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21. Immune mechanisms of inflammation (local and systemic reaction).
22. Physiological mechanisms of regulation of the immune system. Cytokines
(classification according to the function).
23. Defence against extracellular pathogens .
24. Defence against intracellular .
25. Anti-viral defence.
26. Defence against multicellular parasites.
27. Tumour immunology - tumour antigens, mechanisms of defence.
28. Alloimmune reaction. Types of transplantations and immunological
examination before transplantation. Immunologically privileged tissues.
29. Types of graft rejection and their mechanisms. GvH. Principle of materno-
foetal tolerance. Rh incompatibility
Inflammation
Inflammation
Is a physiological response to breach integrity of the
organism, leading to localization of damage, protection
against infection of damaged sites and healing.
Causes of inflammation
 Physical injury
 Infection by pathogens
 Damage caused by chemicals
 Cancer
 Alergic disease
 Autoimmune disease
Inflammation
Inflammation
acute (physiological reactions,
damaged tissue heals completely)
chronic (usually pathological reactions,
destruction of tissue and
compensation with fibrous tissue)
Response of the organism
local
systemic
Local body's response to inflammation
Signs - pain (dolor), heat (calor), redness (rubor), swelling (tumor)
Local inflammation
The first signals for the development of inflammatory
reactions originate from activated phagocytes, mast
cells, complement and substances released from
damaged cells and extracellular matrix components.
Local inflammation
 vasodilation and increased vascular permeability (histamine,
serotonin, bradykinin, complement components C3a, C5a,
leukotrienes, prostaglandins) => redness and swelling
 increased expression of adhesion molecules on endothelial cells
(TNF, IL-1) => capture leukocytes and phagocytes
 influence nociceptors (prostaglandins, ...) => pain
 Increase temperature (IL- 1, IL-6, TNF, prostaglandins)
Local inflammation
Systemic inflammation
 leukocytosis
 fever (TNFa, IL-1, IL-6, IFN)
 ↑ tissue metabolism
 ↑ mobility of leukocytes
 ↑ IFN, cytokines and Ig production
 ↑ expression of Hsp
 acute phase proteins (IL-6, TNF, IL-1)
Acute phase proteins
produced by hepatocytes:
 CRP - opsonization, complement activation
 SAP - opsonization, complement activation
 SAA - attracting leukocytes
 C3, C4
 protease inhibitors - protection against secondary tissue
damage
 serum transport proteins
Systemic inflammation
Septic shock - the massive penetration of microorganisms
into the bloodstream (  TNF)
Anaphylactic shock - basophil degranulation after
contact with allergen (histamin)
Repair of damaged tissue
 after the elimination of pathogens and damaged cells by
phagocytes
 activation of angiogenesis
 regeneration and tissue remodeling (fibroblasts, smooth
muscle cells, keratinocytes, epithelial cells)
 regulated by cytokines: PDGF, TGFb (platelets,
macrophages ...)
Physiological regulation
of the immune system
Physiological regulation
of the immune system
 Regulation by antigen
 Regulation by antibodies
 Regulation by cytokines and cellular contact
 Suppression mediated by T cells
 Other factors influencing the outcome of the immune
response
 Regulation by cytokines
Regulation by antigen
 Immune responses induction and extinction
 Affinity maturation of B lymphocytes
 Maintaining immunological memory
 Antigenic competition
 Threshold density of the complex MHC II-gp Ag on APC
Regulation by antibodies
 Antibodies competes with the BCR for antigen (negative
regulator of B lymphocyte stimulating)
 IgG immune complexes bind to the BCR and FcR on
B cells, resulting in blocking of B cell activation
 Regulation via idiotypic network
Regulation by cytokines and cellular contact
 Interaction APC - T lymphocyte
 Interaction TH1 – macrophages
 Interaction TH2 - B lymphocytes
 Mutual regulation of activity TH1 versus TH2
 Development of leukocyte subpopulations
Negative regulation of effector cells:
 CTLA-4 - T cell inhibitory receptor, binds ligands CD80 and CD86
 Inhibitory receptors of NK cells
 Self-destruction interaction of the apoptotic receptor Fas with
ligand FasL on the surface of activated T lymphocytes
Suppression mediated by T cells
 Mutual negative interactions mediated by Th1 and Th2
