Chapter 69

Rheumatic Fever and RheumaticHeart Disease

L. Guilherme1,3 and J. Kalil1,2,3

1Heart Institute (InCor), School of Medicine, University of Sao Paulo, Sao Paulo, Brazil, 2Clinical Immunology and Allergy Division, School of

Medicine, University of Sao Paulo, Sao Paulo, Brazil, 3Immunology Investigation Institute, National Institute for Science and Technology, University

of Sao Paulo, Sao Paulo, Brazil

Chapter OutlineClinical, Pathological, and Epidemiologic Features 1023

Autoimmune Features 1024

Genetic Features 1024

Innate Immune Response 1024

MBL2 Gene 1024

TLR-2 Gene 1025

Ficolin Gene 1025

FcγRIIA Gene 1025

Adaptive Immune Response 1025

Major Histocompatibility Complex (MHC): DRB1,

DRB3, DQB1, DQA1 Genes 1027

CTLA4 Gene 1027

Both Innate and Adaptive Immune Response 1027

In Vivo and In Vitro Models 1027

In Vivo Model of Myocarditis and Valvulitis 1027

In Vitro Model of Rheumatic Heart Disease Autoimmune

Reactions 1028

Pathologic Effector Mechanisms 1028

Autoantibodies as Potential Immunologic Markers 1029

Concluding Remarks—Future Prospects 1030

References 1030

CLINICAL, PATHOLOGICAL, ANDEPIDEMIOLOGIC FEATURES

The clinical profile of rheumatic fever (RF) was first

described by Cheadle in 1889 and the manifestation of

the disease follows defined criteria established by Jones

in 1944, which were updated in 1992 and remain useful

today (Dajani et al., 1993). Briefly, the disease follows an

untreated S. pyogenes infection in children and teenagers

that present some genetic factors that predispose to the

diverse clinical manifestations. The diagnosis is made on

a clinical basis. The major manifestations include polyar-

thritis, carditis, chorea, subcutaneous nodules, and

erythema marginatum. The minor manifestations are

fever, arthralgia (clinical) and prolonged PR interval,

increased erythrocyte sedimentation rate, and presence of

C-reactive protein.

Polyarthritis and carditis are the most frequent mani-

festations of the disease and occur in around 70% of chil-

dren. Arthritis is one of the earliest and most common

features of the disease, present in 60�80% of patients. It

usually affects the peripheral large joints; small joints and

the axial skeleton are rarely involved. Knees, ankles,

elbows, and wrists are most frequently affected. The

arthritis is usually migratory and very painful. Carditis is

the most serious manifestation of the disease, occurring a

few weeks after the infection, and usually present as a

pancarditis. Endocarditis is the most severe sequel and

frequently leads to chronic rheumathic heart disease

(RHD). Mitral and aortic regurgitation are the most com-

mon events caused by valvulitis. Sydenham’s chorea is

less common (30�40%), characterized by involuntary

movements, especially of the face and limbs, muscular

weakness, and disturbances of speech, gait, and voluntary

movements. It is usually a delayed manifestation, and

often the sole manifestation of acute rheumatic fever.

Other manifestations such as subcutaneous nodules and

erythema marginatum can also occur during RF episodes

and are characterized by nodules on the surface of joints

and skin lesions, respectively (Mota et al., 2009).

1023N. Rose & I. Mackay (Eds): The Autoimmune Diseases, Fifth edition. DOI: http://dx.doi.org/10.1016/B978-0-12-384929-8.00069-1

© 2014 Elsevier Inc. All rights reserved.

Streptococcus pyogenes, or group A streptococcus,

was identified in 1941 by Rebecca Lancefield through

serology based on its cell wall polysaccharide that is

composed of carbohydrates such as N-acetyl β-D-glu-cosamine linked to a polymeric rhamnose backbone.

Group A streptococci contain M, T, and R surface pro-

teins and lipoteichoic acid (LTA), involved in bacterial

adherence to throat epithelial cells. The M protein,

which extends from the cell wall, is composed of two

polypeptide chains with approximately 450 amino acid

residues, in an alpha-helical coiled-coil configuration.

