Transcript receptors - EuroBiotech Project

European Biotechnology project
no 134310-LLP-1-2007-1-ITERASMUS-ENW
Work package: Application of
Immunology in Biotechnology
E. Walajtys-Rode, Rzeszow University
of Technology
With the support of the Lifelong Learning Programme of the European Union
EUROBIOTECH – European Biotechnology Agr. n° 2007-2566 / 001-001 project n° 134310-LLP-1-2007-1-IT-ERASMUS-ENW
This project has been funded with support from the European Commission. This publication reflects the views only of the author, and the Commission
cannot be held responsible for any use which may be made of the information contained therein.
Leader partner:
Prof. Dr Sc. Elzbieta Walajtys -Rode, Rzeszow
University of Technology, PL
Participants:
Prof. Brigitte Schmitz, Reinische Friedrich Wilhelms
University of Bonn, RFN
Prof. Olli Vainio, University of Oulu, SU
Dr Maria Czygier, Rzeszow University of Technology, PL
Dr Maryta Sztukowska, Rzeszow University of
Technology, PL
Dr Magdalena Dabrowska, Nencki Institute of
Experimental Biology, Polish Academy of Sc. PL
E. Walajtys-Rode, Rzeszow University
of Technology
Application of immunology in
biotechnology
course for Bachelor of Biotechnology degree
Lectures for students of Technical
Universities
Prerequisite requirements:
Basic courses of:
•Molecular cell biology with genetics,
•Biochemistry,
•Molecular biology,
E. Walajtys-Rode, Rzeszow University
of Technology
INTRODUCTION
The purpose and rationale
of biotechnology-oriented
immunology course
The modern immunology is an interdisciplinary
science comprising and combining most recent
developments in the fields of biology,
biochemistry, molecular biology, genetics and
genetical engineering
The molecular and cellular mechanisms of
immune response create basis for many
immunological techniques applied in
biotechnology, basic sciences, medicine and
industry
E. Walajtys-Rode, Rzeszow University
of Technology
Levels of todays immunology investigations:
Whole
body
Intercellular
Intracellular
• Clinical immunology:
• Tolerance and acquired immunodeficiences,
• Hypersensitivity and autoimmunity,
• Transplantation and tumor immunology.
• Cellular immunology:
• Proliferation, differentiation lymphocytes (Th1/Th2
subtype),
• Effector cell function: cytotoxic lymphocytes, antibody
production, cytokine production
• Cell biology:
• Signal transduction pathways
• Antigen processing and presentation
• Antigen receptors and accesory molecules
E. Walajtys-Rode, Rzeszow University
of Technology
Levels of todays immunology investigations:
Gene and
genome
Gene and
genome
• Genetics and molecular biology:
• Lymphocyte maturation and expression of
antigen receptor genes
• Congenital immunodeficiences
• Genetic engineering:
• Gene targeting and expression
• Transgenic organisms
E. Walajtys-Rode, Rzeszow University
of Technology
Short story of long experiences and investigations
creating todays immunology
• Immunity comes from Latin immunitas and
means excluded,
• Historically immunity means protection from the
infectious disease,
• The concept of immunity was recognized by
ancient Chinese who protect children against
smallpox by the inhalation of the powdered skin
lessions from recovering patients.
• The term of immunity was mentioned by
Thucydides in Athens at V century BC in
connection with resistance to „plague” (epidemy)
E. Walajtys-Rode, Rzeszow University
of Technology
•First controlled manipulation of the
immunological response belongs to Edward
Jenner, who in 1798 year created concept of
vaccination by protecting people from
smallpox by giving them cowpox pustule
material
•Great progress in the field of vaccination
was made by Ludwig Pasteur, ( 1880) who
investigated and created vacine against
cholera in chicken and rabies vaccine for
human
E. Walajtys-Rode, Rzeszow University
of Technology
Recognition of general properties and
functions of immune response
• The humoral immunity was described by Emil von
Behring and Shibasaburo Kitasato in 1980,
whereas antibody recognition was due to sudies by
Karl Landsteiner and Paul Ehrlich in 1900 who
developed theory for the specificity of antigenantibody reaction
E. Walajtys-Rode, Rzeszow University
of Technology
Recognition of general properties and
functions of immune response
The cellular immunity started with discovery of
phagocytes by Elie Metchnikoff in 1893, developed
by Almroth Wright in 1900 and established by
George Mackaness in 1950 who prooved that
resistance to intracellular bacterium can be
transferred with cells.
E. Walajtys-Rode, Rzeszow University
of Technology
• Further progress in immunology was correlated
with the development and advances in many fields
including recombinant DNA methodology and
genetic engineering creating transgenic and
knock-out animalas, cell cultures,
immunochemistry, x-ray crystalography,
monoclonal antibody production and many others
which allowed to explain the immune sysstem and
its function on the molecular level.
• The combined effords of the investigators provide
concepts of the modern immunology and brougt to
many of them Nobel prise (>30 from the
beginning of 20 century )
E. Walajtys-Rode, Rzeszow University
of Technology
PART I: BASIC MOLECULAR
AND CELLULAR MECHANISMS
OF IMMUNE SYSTEM
This part is devoted to a description of the
components of immune system: cells and tissues ,
their anatomic organization and structure-function
relationships.
General properties and fundamental principles of
immune responses are also introduced.