cytokines (Th2 cells produce IL-4 and IL-10 which suppress the
immune response, based on TH1 cells)
 Clonal elimination or anergition of T cells after antigen
recognition on the surface of other cells than APC (lacking
co-stimulatory signals)
 Regulatory T cells maintain tolerance to autoantigens
(Treg, Tr1)
Factors influencing the outcome of the
immune response
The same antigen can induce an active immune response
or an active state of tolerance, the result of response
depends on many factors:
 State of the immune system
 Properties of antigen
 Dose of antigen
 Route of antigen administration
Cytokines
(Tissue hormones)
Cytokines
 Regulatory proteins and glycoproteins produced by leukocytes and
other cells
 Essential regulators of the immune system
 Apply outside the immune system (angiogenesis, tissue
regeneration, carcinogenesis, treatment of many brain functions,
embryonic development ...)
 Cytokines - secreted
- membrane (CD 80, CD86, CD40L, FasL ..)
Cytokines
 Pleiotropic effect
 Operates in a cascade
 Cytokine Network
 Cytokine system is redundant
 Effects of cytokines - autocrine
- paracrine
- endocrine
 Are known as interleukins (exception: TNF, lymphotoxin, TGF,
interferons, CSF and growth factors)
Distribution of cytokines by function
1) Proinflammatory cytokines (IL-1, IL-6,IL- 8,IL- 12,IL- 18, TNF)
2) Antiinflammatory cytokines (IL-4, IL-10, TGFb)
3) Cytokines with the activity of hematopoietic cells growth
factor (IL-2, 3, 4, 5, 6, 7, 9, 11, 14, 15, CSF, SCF, LIF, EPO)
4) Cytokines applying in TH2 humoral immunity (IL-4, 5, 9, 13)
5) Cytokines applying in the cell-mediated immunity TH1
(IL-2, 12, IFN, GM-CSF, lymphotoxin)
6) Cytokines with antiviral effect (IFN-a, IFN-b , IFN- )
Overview of the most important cytokines
Cytokine Produced
Function
IL-1
MF, N
T cell costimulation, induction of TNF and IL-8, pyrogen
IL-2
Th1
Growth factor for T cells
IL-4
Th2, basophils
Th2 differentiation, B cell stimulation, isotype switching to IgE and IgG4, Th1
inhibition
IL-5
Th2, eosinophils
B cell stimulation, growth factor for eosinophils
IL-6
Th2, MF, N
T and B cell stimulation, stimulation of Ig production, induction of acute phase
proteins synthesis, pyrogen
IL-8
MF, other cells
Granulocyte activation and chemotaxis (primarily neutrophils)
IL-10
Th2,M, Treg
Th1 and MF inhibition, B cell differentiation to plasma cell
IL-12
MF, DC, B
Th1 differentiation, NK stimulation
TNF
M, MF, NK
Induction of local inflammation, endothelium activation, induction of apoptosis
TGFb
T, MF, platelets
The anti-inflammatory effect (control of lymphocyte proliferation, control of Ig
production, control MF activity), stimulation of fibroblasts and osteoblasts,
gain production of extracellular matrix
IFNa
L, M, MF
Inhibition of viral replication
IFNb
Fibroblasts,
epithelial cells
Inhibition of viral replication
IFN
Th1, NK
MF activation, stimulation of MHC gp. expression, Th2 inhibition
MF – macrophages; M – monocytes; N – neutrophils; DC – dendritic cells; NK – natural killers; L – lymphocytes; B – B cell; T – T cell
Cytokine receptors
 Consisting of 2 or 3 subunits
 One subunit binds cytokine, other are associated with cytoplasmic
signaling molecules (protein kinases)
 Signaling subunit is shared by several different cytokine receptors called receptor family
 Signaling through these receptors may lead to proliferation,
differentiation, activation of effector mechanisms or blocking the cell
cycle and induction of apoptosis
Defense against
extracellular pathogens
Defence against extracellular pathogens
 bacteria (gram-negative, gram-positive cocci, bacilli),
unicellular parasites
 pathogens induce inflammation
 removed by phagocytosis - neutrophil granulocytes
 opsonization (IgG and IgA antibodies, C3b, lectins,
CRP...)
Defence against extracellular pathogens
Opsonisation and phagocytosis
Defence against extracellular pathogens
 Phagocytes are attracted to the site of infection by
chemotactic substances (C5a, C3a and chemotactic products
of bacteria…)
 ingested bacteria are destroyed by the microbicidal systems
(products of NADP-H oxidase, hydrolytic enzymes and
bactericidal substances in lysosomes)
 phagocytes produce proinflammatory cytokines
(IL-1, IL-6, TNF)
Defence against extracellular pathogens
 IgM - complement activation
 IgG - activation of complement, opsonization