The amino-terminal (N-terminal) portion is composed of

two regions, A and B, which present variable numbers

of amino acid residues. The A region shows high poly-

morphism and defines the different M types, currently

more than 225 according to CDC (Centers for Disease

Control and Prevention, http://www.cdc.gov/ncidod/bio-

tech/strep/strepblast.htm). The C-terminal portion

(regions C and D) is highly conserved (Smeesters et al.,

2010).

The incidence of ARF in some developing countries

exceeds 50 per 100,000 children. The worldwide inci-

dence of RHD is of at least 15.6 million cases and the

major cause of around 233,000 deaths/year. However,

since these estimates are based on conservative assump-

tions, the actual disease burden is probably substantially

higher. The incidence of ARF can vary from 0.7 to 508

per 100,000 children per year in different populations

from several countries (Carapetis et al., 2005). In Brazil,

according to the WHO epidemiological model and data

from IBGE (Brazilian Institute of Geography and

Statistics), the number of streptococcal pharyngitis

infections is around 10 million cases, which could lead

to 30,000 new cases of RF, of which around 15,000

could develop to cardiac lesions (Barbosa et al., 2009).

AUTOIMMUNE FEATURES

RHD is the most serious complication of RF and depends

on several host factors that mediate a heart tissue-driven

autoimmune response triggered by a defensive immune

response against S. pyogenes.

Genetic predisposition is one of the leading factors

contributing to the development of autoimmunity. In the

last 5 years, using molecular biology tools, several new

single nucleotide polymorphisms of genes involved with

the activation of both innate and adaptive immune

responses were associated with the development of RF/

RHD (see Genetic Features).

The first genetic associations described in the 1980s

focused on HLA class II alleles coded by HLA-DRB1 and

DQB1 genes. The HLA class II molecules are expressed in

the surface of antigen-presenting cells (APCs), e.g., macro-

phages, dendritic cells, and B lymphocytes, and trigger

activation of the immune system. In the case of RF/RHD,

T cell populations activated upon specific self antigen stim-

ulation will trigger autoimmune reactions. The production

of several inflammatory cytokines will perpetuate the

heart-tissue damage. These observations are corroborated

by the fact that during the acute phase of disease,

Aschoff bodies, a granulomatous lesion containing

macrophages, Anitschkow cells, multinucleated cells, and

polymorphonuclear leukocytes develop in the myocardium

and/or endocardium of RHD patients. Inflammatory

cytokines such as IL-1, TNF-alpha, and IL-2 have been

found, depending on the developmental phase of the

Aschoff bodies (Fraser et al., 1997) and as mentioned

above, probably initiate the inflammatory process leading

to heart tissue rheumatic lesions.

More recently, other molecules were described involved

with the inflammatory process like integrins and chemo-

kines and cytokines such as IFN-gamma, IL-23, and IL-17

that play a role in the recruitment of both T and B lympho-

cytes leading to the autoimmune reactions observed in

rheumatic heart lesions (reviewed by Guilherme et al.,

2011). T and B lymphocytes react against self antigens

through molecular mimicry, first in the periphery and later

in the heart tissue. The mechanisms of T cell receptor

degeneracy and epitope spreading amplifies the autoim-

mune reactions (see Pathologic Effector Mechanisms). All

these steps are represented in Figure 69.1.

GENETIC FEATURES

RF and RHD occur in 1 to 5% of untreated children with

genetic predisposition. The disease is associated with sev-

eral genes, some of which are related to the innate or

adaptive immune response or both (Table 69.1).

In order to facilitate the comprehension of the role of

implicated genes known up to now, we describe the asso-

ciated genes/alleles based on their role.

Innate Immune Response

MBL2 Gene

MBL (mannan-binding lectin) is an acute phase inflam-

matory protein and functions as a soluble pathogen rec-

ognition receptor. It binds to a wide variety of sugars on

the surface of pathogens and plays a major role in innate

immunity due to its ability to opsonize pathogens,

enhancing their phagocytosis and activating the comple-

ment cascade via the lectin pathway (Jack et al., 2001).