E. Walajtys-Rode, Rzeszow University
of Technology
The physiological function of the immune system is defence of
the organism against pathogens which depends on the recognition
of forigne and self molecules
Realization of such a purpose requires coordinate action
of all compounds of immune system which create the
innate and adaptive immune response:
Humoral
Adaptive
response
Cytotoxic
Immune
system
Innate
response
E. Walajtys-Rode, Rzeszow University
of Technology
Constitutive
compounds
Organs, tissues, lymphatic vessels, cells and
cytokins comprise immune system which elecit
immune responses
Bone
marrow
and thymus
peripheral organs ,
tissues and lymphatic
vessels
Cells of the immune system
and cytokines
E. Walajtys-Rode, Rzeszow University
of Technology
Cells and tissues of the immune system act in a
coordinate and collective manner (network)
Primary lymphatic
organs: bone marrow
and thymus
Secondary lymphatic organs:
spleen, lymph nodes, BALT, GALT
Cytokines
Cells of the immune
system localized in
organs and tissues
Cells of the immune
system circulating with
lymph and blood
E. Walajtys-Rode, Rzeszow University
of Technology
Cells of the immune system (in adult life) all come from common
precursor: self-renewing bone marrow stem cells which develop into two
progenitor lines: lymphoid and myeloid which differentiate into
various types of cells
Selfrenewing
stem cell
Pluripotent
stem cell
Myeloid
progenitor
Lymphoid
progenitor
E. Walajtys-Rode, Rzeszow University
of Technology
Bone marrow
blood
tissue
immature
dendritic cell
megakaryocyte
mature dendritic
cell( lymph nodes)
platelets
macrophage
monocyte
myeloid
progenitor
erythroblast
neutrophil
basophil
mast cell
eosinophil
Myeloid cell lineage
E. Walajtys-Rode, Rzeszow University
erythrocyte of Technology
Cells derived from the myeloid progenitor:
mononuclear phagocytes
1. Mononuclear phagocytes are devoted to host defense
by phagocytosis. They originate from bone marrow,
circulate in the blood , mautre and become activated in a
various tissues,
• Monocytes are incompletly differentiated cells circulating
in the blood ,
• After entering tissues monocytes mature to macrophages
assuming different morphologic forms named after specific
locations. Macrophages are found in all organs ,
connective tissues, and submucosal layers and belong to
one of the three types of profesional phagocytes. May
remain in the tissue for extended periods and can undergo
cell division.
E. Walajtys-Rode, Rzeszow University
of Technology
Cells derived from the myeloid progenitor:
mononuclear phagocytes
• Related to lineage of mononucler phagocytes are
myeloid dendritic cells: immature cells are located
in epithelia of the skin (Langerhands cells) and
respiratory and gastroinestinal systems, during
migration to the lymph nodes dendritic cells mature
and become proffesional antigen-presenting cells
(APC). They are both phagocytic and macropinocytic
E. Walajtys-Rode, Rzeszow University
of Technology
Maturation of mononuclear phagocytes
Bone marrow
Stem cell
blood
tissues
monocyte
macrophage
Microglia (CNS)
Alveolar
macrophages
(lungs)
Kupfer cells (liver)
Ostoclasts (bones)
Epithelioid cells
(skin)
Immature
dendritic cells
Mature dendritic
cells
E. Walajtys-Rode, Rzeszow University
of Technology
Lymph nodes
Other cells of myeloid lineage
2. Polymorphonuclear leucocytes or granulocytes are
primarily secretory cells releasing contents of their
granules upon activation
Short living cells produced during immune responses, migrating
from blood to sites of infection or inflammation ;
– Neutrophils: third phagocytic cell of immune system,
activates of bactericidal mechanisms. Contains granules
comprising degradative enzymes (lysozyme, collagenase and
elastase) and lysosomes called azurophilic granules .
Neutrophils are produced at 1011 per day and survive for 6 hours,
– Eosinophils: involved in killing of extracellular parasites..
– Basophils: function not well recognized (they are not
precursors of mast cells)
E. Walajtys-Rode, Rzeszow University
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Other cells of myeloid lineage
3. Mast cells: tissue cells releasing the granule
contents and producing many mediators of
inflammatory and allergic reactions
4. Megakaryocytes: precursors of platelets involved in
blood coagulation
5. Erythroblasts: precursors of erythrocytes
E. Walajtys-Rode, Rzeszow University
of Technology
Lymphoid cell lineage
Bone marrow
Thymus
Blood and lymph nodes (peripheral tissues)
Effector cells
T cell
B cell
lymphoid
progenitor
NK cell
E. Walajtys-Rode,
Rzeszow
dendritic
cellUniversity
of Technology
activated T cell
plasma cell (also in
bone marrow)
activated NK cell
Cells derived from the lymphoid progenitor
• Lymphocytes. There are three major classes of
lymphocytes:
1. B lymphocytes or B cells mature in bone marrow and
produce antibodies after activation
2. T lymphocytes or T cells precursors arise in the bone
marrow and then migrate and mature in the thymus. T
lymphocytes consist of two main types:
•Helper T lymphocytes (Th) after activation effect and
stimulate other cells such as B lymphocytes or
macrophages
•Cytotoxic T lymphocytes (CTL) after activation kill
cells infected with intracellular pathogen
Lymphocytes B and T are the only cells capable of
very specific recognition of antigen determinants due
E. Walajtys-Rode,
Rzeszow University
to the presence of unique
variants
of antigen receptors
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Cells derived from the lymphoid progenitor