 IgA - opsonization
sIgA prevents against infection by intestinal and respiratory bacteria
 in the defense against bacterial toxins apply neutralizing
antibodies (Clostridium tetani and botulinum …)
Defence against extracellular pathogens
 "indirect toxins - bacterial Lipopolysaccharide (LPS)
stimulates big number of monocytes to release TNF,
which can cause septic shock
 individuals with immunodeficiency of phagocytes,
complement and antibodies production are especially
at risk of infections with extracellular bacterial
Defense against
intracellular pathogens
Defense against intracellular pathogens
 bacteria, fungi and unicellular parasites
 intracellular parasites are resistant to the microbicidal
mechanisms of phagocytes
 macrophages, which absorbed them, produce IL-12 → TH1
differentiation, production of IFN and membrane TNF →
activation of macrophages and production of NO
Defense against intracellular pathogens
Defense against intracellular pathogens
 TC lymphocytes apply in the defense against intracelular
parasites, which escape from phagolysosomes
 individuals with certain disorders of phagocytes and
defects of T lymphocytes are at risk of infections with
intracellular microorganisms
Defense against intracellular pathogens
Anti-viral defense
Anti-viral defence
 interferons - production of IFNa and IFNb is induced in
infected cells; IFN activates macrophages (iNOS)
 IFNa and IFNb - prevents viral replication
- induce proliferation of NK cells
- increase the expression of HLA-I
Anti-viral defence - interferons
Anti-viral defence
 NK cells - ADCC (Antibody-dependent cell-mediated
cytotoxicity); NK cell bind with CD16 (Fc receptor) to
IgG which has bound to the surface of infected cell and
then NK cell release perforins and granzymes
(degranulation)
 infected macrophages produce IL-12 (a strong activator
of NK cells)
Anti-viral defence - NK cell activation
ADCC
Anti-viral defence
 in the defense against cytopathic viruses applied
antibodies:
 sIgA inhibit mucosal adhesion of viruses (defense
against respiratory viruses and enteroviruses)
 neutralizing IgG and IgM antibodies activate
the classical pathway of complement, that is able
to lyse certain viruses
 opsonized viral particles are phagocytosed
 IgA and IgG have preventive effect in secondary
viral infection
Anti-viral defence - antibodies
Anti-viral defence
 effector TC lymphocytes destroy infected cells in direct contact
(granzym/perforin; FasL) and by produced cytokines (lymfotoxin)
 some viruses after infection integrate into the host genome, where
persist for years (varicella zoster, EBV, papillomavirus)
 individuals with T lymphocyte immunodeficiency and with combined
immune disorders are at risk by viral infections
 increased susceptibility to herpes infections in individuals with
dysfunction of NK cells
Anti-viral defence – NK cells and Tc lymphocytes
Defense against
multicellular parasites
Defense against multicellular parasites
 IgE, mast cells, basophils and eosinophils
 TH2 stimulation under the influence of IL-4 (mast cells
and other APC stimulated by parasite)
 TH2 stimulate B cells with BCR-specific parasite antigens
 isotype switching under the influence of IL-4 to IgE
 IgE bind to FceRI on mast cells and basophils
Defense against multicellular parasites
 multicellular parasite binds to IgE on mast cell→ crosslinking of several molecules FceRI
 initiate mast cell degranulation (release of histamin,
tryptase, serotonin…)
 activation of arachidonic acid metabolism (leukotriene
C4, prostaglandin PGD2) - amplification of inflammatory
responses
 cytokine production by mast cell (TNF, TGFb, IL-4, 5, 6)
Defense against multicellular parasites
Histamine
 vasodilatation, increase vascular permeability (erythema,
edema, itching)
 bronchoconstriction (cough)
 increases intestinal peristalsis (diarrhea)
 increased mucus secretion
This helps eliminate the parasite.
Mast cell activation
Defense against multicellular parasites
 eosinophils fagocyte complexes of parasitic particles
with IgE via their receptors for IgE
 eosinophils use against parasites extracellular
bactericidal substances released from granules
(ECP- eosinophil cationic protein, MBP-major basic protein…)
Defense against multicellular parasites - eosinophils
Tumour immunology
Tumor antigens
a) Tumor – specific antigens (TSA)