Different variants of the promoter and exon 1 regions of

the MBL2 gene, which encodes mannan-binding lectin,

have been reported in patients with RF/RHD.

Interestingly, the A allele that codes for high production

of MBL was associated with development of mitral

1024 PART | 14 Cardiovascular System and Lungs

stenosis (MS) and most of these patients presented high

serum levels of MBL (Messias-Reason et al., 2006). In

contrast, RHD patients with aortic regurgitation (AR)

presented the O allele that codes for low production of

MBL, and the patients presented low serum levels of

MBL (Ramasawmy et al., 2008).

TLR-2 Gene

Toll-like receptors (TLRs) are sensors of foreign micro-

bial products, which initiate host defense responses in

multicellular organisms. A polymorphism of TLR-2 at

codon 753 generally leads to the replacement of arginine

with glutamine. The genotype 753Arg/Gln was more fre-

quent in a Turkish ARF cohort when compared to con-

trols (Berdeli et al., 2005).

Ficolin Gene

Ficolins trigger the innate immune response by either

binding collectin cellular receptors or initiating the com-

plement lectin pathway (Meassias-Reason et al., 2009). In

Brazilian chronic RHD patients, with prolonged time of

infection or repeated streptococcal infections, the haplo-

type G/G/A (-986/-602/-4) was found to be more frequent

than in controls, and was also correlated with low expres-

sion levels of this protein.

FcγRIIA Gene

This protein plays a role in the clearance of immune com-

plexes by macrophages, neutrophils, and platelets (Hirsch

et al., 1996). ARF patients presented histidine (H) in the

codon 131, which typically encodes for argenin (A); con-

sequently RF/RHD patients present a protein with low

binding capacity to the immune complex, favoring the

inflammatory response.

Adaptive Immune Response

The HLA (human leukocytes antigens) system is located

in the short arm of the human chromosome 6 and codes

for diverse proteins; it is considered the most polymorphic

system, composed of several genes with several alleles.

The class I proteins are present in all nucleated cells; how-

ever, the class II are expressed only in specialized cells of

the immune system (B lymphocytes, activated T lympho-

cytes, monocytes/macrophages, and dendritic cells). These

proteins are involved with antigen recognition and presen-

tation of self and foreign (microbes) antigens.

FIGURE 69.1 Acute phase rheumatic lesions (A and B) and cultured intralesional T lymphocytes (C).

1025Chapter | 69 Rheumatic Fever and Rheumatic Heart Disease

TABLE 69.1 Genetic Polymorphism Associated with Development of RF/RHD

Immune

response

Gene Chromosome

localization

Polymorphism Allele/Genotype/

Haplotype associated

with disease

Clinical

picture

Population

studied

References

INNATE MBL-2 10q11.2-q21 2221 X,YA (52C,54G, 57G), O (52T,54A, 57A)

YA/YA, YA/XA RHD-MS Brazilian Messias-Reason et al.(2006)

A (52C, 54G, 57G),O (52T, 54A, 57A)

O, O/O RHD-AR Brazilian Ramasawmyet al. (2008)

TLR-2 4q32 2258A/G (753 Arg/Gln)

753Gln, Arg753Gln ARF Turkish Berdeli et al.(2005)

FCN-2 9q34 2986G/A, 2602G/A, 24G/A

G/G/A RHD Brazilian Messias-Reason et al.(2009)

FCγRIIA 1q21-q23 494A/G (131H/R) 131R, R/R (high risk), R/H (intermediate risk)

ARF Turkish Hirsch et al.(1996)

ADAPTIVE MHC 6p21.31 DRB1, DRB3,DQB1, DQA1

Several alleles RF/RHD Several Guilhermeet al. (2011)(review)

CTLA-4 2q33.2 149A/G G/G RHD Turkish Duzgun et al.(2009)

BOTHINNATE andADAPTIVE

TNF-α 6p21.3 2308G/A A RHD Mexican Hernandez-Pacheco et al.(2003a)