3. NK cells are the third lineage of the lymphoid cells.
This cells lack of the antigen-specific receptor, but instead
they have other receptors recognizing foreign antigens
(PRP receptors) and are the part of innate immune
defence
• Lymphoid dendritic cells: the minor subpopulation of
dendritic cells arising from lymphoid progenitor
E. Walajtys-Rode, Rzeszow University
of Technology
Lymphoid organs and tissues, the site of immune
cell development and maturation
• Generative or primary lymphoid organs
where B and T lymphocytes express their antigen receptors
and attain functional maturity:
– Bone marrow : the site of hematopoiesis ( the
process of generation of all blood cells ) and site of B
cell maturation,
– Thymus: the primary organ of mammals in which T
cells mature and reach a stage of functional
competence.
Cells leaving primary limphatic organs
are naive – without contact with forigne
antigens
E. Walajtys-Rode, Rzeszow University
of Technology
Lymphoid organs and tissues, the site of
immune cell development and maturation
•Peripheral or secondary lymphoid
organs where the lymphocyte response to foreign
antgens is initiated and developed consist:
–Lymphatic sysstem (lymph nodes
and lymph vessels)
–Spleen,
–Cutaneous immune system,
–Mucosal immune system,
–Aggregates of lymphocytes found in in
connective tissue of many organs.
E. Walajtys-Rode, Rzeszow University
of Technology
Structure and function of the primary lymphoid organs
During fetal development the hematopoiesis occurs in
yolk sac and para-aortic mesenchyme and later in
the liver and spleen
Bone marrow
In adults hematopoiesis occurs in bone marrow of flat
bones (sternum, vertebrae, iliac bones and ribs). Under
emergency conditions the liver and spleen can be
involved in hematopoiesis.
• The bone marrow has vascular and extravascular
compartment. The vascular compartment is supplied
by artery which branches into smaller opening into
sinuses from which blood is carried via vein into general
circulation
E. Walajtys-Rode, Rzeszow University
of Technology
• Bone marrow
•The extravascular compartment consists of a stroma
of a fibrous connective tissue , stromal cells
(fibroblasts, fat cells, numerous plasma cells) and
pluripotent stem cells (1 per 104 stromal cells).
•The epithelial cells of the capillary sinuses contain
narrow openings that permit the mature red and white
blood cells to enter sinus space and be transferred into
circulation.
E. Walajtys-Rode, Rzeszow University
of Technology
Structure and function of primary lymphoid
organs: thymus
T lymphocytes mature in the thymus, derived from ectoderm
lobular organ located in the anterior mediastinum, covered by
connective tissue capsule and lobulated by septa originating
from capsule,
• Thymus continues to grow after birth, then undergoes
involution by which the volume of active tissue is reduced,
• Each lobule consists of outer cortex and inner medulla both
containing lymphocytes (thymocytes) of different density.
Thymus contains also:
macrophages ,
epithelio-reticular cells supporting the framework,
dendritic cells (also lymphoid lineage),
E. Walajtys-Rode, Rzeszow University
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Structure and function of primary lymphoid
organs: thymus
• Thymus has rich vascular and lymphatic
veasculature. Trough the former immature T
lymphocytes enter thymic cortex . After maturation
lymphocytes enter medulla and exit the thymus through
efferent lymphatic vessels
• Physical cellular blood-thymus barrier prevent the
contact of immature lymphocytes with blood born
antigens
E. Walajtys-Rode, Rzeszow University
of Technology
Structure and function of peripheral lymphoid
organs: lypmhatic system
• Principial function of lymphatic system is collection of the
antigens from their entry sites an delivery to the lymph
nodes by the lymph vessels
• Lymphatic system present in higher vertebrates is vital to
the development of defence mechanisms and is structurally
and functionally related to the blood vascular system
• Lymphatic system is open-enden and linear without
central driving force, whereas blood vascular system is
closed and blood is pumped by the heart
E. Walajtys-Rode, Rzeszow University
of Technology
Structure and function of peripheral lymphoid
organs: lypmhatic system
• Lymph vessels are present in almost all tissues except
brain, cornea and retina, epidermis and cartilage
• Lymph is an interstitial fluid made of plasma
exudate from blood capillaries which enter the
lymphatic capillaries in peripheral tissues and through
larger lymphatic vessels and thoracic duct flows
back into the blood vascular system for recirculation
• Lymph carries immune cells including lymphocytes
and antigen-presenting cells as well as nutritions
(dietetary fat and fat-soluble vitamins A,D,E and K from
digestive system) and soluble and particulate
E. Walajtys-Rode, Rzeszow University
antigens .
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Function of the lymphatic system
Lymph is collected in peripheral
tissues by lymphatic capillaries
together with soluble and particulate
antigens
Afferent lymphatics carry lymph into
lymph nodes where it percolates
through the nodal stroma. Antigens
are extracted by APC (including B
cells) and presented to T cells.
