood incubated with PBS served as background complement activation.

The blood was incubated at 37oC for 20 mins and then washed 4 times with cold PBS

(containing MgCl and CaCl, Gibco 14040). Packed RBCs were resuspended to a 3% solution

with fresh human AB serum (Appendix F). The samples were then incubated at 37oC for 20 mins

and washed 4 times with cold PBS (containing MgCl and CaCl). Each sample was resuspended

in PBS to a 5% solution. Complement proteins were detected by flow cytometry (Appendix C).

Serology was used to detect the presence of bound IgG before and after serum incubation using a

diagnostic monoclonal anti-human IgG (Immuncor, Lot. NIG04401) [see Table 9].

2.5.1 Flow Cytometric Analysis of Complement Proteins

Flow cytometry was used as a means to detect C3d deposition on RBCs. Briefly, 2.5µl of 5%

RBCs was added to 50µl of SB after which 2µl of mouse-anti-human anti-C3d or –C3a

(Cedarlane, Cat No. C7850-14D2, C7580-13K, respectively) were added. Values were compared

to a mouse IgG1 negative control (AbD Serotec, U.S.A.). Cells labeled with anti-C3a, served as

an internal negative control, since C3a is cleaved from C3b and does not stay bound to the RBC.

All samples were incubated with a secondary FITC-conjugated goat anti-mouse IgG (Jackson

Laboratories, U.S.A., Lot. 71597) to label non-fluorescein-conjugated antibodies. For each

sample, 10 000 events were acquired through a FACSCalibur flow cytometer (Becton Dickinson,

Franklin Lake, NJ) and analysed with CellQuest Pro software.

2.5.2 IVIg anti-A/B Adsorption

The following protocol to remove anti-A/B IgG from IVIg was obtained from Shoham-Kessary

et al.[36] Briefly, washed, packed AB+ RBCs were incubated twice with IVIg (100 mg/ml) in a

34

10% solution 2 hours at 37oC. After incubation, the sample was centrifuged for 10 minutes at

1500 rpm. The supernatant, containing IVIg without anti-A/B IgG, was stored at -20oC until used

when performing the rosette inhibition assay.

2.6 Inhibition of Rosette Formation by IVIg

The rosette inhibition assay protocol employed was modified from the phagocytic assay from

Aslam et al[201]. Briefly, THP-1 cells were washed and concentrated in culture media to obtain

5x105 cells/100μL. The cells were pretreated with soluble IgG at 1, 10 and 100 µg/ml, 69 µg/ml

F(ab‘)2, 47 µg/ml BSA, or no inhibitor for 20 mins at 37oC. Frozen Rh-positive (R2R2) RBCs

were thawed, washed 3 times, and resuspended in PBS to a 10% solution. One hundred μL of the

10% solution was transferred to 5 eppendorf tubes and then sensitized with an equal volume of

either 10 μg/ml final concentration of polyclonal anti-D (WinRho SDF), 20 or 80 mg/ml final

concentration of IVIg, 20 mg/ml adsorbed IVIg, or PBS. RBCs were incubated at 37oC for 45

minutes, washed 3 times with cold PBS at 1000g for 5 mins at 4oC and then resuspended to a

20% solution (400 x 106/200 µl). One hundred μl of THP-1 cells were transferred to 12x75 glass

tubes. Treated RBCs were added to obtain an RBC:THP-1 ratio of 30:1 and then centrifuged on

high for 15 seconds. Samples were incubated at 37oC for 2 hours. Samples were gently

resuspended and rosettes were observed and counted on glass slides using an inverted

microscope.

2.7 RBC Phagocytosis Bioassay

The phagocytosis assay utilized in this study was derived from Aslam et al.[201], and modified

to assess phagocytosis of human RBCs. Briefly, whole blood was washed 3 times at 1000 rpm

for 15 mins to remove platelet rich plasma (PRP). The RBCs were then washed 5 times at 1500

rpm for 15 mins to remove buffy coat. Packed RBCs were resuspended up to a volume of 10 ml

with PBS. The RBC were then labeled with 20 µL of CellTrackerTM

green CMFDA (Invitrogen,

Lot. 432927) and incubated in the dark for 20 mins at 37oC. The RBCs were washed once at

1000 rpm for 10 mins, the supernatant discarded, resuspended up to a volume of 10 mL with

PBS, and incubated for another 15 mins in the dark at 37oC. The cells were washed again at 1000

rpm for 10 mins and stored overnight with PBS supplemented with citrate phosphate dextrose

(CPD) [Sigma, Lot. 127K8612]. RBCs were then counted, readjusted to 400 x 106 cells/200 µL

35

(20% RBC solution) and sensitized (see 2.6.1). THP-1 cells were prepared by washing with PBS

twice and resuspending in 1-5 ml of PBS. The cells were counted and adjusted to 10 x 106

cells/ml. THP-1 cells were then activated with 10 µL of a 1:100 dilution of phorbol-12-

myristate-13-acetate (PMA) [Sigma, P8139-1 MG; stock at 1µg/µl] per 10 x 106 cells/ml. The

cells were incubated at room temperature for 15 mins under constant mixing, then washed once

with PBS, and re-adjusted to 5 x 106 cells/mL with 16% Iscove‘s DMEM (Gibco). A 30:1

RBC:THP-1 ratio was used for the phagocytosis assay. The phagoyctosis reactions took place at

37oC. The controls that were tested were non-opsonized RBCs (background control), C3b alone,

anti-D alone (positive control), and C3b + anti-D. Test samples included RBCs sensitized with

IVIg (at 8, 20, 40, or 80 mg/ml) and with IVIg + C3b. THP-1 cells alone was used as the

negative control. Incubation was for 2 hours in the dark. After incubation, excess fluorescence

was quenched by addition of 100 µl of cold 1:1 PBS:Trypan Blue (Gibco, U.S.A.). Cells were

washed once with cold PBS, followed by treatment with 2 ml of Lysing Solution (BD FACS, Cat

No. 349202) for 15 mins on ice in the dark. The cells were washed and then, THP-1 cells were

labeled with 200 µl LDS (Invitrogen, Cat No. L7595) for 25 mins. FACS analysis was

performed gating LDS-positive cells through the FL3 gate. CMFDA positive cells were acquired

through the FL3 gate in FL1. This was a measure of internal THP-1 fluorescence. Ten thousand

events were acquired through a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lake,

NJ) and analysed with CellQuest Pro software. In some experiments, THP-1 cells were

pretreated with either soluble IgG (100, 10 or 1 µg/ml), F(ab‘)2 (69, 6.9 or 0.69 µg/ml) or BSA

(47 µg/ml) for 20 mins at 37oC. Anti-D RBCs were added directly to the THP-1 cells and the

phagocytosis assay proceeded as outlined above.

2.7.1 Setting the Phagocytosis Assay Controls

Whole blood samples were taken from Rh+ donors (A+, B+). Samples were centrifuged

(1500 rpm, 10 min) and the plasma was separated and stored at 4oC prior to CMFDA labeling

(see 2.6). Subsequent to CMFDA labeling, RBCs were sensitized in the following manner. Two

hundred µl of reconstituted whole blood was added to four 14 ml conical tubes (Falcon). Ten µl

of anti-D (Appendix D) was added to 2 tubes and 10 µl of PBS was added to another 2 tubes. For

the two tubes with anti-D, 2 ml of PBS and 2 ml of 10% sucrose (10g Sucrose, 100 ml distilled

water) was added separately[202]. A 10% sucrose solution provides a low ionic environment that

36

allows C3b to be deposited on the RBC membrane without prior attachment of antibodies

(Appendix E). The same procedure was done for the RBCs incubated with PBS alone. RBCs

incubated at 37oC for 15 mins and then washed 3 times with PBS (Mg

2+, Ca

2+). Packed RBCs

were resuspended to a 20% solution in PBS and then subsequently used in the phagocytic assay.

Thus, the following four controls were available for analysis: anti-D RBCs, anti-D + C3b RBCs,

C3b RBCs, untreated RBCs. IVIg test samples were prepared as in section 2.4; however, packed

RBCs were resuspended to a 20% solution.

2.7.2 Flow Cyometric Analysis of THP-1 Cell Viability

To determine cell viability by flow cytometry the Annexin V-FITC Apoptosis detection kit was

used (BD Pharmingen, Cat. No. 556547). THP-1 cells were adjusted to 2 x 105 cells/100µl with

binding buffer and then stained with 10 µl of FITC-Annexin V and propidium iodide (PI).