A/A, G/G RHD- MVL,MVD

Egyptian Sallakci et al.(2005)

A ARF/RHD Brazilian Ramasawmyet al. (2007)

A ARF/RHD Turkish Berdeli et al.(2006)

2238G/A G, G/G RHD Mexican Hernandez-Pacheco et al.(2003b)

A ARF/RHD Brazilian Ramasawmyet al. (2007)

IL-1RA 2q14.2 A1, A2, A3, A4 A1/A1 RHD Egyptian Settin et al.(2007)

A1, A1/A1 RHD Brazilian Azevedo et al.(2010)

TGF-β1 19q13.1 2509C/T T, T/T RHD Egyptian Kamal et al.(2010)

869T/C C/C RHD Egyptian Chou et al.(2004)

IL-10 1q31-q32 21082G/A G/G RHD-MVD Egyptian Settin et al.(2007)

A/A RHD-MVL Egyptian Settin et al.(2007)

MHC: major histocompatibility complex; TNF-α: tumor necrosis factor alpha; TGF-β: transforming growth factor beta; IL-1RA: IL-1 receptor antagonist;MBL: mannan binding lectin; TLR-2: Toll-like receptor 2; FCN-2: ficolin 2; FCγRIIA: IgG Fc receptor; CTLA-4: cytotoxic T cell lymphocyte antigen 4; ARF:acute rheumatic fever; RHD: rheumatic heart disease; AR: aortic regurgitation; MS: mitral stenosis; MVD: mitral valve disease; MVL: multivalvular lesions.

1026 PART | 14 Cardiovascular System and Lungs

Major Histocompatibility Complex (MHC):DRB1, DRB3, DQB1, DQA1 Genes

Several HLA class II alleles have been described in asso-

ciation with RF/RHD. Patarroyo et al. (1979) described

an alloantigen on the surface of B cells, designated 883,

probably related to the HLA class II molecules, which

was present in a high frequency in RF patients. Later, a

monoclonal antibody (D8/17 MoAb) was produced

against B cells from RF patients bearing the 883 alloanti-

gen. Studies performed by Zabriskie et al. (1985) showed

an increased frequency of this alloantigen in RF patients.

The susceptibility of developing RF/RHD was first

associated with alleles of HLA class II genes (DRB1,

DRB3, DQB, and DQA), which are located on human

chromosome 6 (Table 69.1). Briefly, HLA-DR7 was the

allele most consistently associated with RF (Guilherme

et al., 1991; Ozkan et al., 1993; Weidebach et al., 1994,

Guedez et al., 1999; Visanteiner et al., 2000; Stanevicha

et al., 2003). In addition, the association of DR7 with dif-

ferent DQ-B or DQ-A alleles seems to be related to the

development of multiple valvular lesions (MVL) or mitral

valve regurgitation (MVR) in RHD patients (Guedez

et al., 1999; Stanevicha et al., 2003). HLA-DR53 coded by

the DRB3 gene is another HLA class II molecule in link-

age disequilibrium with HLA-DR4, DR7, and DR9. This

allele was strongly associated with RF/RHD in two studies

with Mulatto Brazilian patients (Guilherme et al., 1991;

Weidebach et al., 1994), but not in Brazilian Caucasian

patients (Visanteiner et al., 2000). Although DR53 has not

been described in previous studies, DR4 and DR9 were

associated with RF in American Caucasian and Arabian

patients (Ayoub, 1984; Rajapkase et al., 1987), whereas in

Egyptian and Latvian patients, DR7 was associated with

the disease (Guedez et al., 1999; Stanevicha et al., 2003)

(Table 69.1). In Japanese RHD patients, susceptibility to

mitral stenosis seems to be in part controlled by the HLA-

DQA gene or by genes in close disequilibrium linkage

with HLA-DQA*0104 and DQB1*05031 (Koyanagi et al.,

1996). HLA-DQA*0501 DQB*0301 with DRB1*1601

(DR2) were associated with RHD in a Mexican Mestizo

population (Hernandez-Pacheco et al., 2003a).

