Innate Immunity -M261 Spring 2005 May 6, 2005 Kathleen A. Kelly

1. Innate Immunity -M261 Spring 2005May 6, 2005 Kathleen A. Kelly Reading: Immunobiology (6th Edition) Janeway, Travers, Walpert & Capra Chapter 2 (p. 37-100), and Chapter 6 (p. 209-212) Fundamental Immunology (5th Edition) Lippincott, Williams & Wilkins Chapter 17 (p.497-517) Janeway, CA, et al. Innate Immune Recognition. Annu. Rev. Immunol. 20:197-252, 2002

2. Innate Immunity -M261 Spring 2004Kathleen A. Kelly Innate immunity predates development of adaptive immunity Does not produce protective immunity No memory response Prerequisite for developing adaptive immunity Non-antigen-specific immunity Found in plants, invertebrates and vertebrates Adaptive immunity is found only in jawed vertebrates and apparently developed by acquiring of the RAG genes. Although innate immunity does not produce Ag-specific or protective immunity it is important because it is a prerequisite for developing an adaptive immune response. Older than the adaptive immunity because it is found in all living organisms. It is a universal form of host defense found in all multicellular organisms. Some of these molecular mechanisms diverged overtime. Adaptive immunity is found only in jawed vertebrates and apparently developed by acquiring of the RAG genes. Although innate immunity does not produce Ag-specific or protective immunity it is important because it is a prerequisite for developing an adaptive immune response. Older than the adaptive immunity because it is found in all living organisms. It is a universal form of host defense found in all multicellular organisms. Some of these molecular mechanisms diverged overtime.

3. Innate Immunity 1. Provides a barrier to prevent the spread of infection Mechanical (tight junctions, movement) Chemical (fatty acids, enzymes, pH, antimicrobial peptides) Microbiological (normal flora) Mucosal surfaces Nasopharyngeal, Oral, Respiratory, Intestinal tract Urogenital tract Skin (epithelial cells) Wounds, burns, insect bites Prevents the pathogen from colonization -To establish an infection the organism must attach the epithelial surfaces. -Infectious disease occurs when a microorganism succeeds in evading or overwhelming innate host defenses to establish a local site of infection and replication that allows its further transmission. Mechanisms -Movement: mucin, cilia, perisalsis -Enzymes: lysozyme, pepsin -Antimicrobial peptides discussed in a later lecture Prevents the pathogen from colonization -To establish an infection the organism must attach the epithelial surfaces. -Infectious disease occurs when a microorganism succeeds in evading or overwhelming innate host defenses to establish a local site of infection and replication that allows its further transmission. Mechanisms -Movement: mucin, cilia, perisalsis -Enzymes: lysozyme, pepsin -Antimicrobial peptides discussed in a later lecture

4. Innate Immunity 2. Identifies and eliminates pathogens Non-adaptive recognition systems Activates molecules that target the microbe and aid in it�s identification. These factors may be expressed at the surface or within cells, released from immune cells or are secreted and present within circulatory system Factors released by immune cells = cytokine/chemokines Factors present in circulatory system = plasma proteins (C� Dr. Morrison)Factors released by immune cells = cytokine/chemokines Factors present in circulatory system = plasma proteins (C� Dr. Morrison)

5. Innate Immunity 3. Initiates an inflammatory response Reaction to injury or infection Trauma to tissues or cells Presence of foreign matter (self vs. non-self) Infectious agents (viruses, bacteria, fungi) Delivers effector molecules & immune cells to the site of infection Components Leukocytes & secreted factors Blood vessels Plasma proteins Innate Immunity can also be referred to as an �Inflammatory Response�.Innate Immunity can also be referred to as an �Inflammatory Response�.

6. Innate Immunity 4. Provides signals to activate and regulate the type of adaptive immune response generated Stimulation of co-stimulatory molecules B7 family (CD80/86, PD-L, ICOSL) TNFR family (OX40L) Induction of a cytokine/chemokine response Cytokines: IL-12, IL-23, IL-4 Chemokines: CXCR1, CXCR2, CCL20 a variety and depends on stimulus Sometimes this can lead to immune-mediated pathology.Sometimes this can lead to immune-mediated pathology.

7. The Phases of Immunity The immune response can be broken into three phases. Dendritic cells are a bridge between the innate and adaptive immune response. -They are the only cell type that can activate na�ve or T cells not yet stimulated by antigen. -They carry the information obtained during the innate immune response to the adaptive immune response.The immune response can be broken into three phases. Dendritic cells are a bridge between the innate and adaptive immune response. -They are the only cell type that can activate na�ve or T cells not yet stimulated by antigen. -They carry the information obtained during the innate immune response to the adaptive immune response.

