Innate Immunity
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always present
responds to microbial substances ONLY
may respond to damaged host cells
powerful early defense mechanism which controls and may eradicate infections
before adaptive response
instructs the adaptive immune system and is enlisted by the adaptive immune
response
Questions:
 How does the innate immune system recognize microbes?
 How do different components of innate immunity function to combat different
kinds of microbes?
 How do innate immune reactions stimulate adaptive immune responses?
Recognition of Microbes by the Innate Immune System
The components of innate immunity recognize structures that are shared by
various classes of microbes and are not present on host cells.
 LPS – lipopolysaccharide
- Present in cell wall of many bacterial species (not produced by
mammalian cells)
 Terminal Mannose Residues
- Typical of bacterial glycoproteins
 Double-stranded RNA (viruses)
 Unmethylated CpG oligonucleotides
- Common in microbial DNA
These microbial molecules that are the targets of the innate immune system are called
pathogen-associated molecular patterns or PAMPs. They are recognized by innate
immune cells through pattern recognition receptors or PRPs.
The components of innate immunity have evolved to recognize structures that are vital
for the survival and infectivity of these microbes.
The innate immune system also recognizes molecules that are released from stressed or
necrotic cells. These molecules are termed damage-associated molecular patterns or
DAMPs.
Where are the receptors of the I.I. system encoded? The GERMILINE!
- unlike adaptive immune system which enlists recombination
- thus, I.I. system recognition is less diverse – less than 100
microbial patterns
- the receptors are non-clonally distributed – identical receptors are
expressed on all cells of a particular type (ie. macrophages)
The I.I. system does not react against the host. WHY?
1. inherent specificity of system for microbial structures
2. mammalian cells express regulatory proteins
The I.I. system usually responds in the same way to repeat encounters with a
microbe.
- unlike the A.I. system, it doesn’t have immunologic memory
2 principal reactions of the innate immune system:
- inflammation – recruitment and activation of leukocytes
- viral defense – defense against intracellular viruses by NK cells
Cellular receptors for microbes:
- expressed on macrphages, dendritic cells, lymphocytes, epithelial and endothelial cells
- these receptors are expressed in different cellular compartments (cell surface, ER,
endosomes, cytosol
THE BIG CHEESES OF RECEPTORS
Toll-like receptors
- specific for different components of microbes
TLR-2: bacterial lipoglycans
TLR-3,7,8: viral nucleic acids (ie. double-stranded RNA
TLR-4: LPS
TLR-5: bacterial flagella
TLR-9: unmethylated CpG oligonucleotides
Signals generated by engaged TLRs activate transcription factors which stimulate
expression of genes coding for cytokines, enzymes, and other proteins involved in
antimicrobial functions of activated phagocytes and dendritic cells.
TWO OF THE MOST IMPORTANT TRANSCRIPTION FACTORS:
- NF- B (nuclear factor B) – promotes expression of cytokines
and endothelial adhesion molecules
- IRF-3 (interferon response factor-3) – stimulates production of
type 1 interferons (cytokines that block viral replication)
Other types of receptors:
- receptor for N-methyl methionine (particular to bacterial proteins)
- receptor for terminal mannose residues (involved in the
phagocytosis of bacteria)
There are also several cytoplasmic receptors that recognize viral nucleic acids or bacterial
peptides. Some cytoplasmic receptors associate with inflammasomes.
What is an inflammasome? A multi-protein complex which transmits signals that
activate an enzyme to cleave a precursor of the cytokine IL-1. IL-1 is a powerful
inducer of the inflammatory reaction to microbes and damaged tissues.
Components of Innate Immunity
The innate immune system consists of epithelia which provide barriers to
infections, cells in the circulation and tissues, and several plasma proteins.
Epithelial Barriers:
COMMON PORTALS OF ENTRY BY MICROBES: skin, GI tract, respiratory tract
These 3 portals are lined by continuous epithelia. These cells also produce peptide
antibiotics. In addition, intraepithelial lymphocytes (T cell lineage) express antigen
receptors of limited diversity – common class is  T cells (have  and  chains which are
similar to  T cell receptors)
Intraepithelial lymphocytes recognize microbial structures and serve as sentinels
against agents trying to breach epithelia.
Phagocytes: Neutrophils and monocytes/macrophages:
- 2 types of circulating phagocytes
- recruited to site of infection
- recognize and ingest microbes for intracellular killing
Neutrophils (a.k.a polymorphonuclear leukocytes)
 most abundant leukocytes in blood
 rapidly increase in number in response to infection
 stimulated by cytokines known as colony-stimulating
factors
- NEUTROPHILS ARE THE FIRST CELL TYPE TO RESPOND
TO MOST INFECTIONS, PARTICULARILY BACTERIAL
AND FUNGAL
 ingest microbes in circulation, enter extra-vascular sites of
infection and die within a few days
Monocytes (which differentiate in macrophages in the tissues)
 less abundant
 ingest microbes
 monocytes that enter EV tissues (and thus become
macrophages) stay for long periods of time
 Monocytes and macrophages are two types of the same cell
lineage known as the mono-nuclear phagocyte system.