The molecular mechanism by which MHC class II

molecules confer susceptibility to autoimmune diseases is

not clear. However, since the role of HLA molecules is to

present antigens to the T cell receptor, it is probable that

the associated alleles facilitate the presentation of some

streptococcal peptides that will later trigger autoimmune

reactions mediated by molecular mimicry mechanisms.

CTLA-4 Gene

This gene is an essential inhibitor of T cell responses. It

is a strong candidate susceptibility gene in autoimmunity

and several studies suggest disease-associated polymorph-

isms (reviewed by Gough et al., 2005).

Both Innate and Adaptive Immune Response

More recently, with new technologies that have allowed

the description of gene variability by single nucleotide

polymorphisms (SNPs), other associations have been

established that could clarify some reactions related to

both the innate and the adaptive immune response leading

the autoimmune reactions in RF/RHD.

� TNF-α gene, also located in the chromosome 6,

between HLA class I and II genes, codes for a proin-

flammatory cytokine that plays a role during the S.

pyogenes infection and later in the inflammatory pro-

cess in the valves. Polymorphisms at 2308 G/A and

2238 G/A were associated with the susceptibility of

RHD patients from several countries (Hernandez-

Pacheco et al., 2003b; Sallakci et al., 2005; Berdeli

et al., 2006; Ramasawmy et al., 2007).� IL-10 gene is responsible for the production of IL-10, an

anti-inflammatory cytokine. The genotype 21082 G/A,

misrepresented in RHD patients, is apparently associ-

ated with the development of multivalvular lesions

(MVL) and with the severity of RHD (Settin et al.,

2007).� TGF-B1 is a gene that controls the proliferation and

differentiation of cells. The polymorphisms of both

the SNPs 869 T and 2509 T alleles were considered

as possible risk factors for the development of valvu-

lar RHD lesions in Egyptian and Taiwanese RHD

patients (Chou et al., 2004; Kamal et al., 2010).� IL-1Ra gene, for which the most frequent alleles are

1 and 2, encodes the antagonist of IL-1α and IL-1β,which are inflammatory cytokines. Two studies in

Brazilian and Egyptian RHD patients with severe

carditis showed low frequencies of allele 1, suggest-

ing lack of inflammatory control (Settin et al., 2007;

Azevedo et al., 2010).

IN VIVO AND IN VITRO MODELS

In Vivo Model of Myocarditis and Valvulitis

Humans are unique hosts for S. pyogenes infections.

However, several studies have been performed to deter-

mine a suitable animal model and numerous different

species (mice, rats, hamsters, rabbits, and primates)

have been tested for the development of autoimmune

reactions that resemble those observed in RF/RHD

patients (Unny and Middlebrooks, 1983), all with little

success.

In the last decade, a model that appears to be useful

for the study of RF/RHD has been developed with Lewis

1027Chapter | 69 Rheumatic Fever and Rheumatic Heart Disease

rats. These rats have already been used to induce experi-

mental autoimmune myocarditis and to study the patho-

genesis of RF/RHD (Li et al., 2004)

Immunization of Lewis rats with recombinant M6 pro-

tein induced focal myocarditis, myocyte necrosis, and val-

vular heart lesions in three out of six animals. The

disease in these animals included verruca-like nodules

and the presence of Anitschkow cells, which are large

macrophages (also known as caterpillar cells), in mitral

valves. Lymph node cells from these animals showed a

proliferative response against cardiac myosin, but not

skeletal myosin or actin. A CD41 T cell line responsive

to both the M protein and cardiac myosin was also

obtained. Taken together, these results confirmed the

cross-reactivity between the M protein and cardiac myo-

sin triggered by molecular mimicry, as observed in

humans, possibly causing a break in tolerance and conse-

quently leading to autoimmunity (Quin et al., 2001).