complexes of MHCgp I with abnormal fragments of cellular proteins
(chemically induced tumors, leukemia with chromosomal translocation)

complexes of MHC gp with fragments of oncogenic viruses proteins
(tumors caused by viruses: EBV, SV40, polyomavirus…)

abnormal forms of glycoproteins
(sialylation of surface proteins of tumor cells)

idiotypes of myeloma and lymphoma
(clonotyping TCR and BCR)
Tumor antigens
b) Tumor - associated antigens (TAA)
 present also on normal cells
 differences in quantity, time and local expression
 auxiliary diagnostic markers
Tumor - associated antigens

onkofetal antigens -on normal embryonic cells and some tumor cells
 a-fetoprotein (AFP) - hepatom
 carcinoembryonic antigen (CEA) - colon cancer

melanoma antigens - MAGE-1, Melan-A

antigen HER2/neu -receptor for epithelial growth factor, mammary
carcinoma

EPCAM – epithelial cell adhesion molecule, metastases

differentiation antigens of leukemic cells - present on normal cells of
leukocytes linage
 CALLA -acute lymphoblastic leukemia (CD10 pre-B cells)
Anti-tumor immune mechanisms
Immune control
 tumor cells normally arise in tissues
and are eliminated by T cells
Anti – tumor immune response
 tumor cells are weakly immunogenic
 occurs when tumor antigens are presented to T lymphocytes
by dendritic cells activated in the inflammatory environment
Anti-tumor immune mechanisms
If tumor cells are detected, in defense may be
involved non-specific mechanisms (neutrophilic
granulocytes, macrophages, NK cells, complement) and
antigen-specific mechanisms (TH1 and TC cells,
antibodies).
Anti-tumor immune mechanisms
 DC are necessary for activation of antigen specific mechanisms
 predominance of TH1 (IFN , TNFa)
 specific cell-mediated cytotoxic reactivity – TC
 activation of TH2 → stimulation of B cells→ tumor specific
antibodies production (involved in the ADCC)
 tumor cells are destroyed by cytotoxic NK cells (ADCC)
 interferons - antiproliferative, cytotoxic effect on tumor cells
- INF - DC maturation
Regulatory T cells prevents removal of cancer cells and
thus contribute to the development of the tumor.
Mechanisms of tumor resistance to the
immune system
 high variability of tumor cells
 low expression of tumor antigens
 sialylation
 some anticancer substances have a stimulating effect
 production of factors inactivating T lymphocytes
 expression of FasL → T lymphocyte apoptosis
 inhibition of the function or durability dendritic cells (NO, IL-10,
TGF-b)
Transplantation
Transplantation
= transfer of tissue or organ
 autologous - donor = recipient
 syngeneic - genetically identical donor and recipient
(identical twins)
 allogeneic - genetically nonidentical donor of the
same species
 xenogenic - the donor of another species
 implant - artificial tissue compensation
Allotransplantation