 Resident macrophages are found in connective tissue and
in every organ
Monocytes and neutrophils migrate to extravascular sites of infection by binding to
epithelial adhesion molecules.
1st step: loose attachment of leukocytes to endothelial cells
2nd step: firm adhesion and transmigration through epithelium
If an infectious microbe breaches an epithelium and enters subepithelial tissue,
tissue, resident macrophages recognize the microbe and respond by producing
cytokines.
 TNF (tumour necrosis factor)
 IL-1
These stimulate endothelial cells to express E-selectin and P-selectin
- circulating N’s and M’s weakly bind to the selectins
- “rolling occurs”
Leukocytes express another set of adhesion molecules: the integrins
- integrins “integrate” signals into cytoskeletal alterations
- as these cells are rolling, both the macrophages and endothelial cell
produce chemokines
 bind to glycoproteins on luminal surface of endothelial
cells and are displayed at high concentration to rolling
leukocytes.
- chemokines stimulate rapid increase in affinity of leukocyte
integrins for their ligand.
AT THE SAME TIME
- TNF and IL-1 act on endothelial cells to express ligand for
integrins
BINDING OCCURS!
- this arrests leukocyte rolling
- cytoskeleton is reorganized
- cells spread out on endothelial surface
- chemokines stimulate the motility of leukocytes
Leukocytes begin to migrate between endo. cells through vessel wall and towards site of
infection
LEADS TO MIGRATION TO AN EXTRAVASCULAR SITE IN MINUTES!
What is inflammation? The accumulation of leukocytes at sites of infection with
vascular dilation and increased leakage of fluids and proteins
- n.b. inherited deficiencies in integrins and selectins lead to
increased susceptibility to infections – leukocyte adhesion
deficiencies
How do neutrophils and macrophages recognize microbes? RECEPTORS!!
Receptors involved in activating phagocytes: TLRs, formyl methionine
receptors, receptors for IFN- and chemokines
Receptors involved in phagocytosis and activation of phagocytes: mannose
receptors, scavenger receptors, receptors for products of compliment activation
What is opsonization? the process of coating microbes for efficient recognition by
phagocytes
PHAGOCYTOSIS
- membrane receptors bind to microbe
- extension of phagocytic membrane around microbe
- membrane closes and pinches off
- microbe is internalized in a membrane-bound phagosome
(phagosomes can fuse with lysosomes to form phagolysosomes)
-
at the same time, receptors deliver signals that activate enzymes
in phagolysosomes
What do we get??
 ROS (reactive oxygen species)
 NO (nitric oxide)
 Lysosomal proteases
**sometimes these products may leak into EC space and damage host tissue.
Chronic Granulomatous Disease: Phagocytes unable to eradicate IC microbes, host
calls more macrophages and lymphocytes resulting in collections of cells around the
microbe (granuloma)
What else to macrophages do? Produce cytokines to recruit and activate other
leukocytes! They can also:
- secrete growth factors and enzymes to repair tissue
- stimulate T lymphocytes
- respond to products of T lymphocytes and act as effector cells of
cell-mediated immunity
Dendritic Cells
-
respond to microbes by producing cytokines to recruit leukocytes
– initiates adaptive immune response
APC
Natural Killer Cells
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recognize infected and distressed cells and KILL THEM!! How?
By secreting macrophage-activating cytokine IFN-
NKs contain abundant cytoplasmic granules
DO NOT EXPRESS Igs or T CELL RECEPTORS
Activation triggers discharge of granules toward infected cells. These granules
enter infected cells and activate enzymes that induce apoptotic death.
- these are the same mechanisms used by CTLs
NK CELLS KILL INFECTED HOST CELLS THUS ELIMINATING MICROBIAL
RESERVOIR
- these cells also secrete IFN- which activates macrophages to
become more efficient at killing phagocytosed microbes – NKs
and macrophages function cooperatively:
Macs ingest microbes and make IL-12. IL-12 activates NK cells to make IFN-, and
IFN- aciivates microbes to kill ingested microbes. BEAUTIFUL!!