In another study done by the same group, Lewis rats

were immunized with a pool of synthetic peptides from

the conserved region of the M5 protein. Mononuclear

spleen cells from these animals were able to proliferate in

response to peptides from both the C-terminal region of

M5 protein and the N-terminal region of a heterologous

protein (M1) and myosin. These rats developed focal

infiltration of mononuclear cells predominantly in the aor-

tic valve, although no evidence of Aschoff bodies, the

hallmark of RF lesions, or Anitschkow cells was observed

(Lymbury et al., 2003).

Another study immunized Lewis rats with recombi-

nant M5 or synthetic peptides from the B- and C-regions

of GAS M5 (Gorton et al., 2009). Sera and T cells from

these animals recognized a peptide (M5-B.6) from the

B-repeat of the N-terminal portion of M5 protein and

induced heart lesions (Gorton et al., 2010), confirming

the previous results. The immunized rats (five out of

seven) developed mononuclear cell infiltration in the

myocardial or valvular tissue. Histopathological analysis

of valve lesions showed the presence of both CD41 T

cells and CD681 macrophages (Gorton et al., 2010),

consistent with human studies (Guilherme et al., 1995).

Altogether, these studies indicated that the Lewis rats

could be a model of autoimmune valvulitis.

In Vitro Model of Rheumatic Heart DiseaseAutoimmune Reactions

The major sequels of rheumatic fever are heart tissue

lesions that lead to chronic rheumatic heart disease, which

is characterized by permanent valvular lesions. The heart

disease starts by pericarditis, followed by myocarditis epi-

sodes in which the healing process results in varied

degrees of valvular damage (Mota et al., 2009).

By isolating infiltrating T lymphocytes from damaged

valvular tissue, we could establish the mechanism by

which the immune response in the heart leads to autoim-

mune reactions (Guilherme et al., 1995). Figure 69.1

shows a damaged mitral valve in which verrucae lesions

are observed, indicative of an acute rheumatic fever epi-

sode. Furthermore, the presence of Aschoff bodies in the

myocardium tissue allowed for histological diagnosis of

an active episode of rheumatic disease. In vitro tissue cul-

ture of small pieces of the surgical fragment allowed the

isolation of infiltrating T cells.

The in vitro analysis of these tissue infiltrating T cells

showed their ability to recognize several streptococcal-M

protein peptides and self antigens by molecular mimicry

mechanisms. We identified some mitral valve-derived pro-

teins such as vimentin, PDIA3 (protein disulfide isomerase

ER-60 precursor), and HSPA5 (78 kDa glucose-regulated

protein precursor) that were recognized by both peripheral

and intralesional T cell clones (Fae et al., 2008).

The identification of heart-M protein cross-reactive

T cell clones directly from rheumatic valvular lesions

established their involvement in the pathogenesis of the

disease.

PATHOLOGIC EFFECTOR MECHANISMS

The term “molecular mimicry” was introduced in 1964

by Damian to define the mechanism by which self anti-

gens are recognized after an infection by cross-reactivity

(Damian, 1964).

Pathogen and self antigens can be recognized by T lym-

phocytes and antibodies through molecular mimicry by

four different mechanisms. They can recognize (1) identical

amino acid sequences, (2) homologous but non-identical

sequences, (3) common or similar amino acid sequences of

different molecules (proteins, carbohydrates), and (4) struc-

tural similarities between the microbe or environmental

agent and its host (Peterson and Fujinami, 2007).

RF/RHD is the most convincing example of molecu-

lar mimicry in human pathological autoimmunity, in

light of the cross-reactions between streptococcal anti-

gens and human tissue proteins, mainly heart tissue pro-

teins, that follow throat infection by S. pyogenes in

susceptible individuals.

The inflammatory process that follows an S. pyogenes

throat infection in individuals with genetic predisposition

leads to intense cytokine production by monocytes and

macrophages that trigger the activation of B and T

lymphocytes.

Several heart-reactive antibodies described from 1945

until nowadays (reviewed by Cunningham, 2000 and

Guilherme et al., 2011) also play role in the development

of the disease.