Efferent lymphtics may become afferent for
another node. Lymph carrying lymphocytes
is collected in larger lymphatic vessels and
into thoracic duct and emptied into superior
E. Walajtys-Rode,
Rzeszow
University
vena cava , returning
to the
blood
stream
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Lypmhatic system : lymph nodes
and lymphatic vessels
• Lymph nodes are the sites where the adaptative immune
responses are initiated and are the most organized of the
lymphatic organs. Are localized along lymphatic vessels,
• Lymph nodes are enveloped by the fibrous capsule, the
entry of afferent lymphatic vessels which empty the
lymph into marginal sinus
• Lymph passes the node into medullary sinus and leaves
the node through the efferent lymphatic vesels in the
hilum,
E. Walajtys-Rode, Rzeszow University
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The distribution of lymphoid tissues in the body
tonsils
adenoids
cervical nodes
thymus
axillary nodes
Intercostal
nodes
appendix
Peyer’s patch
spleen
inguinal nodes
bone marrow
E. Walajtys-Rode, Rzeszow University
of Technology
Lypmhatic system : lymph nodes and
lymphatic vessels
• Interior architecture of lymphatic node is made of outer
cortex and inner paracortex beneth which is medulla
consisting of medullary cords that lead to to the
medullary sinus and collagen fibers on which
lymphocytes are localized
• Cortex contains bulk of lymphatic tissue diffused and
organized into primary and secondary follicles
(germinal centers). Paracortex containis mostly T
lymphocytes, dendritic cells and macrophages
• Medulla consists medullary cords populated by
macrophages and plasma cells and medullary sinus.
E. Walajtys-Rode, Rzeszow University
of Technology
The lymph node organization
primary lymphoid
follicle
germinal
center
marginal sinus
Secondary
lymphoid
follicle
cortex
paracortical
area
medullary
cords
afferent
lymphatic
vessel
efferent lymphatic
vessel
medullary
sinus
E. Walajtys-Rode, Rzeszow University
ofvein
Technology
artery
The different classes of lymphocytes undergo
Sequestration in the specific areas of the lymph
node:
Primary follicles
Secondary
follicles, germinal
centers
Paracortex
• Mature, naive B cells
• Proliferating B cells,
• Producing antigens B cells
• Memory B cells
• T cells
E. Walajtys-Rode, Rzeszow University
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Lymph and blood flow through lymph nodes
carrying antigens and naive and activated cells
1. Lymph flow:
• afferent lymphatic vessels bring lymph to the node
from periphery towards the hilus where it drains via
efferent lymphatic vessels
2. Blood flow:
• Blood enters via arteriole at the hilus and through
capillaries into the outer cortex and leaves via vein at
the hilus.
• Lymphocytes carried in the blood enter the node and
return to the circulation via the lymphatic vessels.
E. Walajtys-Rode, Rzeszow University
of Technology
The lymphatic system
cervical nodes
subclavian
trunk
thoracic duct
axillary
nodes
vessels from stomach
vessels from large
intestine
cisterna
chyli
inguinal
nodes
vessels from small
intestine
E. Walajtys-Rode, Rzeszow University
of Technology
Structure and function of peripheral lymphoid organs:
spleen
• The spleen is part of the circulatory system since it is a
storage and processing station for erythrocytes. It
contains also a very large population of lymphocytes and
is a major site of immune responses to blood-borne
antigens,
• The spleen is surrounded by the collagenous capsule
from which arise fibrous trabeculae subdividing the
interior volume of the organ,
• Interior of spleen comprises the aggregated masses of
cels:
the „red pulp” contains mostly erythrocytes,
macrophages, dendritic and plasma cells, splenic cords
Walajtys-Rode, Rzeszow University
and blood sinuses E.between
them,
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Structure and function of peripheral lymphoid
organs: spleen
• The „white pulp” comprises the lymphocyte population
in the form of periarteriolar lymphocyte sheath
(PALS) containing mainly T lymphocytes. To the
PALS are attached lymphoid follicles, some of them
contains germinal centers, which contains
predominantly B cells. These structures are
surrounded by perifollicular zone comprising
lymphocytes and macrophages
• Antigens and lymphocytes enter spleen through the
splenic arteria into vascular sinusoids and leave
organ through splenic
vein.
E. Walajtys-Rode, Rzeszow University
of Technology
Function of the spleen
Blood carrying lymphocytes and antigens enter
spleen through splenic artery, which divides into
progressively smaller branches. The arterioles end in
vascular sinusoids scattered with erythrocytes,
macrophages, dendritic and plasma cells (red pulp).
The spleen is the major site for clearing the
blood of microbes by red pulp macrophages
Through the small arterioles antigens are
delivered to T lymphocytes (PALS) and B
lymphocytes (lymphoid follicles and germinal
centers )
Anatomical organization of spleen allowes for
very efficient interaction and activation of B
and T lymphocytes and antigen presenting cells
E. Walajtys-Rode, Rzeszow University
of Technology
Cutaneous immune system
• Skin creates the largest physical barrier between
external environment and organisms and is a site
of many immune responses to foreign antigens,
• This purpose is realized by the specific cell populations
present within epidermis and dermis.
• Immature dendritic cells (Langerhans cells) form
almost continuous meshwork in epidermis and capture
antigens entering through the skin. After stimulation by
cytokines Langehrans cells migrate into the dermis and
to the lymph nodes via lymphatic vessels
E. Walajtys-Rode, Rzeszow University
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Cutaneous immune system
•Many intraepidermal T cells express  antigen
receptor, an uncommon type, not clearly definied yet.