Activating and Inhibitory receptors on NK cells
ACTIVATING RECEPTORS recognize cell surface molecules on stressed cells
(viruses, IC bacteria, DNA damage, malignant transformation, tumour cells)
 eg NKG2D – responds to MHC class I
 antibody coated cells – results in antibody dependent
cellular cytotoxicity (ADCC)
NK CELLS ARE THE PRINCIPAL MEDIATORS OF ADCC
What are ITAMs? Immunoreceptor tyrosine-based activation motifs. These are
contained in activating receptors
- are phosphorylated when receptors bind ligands – activates several other
substrates downstream resulting in cytotoxic endocytosis and production of IFN-
Inhibitory Receptors
- specific for class I MHCs
- KIR (killer cell Ig-like receptors) – composed of CD94 and NKG2
- both contain ITIMs (immunoreceptor tyrosine-based inhibitory
molecules
- cause the NK cells to be shut off
Viruses have mechanisms which block expression of MHC class I molecules. This
mechanism evades CD8+ CTLs but inhibits the inhibitory NK receptors. Thus, NK cells
are still activated and continue to eliminate virus-infected cells.
NK-activating cytokines: IL-15, type 1 interferons, IL-12
- IL-15 important for development and maturation
- IFNs and IL-12 important to enhance killing function
Other classes of lymphocytes
NK-T cells – found in epithelia and lymphoid organs
- recognize microbial lipids bound to MHC II
B-1 cells
- found in peritoneal cavity and mucosa
- produce antibodies in response to microbes/microbe products that
pass through walls of intestine
- most of IgM found in blood (natural antibodies) are products of
B-1 cells
- many specific for carbs in bacterial cell walls
Marginal Zone B cells
- present at edges of lymphoid follicles in spleen, other organs
- also involved in Ab response to blood-borne carb-rich microbes
These lymphocytes have characteristics of adaptive immunity (Ab production) but also
have features of innate immunity
Complement System
- collection of circulating membrane associated proteins (mainly
proteolytic enzymes)
**The following pathways have different beginnings but chare same late steps and
effector functions**
Alternative pathway (innate) is triggered when complement proteins are
activated on microbial surfaces. Microbes don’t have regulatory proteins and can’t
control it.
Classical pathway (humoral adaptive) is triggered after Abs bind to microbes and
other Ags
Lectin pathway (innate immunity) activated when mannose-binding lectin binds
to terminal mannose residues on microbial surface glycoproteins (activates proteins of the
classical pathway but is innate)
THE CENTRAL COMPONENT OF COMPLEMENT IS C3
CD3b is the major proteolytic fragment
-activates downstream proteins on microbial surface
3 functions of complement system:
1. C3b coats microbes (opsonization)
2. some fragments are chemoattractants for phagocytes and eventually other
leukocytes (ie. C5a, C3a)
3. culminates in polymeric protein complex that penetrates the cell membrane
and causes either lysis or microbial apoptotic death
Cytokines of Innate Immunity
Interleukins - most are produced by leukocytes, act on leukocytes (though not
all)
The principal source of cytokines are activated dendritic cells and
macrophages.
- most produced in small amounts
- bind with high affinity to target cells
- can act distant from site of secretion
Cytokines serve various functions in host defense:
TNF, IL-1 recuit blood neutrophils and monocytes to infection (TNF can also
produce thrombus formation, reduced myocardial contractility, vascular dilation,
leakiness and shock
Septic shock can be produced by disseminated gram-neg infections. One sees low blood
pressure, coagulation, metabolic disturbance – in septic shock, one sees high TNF levels
IL-12 – produced by macs and dends in response to LPS, other microbial
products
IFN  - produced by NK cells, T cells – both innate and adaptive
Other Plasma Proteins
Plasma mannose-binding lectin (MBL) – opsonizes and activates complement
C-reactive protein (CRP) – opsonization
Circulating levels of many plasma proteins rapidly increase after infection – acute phase
response
To review…
Extracellular bacteria and fungi combated by phagocytes, complement, acute phase
proteins
Intracellular bacteria and viruses combated by phagocytes, dendritic cells, NK cells
with help from cytokines
Evasion of I.I. by Microbes
- produce protein that allows escape from phagocytic vesicles into
cytoplasm (although Dr. Madrenas said this didn’t occur)
- cell walls containing lipid that avoids fusion with vesicles
Role of I.I. in Stimulating Adaptive Immunity
I.I. responses generate molecules that serve as second signals (together with antigens) to
activate B and T lymphocytes. This microbe-dependent second signal requirement
ensures no response to harmless substances.
What is an adjuvant? A substance administered with an antigen (in a vaccination) to
elicit similar response to an infection.
Microbes stimulate dends and macs to produce second signals that stimulate T
lymphocytes:
- dends and macs express costimulators
- these bind to receptors on naïve T cells – Ag + costimulation
activates T cells
- ALSO dends and macs secrete IL-12 which stimulates T cell
differentiation
How else?
-
B lymphocytes recognize C3d on microbes from alternative
pathway
there is a receptor for C3d on B cell which serves as costimulator
These second signals not only stimulate A.I. but also guide the response
IC pathogens need to be eliminated by T cells thus T cells are stimulated by “Tcell costimulators” on dendritic cells and macrophages.
EC pathogens (ie. blood-borne) need to stimulate B cells and do so by
“complement system co-stimulators”