8. Identification of Microbes Recognition Receptors � Pattern Recognition Receptors (PRRs) Fixed in the genome, ie gene rearrangement is not needed Distribution Non-clonal, ie all cells of a class are identical Differentiation Pathogen vs. Commensal Recognition of microbe is a primary function for innate immunity. All microbes must be recognized, pathogens and commensals. The mechanism by which this occurs in not well understood. How does the host differentiate a pathogen from a commensal? Anti-inflammatory cytokines (IL-10, TGFb) and compartmentalization are important in preventing the triggering of the innate immune response. Recognition of microbe is a primary function for innate immunity. All microbes must be recognized, pathogens and commensals. The mechanism by which this occurs in not well understood. How does the host differentiate a pathogen from a commensal? Anti-inflammatory cytokines (IL-10, TGFb) and compartmentalization are important in preventing the triggering of the innate immune response.

9. Identification of Microbes PRR Recognize conserved molecular patterns on microbes called microbe associated molecular patterns (MAMPs) which are not present on the host Not limited to pathogens Identify a class of microbes LPS, LTA, peptidoglycan, lipoarabinomannan, dsRNA, mannans, b-glycans MAMPs are often essential for microbe survival Action Time Immediate activation of effectors Delays need for adaptive immunity MAMPS � microbe associated molecular pattterns LPS � from Gram negative LTA � lipotechoic acid � from Gram positive Peptidoglycans Gram+: thick layer Gram-: Thin layer Lipoarabinomannan - from microbacteria) dsRNA -produced by most viruses Mannans � from fungi B-glucans � from fungi MAMPS � microbe associated molecular pattterns LPS � from Gram negative LTA � lipotechoic acid � from Gram positive Peptidoglycans Gram+: thick layer Gram-: Thin layer Lipoarabinomannan - from microbacteria) dsRNA -produced by most viruses Mannans � from fungi B-glucans � from fungi

10. Pattern Recognition Receptors (PRRs) Three broad classes of PRRs based on expression profile, localization, function 1) PRRs that signal an infection Include the Toll Receptor Family Expressed external or internally Activation of �pro-inflammatory� signaling pathways NFkB and MAP kinase signaling pathways Antimicrobial peptides (Defensins) / lysozyme, Inflammatory cytokines (TNFa, IL-8, IL-1) Regulate activation of adaptive immune response co-stimulatory molecules These signaling pathways are evolutionarily conserved. These signaling pathways are evolutionarily conserved.

11. 2) Phagocytic (endocytic) PRRs Expressed on the surface of phagocytic cells (MQs, PMNs, DCs) Mediate uptake of microbe into phagocytes 3) Secreted PRRs Secreted by MQs, epithelial cells, liver Activate C�, opsonize microbial cells, function as accessory proteins for MAMP recognition Pattern Recognition Receptors (PRRs) Proteins secreted by MQ & epithelial cells appear in tissue fluids while those from the liver appear in the serum.Proteins secreted by MQ & epithelial cells appear in tissue fluids while those from the liver appear in the serum.

12. Toll-like Receptor Family

13. Intracellular PRRs:Present in the Cytosol of Host Cells 1. Protein kinase receptor (PKR) Activated upon binding to dsRNA (viruses) Blocks viral & cellular protein synthesis (eIF2a) Activates NFkB, MAP kinase STATs & IRF signaling pathways Induces apoptosis & IFNa/b production of infected cells 2. 2�-5� Oligoadenylate Synthase & RNaseL Family of IFN-inducible enzymes dsRNA activates OAS RNaseL degrades viral and host RNA Induces apoptosis Mammalian surveillance mechanism that senses and responds to bacteria in the cytosal. STATs (signal-transducing activators of transcription) IRF (interferon responsive factor)Mammalian surveillance mechanism that senses and responds to bacteria in the cytosal. STATs (signal-transducing activators of transcription) IRF (interferon responsive factor)