Unstained THP-1 cells were used to set the boundaries for positive staining, while THP-1 cells

stained with either FITC-Annexin V or PI were used as negative controls. Samples were

acquired through a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lake, NJ) and

analysed with CellQuest Pro software.

2.8 Statistical Analysis

All statistical analyses were performed using Microsoft excel worksheet. A paired student t-test

was performed to determine if the level of C3d-positive RBCs treated with 80 mg/ml of IVIg

compared to those treated with 40 mg/ml of IVIg differed significantly. The same test was

employed to determine if the phagocytosis of anti-D RBCs was significantly inhibited by THP-1

pretreated with soluble IgG, IgG F(ab‘)2, or BSA. Additionally, this statistical test was used to

determine if C3b deposition significantly enhanced RBC phagocytosis. Furthermore, the t-test

was employed to evaluate whether C3b deposition on sucrose versus PBS treated RBCs was

significant, as well as, to determine if IgG binding was significantly altered by sucrose or PBS

treatment. P values less than 0.05 were considered to be statistically significant.

37

Chapter 3

Results

38

3.1 Characteristics of the antibodies in IVIg: Clinical Laboratory Studies

Scientific literature has revealed that administration of IVIg can lead to rare (with a frequency of

approximately 0.1%), but significant hemolysis. The majority of patients who experience

hemolytic reactions in response to IVIg treatment are either blood group A , B or, AB.

Infrequently, group O patients suffer from IVIg-induced hemolysis[62]. As a result of these

published observations, studies have focused on the anti-A and anti-B contained in IVIg

preparations[30;34-36]. The properties of blood group immunoglobulins were investigated and

are reported herein. Clinical laboratory studies performed on Gamunex IVIg directly were done

to evaluate the characteristics of this specific manufacturer‘s preparation (Section 2.3). Further,

in vitro tests were performed by the MSc candidate to determine the presence of anti-A/B and

relative titres after the addition of IVIg to whole blood—based on plasma levels obtained after

therapeutic doses were given[198]—the ability of bound antibody to activate complement, and

whether or not anti-A/B mediates FcγR-dependent phagocytosis.

3.1.1 ABO antibodies in IVIg

The presence of anti-A/B in Gamunex IVIg was investigated directly. Based on the observations

reported in scientific literature, a routine reverse A and B blood group test was used to evaluate

the presence of IVIg anti-A/B by adding commercially available A1 and B red cells (Ortho

Diagnostics) to dilutions of IVIg and examining direct hemagglutination. To evaluate IVIg for

anti-A/B hemolysin activity, additional aliquots of pooled A1 and B red cells were directly

sensitized with IVIg, washed, incubated in fresh A serum at 37oC for 1hour, centrifuged, and the

supernatant was examined for hemolysis[15]. The concentrations of IVIg used were based on the

IgG changes in plasma post IVIg infusion[198]. Table 2 shows that IVIg contains antibodies

towards ABO blood group antigens that bind in a dose dependent manner; however, the

antibodies do not have hemolysin activity. A human serum known to contain anti-A hemolysin

activity was used as a positive control.

To evaluate IVIg for other clinically significant blood group alloantibody activity, an antibody

screen was performed using a saline indirect antiglobulin test (tube method). The IVIg

concentrations used here correlated to the doses employed in the clinical setting: 8, 20, 40, and

39

80 mg/ml. Table 3 demonstrates that IVIg does not contain clinically significant antibodies other

than anti-A/B when evaluated at 8, 20, 40 and 80 mg/ml.

Table 2. ABO antibodies in IVIg

Sample 2mg/ml 5mg/ml 10mg/ml 20mg/ml

A1 W+ 1+ 1+ 2+

B W+ W+ 1+ 2+

hemolysin activity neg* neg neg neg

hemolysin control** pos*

*neg = negative, pos = positive result for hemolysin activity

** the positive control was a serum known to contain anti-A hemolysin activity

Table 3. Blood Group antibodies other than anti-A/B in IVIg

*neg = negative result

3.1.2 Antibody Titres in IVIg

Studies have shown that IVIg contains anti-A/B and previous clinical experiments have

demonstrated greater IVIg binding to red cells when the concentration of IVIg is elevated

(Section 3.1.1.) Normal human plasma contains an average anti-A titre of 16 (Figure 4.1;

Mollison PL)[15]. Since there are no Health Canada guidelines on the appropriate levels of anti-

A/B that IVIg should contain, the European Pharmacopeia is followed, which states that IVIg

should have an anti-A/B titre of <32 in a 3% w/v solution. Therefore, the anti-A titre in IVIg at

20 mg/ml (the highest therapeutic dose) was examined with four lot numbers of Gamunex IVIg

and analysed using a standardized in vitro double dilution indirect antigloubin test and

commercially available A1 red cells (Ortho Diagnostics). The results are expressed as the inverse

of the last double dilution that was positive for macroscopic agglutination. One IVIg lot was

further examined for anti-A titre at various concentrations to evaluate the effect of IVIg

concentration on anti-A titre. Table 4 demonstrates that the anti-A titre in Gamunex IVIg is 16

and that this titre is identical between the 4 IVIg lots tested. Table 4 also shows that anti-A titre

(IVIg Lot: 7321) increases in a dose-dependent manner.

Test 8mg/ml 20mg/ml 40mg/ml 80mg/ml

Ab screen neg* neg neg neg

40

Table 4. Anti-A Titres in Gamunex IVIg

3.1.3 IVIg Anti-A/B and Complement Activation

Clinical laboratory studies were performed to evaluate whether IVIg can fix complement C3

component (C3b) onto red cells in vitro. Commercially available A1 red cells (Ortho

Diagnostics) were incubated with IVIg at 4 concentrations of IVIg in fresh A serum at 37oC for

20 minutes. Again, the concentrations used were based on IgG changes in plasma post IVIg

infusion at therapeutic doses.[198] After washing the red cells, they were incubated in a single

source of fresh AB serum for 30 minutes at 37oC and then washed 4 times again. Membrane

bound IgG and C3b was determined using a diagnostic anti-C3b/d reagent (Bioclone) and a

monoclonal anti-IgG reagent (Novaclone). Table 5 demonstrates that IVIg can fix complement

C3b proteins onto the RBC membrane at concentrations of 10 and 20 mg/ml.

Table 5. Complement Activation and Fixation by IVIg

Sample 2mg/ml 5mg/ml 10mg/ml 20mg/ml

A1, IgG 2+ 3+ 3+ 3+

A1, C3b N N W+ 1+

3.2 In Vitro studies on IVIg with Whole Blood

The clinical laboratory investigations performed directly on Gamunex IVIg provided the

rationale for a study, which was designed to evaluate IVIg anti-A/B-induced complement

activation and subsequent RBC hemolysis on whole blood. In order to mimic the activities of

IVIg in vivo, an in vitro method was employed in which IVIg was added to whole blood and

examined for different parameters. Anti-A/B binding to red cells and residual anti-A/B in plasma

was evaluated after the addition of IVIg at various concentrations to whole blood samples. In

subsequent experiments, samples representing four blood groups were further examined for

IVIg-induced complement activation and in other experiments, the ability of IVIg sensitized red

cells to be phagocytosed in vitro using a monocyte/macrophage cell line, THP-1.

IVIg Lots at 20mg/ml IVIg Lot: 7321

7321 9T71 9T21 9V11 2mg/ml 5mg/ml 10mg/ml 20mg/ml

Titre 16 16 16 16 1 2 4 8

41

3.2.1 The Effect of IVIg concentration on Whole Blood

Clinical studies have shown that IVIg causes a positive DAT at therapeutic doses and elution

studies have shown that anti-A/B can be eluted from the RBC membrane[60;62;203]. The

experiments performed here were designed to measure both red cell bound and free plasma anti-

A/B after IVIg is added to whole blood at various concentrations. When IVIg is incubated with

RBCs, the A/B antibodies from IVIg bind to their respective antigens on the red cell membrane.

If anti-A/B are in excess, then those antibodies that are not bound will be found in the plasma.

3.2.1.1 The Relationship Between Blood Group and IVIg Dose on Anti-A/B Binding

In order to test the relative level of anti-A/B binding on RBCs according to IVIg dose, an in vitro

assay was employed that used whole blood to dilute IVIg to mimic the final concentration after

IVIg administration in vivo[198]. Four aliquots of the 54 blood samples were incubated with 8,

20, 40 and 80 mg/ml final concentration of IVIg, which is equivalent to 0.4, 1, 2, and 4 g/Kg of

IVIg administered, respectively[198]. Then, the DAT was used to assess relative antibody

binding to A, B, AB, and O red cells. Table 6 demonstrates that anti-A/B binding occurs in a

dose dependant manner on the basis of increasing DAT scores. However, some samples show a

gradual increase whereas, other samples show an exponential increase. Table 6 also shows that

IVIg binding to red cells is ABO blood group-dependent since group O RBCs failed to show

IVIg binding.