1028 PART | 14 Cardiovascular System and Lungs

Streptococcal and heart tissue cross-reactive antibo-

dies activate the heart tissue valvular endothelial cells,

increasing the expression of adhesion molecules such as

VCAM1, which facilitates cellular infiltration by neutro-

phils, monocytes, B and T cells (Yegin et al., 1997) The

“rolling” of leukocytes through vessels is triggered by

chemokines expressed by activated endothelial cells that

induce the expression of integrins, selectins, and subse-

quent trans-endothelial migration. Recently we identified

increased expression of ICAM, another adhesion mole-

cule, and a few chemokines (CCL-1, CCL-3, and CCL9),

as well as some integrins (P- and E-selectins) in the myo-

cardium and valvular tissue of RHD patients (Guilherme,

L. in preparation). All of these molecules are involved

with the inflammatory process and T and B lymphocyte

infiltration leading to rheumatic valvular tissue damage.

CD41 infiltrating T cells are predominant in heart

rheumatic lesions (Raizada et al., 1983; Kemeny et al.,

1989), and the first evidence of the molecular mimicry

between streptococcus and heart tissue was obtained

through an analysis of these heart tissue-infiltrating T

cells. Three immunodominant regions of the M5 protein

(residues 1�25, 81�103, and 163�177), heart tissue pro-

teins (myocardium and valve-derived proteins, as well as

vimentin), and synthetic peptides of the beta chain of car-

diac myosin-light meromyosin region (LMM) were recog-

nized by cross-reactivity by intralesional T cell clones

(Guilherme et al., 1995, 2001; Ellis et al., 2005; Fae

et al., 2006). Peripheral T cell clones also recognized

human purified myosin, tropomyosin, laminin, and car-

diac myosin-derived peptides from LMM and S2 regions

(Guilherme et al., 1995).

Employing a proteomics approach, we characterized a

number of mitral valve proteins identified by molecular

weight (MW) and isoelectric point (pI). Four valve-

derived proteins with molecular masses ranging between

52 and 79 kDa and different pI cross-reacted with the

M5 immunodominant peptides, and were recognized in

proliferation assays by intralesional T cell clones from

patients with severe RHD. Vimentin was one of the iden-

tified proteins, a result that reinforces the role of this pro-

tein as a putative autoantigen involved in the rheumatic

lesions. Novel heart tissue proteins were also identified,

including disulfide isomerase ER-60 precursor (PDIA3)

protein and a 78-kDa glucose-regulated protein precursor

(HSPA5). The role of PDIA3 in RHD pathogenesis and

other autoimmune diseases is not clear (Table 69.2) (Fae

et al., 2008).

The analysis of the T cell receptors (TCRs) of auto-

reactive T lymphocytes that infiltrate both myocardium

and valves allowed us to evaluate the Vβ chains usage of

TCR and the degree of clonality of heart tissue infiltrating

T cells (Guilherme et al., 2000). In the heart tissue (myo-

cardium and valves) of both chronic and acute RHD

patients, several expanded T cell populations with an oli-

goclonal profile were found. Such oligoclonal expansions

were identified by T cell receptor (TCR) analyses

(Guilherme et al., 2000). The finding of oligoclonal T

cell populations is in contrast with the peripheral blood

scenario, which contains polyclonal TCR-BV families.

The fact that a high number of T cell oligoclonal expan-

sions could be found in the valvular tissue indicates that

specific and cross-reactive T cells migrate to the valves

(Guilherme et al., 2000) and proliferate upon specific

cytokine stimulation at the site of the lesions.

Cytokines are important secondary signals following

an infection because they trigger effective immune

responses in most individuals and probably deleterious

responses in patients with autoimmune diseases. Three

subsets of T helper cytokines are currently described.

Antigen-activated CD41 T cells polarize to the Th1, Th2,

or Th17 subsets, depending on the cytokine secreted. Th1

is involved with the cellular immune response and pro-

duces IL-2, IFN-γ, and TNF-α. Th2 cells mediate humoral

and allergic immune responses and produce IL-4, IL-5,

and IL-13.

