CELLULAR AND HUMORAL MECHANISMS OF
INNATE IMMUNITY
PHAGOCYTOSIS
Phagocytosis
Intracellular killing
Phagocyte
Bacterium
NK-CELLS
NK-cell
Lysis of infected cell
Virus-infected
cell
INFLAMMATION
Cytokines
IL-12
Bacterium
LPS
COMPLEMENT
TNF
IFN
Complement proteins
Neutrophil
NK-cell
Macrophage
Lysis of bacteria
Inflammation
Bacterium
Complement-dependent phagocytosis
DEVELOPMENT OF MACROPHAGES
Stem cell
Bone marrow
Szerv/szövet
Makrofág populáció
Bone
PU-1
Osteoclast
Central nervous
system
Microglia
Connective
tissue
Placenta
Histiocytes
Hofbauer cells
Vessel
Monocyte
Kidney
Liver
Peritoneum
Tissues
organs
Macrophage
Mesengial cells
Kupffer cells
Peritoneal macrophages
Lung
Alveolar macrophages
Skin
Epidermal and dermal
macrophages
Macrophages can act as stromal cells to help the differentiation of other cells.
RECEPTORS AND OTHER MOLEKULES OF
MACROPHAGES
LPS receptor (CD14) + TLR4
Scavanger receptor
Mannose receptor
MHCI
TLR – pathogen
pattern
FcRI (CD64)
Ag + IgG
complex
FcRII (CD32)
FcRIII (CD16)
LFA1 (CD11a/CD18)
peroxidáz
hidroláz
MHCII
CR1 (CD35)
CR3 (CD11b/CD18)
Activation of macrophages
Dale, D. C. et al. Blood 2008;112:935-945
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Receptors and molecules 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
Activation of macrophages
Activation of macrophages
Inflammatory cytokines
Microorganism
TNF
IL-6 IL-12
Inflammatory cytokines
Antimicrobial substances
IL-12
IL-18
Th 1 cell
NK cell
IFN
Inactivation
IL-4
IL-10
T cell
APC
IL-13
Th 2 cell
Alternative activation:
Mannose receptor – endocytosis
Th2 chemokines
NOS inhibition
Tissue regeneration
Functions of activated macrophages
in anti-bacterial immunity
Macrophage Response**
Role in Cell-mediated Immunity
Production of reactive oxygen intermediates,
nitric oxide; increased lysosomal enzymes
Killing of microbes in phagolysomes
(effector function of macrophages)
Secretion of Cytokines
(TNF-a, IL-1, IL-12)
TNF-a, IL-1: leukocyte recruitment
(inflammation)
IL-12: TH-1 differentiation, IFN- production
(induction of response)
Increased expression of:
CD80, CD86
Class I, Class II MHC
Increased T cell activation
(amplification)
**
These macrophage responses are induced by CD40 ligation to CD154 (CD40L) and T cellderived 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.
Reactive Oxygen Intermediate (ROI) production is initiated by membranebound NADPH oxidase, which is activated by IFN-.
NADPH Oxidase
O2 + NADPH
NADP + O2- + H+
O2- is further metabolized by superoxide dismutase (SOD).
SOD
-
O2 +
H+
O2 + H2O2
Intracellular bacterial killing
Reactive oxygen species
In the presence of appropriate iron catalysts, the Haber-Weiss reaction
takes place:
O2- + Fe3+
H2O2 + Fe2+
O2- + H2O2
O2 + Fe2+
OH + OH- + Fe3+
OH +OH- + O2
O2- is transformed into 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
1O – singlet oxygen, a highly reactive form of O
2
2
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-
MPO
OCl- + H2O
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).
Dale, D. C. et al. Blood 2008;112:935-945
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.
Intracellular bacterial killing
Reactive 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 species
NO 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.
Effects of reactive species
O2iNOS
0NO2-
NO
Destruction of
mitochondria
DNA destruction
Protein destruction
S-nitrosilation
PARP activation
Producing ROI
1 NAD  ADP – ribose + NAM
Decreased energy
4 ATP
CELL DEATH
Detection of mediators produced by macrophages
Phagocytosis assay:
yeast,uptake of fluorescent beads,
Preopsonized FITC labeled E. coli (FACS)
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)
Citokines : TNFa, TGFb (ELISA, ELISPOT)
ROI:
NBT reduction assay
Hydrogen peroxide assay
Citochrome c reduction assay
Intracellular bacterial evasion of
killing in phagocytes
Intracellular bacteria have evolved strategies to evade killing by the
mechanisms available to the phagocyte.
Macrophage effector capacity
Microbial evasion mechanism
Defensins
Unknown
Phagosome acidification
Phagosome neutralization
Phagosome–lysosome fusion
Lysosomal enzymes
Inhibition of phagosome–lysosome fusion
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
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
Granulomas
syncytium (multinucleated giant cells)
lymphocytes
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.
Tuberculosis (TBC)
Phatogen:
Mycobacterium tuberculosis
Mycobacterium 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.
2.000.000.000 infected worldwide
Mycobacterium infection
3. T cell and macrophage
activation
IFN-
TNF
2. Antigen presentation
IL-12
1.Infection of
macrophages
Macrophage
CD4+
T cell
Macrophage
DS
CD8+
T cell
Macrophage
perforin
granulysin
Mycobacterium infection
Symptom-free carriers
90%
IFN-
TNF
IL-12
CD4+
T cell
MTb. remains in granulomas
Macrophage
DS
Macrophage
Healing (?)
CD8+
T cell
perforin
granulysin
Reactivation
(10%)
Reinfection
HIV infection:
800x more
tuberculosis
Acute tuberculosis - 10%
(HIV infection)
Other
immune suppression
Dissemination
Transmission
Appearance and frequency of TBC
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
Morbus hungaricus
Morbidity 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
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
Diagnostic testing of tuberculosis
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)
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