•Dermal T lymphocytes (both CD4+ and CD8+
activated and memory cells) are located mostly
around venules and lymphatic vessels together
with macrophages
E. Walajtys-Rode, Rzeszow University
of Technology
Cellular components of the cutaneous immune
system
Epidermis
Dermis
• Keratinocytes (produce cytokines)
• Langerhans cells (immature dendritic
cells)
• Intraepidermal lymphocytes (mostly
CD8+, also  T cells)
• Lymphocytes predominantly perivascular
(CD4+ and CD8+)
• Macrophages perivascular and scattered
E. Walajtys-Rode, Rzeszow University
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Mucosal immune system
Mucosal epithelia are barriers between the internal
and external environments and create first line of
defence against forigne antigens due to specialized
cell populations of different locations and distinct
phenotypic and functional characteristic
E. Walajtys-Rode, Rzeszow University
of Technology
Mucosal immune system
Gut associated lymphoid tissue:
In the mucosa of the gastrointestinal tract lymphocytes are
located :
within epithelial layer ( mostly CD8+, also T cells
expressing  antigen receptor)
scattered throughout the lamina propria,
In the lamina propria as aggregates in the small
intestines (Peyer’s patches). Some epithelial cells of
Peyer’s patches are specialized M cells
(multifenestrated cells), which are pinocytic and
transport antigens from the intestinal lumen into
subepithelial tissues. Similar structures are found in
appendix, tonsils and adenoids
E. Walajtys-Rode, Rzeszow University
of Technology
Organized lymphoid tissue of the mucosal immune system
comprises mucosal follicles reach in B cells and often
containing germinal centers and interfollicular regions
which are T cell regions.
The same basic architecture share other mucosal systems:
• Bronchial associated lymphoid tissue (BALT)
• Mucosal-associated lymphoid tissue (MALT)
gut lumen
epithelium
M cells
gut wall
T cell area
follicle
B cell area
germinal center
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Lymphocytes circulate between blood and lymph
• Effective immune response to foreign antigens requiers
lymphocyte recirculation via the blood stream and
lymphatics among peripheral lymphoid tissues and
peripheral sites of antigen entry and inflammatory
reaction.
• Recruitment of lymphocytes into specific tissue location
(lymphocyte homing) depends on adhesive interactions :
– between lymphocytes and specialized vascular
endothelil cells (high endotelial venules(HEV), the site
of lymphocyte extravasation into tissue
– between lymphocytes and extravascular connective
E. Walajtys-Rode, Rzeszow University
tissue components
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Lymphocytes circulate between blood and
lymph
• The process of lymphocyte recirculation is due to
adhesion molecules responsible for interactions
between lymphocytes and vascular endothelial cells:
 the molecules on lymphocytes are homing
receptors
 the different ligands for homing receptors on
endothelial cells are adressins
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vein
thoracic duct
efferent
lymphatic
Superior
vena
cava
artery
arteriole
aorta
heart
venule
lymph
node
lymph
venous blood
afferent
lymphatic
arterial blood
ativated and
memory T cell
naive T cells
peripheral tissue
Pathwys of T lymphocyte circulation
E. Walajtys-Rode, Rzeszow University
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Innate and adaptive immunity
Defence against microbes and other forigne antigens
is mediated by the early reactions of innate
immunity and the later responses of adaptive
immunity
The various components of the mammalian immune
system developed during phylogeny and become
increasingly specialized with evolution
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Evolution of the immune system
All organisms have mechanisms defending host
organism against forigne invaders
Bacteria protect themself from parasites (plasmids) and
pathogens (bacteriphages) with restriction nucleases
and against competing bacteria with atimicrobial
peptides
Innate immunity:
Invertebrates respond to microbes and forigne antigens
by phagocytosis with specialized cells and by
antimicrobial peptides and also by the ancestral
three-compound complement system
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Innate immunity:
Fruit fly Drosophila melanogaster have cell surface
receptors (Toll receptors) activating expression of
antimicrobial peptides found also in vertebrates, which
suggests presence of the common signalling pathway for
innate immune response .
Mammals have more than 10 Toll-like receptors
activating different signalling pathways of innate immune
response
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Adaptive immunity:
• Jawed fishes and higher vertebrates developed antibody
molecules
Fishes have only IgM type, amphibians , reptiles and
birds have 3 classes of immunoglobulins and 7-8 antibody
classes are found in mammals
• Jawed fishes and higher vertebrates have also both major
histocompatibility complexes (MHC I and II class)
• Lymphocytes of general characteristics are present in all
vertebrates and become specialized into T i B class in
amphibians, birds and mammals
• The lymph nodes are found in some birds and in
mammals
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Specificity of innate and adaptive immunity:
both systems recognize self from nonself
Innate
immunity
Adaptive
immunity
• Recognize „molecular patterns” of
different types of microbes
• Receptors encoded in the germline,
nonclonally distributed
• Recognize structural details of
microbial and nonmicrobial antigens
• Receptors encoded by gene segments
rearrangement during development
(somatic recombinations), clonally
distributed
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Specificity and function of innate immunity
• The principal feature of innate immunity is constitutive
presence of all components and mechanisms and lack of
memory,
• The innate immunity , phylogenetically oldest
mechanisms, provide the first line of defence against
infection by the common pathogen organisms and can
elimnate the microbes.