Another lineage of CD41 T cells, namely Th17 cells,

have been more recently described and produce a com-

plex set of cytokines initially identified as IL-17, TGF-β,IL-6, and IL-23. This subset of cells has been described

in and associated with several autoimmune diseases

(reviewed by Volin and Shahrara, 2011).

In RHD in both myocardium and valvular tissue, we

found large numbers of infiltrating mononuclear cells

secreting the inflammatory cytokines IFN-γ and

TNF-α. However, mononuclear cells secreting IL-10

and IL-4, which are regulatory cytokines, were also

found in the myocardium tissue; nonetheless, in the

valvular tissue, only a few cells secrete IL-4, suggest-

ing that low numbers of IL4-producing cells may con-

tribute to the progression of valvular RHD lesions

(Guilherme et al., 2004).

Recently, using immunohistochemistry, we identified

IL-171 and IL-231 infiltrating cells in both myocardium

and valvular tissue. The expression of these cytokines

was also observed in the valvular endothelium (manu-

script in preparation), confirming that Th17 cells also

play an important role in the inflammatory process in

RHD heart lesions.

AUTOANTIBODIES AS POTENTIALIMMUNOLOGIC MARKERS

Several streptococcal and human cross-reactive antibodies

have been found in the sera of RF patients and immu-

nized rabbits and mice over the last 50 years and have

been recently reviewed (Cunningham, 2000; Guilherme

1029Chapter | 69 Rheumatic Fever and Rheumatic Heart Disease

et al., 2004). N-acetyl β-D-glucosamine, which is present

in both the streptococcal cell wall and heart valvular tis-

sue, is one of the major targets of the humoral response in

RF/RHD, and antibodies against this polysaccharide dis-

played cross-reactivity with laminin, an extracellular

matrix alpha-helical coiled-coil protein that surrounds

heart cells and is also present in the valves (Cunningham

et al., 1989; Cunningham, 2000).

Cardiac myosin is the most important protein in the

myocardium and by using affinity purified anti-myosin

antibodies, Cunningham’s group identified a five amino

acid residue (Gln-Lys-Ser-Lys-Gln) epitope of the N-

terminal M5 and M6 proteins as being cross-reactive with

cardiac myosin (Cunningham et al., 1989).

The permanent rheumatic lesions that damage the

valves and antibodies against vimentin, an abundant pro-

tein in the valvular tissue, probably play a role in the val-

vular lesions (Cunningham, 2000).

In conclusion, antibodies against N-acetyl β-D-glucos-amine, some epitopes of cardiac myosin, and vimentin

can be considered as immunological markers of the

disease.

CONCLUDING REMARKS—FUTUREPROSPECTS

RF/RHD is the most convincing example of molecular

mimicry in which the response against S. pyogenes trig-

gers autoimmune reactions with human tissues. RF/

RHD lesions result from a complex network of several

genes that control both innate and adaptive immune

responses after an S. pyogenes throat infection. An

inflammatory process permeates the development of

heart lesions, in which adhesion molecules and specific

chemokines facilitate the valvular tissue infiltration by

B and T cells. CD41 T lymphocytes are the prime

effectors of heart lesions. Several self antigens such as

vimentin, myosin, and other mitral valve-derived pro-

teins are recognized by molecular mimicry of strepto-

coccal immunodominant peptides, particularly in

individuals with genetic predisposition. Production of

inflammatory cytokines (IFN-γ, TNF-α, IL-17, and IL-

23), and low numbers of IL-4 producing cells, a regula-

tory cytokine, lead to local inflammation.

All this information creates a new scenario for the

development of RHD, opening new possibilities for

immunotherapy. Molecular knowledge of the autoimmune

reactions mediated by intralesional T cells will certainly

assist in the choice of streptococcal protective epitopes

for the construction of an effective and safe vaccine.

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TABLE 69.2 Mitral Valve Proteins Identified by 2D Gel Electrophoresis and Mass Spectrometry Analysis Recognized

by Peripheral and Intralesional T Cells

Protein Accession number Coverage (%) Masses matched/total MW/pI

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