NK-CELLSVirus-infected

cell

NK-cell Lysis of infected cell

INFLAMMATION

BacteriumLPS

Cytokines

PhagocytosisIntracellular killing

PHAGOCYTOSIS Phagocyte

Bacterium

Neutrophil

NK-cell

Macrophage

TNF

IL-12

Bacterium

Complement proteins Lysis of bacteria

Inflammation

Complement-dependent phagocytosis

IFN

COMPLEMENT

CELLULAR AND HUMORAL MECHANISMS OF INNATE IMMUNITY

Vessel

Bone marrow

Stem cell

PU-1

Szerv/szövet Makrofág populáció

Bone Osteoclast

Central nervous system

Microglia

Connective tissue

Histiocytes

Placenta Hofbauer cells

Kidney Mesengial cells

Liver Kupffer cells

Peritoneum Peritoneal macrophages

Lung Alveolar macrophages

Skin Epidermal and dermal macrophages

Macrophages can act as stromal cells to help the differentiation of other cells.

Monocyte

Macrophage

Tissuesorgans

DEVELOPMENT OF MACROPHAGES

RECEPTORS AND OTHER MOLEKULES OF MACROPHAGES

LPS receptor (CD14) + TLR4

MHCI

MHCII

TLR – pathogen pattern

CR1 (CD35)

CR3 (CD11b/CD18)

LFA1 (CD11a/CD18)

FcRIII (CD16)

FcRII (CD32)

FcRI (CD64)

Ag + IgG complex

Mannose receptor

Scavanger receptor

peroxidázhidroláz

Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

Dale, D. C. et al. Blood 2008;112:935-945

Activation of macrophages

RECEPTOR LIGAND FUNCTION

FcR IgG, IgE Opsonized phagocytosis, ADCC, release of inflammatory mediators

CR3 iC3B, ICAM-1 Opsonized phagocytosis

Macrophage Mannose Receptor

Lectin, Endocytosis, phagocytosis, antigen capture and transport

SR-A LPS, polianions, lipoteikolic acid

Endocytosis, phagocytosis, adhesion

CD14 LPS Transduces LPS aktivation , TNFa release

CCR1 MIP1a, MCP-3 Recruitment, migration of monocytes

CCR3 Eotaxin Haematopoiesis, HIV-1 coreceptor

CCR5 MIP1 Haematopoiesis, HIV-1 coreceptor

CXCR4 SDF-1a Haematopoiesis, HIV-1 coreceptor

Receptors and molecules of macrophages

Activation of macrophages

IFN

IL-12IL-18

Th 1 cellNK cell

Inflammatory cytokines Antimicrobial substances

Alternative activation: Mannose receptor – endocytosis Th2 chemokines NOS inhibition Tissue regeneration

IL-4IL-13 Th 2 cell

MicroorganismTNF

IL-6 IL-12

IL-10

T cellAPC

Inactivation

Activation of macrophages

Inflammatory cytokines

Functions of activated macrophagesin anti-bacterial immunity

Macrophage Response** Role in Cell-mediated Immunity

Production of reactive oxygen intermediates, Killing of microbes in phagolysomesnitric oxide; increased lysosomal enzymes (effector function of macrophages)

Secretion of Cytokines TNF-, IL-1: leukocyte recruitment(TNF-, IL-1, IL-12) (inflammation)

IL-12: TH-1 differentiation, IFN- production(induction of response)

Increased expression of: Increased T cell activationCD80, CD86 (amplification)

Class I, Class II MHC

** These macrophage responses are induced by CD40 ligation to CD154 (CD40L) and T cell-derived IFN- in cell-mediated immunity; similar responses are induced by microbial products, particularly LPS, and NK cell-derived IFN- in innate immunity.

Phagocytosis

Intracellular Bacterial Killing

Intracellular bacterial killing

Reactive oxygen species

Intracellular bacteria in phagolysosomes are susceptible to reactive oxygen species, which damage cell wall components and fragment genomic DNA.

O2 + NADPHNADPH Oxidase

NADP + O2- + H+

O2- + H+

SODO2 + H2O2

Reactive Oxygen Intermediate (ROI) production is initiated by membrane-bound NADPH oxidase, which is activated by IFN-.

O2- is further metabolized by superoxide dismutase (SOD).

Intracellular bacterial killing

Reactive oxygen speciesIn the presence of appropriate iron catalysts, the Haber-Weiss reaction takes place:

O2- + Fe3+ O2 + Fe2+

H2O2 + Fe2+ OH + OH- + Fe3+

O2- + H2O2 OH +OH- + O2

O2- is transformed into 1O2. 1O2 and OH are short-lived, powerful

oxidants with high antibacterial activity, causing damage to DNA, membrane lipids, and proteins.