• The cells of innate immune system play a crucial part
in the initiation and subsequent direction of the
adaptative immunity mechanisms and cooperate in
elimination of microbes by phagocytosis and complement
system
E. Walajtys-Rode, Rzeszow University
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Components of innate immune system
• Epidermal and mucosal surface barriers consist
epithelil cell layers, produced by epithelial cells peptides
–defensins and cryptocidins (acting as antibiotics )
and mucus entrapping bacteria
• Efector cells of innate imunity: neutrophils,
mononuclear phagocytes and natural killer (NK) cells
recognize, attack and destroy microbes that entered into
circulation or tissue.
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Components of innate immune system
•Effector circulating proteins: complement system,
mannose-binding lectin (collectin), C-reactive protein
(pentraxin) and other mediators of inflammation,
coagulation factors
•Cytokines and chemokines - protein molecules
produced by activated cells which coordinate immune
response
Same components of innate immune system are
functioning all the time (surface barriers), other ,
normally inactive ,become rapidly activated in the
presence of microbes (phagocytosis, complement
E. Walajtys-Rode, Rzeszow University
system).
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The major family of phagocytes: macrophages and
neutrophils play a key role in innate immunity.
They can recognize, ingest and destroy pathogens
without the aid of an adaptive immunity
• Both macrophages and neutrophils have surface
receptors of phagocytosis recognizing molecules
displayed by pathogens:
Ligation of phagocytic receptors induce
internalization of the pathogen in phagosome, and
fusion with lysosomes to generate phagolysosome
in which pathogen undergos destruction by
lysosomal enzymes.
E. Walajtys-Rode, Rzeszow University
of Technology
Phagocytes after activation produce a variety of
toxic products that help kill engulfed organisms
1. Reactive oxygen intermediates(ROI) toxic to the cells:
Superoxide anion radical O 2, hydrogen peroxide H2O2,
singlet oxygen 1O2 , hydroxyl radical OH , hypohalite OCl
produced as a result of activation of NADPH oxidase by
microbial antigens
2. Reactive nitrogen intermediates (RNI) toxic to the
cells:
Nitric oxide free radical NO , nitrosonium ion NO+ , nitroxyl
anion NO, nitrite NO2 , nitrogen dioxide NO2,
Excessive formation of nitric oxide is due to activation of
inducible isoenzyme of nitric oxide synthase (iNOS) during
host injury and inflammatory processes
E. Walajtys-Rode, Rzeszow University
of Technology
Phagocytes after activation produce a variety
of toxic products that help kill engulfed
organisms
Superoxide anion radical and nitric oxide react to
give peroxynitrite ONOO a potent factor capable
of protein nitration and oxidation and inducer of
cell death.
3. Antimicrobial peptides: defensins and cationic
peptides
4. Enzymes: lysozyme disrupting proteoglicans cel wall
Gram-positive bacteria, acid hydrolases –digest bacteria
5. Competitors binding nutritiens essential for bacteria:
lactoferrin (binds Fe 2+), vitamin B12 –binding proteins.
E. Walajtys-Rode, Rzeszow University
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Extracellular and intracellular microbicidal
mechanisms of neutrophils and macrophages
Extracellular
microbes
NADPH
oxidase
O2
Phagosome
with ingested
microbes
ROIs
NO
arginine
ROIs
oxidase
lysosome with
enzymes
phagolysosome with ingested
microbes
E. Walajtys-Rode, Rzeszow University
of Technology
iNOS
Pathogen recognition and tissue damage
initiate inflammatory response
Infalammation plays the essential role in defence
against infection by two general mechanisms:
• providing physical barrier against spreading of the
microbes via the blood stream by initiation of
microcoagulation process
• accumulation effector cells at the site of infection
E. Walajtys-Rode, Rzeszow University
of Technology
Inflammation is accompanied by pain, redness , heat
and swelling at the site of infection due to release of
variety of inflammatory mediators acting on
different cell types,
•Under influence of the inflammatory mediators
endothelial cells lining the blood vessels become
separated leading to an exit of fluid from the blood and
express adhesion molecules promoting binding and
migration of circulating leucocytes into tissues in the
process called extravasation
E. Walajtys-Rode, Rzeszow University
of Technology
Mediators released by macrophages after
recognition of pathogens involved in innate
response
1. Cytokines:
major cytokines of inflammatory reaction: Interleukin-1,
Interleukin-6 and Tumor necrosis factor -  (TNF-),
Interleukin-12: activator of NK cells,
2. Chemokines:
Interleukine-8: chemoattraktant for neutrophils,
3. Lipid mediators of inflammation produced
enzymatically by the degradation of plasma membrane
phospholipids: prostaglandins, leukotrienes,
thromboxanes, platelet-activating factor (PAF)
E. Walajtys-Rode, Rzeszow University
of Technology
Inflammatory mediators released by
macrophages after recognition of pathogens
during innate response
Cytokines:IL-1, IL-6
TNF-, IL-12
Chemokines: IL-8
Lipid mediators:
prostaglandins,
leukotrienes,
tromboxanes, PAF