3.2.1.2 The Effect of A-antigen quantity on IVIg anti-A binding

It was shown that IVIg anti-A/B binding increased in a dose-dependent manner. However, just as

the level of antibody can affect IVIg binding, the quantity of A-antigen present on the RBC

membrane may also influence anti-A binding. To understand possible relationships between anti-

A binding and blood group allelic variants or Group A zygosity, all of the group A blood

samples were evaluated for allele zygosity and A2 expression using an anti-A1 lectin (extract of

Dolichos biflorus seeds), which only binds to the A1 antigen. The zygosity was of the A1 allele

was determined by PCR-RFLP as described in sections 2.3.1.1 and 2.3.1.2.

42

Table 6. IVIg Anti-A/B Binding Associated with Blood Group, A subclass and Genotype

*genotypes determined by PCR-RFLP, sections 2.3.1.1 and 2.3.1.2.

** neg = negative result; pos = positive result

Group **A1 Expression *Genotype

(zygosity)

Final IVIg Concentration in Whole Blood

8mg/ml 20mg/ml 40mg/ml 80mg/ml A pos A1A1 W+ W+ 2+ 2+ A pos A1A1 W+ 1+ 3+ 4+ A neg A2O W+ 1+ 2+ 3+

A neg A2O W+ 1+ 1+ 2+

A neg A2O N W+ 2+ 4+

A pos A1O W+ 1+ 2+ 3+ A pos A1O 1+ 1+ 2+ 2+ A pos A1O 1+ 1+ 2+ 3+ A pos A1O W+ W+ 2+ 3+ A pos A1O N N W+ 4+ A pos A1O N N 3+ 3+ A pos A1O W+ W+ 1+ 2+ A pos A1O N N 1+ 1+ A pos A1O N W+ 1+ 2+ A pos A1O W+ 2+ 3+ 4+ A pos A1O 1+ 3+ 3+ 4+ A pos A1O W+ 1+ 1+ 3+ A pos A1O 1+ 1+ 2+ 4+ A pos A1O N 2+ 2+ 3+ A pos A1O W+ 1+ 3+ 4+ A pos A1O 1+ 2+ 3+ 3+ A pos A1O W+ 2+ 2+ 3+ A pos A1O 1+ 2+ 3+ 3+ A pos A1O 1+ 2+ 3+ 4+ A pos A1O W+ 2+ 2+ 3+ A pos A1O N 1+ 2+ 3+ A pos A1O 1+ 2+ 2+ 3+ A pos A1O 1+ 2+ 2+ 3+ A pos A1O W+ 1+ 2+ 2+ A pos A1O N 2+ 2+ 3+ A pos A1O W+ 1+ 2+ 4+ A pos A1O W+ 1+ 2+ 3+ A pos A1O 1+ 1+ 2+ 3+ A pos A1O 1+ 2+ 2+ 3+ A pos A1O W+ 2+ 2+ 3+ A pos A1O 1+ 2+ 2+ 3+ A pos A1O W+ 2+ 2+ 2+ A pos A1O N N W+ 1+

43

Table 6. Continued.

*genotypes determined by PCR-RFLP, sections 2.3.1.1 and 2.3.1.2.

** neg = negative result; pos = positive result

Group **A1 Expression *Genotype

(zygosity)

Final IVIg Concentration in Whole Blood

8mg/ml 20mg/ml 40mg/ml 80mg/ml

B neg BO1 W+ W+ 2+ 2+

B neg BO1 N N 1+ 3+

B neg BO1 N 1+ 2+ 2+

B neg BO1 W+ W+ 2+ 3+

B neg BO1 1+ 1+ 2+ 4+

B neg BO1 W+ 1+ 2+ 3+

B neg BO1 W+ 1+ 1+ 3+

AB pos A1B 1+ 2+ 3+ 3+

AB neg A2B W 2+ 2+ 4+

AB pos A1B W 1+ 1+ 4+

AB pos A1B 1+ 2+ 4+ 4+

AB pos A1B N 2+ 2+ 3+

AB pos A1B W+ 2+ 3+ 4+

O neg O1O1 N N N N

O neg O1O1 N N N N

O neg O1O1 N N N N

44

3.2.1.3 The Effect of Membrane-bound A/B Antigens on Anti-A/B Binding

The group A samples shown in Table 6 demonstrate variation in IVIg anti-A binding at each

IVIg dose. To determine if the quantity of A antigens influenced IVIg anti-A binding, all 38

group A, (as well as one O, one B and 2 AB) samples were genotyped using PCR RFLP. The

genotypes that were discovered were A1A1, A1O, A2O, A1B, A2B, BO1, and O1O1. A

corresponding restriction enzyme digestion gel can be seen in Appendix B. Flow cytometric

analysis of A-antigen was also performed to display the relative amount of A-antigen according

to genotype (Figure 4). A1 red cells (A1A1, A1O, or A1B) have approximately 4 times the amount

of membrane-bound A-antigens than do A2 red cells. In addition, the zygosity of the A1 allele

does not have an influence on the amount of A antigen on the red cell membrane. Consequently,

this has no effect on IVIg anti-A binding (Table 6). Furthermore, the A2 allele confers reduced

anti-A binding (Table 6).

45

Figure 4. Quantification of A-antigen by flow cytometric analysis. The histogram overlay

shows the relative amount of A antigen associated with genotype. The genotype for each

histogram is labelled with an arrow. The greater the fluorescence signal, the greater amount of

A-antigen present on the RBC membrane. RBCs were treated with a FITC human anti-A IgG.

Group O red cells served as the negative control.

O1O1

BO1

A2B A2O1

A1O1

A1A1

A1B

46

3.2.1.4 The Effect of Soluble A Substance on Anti-A Binding

ABO antigens have both membrane bound and soluble forms. The amount of membrane bound

A antigen was not observed to have an influence on IVIg anti-A binding. Therefore, to determine

if the variation in the amount of soluble A antigens will affect anti-A binding to red cells, the

secretor status on each A1O sample was determined and compared to the IVIg binding DATs.

Similar to Group A zygosity, the secretor status did not considereably influence the strength of

the DAT score following IVIg incubation in whole blood. (Table 7).

Table 7. Anti-A Binding in Association with Secretor Status

Group *Anti-

Le(a)

*Anti-

Le(b)

Lewis

Phenotype

**Secretor

Status

Final IVIg Concentrations in Whole Blood

8mg/ml 20mg/ml 40mg/ml 80mg/ml A neg 1+ (a-b+) secretor W+ 2+ 3+ 4+

A neg 1+ (a-b+) secretor 1+ 3+ 3+ 4+

A neg 1+ (a-b+) secretor W+ 1+ 1+ 3+

A neg 1+ (a-b+) secretor 1+ 1+ 2+ 4+

A neg 1+ (a-b+) secretor W+ 1+ 3+ 4+

A neg 1+ (a-b+) secretor 1+ 2+ 3+ 3+

A neg 1+ (a-b+) secretor W+ 2+ 2+ 3+

A neg 1+ (a-b+) secretor 1+ 2+ 3+ 4+

A neg 1+ (a-b+) secretor W+ 2+ 2+ 3+

A neg 1+ (a-b+) secretor 1+ 2+ 2+ 3+

A neg 1+ (a-b+) secretor W+ 1+ 2+ 4+

A neg 1+ (a-b+) secretor W+ 2+ 2+ 3+

A 2+ neg (a+b-) nonsecretor 1+ 1+ 2+ 3+

A 2+ neg (a+b-) nonsecretor N N 3+ 3+

A 2+ neg (a+b-) nonsecretor 1+ 2+ 3+ 3+

A 2+ neg (a+b-) nonsecretor N 1+ 2+ 3+

A 2+ neg (a+b-) nonsecretor 1+ 2+ 2+ 3+

A 1+ neg (a+b-) nonsecretor N 2+ 2+ 3+

*neg = negative result

** samples are either a secretor or nonsecretor of ABO antigens

47

3.2.1.5 The Effect of IVIg dose on the Presence of Anti-A/B in the Plasma

In addition to evaluating IVIg anti-A/B binding on red cells, residual IVIg anti-A/B in the plasma

was examined after IVIg was added to whole blood samples. Therefore, a standardized reverse

blood group test was employed to determine the dose at which excess IVIg anti-A/B appears in

the plasma. IVIg was added to whole blood at four different concentrations, 8, 20, 40, and 80

mg/ml. The samples were centrifuged and the plasma was analyzed for residual anti-A/B

reactivity using the reverse blood group test. Table 8 demonstrates that at therapeutic doses of

IVIg (8, 20, 40 mg/ml), there is no excess anti-A/B in the plasma. However, at 80mg/ml of IVIg,

excess anti-A/B appeared in the plasma as was demonstrated with the A and AB samples.