Nomenclature

O2- - superoxide anion

OH – hydroxyl radical containing a free electron 1O2 – singlet oxygen, a highly reactive form of O2

Intracellular bacterial killing

Reactive oxygen species

myeloperoxidase-dependent killing

The lysosomes of granulocytes and monocytes/macrophages contain the enzyme myeloperoxidase (MPO). This enzyme catalyzes the following reaction:

H2O2 + Cl- OCl- + H2OMPO

Hypochlorous acid and chloramines are formed – both agents further increase the bactericidal power of the ROI system by destroying biologically important proteins through chlorination (Halogenation).

Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

Dale, D. C. et al. Blood 2008;112:935-945

Intracellular bacterial killingReactive nitrogen species

Phagocytes possess an additional pathway for generating reactive species that possess bactericidal activity. These species are the reactive nitrogen intermediates (RNI).

The principal RNI is nitric oxide (NO), which is derived from the terminal guanidino-nitrogen atom of L-arginine. The reaction is catalyzed by the inducible form of nitric oxide synthase (iNOS; NOS2), leading to the formation of L-citrulline and NO.

Intracellular bacterial killing

Reactive nitrogen speciesNO can act as an oxidizing agent alone, or it interacts with O2

- to form unstable peroxynitrite (ONOO-). This may be transformed to the more stable anions, NO2

- and NO3-, or decomposed to NO.

O2- + NO ONOO-

ONOO- + H+ NO2- + .OH

NO2- + .OH NO3

- + H+

ONOO- + H+ .OH + NO.

NO· and ONOO- are highly reactive antimicrobial agents. NO· may be transformed to nitrosothiols expressing the most potent antimicrobial activity. In contrast, NO2

- and NO3- are without

notable effects on microorganisms.

NO

O2-

0NO2-

Destruction of mitochondriaDNA destruction

PARP activation

CELL DEATH

Producing ROI

Decreased energy1 NAD ADP – ribose + NAM

4 ATP

Protein destruction

iNOS

S-nitrosilation

Effects of reactive species

NO : sera, (clinical)

Gries Ilosvay assay (reduction of nitrit and nitrate to NO), Arginin –Citrulin reaction, detection of iNOS activity (IHC, Western blot), measure of released NO by DAF (FACS)

Detection of mediators produced by macrophages

Phagocytosis assay:

yeast,uptake of fluorescent beads,Preopsonized FITC labeled E. coli (FACS)

Citokines : TNFa, TGFb (ELISA, ELISPOT)

ROI:NBT reduction assayHydrogen peroxide assayCitochrome c reduction assay

Intracellular bacterial evasion ofkilling in phagocytes

Intracellular bacteria have evolved strategies to evade killing by the mechanisms available to the phagocyte.

Defensins Unknown

Phagosome acidification Phagosome neutralization

Phagosome–lysosome fusion Inhibition of phagosome–lysosome fusionLysosomal enzymes Resistance against enzymes

Intraphagolysosomal killing Evasion into cytosol Robust cell wall

C3b receptor-mediated uptake,

ROI ROI detoxifiers, ROI scavengers

RNI Unknown (ROI detoxifiers probably interfere with RNI)

Iron starvation Microbial iron scavengers (e.g., siderophores)

Tryptophan starvation Unknown

Macrophage effector capacity Microbial evasion mechanism

Diseases in which macrophages play a significant role

Type Example Mechanism

Lysosomal storage diseases

Gaucher-syndrome

Genetically coded, disfunction of glucocerebrozidase

Niemann-Pick-syndrome

Lack of sphyngomyelinase or disfunction of cholesterol estherization and –transport sphyngomyelin and cholesterol accumulation

Tay –Sachs-syndrome

Most prominent gangliosidosis, lack of hexose-aminidase-A, gangliosides accumulation in CNS

Diseases in which macrophages play a significant role

Type Example Mechanism

Infections AIDS Cellular immunodeficiency, lack of CD4+ T cells and macrophages

Malaria Host mononuclear phagocyte system hyperplasia, massive splenomegaly

Cerebrospinal diseases

Alzheimer-dysease

Senilis cerebralis amiloidosis, caused by improper elimination of amyloid-associated protein because of defects in macrophage enzymes

Diseases in which macrophages play a significant role

Type Example Mechanism

Chronic inflammation

Silicosis Chrystal quartz powder phagocytosed by alveolar macrophages - progrediated nodular fibrotizing pneumoconiosis

Asbestosis Asbestos filaments phagocytosed by alveolar macrophages - chromic desquamative alveolitis and interstitial inflammation become fibrosis

Atherosclerosis

Monocytes exit to the intima from the blood, become macrophages and store fat cytoplasmatically: foamy cells - inflammation

Granulomatosus inflammation

- chronic inflammation- epitheloid cells in the infiltrate, these are modified macrophages whit pale cytoplasm and nucleus - cells with no intercellular substances (epithelial cell-like tight connections)

- cells become multinucleated Langhans type giant cells

↓Granulomatosus inflammation:

granuloma formation with cell death

lymphocytes

syncytium (multinucleated giant cells)

Granulomas

Periapical granuloma = dental granuloma

Modified granulation tissue containing elements of chronic inflammation, located adjacent to the root apex of a tooth with infected, necrotic pulp.