INFLAMMATION
E. Walajtys-Rode, Rzeszow University
of Technology
Infection stimulates macrophages to initiate
inflammatory response
Activated
macrophage
cytokines
Expression of
adhesion
molecules

Adhesion of circulating
leucocytes to the
endothelial cells
chemokines
Blood vessel
Blood clotting in
the microvessels
Extravasation
of leukocytes
E. Walajtys-Rode, Rzeszow University
of Technology
Cytokines released by phagocytes (IL-1, IL-6 and TNF-α )
activate the systemic response that contribute to host defence by:
1. Acting on hypothalamus (endogenous pyrogens) and on muscle and fat cells
altering energy mobilization to increase the body temperature. The
decrease bacterial and viral replication
2. Stimulating synthesis
plasma:
fever
of acute phase proteins by liver and release
into
C-rective protein – pentraxin, comprising five identical subunit which
bind to phosphocholine portion of certain bacterial and fungal cell-wall
lipopolisaccharides and activate the complement system
Mannose binding lectin and pulmonary surfactant proteins
SP-A and SP-D promoting phagocytosis of pathogens
3. Inducing leucocytosis (an increase in circulating neutrophils) and migration
(maturation) of dendritic cells from peripheral tissues to lymph nodes
E. Walajtys-Rode, Rzeszow University
of Technology
The phagocyte cytokines have a wide spectrum of
biological activities coordinating the body response to
infection
IL-1/IL-6/TNF-α
Liver
Bone marrow
endothelium Hypothalamus
Acute phase
proteins:
C-reactive
protein
Mannose –
binding lectin
Neutrophil
mobilization
Activation of
complement
Phagocytosis
Increased body
temperature
Fat, muscle
Protein and
energy
mobilization
Decreased viral and bacterial
replication. Increased antigen
processing
E. Walajtys-Rode, Rzeszow University
of Technology
Dendritic cells
TNF-α stimulates
migration to lymph
nodes and
maturation
Initiation of
adaptative immune
response
Complement system consists of many proteins
distributed through body fluids and tissues that
interact with pathogens to mark them for
destruction by phagocytes
• Complement components are activated by induced
proteolytic enzyme cascade
• There are three distinct pathways of the complement
activation depending on different molecules of pathogen
surfaces but as a result the same set of effector molecules
are generated for protection of the organism against
infection:
E. Walajtys-Rode, Rzeszow University
of Technology
1. Activated complement components bind to
pathogens, opsonizing them for receptordependent phagocytosis
2. Activated complement components act as
chemoattractants for phagocytes and activate
them
3. Terminal components of complement system act
directly on bacteria by creating pores in the
bacterial membrane
E. Walajtys-Rode, Rzeszow University
of Technology
Three pathways of complement activation
CLASSICAL
PATHWAY
MB-LECTIN
PATHWAY
ALTERNATIVE
PATHWAY
Antigen-antibody
complexes
Lectin binding to
pathogen surface
Pathogen surface
molecules
COMPLEMENT
ACTIVATION
Opsonization and
phagocytosis of
pathogens
Recruitment of cells to
the site of infection
E. Walajtys-Rode, Rzeszow University
of Technology
Direct killing of
pathogens
Complement system consists a few functional
classes of proteins
• Binding to pathogen surfaces ant antigen-antibody
complexes (C1q)
• Binding to mannose compound of bacterial cell-wall
(MBL)
• Enzymes activating components of complement
(serine proteases, convertases)
• Membrane binding proteins (C4b, C3b)
• Mediators of inflammation-peptides (C5a, C4a, C3a)
• Peptides forming pore: membrane attack proteins
(MAC: C5b, C6, C7, C8, C9)
E. Walajtys-Rode, Rzeszow University
of Technology
Assamble of the membrane attack complex generates pore
in the lipid bilayer membrane ( 10 nm)
C7
C8
C6
C9
C5b67
complex
C5b
Lipid bilayer
Pathogen
E. Walajtys-Rode, Rzeszow University
of Technology
10-16 molecules of
C9 bind to form a
pore in the
membrane
Main components and effector actions of complement system
CLASSICAL PATHWAY
MB-LECTIN PATHWAY
First complement protein
C1q binds to pathogen
surfaces through pathogenbinding protein ( Cprotein)and antigen/
antibody complexes and
initiates activation of
enzymes C1r and C1s
Mannose binding lectins
bind mannose on
pathogens and initiates
activation of enzymes
MASP-1 and MASP-2
ALTERNATIVE
PATHWAY
Many pathogen surfaces
initiates directly
spontaneous hydrolysis of
C3 generating C3
convertase
C3 convertase
C3a, C5a
Peptide mediators of
inflammation
C3b
C5b, C6, C7,
C8, C9
Opsonization of
Membrane attack complex
pathogens
Lysis pathogens and cells
Binds to complement
E. Walajtys-Rode, Rzeszow University
receptors
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Complement system is involved in innate as well as
adaptative immune response
Pathogens marked by opsonization with complement are
recognized by cells with surface receptors for complement
compounds and stimulate different cell functions:
• Stimulation of phagocytosis by macrophages,
monocytes, polymorphonuclear leucocytes, dendritic
cells, B cells, erythrocytes (transport of immune
complexes) CR1, CR 3 and CR4
• Part of antigen receptor complex of B cell (CR2)
• Receptors for C3a and C5a activate endothelial cells and
mast cells
E. Walajtys-Rode, Rzeszow University
of Technology