Table 8. Presence of anti-A/B in the Plasma After the

Addition of IVIg at the concentrations Indicated

Sample 8mg/ml 20mg/ml 40mg/ml 80mg/ml

RA RB RA RB RA RB RA* RB*

A N 2+ N 3+ W+ 4+ W+ 4+

B 3+ N 4+ N 4+ N 4+ N

AB N N N N N N 2+ W+

RA and RB refer to reverse A and reverse B blood group

48

3.3 The Ability of IVIg Anti-A/B to Activate the Classical Complement Cascade

Previous clinical studies using pooled group A cells demonstrated that IVIg directly bound to red

cells can activate and fix complement (C3b) onto the RBC membrane (Table 5). Based on these

results, complement activation was examined after incubating whole blood samples with

Gamunex IVIg. This in vitro method was employed to mimic in vivo complement activation by

IVIg. First, IVIg at various concentrations was added to Group A whole blood and the ability of

sensitized red cells to activate complement was assessed by the addition of fresh AB serum (see

methods 2.5 and 2.5.1) Flow cytometric analysis was used to measure C3d deposition. C3b

quickly decomposes into C3d as was detected in preliminary work in which IVIg was added

directly to red cells (Appendix C). Therefore, in subsequent complement activation assays,

detection of C3d was analysed after the addition of IVIg to whole blood. The values in Figure 5

were adjusted by subtracting the mean percent of C3d-positive cells (6.5%) of PBS-treated RBCs

from all other values. Figure 5 demonstrates that IVIg-induced complement deposition is greatest

at the highest IVIg dose (80 mg/ml); only Group A cells demonstrated a significant increase. C3d

binding increased 11-18% above background. Figure 5 also illustrates that IVIg-induced

complement deposition is not blood group dependent at the highest IVIg dose as group O RBCs

also activated complement.

49

Figure 5. Complement Activation by IVIg. Samples of each blood group (A, B, AB, O) were

treated with IVIg at various concentrations correlating to therapeutic (8, 20, 40 mg/ml) and high

(80 mg/ml) doses. Samples were washed and then incubated with AB serum. Bar graphs

represent net mean percent of C3d-positive cells. Error bars represent standard error of the mean

(SEM, n=3). C3d is a breakdown product of C3b, the active complement protein. An (*)

indicates a significant increase in C3d deposition on RBCs treated with 80 mg/ml IVIg compared

to 40 mg/ml IVIg. Only Group A cells demonstrated a significant increase in C3d deposition

when cells were treated with 80 mg/ml of IVIg (p value < 0.05). B cells p = 0.06,

AB cells p = 0.19, O cells p = 0.22.

IVIg dose

*

50

3.4 The Interaction Between Red Blood Cells and Monocytes: Inhibition of Rosette

Formation

Red cells sensitized with IgG bind, specifically, to FcγRs present on the monocyte

membrane[146]. When these receptors are occupied, binding, and subsequent phagocytosis, is

inhibited. Using an in vitro phagocytic model and a chemiluminescence assay to detect reactive

oxygen species (a byproduct of monocyte phagocytic activity), it was shown that phagocytosis of

IgG coated RBCs is inhibited in a dose-dependent manner when monocytes are pre-treated with

soluble IgG[204]. The following experiments use a rosette forming assay and an in vitro

phagocytic model to demonstrate that the interaction between RBCs and model human

monocytes (THP-1) is FcγR-dependent.

3.4.1 THP-1 Cell and IgG-sensitized RBC Interaction: Inhibition of Rosette Formation

Before THP-1 cell phagocytosis of RBCs could be evaluated, the in vitro THP-1:RBC interaction

was examined using a rosetting assay. Rosettes formed when RBCs were treated with anti-D or

IVIg. No rosettes were seen when RBCs were ‗treated‘ with PBS or, when treated with Group

A/B adsorbed IVIg (Figure 6). Figure 7 summarizes rosette formation quantitatively. The most

rosettes formed when RBCs were treated with anti-D. Rosette formation was greater for RBCs

treated with 80 mg/ml of IVIg compared to 20 mg/ml of IVIg. When THP-1 cells were pre-

treated with increasing amounts of soluble IgG, the number of rosettes formed by anti-D/IVIg

coated RBCs decreased in a dose-dependent manner. For THP-1 cells pretreated with the F(ab‘)2

portion of IgG or BSA (an irrelevant protein), the number of rosettes formed was relatively equal

to that formed with untreated RBCs.

51

Figure 6. Images of rosette formation under an inverted microscope (Magnification 40X).

A) PBS treated or adsorbed IVIg treated RBCs + THP-1 cells[with whole IgG, F(ab‘)2, PBS, or

BSA as an inhibitor]: no rosettes observed (representative field of 10); B) anti-D treated RBCs +

THP-1 cells[with F(ab‘)2, PBS, or BSA]: rosettes observed (arrows, representative field of 10).

No rosettes were seen when anti-D treated RBCs + THP-1 cells were incubated with whole IgG.

(observations identical to the representative field in A).

A

100µm

100µm

B

52

Figure 7. Rosette Inhibition Assay. The graph is representative of 2 experiments. Group A Rh+

RBCs were treated with PBS, anti-D, IVIg (20 and 80 mg/ml), or absorbed IVIg (20 mg/ml).

THP-1 cells were pretreated with soluble IgG (100, 10, and 1 µg/ml), F(ab‘)2 (69 µg/ml), BSA

(47 µg/ml) or, left untreated (THP-1 alone). THP-1 cells and RBCs were incubated for 2 hours at

37oC in a 1:30 ratio. Number of rosettes were counted on a glass slide using an inverted

microscope. An (*) indicates that no rosettes were observed when RBCs were treated with anti-

A/B adsorbed IVIg or PBS. Rosettes were averaged from 10 random fields of view.

53

3.4.2 THP-1 Cell and RBC Interaction is Fcγ-receptor Dependent

It has been previously reported that the interaction between peripheral blood mononuclear cells

(PBMC) and IgG-coated RBCs can be inhibited by free IgG blocking FcγRs[204]. In the present

study, an experiment was designed to evaluate the interaction between anti-D coated RBCs and a

monocytic cell line, THP-1. Similar to what was observed, phagocytosis of anti-D sensitized

RBCs decreased significantly in a dose-dependent manner when THP-1 cells were treated with

increasing concentrations of soluble IgG. Figure 8 demonstrates the corresponding histograms to

Figure 9, which shows percent phagocytosis above the negative control. The values were

normalized to anti-D RBCs + THP-1 cells alone, which was set to 100% phagocytosis.

Phagocytosis levels decreased, but not significantly, when THP-1 cells were treated with 1 µg/ml

of soluble IgG. However, a significant decrease in phagocytosis was observed when THP-1 cells

were pretreated with either 10 or 100 µg/ml of soluble IgG. When THP-1 cells were pretreated

with the F(ab‘)2 portion of the IgG molecule, or BSA, phagocytosis levels was not significantly

different from that of untreated THP-1 cells. Based on these observations, Figure 9 demonstrates

that the interaction between RBCs and THP-1 cells is Fcγ-receptor dependent.

54

Figure 8. Inhibition of Phagocytosis by Soluble IgG. Histograms are representative of 3

experiments. The values (not normalized) represent the percentage of THP-1 cells that are

CMFDA positive compared to THP-1 cells alone (negative control). A. THP-1 alone (negative

control); B. THP-1 cells + non-opsonzised RBCs; C. THP-1 cells + anti-D coated RBCs; D.

THP-1 cells pretreated with 100 µg/ml soluble IgG; E. THP-1 cells pretreated with 69 µg/ml IgG

F(ab‘)2; F. THP-1 cells pretreated with 47 µg/ml BSA.