differences in donor-recipient MHC gp and secondary
histocompatibility Ag
 alloreactivity of T lymphocytes - the risk of rejection and
graft-versus-host disease
Tests prior to transplantation
 ABO compatibility (matching blood group)
-risk of hyperacute or accelerated rejection
(= formation of Ab against A or B Ag on graft vascular endothelium)
 HLA typing (matching tissue type)
- determining of HLA alelic forms by phenotyping or genotyping
 Cross-match - detection of preformed alloantibodies
(after blood transfusions, transplantation, repeated childbirth)
 Mixed lymphocyte reaction - testing of T lymphocytes
alloreactivity
Tests prior to transplantation
HLA typing
1) Serotyping (microlymfocytotoxic test)
Allospecific serums (obtained from multiple natal to 6 weeks after
birth, or commercially prepared sets of typing serums (monoclonal
antibodies))
Principle - the incubation of lymphocytes with typing serums in the
presence of rabbit complement, then is added the vital
dye which stained dead cells
- cells carrying specific HLA are killed by complement
Number of death cells is a measure of serum toxicity, positive reaction
is considered more than 10% dead cells
HLA - serotyping
Tests prior to transplantation
HLA typing
2) Molecular genetic methods
2a) PCR-SSP
= Polymerase chain reaction with sequential specific primers
 Extracted DNA is used as a substrate in a set of PCR reactions
 Each PCR reaction contains primers pair specific for a certain allele
(or group of alleles)
 Positive and negative reactions are evaluated by electrophoresis,
each combination of alleles has a specific electrophoretic pattern
Tests prior to transplantation
2b) PCR-SSO
HLA typing
 PCR reaction with sequence-specific oligonucleotides
Multiplication of hypervariable sections of genes coding HLA
 Hybridization with enzyme or radiolabeled DNA probes specific for
individual alleles
2c) PCR-SBT
 Sequencing based typing
 The most accurate method of HLA typing
 We get the exact sequence of nucleotides, which compares
with a database of known sequences of HLA alleles
Tests prior to transplantation
Cross-match testing
 determination of preformed alloantibodies
 recipient serum + donor lymphocytes + rabbit complement
→ if cytotoxic Ab against donor HLA Ag are present in recipient serum ,
Ab activate complement → lysis of donor lymphocytes.
Dye penetration into lysis cells.
 positive test = the presence of preformed Ab → risk of hyperacute
rejection! → contraindication to transplantation
Tests prior to transplantation
Mixed lymphocyte reaction (MRL)
 determination of T lymphocytes alloreactivity
 mixed donor and recipient lymphocytes → T lymphocytes
after recognition of allogeneic MHC gp activate and proliferate
One-way MRL
 determination of recipient T lymphocytes reactivity against donor cells
 donor cells treated with chemotherapy or irradiated lose the ability of
proliferation
One-way MRL
Immunologically privileged sites and tissues
 Transplantation of some tissues don´t lead to the induction
of allogeneic reactivity
 Evolutionarily significant, protection of vital organs (brain,
eye, gonads)
 Factors protecting immunologically privileged structures
 isolation from the immune system (minimal content of lecocytes)
 preference of TH2 reactoin, supression of TH1 reaction
 FasL expression
 production of TGFb
Rejection
 hyperacute
 accelerated
 acute
 chronic
Hyperacute rejection
 minutes to hours after transplantation
 humoral mediated immune response
mechanism:
 if in recipients blood are present preformed or natural Ab (IgM anti-
carbohydrate Ag) before transplantation → Ab + Ag of graft (MHC gp
or endothelial Ag) → graft damage by activated complement
 the graft endothelium: activation of coagulation factors and platelets,
formation thrombi, accumulation of neutrophil granulocytes
prevention:
 negative cross match before transplantation, ABO compatibility
Accelerated rejection
 3 to 5 days after transplantation
 caused by antibodies that don´t activate complement
 cytotoxic and inflammatory responses triggered by
binding of antibodies to Fc-receptors on phagocytes and
NK cells
prevention:
 negative cross match before transplantation,
ABO compatibility
Acute rejection
 days to weeks after the transplantation or after a lack of
immunosuppressive treatment
 cell-mediated immune response