Phatogen:Mycobacterium tuberculosisMycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti can also cause tuberculosis, but these species do not usually infect healthy adults

Tuberculosis most commonly attacks:• the lungs (as pulmonary TB)•central nervous system (meningitidis) • lymphatic and circulatory system (miliary TB) •genitourinary system, •bones, joints• skin

From 2000 to 2004, 20% of TB cases being resistant to standard treatments and 2% resistant to second-line drugs.

Tuberculosis (TBC)

2.000.000.000 infected worldwide

Mycobacterium infection

DS

1.Infection of macrophages

CD8+ T cell

CD4+ T cell

IL-12

2. Antigen presentation

Macrophage

perforingranulysin

IFN-TNF

3. T cell and macrophage activation

Macrophage

Macrophage

Mycobacterium infection

Healing (?)

Acute tuberculosis - 10% (HIV infection)

Symptom-free carriers90%

MTb. remains in granulomas

Reinfection

Dissemination

Transmission

Reactivation(10%)

HIV infection:800x more tuberculosis

Other immune suppression

DS

CD8+ T cell

CD4+ T cell

IL-12

Macrophage perforingranulysin

IFN-TNF

Macrophage

Morbus hungaricusMorbidity of TBC in Hungary:

• first decades of the later century: 340-380/100000 citizens

• 1955: 30/100000 citizens (lower than the European average!)

reason: regular screening, vaccination, up-to-date therapy

• In last years: increasing numbers of TBC

reason: optimistic attitude, ease of strict control

The 90% of people infected with bacteria are symptome-free, living with latent TBC (LTBI), their opportunity is 10% to develop disease.Without treatment, 50% of TBC diseases are lethal.

TBC is one of the three most dangerous infectious diseases worldwide, mortality is two times higher than to malaria.

2.000.000.000 infected persons

Appearance and frequency of TBC

Skin and bone - tuberculosis

Primary infection

Miliaris tuberculosis

CNS tuberculosis

Lymphnode tuberculosis

Diagnostic testing of tuberculosis

Tuberculin skin test(TST) Most often applied tuberculin test:

Mantoux’s test PPD (purified protein derivative) Size of induratio (after 48h)

Disadvantages:

• Not specific for M. tuberculosis

• Positiv reaction: in case of atypical Mycobacterial diseases and BCG vaccination also

IFNγ release assay (IGRA) - ELISPOT ESAT-6 (early secrete antigen target 6) and CFP-10 (culture filtrate

protein) stimulatory antigens Measuring: release of IFNγ by T cells Results: SFU (Spot Forming Unit)

Advantages:

• More specific than TST

• Can be repeated

• The testing protocol requires only one visit

Disadvantages:Reversion: a previously positive IGRA results becomes negative upon revers testing, due to• clearing of TB infection (spontaneous or due to treatment) • biological variations among IGRA+ individuals• the life cycle of M. tuberculosis, where the Mycobacterium enters a dormant state in which it may not be secrete ESAT-6 and CFP-10 antigens (but instead secrete other antigens which are not used in currently available IGRAs)

Diagnostic testing of tuberculosis

Treatment

First line tuberculosis drugs

3-letter 1-letter Drug

EMB E Ethambutol

INH H Isoniazid

PZA Z Pyrazinamide

RMP R Rifampicin

STM S streptomycin

Second line tuberculosis drugs

CIP (none) Ciprofloxacin

MXF (none) Moxifloxacin

PAS P p-aminosalicylic acid

All first-line anti-tuberculous drug names have a standard three-letter and a single-letter abbreviation:•Streptomycin is STM or S, •isoniazid is INH or H, •rifampicin is RMP or R, •ethambutol is EMB or E, •pyrazinamide is PZA or Z. The US commonly uses abbreviations and names that are not internationally recognised: rifampicin is called rifampin and abbreviated RIF; streptomycin is commonly abbreviated SM.

The standard "short" course treatment for tuberculosis (TB):

2 months: isoniazid, rifampicin, pyrazinamide, and ethambutol+4 months: isoniazid and rifampicin alone

For latent tuberculosis, the standard treatment is six to nine months of isoniazid alone