0.80% 4.18%

52.30%

44.41%

A B

C D

E F

2.90%

30.01%

55

Figure 9. Inhibition of FcγR-mediated phagocytosis. Values represent mean percent

phagocytosis above negative control (THP-1 cells + no inhibitor) with standard error of the mean

(SEM, n=3). Values were normalized to THP-1 cells + anti-D RBCs (THP + no inhibitor), which

was set to 100% phagocytosis. RBCs were treated with either PBS (background) or anti-D. THP-

1 cells were treated with either soluble IgG at 100, 10, 1 µg/ml, IgG F(ab‘)2 (69, 6.9, 0.69 µg/ml)

or, BSA (47µg/ml). FcγR-mediated phagocytosis was significantly inhibited in a dose-dependant

manner with soluble IgG. IgG F(ab‘)2 or BSA did not have a significant effect on the phagocytic

process. Therefore, phagocytosis of RBCs by THP-1 cells is Fcγ-receptor dependant. An (*)

indicates significant difference from THP + no inhibitor [100% phagocytosis] (p value < 0.05).

* * *

56

3.4.3 IVIg-induced RBC Phagocytosis with and without Complement Activation

To determine if IVIg-induced complement activation would enhance RBC destruction via

phagocytosis, an in vitro assay adopted from Aslam et al.[201] was employed. Flow cytometric

analysis was used to measure THP-1 internal fluorescence. Figure 10 displays the representative

histograms for the controls used in the assay. The respective values in Figure 11 were normalized

to anti-D RBCs + THP-1 cells (anti-D), which was set to 100% phagocytosis. Fluorescence was

gated above CMFDA negative THP-1 cells. RBCs coated with IgG (anti-D), or C3b, or both,

were used as controls in the phagocytic assay. Figure 11 demonstrates that the phagocytosis of

anti-D and C3b coated-RBCs was greater than that of anti-D or C3b-coated RBCs alone. RBCs

opsonized with IVIg alone did not show a dose-dependent increase in phagocytosis in spite of

the increase in DATs noted in Table 6. In addition, at three IVIg doses (8, 20, and 40 mg/ml)

C3b deposition did not show an increased phagocytosis. At the highest IVIg dose (80 mg/ml),

C3b deposition marginally elevated phagocytosis. Based on these results, IVIg sensitization did

not result in increase phagocytosis and C3b deposition noted in Figure 5 did not significantly

enhance phagocytosis of IVIg opsonized RBCs.

Subsequent to conducting the phagocytic studies, IVIg anti-A/B binding was assessed using the

DAT before and after incubation with fresh human serum for the timeframe that occurred in the

phagocytic assay. It was observed that following serum incubation, the DAT score decreased, by

two or more scores, for A, B, and AB red cells; thus, anti-A/B was eluted off of the RBC

membrane during the incubation with serum (Table 9). Group O cells failed to bind to IVIg anti-

A/B both before and after serum incubation. Conversely, anti-D coated Group A Rh+ red cells

remained opsonized with IVIg anti-A as the DAT score was unchanged after serum incubation.

57

Figure 10. Experimental Controls for Phagocytosis Bioassay. Histograms are representative

of 3 experiments. Values (not normalized) represent percent of CMFDA positive THP-1 cells

above negative control. A. THP-1 alone (negative control); B. THP-1 + PBS RBCs (background

control); C. THP-1 + C3b RBCs; D. THP-1 + anti-D RBCs (positive control); E. THP-1 + C3b

and anti-D RBCs.

A B

C D

E

4.5%

58

Figure 11. IVIg-induced complement-mediated RBC phagocytosis. Bars represent mean

percent of gated cells above negative control (THP-1cells alone), with standard error of the mean

(SEM, n=3). Values were normalized to anti-D RBCs + THP-1 cells (anti-D), which was set to

100% phagocytosis. Assay controls include C3b, anti-D, and anti-D+C3b. C3b deposition did

not significantly increase phagocytosis of IVIg opsonized RBCs (p value > 0.05). C3b was

deposited on anti-D RBCs by incubating whole blood in a 10% sucrose solution, while serum

incubation allowed for C3b deposition on IVIg RBCs.

59

Table 9. The Effect of Serum Incubation on the Binding of IVIg anti-A/B

*scores obtained according to the AABB Technical Manual (Appendix A)

RBC Blood

Group

Treatment *IgG Before serum

incubation

*IgG After serum

incubation

O PBS N N

O 40 mg/ml N N

O 80 mg/ml N N

A PBS N N

A 40 mg/ml 3+ W+

A 80 mg/ml 4+ 1+

A anti-D 4+ 4+

B PBS N N

B 40 mg/ml 2+ W+

B 80 mg/ml 3+ 1+

AB PBS N N

AB 40 mg/ml 3+ 1+

AB 80 mg/ml 4+ 2+

60

Chapter 4

Discussion

61

4.1 Reviewing ABO Blood Group Antibodies and Hemolysis

A rare, but harmful, adverse effect of Intravenous Immunoglobulin (IVIg) is extravascular

hemolysis. When ABO blood group antibodies bind to their respective antigens in vitro,

detectable amounts of IgG can be detected using the indirect agglutination of RBCs. In vivo,

however, other factors (ie. the Complement system) are present within the blood, which may

enhance RBC destruction, either by extravascular or intravascular hemolysis. The determining

factors that lead to extravascular or intravascular hemolysis or, the severity of the hemolytic

episode may include: quantity of antibody, the presence of IgM and/or IgG, activation of the

classical complement pathway to C3b or C9, presence of complement regulators, and variants of

monocyte/macrophage (mø) receptors that play a role in RBC destruction. The purpose of this

study was to determine whether IVIg affects complement activation, which may have

implications for RBC phagocytosis. The study was initiated due to the observation that IVIg,

when added to reagent red cells, can activate complement in vitro (Table 5).

4.2 Assessment of IVIg and Complement Activation: Clinical Studies

Clinical laboratory studies with Gamunex IVIg (Tables 2 through 5) were performed to analyze

the characteristics of the antibodies, specifically anti-A and anti-B, which are present within this

IVIg preparation. The studies confirmed literature findings that IVIg contains clinically

significant blood group A and B antibodies (Table 2) and that they bind to red cells in a dose

dependent manner (Table 4). Although these antibodies were not observed to be hemolytic, they

can fix complement proteins onto the red cell membrane (Figure 5). Additionally, it was

observed that Gamunex IVIg contains no clinically significant red cell antibodies other than anti-

A/B (Table 3). These clinical findings provided a basis for the following research, which

expanded to analyzing the ability of IVIg, when added to whole blood, can induce RBC

phagocytosis in the presence or absence of complement protein, C3b. The clinical studies

assessed the characteristics of IVIg directly, using pooled RBCs. However, the in vitro methods

utilized in this study were designed to mimic in vivo situations by employing a human monocytic

cell line, THP-1, and whole blood from donated human blood samples.

62

4.3 The Effect of IVIg Dose and ABO Genotype on Anti-A/B Binding

IVIg has been shown to contain ABO blood group antibodies[36] and is non-reactive with

Group O red cells (Table 2). The binding of the A/B agglutinins provides the mechanism why

the majority of patients, who experience hemolytic episodes with IVIg therapy, are

predominantly blood group A or B[62]. The severity of the hemolytic episode has been

associated with high dose regiments[37;61;62] and the dose-dependent increase in anti-A/B

binding observed in these studies supports the published observations (Table 2 and 6). The

amount of membrane-ABO antigens is genetically controlled and it was hypothesized early in

this work that the amount of A or B antigens would cause differences in the binding of IVIg to

RBCs. From the DAT results of this study (Table 6), it cannot be ruled out that ABO zygosity

does not affect the binding of ABO antibodies in IVIg. According to the DAT, whether a patient

is heterozygous or homozygous for the A1 antigen has no consequence to the level of anti-A

binding in response to IVIg dose. However, flow cytometric analysis has revealed that,

quantitatively, the level of A-antigens on A1A1 RBCs is greater than A1O RBCs, which is

consistent with what is stated in the literature[205]. In addition, the quantity of A-antigens on A2

RBCs is approximately 1/4 that of A1 RBCs (Figure 4). Consequently, less anti-A will bind,

which was observed using the DAT (Table 6). These results potentiate the possibility that A1

zygosity and the presence of the A1 allele versus the A2 allele may be a minor risk factor in IVIg

anti-A binding and subsequent hemolysis. Therefore, patients who are typed A2O may be less

likely to experience a severe hemolytic episode due to IVIg treatment.