mechanism:
 reaction of recipient TH1 and TC cells against Ag
of graft tissue
 infiltration by lymphocytes, monocytes, granulocytes
around small vessels → destruction of tissue transplant
Chronic rejection
 from 2 months after transplantation
 the most common cause of graft failure
mechanism is not fully understood:
 non-immunological factors (tissue ischemia) and TH2 response
with production alloantibodies, pathogenetic role of cytokines
and growth factors (TGFβ)
 fibrosis of the internal blood vessels of the transplanted tissue,
endothelial damage →impaired perfusion of graft → gradual loss of
its function
 dominating findings: vascular damage
Rejection
Factors:
 The
genetic difference between donor and recipient,
especially in the genes coding for MHC gp (HLA)
 Type
of tissue / organ - the strongest reactions against
vascularized tissues containing many APC (skin)
 The
activity of the recipient immune system - the
immunodeficiency recipient has a smaller rejection reaction;
immunosuppressive therapy after transplantation –
suppression of rejection
 Status
of transplanted organ - the length of ischemia, the
method of preservation, traumatization of organ at collection
Bone Marrow Transplantation
 Removal of hematopoietic stem cells
 Myeloablation
 Transplantation
 Engraftment
 Rejection
 Graft-versus-host reaction
Graft-versus-host (GvH) disease
 after bone marrow transplantation
 GvH also after blood transfusion to immunodeficiency
recipients
 T-lymphocytes in the graft bone marrow recognize
recipient tissue Ag as foreign (alloreactivity)
Acute GvH disease
 days to weeks after the transplantation of stem cells
 damage of liver, skin and intestinal mucosa
 prevention: appropriate donor selection, the removal
of T lymphocytes from the graft and effective
immunosuppression
Chonic GvH disease
 months to years after transplantation
 infiltration of tissues and organs by TH2 lymphocytes,
production of alloantibodies and cytokines → fibrosis
 process like autoimmune disease: vasculitis, scleroderma,
sicca-syndrome
 chronic inflammation of blood vessels, skin, internal
organs and glands, which leads to fibrosis, blood circulation disorders
and loss of function
Graft versus leukemia effect (GvL)
 donor T lymphocytes react against residual
leukemick cells of recipient (setpoint response)
 mechanism is consistent with acute GvH
 associated with a certain degree of GvH (adverse
reactions)
Immunologic relationship
between mother and allogenic
fetus
Immunologic relationship between
mother and allogenic fetus
 fetal cells have on the surface alloantigens
inherited from his father
Tolerance of fetus by mother allow the following mechanisms:
 the relative isolation of the fetus from maternal immune
system (no mixing of blood circulation)
 trophoblast - immune barrier witch protects against mother
alloreactive T lymphocytes (don´t express classical MHC gp, expresses
non-classical HLA-E and HLA-G)
suppressin of TH1 and preference of TH2 immune mechanisms
in pregnancy
• transfer of small doses of fetal antigens in
maternal circulation causes tolerance ...
Rh incompatibility
 Complications in pregnancy: production of anti-RhD
antibodies by RhD- mother carrying an RhD+ fetus
(hemolytic disease of newborns)
 During childbirth or abortion (after 8 weeks of
gestation) fetal erythrocytes can penetrate into the
bloodstream of mother → immunization, formation of
anti-RhD antibodies
Rh incompatibility
Rh incompatibility
 After childbirth, investigate Rh factor of born child, if is child Rh+,
mother gets up to 72 hours after birth injection of anti-RhD
antibodies (administered after abortion too)
 Anti-Rh(D) antibodies bind to RhD Ag on baby´s red blood cells,
this Ag than can´t bind to BCR and can´t activate B lymphocytes,
this immune comlexes also inhibit B lymphocytes
Rh incompatibility
 During next childbirths, if fetus is Rh+ and mother
produce anti-Rh antibodies, this Abb destroy red blood
cells of fetus, which can lead to fetal death, or in severe
postpartum anemia (anemia neonatorum) and neonatal
jaundice (icterus gravis neonatorum)

For each pregnant woman during the first trimester investigate blod Rh
factor and the presence of antibodies, in Rh- women performed a test for
antibodies also in II. and III. trimester
Thank you for your attention