Recognition of the self from nonself by the
cells of innate immune system is due to the
molecular pattern recognizing receptors
• Activated macrophages and neutrophils destroy
pathogens through phagocytosis and/or by initiation of
the inflammatory response
Both patways are dependent on the binding specific
pathogen molecules to the various surface receptors:
Soluble receptors :
• mannose binding lectin (MBL) initiating lectindependend pathway of complement activation,
• collectins opsonizing bacteria
E. Walajtys-Rode, Rzeszow University
of Technology
 Cell-surface receptors
 Fagocytic receptors:
• Macrophage mannose receptor: cell bound lectins with
several carbohydrate recognition domains rcognizing
sugars (mannose, fucose) present on the surface many
bacteria and some viruses
• Scavanger receptors recognize anionic polymers and
acetylated low-density lipoproteins. Some of these
receptors recognize structures of normal host cells
depleted of sialic acid (eg. senescent red blood cell
•
Receptors for opsonins:
Receptors FcγR1 for Fc region of IgG antibodies
coating microbes
Receptors for components of the complement
system opsonizing E.bacteria
( especially C3)
Walajtys-Rode, Rzeszow University
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 Receptors resposible for migration cells into tissue
(receptors dependent on protein G):
• chemotactic receptors:
receptors recognizing N-formylated bacterial (also mitochondrial)
peptides, chemokines (IL-8) and lipid mediators of inflammation
(PAF, prostaglandin E)
 Receptors for cytokins produced by cells of the innate
and adaptive response following activation by
various effectors
• Receptors for cytokines of inflammatory reaction
(TNF-α, IL-6 and IL-1)
• Receptors for cytokines produced by lymphocytes
(interferon- INF- expressed during innate immune response by
lymphocytes NK and by lymphocytes T during cell-mediated
adaptive reactions E. Walajtys-Rode, Rzeszow University
of Technology
 Signalling Toll-like (TLR) receptors:
First Toll receptors were described in the fruit fly
where they trigger (in adult fly) formation of
antimicrobial peptides in response to infection.
Homologous proteins in mammals are named Tolllike receptors.
So far 10 genes are indentified in mice and humans
encoding TLR recognizing „molecular pattern” – a
particular molecular structures that are components
of pathogenic microorganisms but are not present in
vertebrates
E. Walajtys-Rode, Rzeszow University
of Technology
Toll-like receptors are founded on innate
response vertebrate cells and also in plant
defence mechanisms against viruses and
other pathogens. This signalling pathway is
probably present in all multicellular
organisms
E. Walajtys-Rode, Rzeszow University
of Technology
Molecular pattern of microorganisms comprises
repeating structural motifs of surface molecules
and nucleic acids
• bacterial DNA contains unmethylated repeats of
CpG dinucleotide
• viruses express double – strainded dsRNA as a part
of life cycle
• Gram –negative bacteria have lipoplisaccharide
(LPS) as cell-wall component
• bacterial flagella are composed of specific protein
(flagellin)
• Gram-positive bacteria express peptidoglycans as a
cell wall component
E. Walajtys-Rode, Rzeszow University
of Technology
Molecular partterns recognized by Toll-like receptors
(acc. to Beutler et al.2005)
Toll-like receptor
Ligand
TLR-1 dimer
Peptidoglicans
Lipoproteins
Lipoarabinomannan
(mycobacteria)
(glycosylphosphatidylinositol
(T.cruzi)
Zymosan (yeast)
TLR-2/TLR-6 dimer
TLR-3
dsRNA
TLR-4 dimer
Plus CD14
LPS (Gram-negative bacteria)
TLR-5
Fagellin
TLR-9
E. Walajtys-Rode, Rzeszow University
of Technology
Unmethylated CpG DNA
Chemokines
N-formylmethionyl peptides
Lipid mediators
Microbe specific
molecular
pattern
Chemokine
receptors
Toll-like
receptors
Integrin
Expression
Cytoskeletal
reorganization
Migration into
tissue
Opsonized
microbes
mannose , fucose
LDL
Generation of
oxygen and nitrogen
active metabolites
Gene expression
Phagocytic
receptors
phagocytosis
microbes
into phagolysosome
Killing
microbes
Killing
microbes
Receptors and functional response of phagocytes
E. Walajtys-Rode, Rzeszow University
of Technology
Characteristics of the innate immunity
receptors
1. Specificity of the receptors is inherited in the
genome
2. Receptors are expressed by all cells of
particular type
3. Broad classes of pathogen are recognized
4. Receptors interacts with a range of molecular
structures of given type
5. Receptor activation triggers immediate
response
E. Walajtys-Rode, Rzeszow University
of Technology
Natural killer cells - subset NK lymphocytes that
kill intracellular microbe infected and cancer cells
and secrete cytokines
 NK cells do not express specific lymphocyte lineage cells
B or T antigen receptors
 NK cells are activated by specific set of receptors
recognizing:
• Antibody coated cells by FcRIIIa low affinity
receptor for Fc region of IgG antybody
• Cells infected with viruses and other
intracellular pathogens possibly by recognizing
adhesion molecules (integrins)
• Cells lacking class MHC I molecules by complex
mechanism including activating (lack of MHC I
molecules) and inhibitory ( presence of MHC I
E. Walajtys-Rode, Rzeszow University
molecules ) receptors
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