4.3.1 The Effect of IVIg Binding in the Presence of Soluble A Substance

Evaluation of A-antigen density has revealed that there are more A-antigens on A1 versus A2

RBCs[206] and, as expected, A1 plasma of an ABO secretor has considerably more soluble A

substance than A2 plasma (Figure 3)[195]. Therefore, it was hypothesized that as the dose of

IVIg increased, the anti-A would be neutralized by the soluble A substances in the plasma of an

ABO secretor before binding to the RBC membrane. In addition, IVIg anti-A binding would be

greater for RBCs of an ABO non-secretor since there are no soluble A-substances to neutralize

the anti-A. However, this study has observed that the secretor status of an individual does not

play a role in in vitro IVIg anti-A binding (Table 7). Therefore, the variable levels of soluble A

substance in plasma are not a significant contributor to the level of anti-A binding on red cells.

63

4.4 Activation of the Classical Complement Cascade by IVIg

Shoham Kessary et al.[36] have demonstrated that RBCs can bind IgG and complement (C3d) in

vivo after IVIg treatment. High dose (80 mg/ml) IVIg induced complement C3b deposition on

the RBC membrane equally among groups A, B, AB, and O (Figure 5). A dose-response in

complement deposition to IVIg dose was not observed; however, there seems to be a threshold

dose between 40 and 80 mg/ml that facilitates complement activation. Furthermore, C1qrs

requires the Fc portion of IgG molecules to be no more than 20 nm apart[15]. Therefore, the

therapeutic doses of IVIg, may not provide enough anti-A/B to bind to the RBC membrane; ie,

not providing the C1qrs molecule an opportunity to initiate the complement cascade. Thus, the

contributing factor for complement activation in this model may be the difference in anti-A/B

titre between the 40 mg/ml and 80 mg/ml dose of IVIg.

4.4.1 The Effect of Soluble A Substance on Complement Activation

Interestingly, previous experiments have shown that anti-A/B binding post-serum incubation

decreases (Table 9). One reason could be that soluble A/B substances in serum added as a source

of complement are competing with membrane-bound antigens for anti-A/B IgG and a new

equilibrium is established. Therefore, at low therapeutic doses of IVIg, anti-A/B become

unbound and is unable to deposite C3b on the RBC membrane. At high IVIg doses (80mg/ml) IC

may form either between IVIg molecules or, IVIg and plasma proteins, which can deposit C3b

onto the RBC membrane, regardless of the ABO blood group or presence of A/B soluble

substnaces. Additionally, non-ABO antibodies either within IVIg or, autologous may bind to the

RBC membrane and activate complement[207-209]. These observations suggest that the in vitro

model to evaluate complement activation is sub-optimal and may not reflect complement

activation during high dose IVIg therapy.

4.4.2 Complement Activation on Group O RBCs

An interesting finding in this work was that complement deposition was observed when O cells

were treated with high dose IVIg (80 mg/ml). To explain this observation, it was hypothesized

that when IVIg is added to whole blood in vitro, IC formed, which bound to RBCs and activated

complement on the RBC membrane. Previous studies have reported that IVIg forms IC either as

IgG dimers[21] or auto-IC[51] when added to whole blood. These observations lead to the

64

conclusion that complement activation is not blood group dependant. Additionally, they are

supported by Daw et al.[62] who reported one group O patient that experienced a hemolytic

episode of IVIg at high doses.

4.4.3 The Role of Complement Regulators on C3b Deposition

This study did not assess the influence that complement regulators may have on complement

activation. Negative complement regulators, such as CR1 and DAF play a role in controlling the

amount of C3b/C3d that is deposited on the RBC membrane. The presence or absence of these

regulators may predispose an individual to various levels of complement activation. For instance,

patients who suffer from PNH lack all GPI-linked membrane proteins, including DAF[132].

DAF-deficient red cells are, therefore, susceptible to complement mediated lysis. When

considering complement regulators as contributors to complement deposition, the antigens

carried on such proteins may be useful markers for determing the relative quantity of

complement that may be deposited. The null phenotype for the Knops antigens is associated with

low CR1 expression levels.[108] Low CR1 on the RBC members will allow C3b molecules to

remain active (ie. not be degraded to C3d), therefore, RBCs will maintain their ability to bind to

macrophage CR1. In addition the Cromer antigen Dra-

is associated with low DAF expression.

This phenotype may help to identify those patients who may not have stable complement

regulation.

4.5 The Interaction Between Monocytes and RBCs is FcγR-dependant

During an extravascular hemolytic reaction, RBCs are destroyed via the MPS. This system

involves the interaction between antibodies, which opsonize target cells, and receptors, which

activate internal cell signaling pathways required for target destruction. The interaction between

monocytes and RBCs was evaluated using the THP-1 monocytic cell line and an in vitro assay to

evaluate rosette formation and RBC phagocytosis. Anti-D sensitized RBCs served as the positive

control since purified human anti-A is not available. A mouse anti-A could have been used

instead, however, the ability to bind to human FcγRI and II would require investigation since

certain polymorphisms of human monocyte FcγRs bind to different murine monoclonal IgG

subclasses[210]. Therefore, the focus of the phagocytic studies would not have been on IVIg

65

anti-A/B, but on the polymorphisms of THP-1 cell FcγRs. The current findings confirm that the

interaction between IgG-sensitized RBCs and monocytes is FcγR-dependent (Figures 6, 7 and 9).

Soluble IgG blocks FcγRs, which inhibits the interaction between IgG-opsonized RBCs and

monocytes[204]. Blockade of the MPS is one hypothesis for the amelioration of ITP since IVIg

was implicated as a definite therapeutic tool for the disease.[44] The data in this study supports

the FcγR blockade hypothesis as an immediate effect but, does not explain the long term

observations of the therapeutic effects of IVIg[211;212].

4.6 Studies on Complement-mediated Synergism of IgG Sensitized RBC Phagocytosis

The current study has implications for the pathophysiology of extravascular hemolysis involving

complement activation. Previous studies have shown that complement deposition enhances

phagocytosis of IgG-sensitized RBCs[125;126;213]. Indeed, this work demonstrated that C3b

deposition can potentially synergize with IgG to enhance RBC phagocytosis (see anti-D + C3b

control in Figure 11). This work supports the previous observation for the role of both CR1 and

FcγRs in RBC destruction[127]. Alternatively, the methodology used to sensitize RBCs with

both anti-D and C3b may artificially increase the amount of IgG on RBCs and account for the

increased phagocytosis observed. Since anti-D does not activate complement[15], Rh immune

globulin was added to whole blood and then incubated in a 10% sucrose solution, which may

have caused the adsorption of more IgG onto the cells (Appendix E). Therefore, an alternate

method of anti-D+C3b coated RBCs should be explored before conclusive evidence of IgG +

C3b synergism can be confirmed in these studies. Essentially, anti-D sensitization should

precede 10% sucrose incubation. With IVIg anti-A/B binding alone, phagocytosis was increased

above background but, not in a dose dependent manner as expected. It is thought that soluble

A/B substances present in the serum compete with membrane bound antigens for the binding

sites on IVIg anti-A/B. Therefore, anti-A/B elutes off of the RBC membrane and remains in a

new equilibrium where less IgG is bound to the red cell (Table 9). With the pre-incubation of

fresh sera to allow for C3b deposition, phagocytosis was decreased and for the highest dose of

IVIg, phagocytosis was equal to that of IVIg alone (Figure 11). Previous experiments showed

that IVIg induced complement deposition only at a high dose (80 mg/ml) [Figure 3]. This

observation infers that the amount of C3b deposited may be insufficient to cause synergistic

enhancement of RBC phagocytosis.

66

The phagocytosis of IVIg sensitized RBCs in this study was low compared to that of the

controls. Perhaps these observations illustrate the results Craig et al.[214;215] reported with their

studies on the transfer of RBC membrane during monocyte and RBC interactions. It was

observed that when RBCs are bound to IC via CR1, the IC, as well as CR1 are removed by

(transferred to) activated monocytes. In addition, IC and C3b bound via non-CR1 proteins such

as, glycophorin A (GPA), were also transferred from the RBC membrane to the monocytes. The

low levels of phagocytosis seen in this study may reflect complement transfer, not phagocytosis.

It is uncertain whether the current model can detect ‗partial‘ phagocytosis in which pieces of the

RBC membrane are removed, rendering the RBCs spherocytic[216]. Alternatively, RBCs can be

eliminated through ADCC[216]. THP-1 cells were shown to be cytotoxic to chicken erythrocytes

via FcγRs I and II, which included FcγRIII upon stimulation with recombinant interferon

gamma.[217] Bound RBCs, via IgG and/or C3b, are destroyed without being ingested by the

macrophage, which would make it impossible to detect RBC membrane within THP-1 cells

using the phagocytic bioassay.

4.6.1 The Effect of Receptor Expression on THP-1 cells on RBC Phagocytosis

It has been suggested that an inflammatory environment is correlated to the incidence of

hemolysis associated with IVIg therapy[62]. An inflammatory environment may influence the

receptor expression on monocytes/macrophages, rendering them more or less phagocytic.

Therefore, in preparation for the phagocytic studies, the effect of PMA on THP-l cell receptor

expression was investigated. THP-1 cells express FcγRI and II and CR1[196]. However, in the

presence of PMA, the expression of FcγRI and II decreases while that of CR1increases[218;219].

It was explained by Auwerx et al.[218] that the changes in receptor expression are consistent

with that of THP-1 cell growth arrest and differentiation into a more macrophage-like cell. The

decrease in FcγRs may explain why RBCs, sensitized with an increasing dose of IVIg, were not

phagocytosed in a dose-dependent manner. THP-1 cell FcγR and CR1 expression was analyzed

by flow cytometry (Appendix G). Both FcγRI and CR1 expression decreased whereas, FcγRII

expression remained unchanged by PMA treatment. These observations are contrary to those

observed by Auwerx et al. Therefore, the enhancement of RBC phagocytosis will not be

observed if both FcγR-mediated and CR1-mediated destruction of RBCs is reduced. These points

raise the question of whether or not THP-1 cells are a suitable cell line to correlate pro-

67

inflammatory signaling with phagocytosis. Alternatively, the pretreatment of THP-1 cells with

PMA requires re-evaluation. Auwerx et al.[219] treated THP-1 cells with 1.6 x 10-7

M of PMA

for 48 hours, which caused the cells to become adherent. In this study, THP-1 cells were treated

with 1.6 x 10-9

M of PMA for 15 minutes and cells remained in suspension.

4.6.2 The Presence of Complement Regulators and Their Influence on RBC Phagocytosis

As previously mentioned, the presence of complement regulators on the RBC membrane can

influence the extent of complement protein deposition. RBCs with high levels of CR1 and DAF

will be more apt to inactivate (C3b C3d) [in the presence of Factor I], remove deposited

complement proteins, and prevent C3b deposition (by degenerating C3 convertases)[220-222].

As a result, there will be less interaction with monocyte CR1 as this receptor does not bind to

C3d. The lack of synergism between IVIg and complement seen in Figure 11 can be due to the

inactivation of C3b to C3d. Conversely, RBCs with low levels of CR1 and DAF will be less

likely to control the amount of C3b deposition and therefore, will be more susceptible to

complement-mediated destruction as it is observed in patients with PNH. It is probable that the

donors used in this study for the phagocytic assay have normal CR1 and DAF levels, and that the

levels of these receptors were not sufficiently different to show individual variances in C3b

deposition. In Figure 11, at the lower doses of IVIg, if C3b was activated, it was not deposited

enough or inactivated to C3d, thus, preventing RBCs from interacting with THP-1 cell CR1.

The in vivo studies that report hemolysis following administration of high IVIg doses[37;60;62],

use IVIg at 20 or 40 mg/ml. It is very rare that a total dose of 80 mg/ml of IVIg is used for

treatment, although the cumulative effect of repeated doses could conceivably reach 80

mg/ml.[61] Coupled with the fact that hemolysis associated with IVIg is an uncommon adverse

event, a good model to evaluate the effect of high doses of IVIg remains to be resolved.

4.7 Summary

Hemolysis is a rare but adverse reaction to IVIg. The majority of patients treated with IVIg do

not experience ill effects; however, patients receiving high dose regiments of IVIg have been

documented to experience hemolytic reactions. The factors that delineate the severity of such

reactions remain unresolved. The purpose of this study was to determine whether IVIg dose

68

affects complement activation, which may have implications for RBC destruction. The

conditions that allow for IVIg to exert its hemolytic effects were studied in vitro, with the hope

that the findings could be implicated for the in vivo activity of IVIg.

Based on the observations made by clinical studies using in vitro diagnostic tools, the present

research involved the use of whole blood in in vitro assays in order to evaluate the effects of

Gamunex IVIg. This study confirmed that the isoagglutinins, anti-A/B, in IVIg bind to RBCs in a

dose dependent and blood group dependent manner. The zygosity of the A1 gene, as well as the

secretor status, does not seem to have an influence on IVIg binding to red cells in response to

IVIg dose according to the DAT. However, since the amount of A-antigens on A2 red cells is

significantly less than on A1 red cells, which can be seen using flow cytometry, then this work

predicts that the elimination of A2 cells by the MPS may not be as pronounced. A1 red cells, on

the contrary, can bind more anti-A and therefore, will most likely undergo phagocytosis by the

MPS. It will be beneficial to subgroup A and AB patients (A1 vs. A2) to support or refute the

hypothesis that A2 is a negative risk factor for IVIg hemolysis.

Since hemolysis is an immunologically-mediated condition, the activation of the complement

pathway by IVIg was examined. IVIg-induced complement activation was neither dose-

dependent nor, blood-group dependent. However, significant complement activation (as detected

by flow cytometry) was observed at the highest dose of IVIg, 80 mg/ml for Group A red cells.

IVIg may be creating IC with itself or, creating IC with plasma proteins, both of which can

activate complement and deposit C3b onto the RBC membrane. Alternatively, there may be IgG

within IVIg or, auto-Ab that bind to non-ABO antigens. The fact that complement activation was

barely observed at the therapeutic doses of IVIg (8, 20, and 40 mg/ml), likely illustrates that

complement regulators such as, CR1 and DAF, are able to regulate the deposition of C3b or that

C3 is not significantly activated to form C3b.

The present study used an in vitro model using the THP-1 cell line in order to mimic the MPS in

vivo. This work confirmed that the short-term interaction between monocytes and IgG-sensitized

RBCs is FcγR-dependent. This study confirms previous in vitro studies that show that FcγR-

mediated phagocytosis can be augmented by complement C3b on red cells (controls in Figure

69

11). However, for all IVIg doses, the pre-incubation with fresh serum to allow for C3b

deposition did not enhance IgG-sensitized red cell phagocytosis. It was speculated that soluble

A/B substances are competing with membrane bound A/B antigens for the binding site on IVIg

anti-A/B; therefore, creating a new equilibrium in which less antibody is bound to the red cell

membrane. Consequently, the C1qrs molecule is not given an opportunity to become activated

and opsonize the red cells with C3b. Since it was only the highest dose of IVIg that activated

complement, the phagocytic assay suggests that the amount of C3b activation is insufficient to

enhance RBC phagocytosis.

4.8 Future Directions

The findings of the current study have illustrated the complexity of the mechanisms of action of

IVIg. Although many mechanisms have been suggested, it is still unclear how IVIg illicits its

adverse effect. Additionally, whether therapeutic doses of IVIg infuse sufficient anti-A/B to

activate the classical complement pathway appears unlikely from these in vitro studies, but

remains to be explored further.

The phagocytosis model used in this investigation requires further optomization in order to

create an in vitro system that addresses the limitations observed to effectively mimic the anti-

A/B activity of IVIg in vivo. The experimental techniques used herein need to be re-evaluated in

order to better assess complement activation and C3b synergism with FcγR-mediated

phagocytosis at low and high doses of IVIg. It appears that, when whole blood is incubated with

IVIg, anti-A/B bind to their respective antigens (Table 6). However, when the RBCs are

prepared for further analysis (C3 activation and RBC phagocytosis) anti-A/B (contrary to the

anti-D control) begins to elute off of the red cells (Table 9). Therefore, C3 activation and IgG

anti-A/B interaction with monocyte FcγRs cannot accurately reflect the in vivo interaction. IVIg

therapy does not include a ‗washout‘ of the unbound IVIg anti-A/B remaining in the plasma. As

a result, it may not be possible to optimize an in vitro model to include low affinity antibodies

and classical complement activation. Alternatively, a transgenic mouse model where the human

ABO blood group is expressed can be used. Testing the effects of IVIg dose in an animal model

circumvents the difficulties in preparing C3b and/or IgG sensitized RBCs, since the in vivo

environment provides optimal conditions that an in vitro assay is unable to provide.

70

This study has demonstrated that the level of membrane bound or soluble ABO antigens may

have contribute to the overall binding of anti-A/B relative to IVIg dose, and subsequent

phagocytosis. A comparison of the phagocytosis of A1 red cells versus A2 red cells sensitized

with IVIg would be essential to validate the clinical significance of subtyping A or AB patients

to predict the severity of IVIg-associated hemolysis. Studies that evaluate Group A subclass,

zygosity, and soluble A/B substances on antibody sensitization and subsequent phagocytosis,

could be accomplished independent of the elution problems observed with IVIg anti-A/B. A high

affinity murine anti-A could be used to evaluate Group A1 zygosity and the presence of soluble

A substance in whole blood given a large enough sample population to explore the changes these

variable have on IVIg binding.

In addition, RBCs with high or low levels of CR1 and DAF, which may be identified through

phenotyping patients for Cromer and Knops antigens, should be analyzed to confirm and address

the role of complement regulators in RBC phagocytosis. These RBCs may also be compared to

PNH RBCs, which have no DAF present on the membrane. Furthermore, RBCs that are Dra-

have 40% of DAF expressed compared to Dra+

RBCs[223]. Although these RBCs do not show

increased sensitivity to complement lysis in vitro, they still may be at risk for IVIg-induced

hemolysis provided that other factors are involved such as, the presence of the A1 allele and high

affinity FcγRs.

The phagocytosis of RBCs depends on many factors. This study examined one side of the

RBC:monocyte interaction; that is, the opsonization of RBCs (ie. IgG, C3b). However,

hemolysis can also be affected by the receptors on monocytes that initiate phagocytosis, namely,

the FcγRs. Therefore, this study emphasizes that the hemolysis experienced by patients who

receive IVIg therapy, may be due to individual variances. For example, it has been previously

demonstrated that monocytes from different individuals possess variable ability to engage in

RBC phagocytosis; possibly due to the fact that there are functional variants of FcγRs[224]. It

has been shown that RBCs opsonized with either IgG1 or IgG3 molecules may be more or less

susceptible to FcγR-mediated phagocytosis[224]. In addition, a proinflammatory environment

may augment the effect of FcγR variants in mediating RBC destruction as different cytokines,

namely interferon gamma (IFNγ), have been shown to increase the phagocytic activity of

71

monocytes[224]. The effect of FcγR variants and the in vitro pro-inflammatory activation of

human monocytes on the phagocytosis of IVIg sensitized RBCs is a worthwhile investigation.

72

Chapter 5

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73

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Chapter 6

Appendices

92

Appendix A. Scores for grading agglutination by direct antiglobulin tests. (AABB

Technical Manual, 2005).

Score Appearance of agglutination

4+ One large agglutinate, clear background

3+ Several large agglutinates, clear background

2+ Many small agglutinate, clear background

1+ Many small agglutinates, turbid background

W+ Very small agglutinates, turbid background

N No agglutinates

93

Appendix B. ABO Exon 6 and 7 Restriction Enzyme Digestion. A. Exon 6. B. Exon 7. Lane 1

is the DNA ladder, 50 bp; lane 2 is the undigested PCR product (digestion control); lanes 3-9

represent different blood group genotypes as denoted above the lanes in the figure. PCR and

restriction enzyme digestion were performed as described in sections 2.3.1.1. and 2.3.1.2. Band

sizes are shown with arrows and the corresponding size on the right hand side of each figure.

252

164

87

309

223 204

150 137 119 96

O1O1 A1A1 A1O A2O BO1 A1B A2B O1O1 A1A1 A1O A2O BO1 A1B A2B

A B

94

D E F

0.06% 2.49% 7.48%

G H I

0.11% 2.55% 5.77%

J K L

0.31% 8.04% 17.38%

0.14% 2.64% 5.44%

A B C

M N O

0.05% 2.47% 6.30%

95

Appendix C. Deposition of Complement Proteins. Group B Rh+ blood samples were

incubated at 4 concentrations of IVIg—8 mg/ml: A, B, C; 20mg/ml: D, E, F; 40mg/ml: G, H, I;

80mg/ml: J, K, L; or, PBS: M, N, O—washed, and subsequently incubated in fresh AB serum to

activate complement. Samples were washed and prepared for flow cytometric analysis for C3a

(negative control)[C3a is a fragment of C3b that does not stay bound to the cell]: A, D, G, J, M;

C3b: B, E, H, K, N; and C3d (the final breakdown product of C3b that remains bound to the

cell): C, F, I, L, O. C3d detection was consistently greater than that of C3b, which illustrates how

quickly C3b decomposes. Complement deposition on PBS treated RBCs served as the

background control.

96

Appendix D. Binding of anti-D to RBCs. RBCs at 200x106 cells/100µl (20% RBCs) were

sensitized with 2.5, 5, 7.5, and 10 µl of anti-D (WinRho SDF) at 120 µg/ml. Non-opsonized

RBCs, incubated with PBS alone, served as the negative control. Anti-D binding was detected

using a FITC-mouse-anti-human IgG secondary antibody. A) Bar graph represents mean

fluorescent intensity for anti-D at various volumes B) Histogram overlay of mean fluorescence.

An anti-D volume of 10 µl gave the highest mean fluorescence and was therefore the amount of

anti-D used to maximally sensitize a 20% RBC solution in subsequent experiments. In

experiments where reconstituted whole blood (40% RBC solution) was used, RBCs were

sensitized with 10 µl of anti-D to avoid maximal sensitization.

A

B

97

Appendix E. C3b Deposition and IgG Binding of RBCs Treated with Sucrose. Rh+ RBCs

were treated with either PBS or anti-D. Aliquots were then incubated in PBS or a 10% sucrose

solution. RBCs were preapred for cytometric analysis of C3b deposition and IgG binding. Values

represent mean fluorescent intensity. An (*) indicates a statistically significant difference

between complement deposition on RBCs incubated with sucrose compared to PBS. An (**)

indicates a statistically significant difference in IgG binding of anti-D coated RBCs versus RBCs

treated with PBS (with or without incubation in sucrose) [p values < 0.05]. There was no

statistical difference of IgG binding between anti-D coated RBCs incubated in PBS versus

sucrose.

98

Appendix F. Activation of complement with fresh human serum. Rh+ RBCs were opsonized

with IVIg (80mg/ml) or PBS, washed and then aliquots were incubated in PBS or fresh AB

serum. Samples were washed again and then prepared for flow cytometric analysis for C3d

detection. Values represent percent C3d positive cells with standard error of the mean (SEM,

n=3). A mouse-anti-human IgG1 antibody was used as the isotype control to evaluate anti-C3d

non-specific binding. An (*) indicates a significant increase in C3d deposition in the presence of

serum (p value < 0.05). To confirm complement activity in serum, 50 µl of a 5% suspension of

Group A RBCs was incubated in AB serum (0.5 ml). Using a diagnostic anti-C3b/C3d (Ortho

Diagnostics), the agglutination score was 2+ after appropriate washes (data not shown).

*

99

Appendix G. Receptor Expression on THP-1 cells. First two rows represent dot plots for THP-

1 receptor expression. Bottom row represents the corresponding histograms. THP-1 cells, at

2x105/100µl, were incubated with either A) a human anti-CR1, B) a PE-conjugated human anti-

CD64 (FcγRI) or C) a PE-conjugated human anti-CD32 (FcγRII). A mouse anti-human IgG1 was

used as the isotype control. A FITC mouse anti-human IgG was used as a secondary antibody to

label non-fluorescein antibodies. A thin black line denotes the isotype control, a thick black line

is the receptor expression for untreated THP-1 cells and a dotted line is the receptor expression

for PMA treated THP-1 cells. Cells were acquired in BD FACSCalibur and analysed with Cell

QuestPro Software (% indicates positive cells above background).

0.87% 1.06%

83.90% 99.81%

A B C

90.88%

0.61%

CR1 FcγRI FγcRII

100

Appendix H. THP-1 Cell Viability. THP-1 cell viability was determined using propidium

iodide (PI) exclusion and Annexin V binding. Cells were analyzed by flow cytometry. Briefly, 2

x 105 cells/100 µl were suspended in 1x binding buffer. Ten µl of either PI or Annexin V were

added to the cells. A. unstained THP-1. B. THP-1 stained with Annexin V. C. THP-1 stained

with PI. D. THP-1 stained with both PI and Annexin V. THP-1 cells stained with either PI or

Annexin V alone served as negative controls. Percent values represent PI and Annexin V

negative THP-1 cells (seen in the lower left hand corner of each quadrant).

A B

C D

97% 80%

73% 77%