14. Intracellular PRRs:Present in the Cytosol of Host Cells 3. NOD proteins or nucleotide-binding oligomerization domain Recognize intracellular peptidoglycan-derived MAMPs and transduce signals three distinct functional domains carboxy-terminal ligand-recognition domain (LRD) centrally located NOD amino-terminal effector-binding domain (EBD) CARD domains in mammals Interacts and activates RIP2 inducing NFkB and MAP-kinase pathways LRD region contains a leucine rich repeat. The centrally located domain is necessary for oligomerization. Effector-binding domain is a CARD domain in mammals. Activates transcriptional pathways that are antimicrobial In plants the resistance genes interact with pathogens and induce the equivalent of an inflammatory response. Nucleotide binding domain � NBD Leucine rich repeat � LLR Caspase activation and recruitment domain - CARD Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003 May;3(5):371-82. Review. PMID: 12766759 [PubMed - indexed for MEDLINE] LRD region contains a leucine rich repeat. The centrally located domain is necessary for oligomerization. Effector-binding domain is a CARD domain in mammals. Activates transcriptional pathways that are antimicrobial In plants the resistance genes interact with pathogens and induce the equivalent of an inflammatory response. Nucleotide binding domain � NBD Leucine rich repeat � LLR Caspase activation and recruitment domain - CARD Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003 May;3(5):371-82. Review. PMID: 12766759 [PubMed - indexed for MEDLINE]

15. Structure of NOD Proteins Example of the domains of a NOD The NOD proteins signal as a dimer. Example of the domains of a NOD The NOD proteins signal as a dimer.

16. NOD Proteins In this model, phagocytosed microorganisms, including bacteria release ligands that are recognized by NOD proteins. Recognition of intracellular ligands by NOD proteins and extracellular ligands by Toll-like receptors (TLRs) mediates the activation of signalling pathways including those downstream of nuclear factor- B (NF- B). These intracellular events induce the secretion of cytokines by the antigen-presenting cell (APC), as well as the expression of co-stimulatory molecules (for example, CD80 and CD86) on the surface of the APC. Signaling through the T-cell receptor (TCR) and co-stimulatory receptors, such as CD28, leads to the development of antigen-specific effector T cells. CIITA, MHC class II transactivator; NOD, nucleotide-binding oligomerization domain. Function is not defined in humans but possibly these proteins play an important role in innate immunity and function as intracellular receptors for pathogens Structurally similar to family of resistance gene products in plants Activate NFkB and MAP kinases signaling pathways Some NOD family proteins implicated in pathogenesis of inflammatory diseases (NOD2/Crohn�s Dis.) In this model, phagocytosed microorganisms, including bacteria release ligands that are recognized by NOD proteins. Recognition of intracellular ligands by NOD proteins and extracellular ligands by Toll-like receptors (TLRs) mediates the activation of signalling pathways including those downstream of nuclear factor- B (NF- B). These intracellular events induce the secretion of cytokines by the antigen-presenting cell (APC), as well as the expression of co-stimulatory molecules (for example, CD80 and CD86) on the surface of the APC. Signaling through the T-cell receptor (TCR) and co-stimulatory receptors, such as CD28, leads to the development of antigen-specific effector T cells. CIITA, MHC class II transactivator; NOD, nucleotide-binding oligomerization domain. Function is not defined in humans but possibly these proteins play an important role in innate immunity and function as intracellular receptors for pathogens Structurally similar to family of resistance gene products in plants Activate NFkB and MAP kinases signaling pathways Some NOD family proteins implicated in pathogenesis of inflammatory diseases (NOD2/Crohn�s Dis.)

17. Phagocytic (endocytic) PRRsBind Carbohydrates 1. Macrophage Mannose Receptor (C-type lectin) Type 1 transmembrane receptor Recognizes patterns of mannose residues in a certain spatial orientation unique to microbes (CRD) Only found on macrophages (not monocytes or PMNs) 2. Glucan Receptor (Dectin-1) Type 2 transmembrane receptor Recognizes b-1,3 & b-1,6 linked glycans Present on all phagocytes These CHO may also be expressed on host cells. Signaling by be due to the co-recognition by other PRRs (TLRs) Updates: Mention that alleles of MBP can alter the concentration of MBP and enhance development of diseases associated with chronic infections (CAD, asthma, rheumatoid arthritis and SLE).These CHO may also be expressed on host cells. Signaling by be due to the co-recognition by other PRRs (TLRs) Updates: Mention that alleles of MBP can alter the concentration of MBP and enhance development of diseases associated with chronic infections (CAD, asthma, rheumatoid arthritis and SLE).

18. Phagocytic (endocytic) PRRs: Cont. 3. Scavenger Receptors Recognize charged ligands Polyanionic ligands (ds-RNA, LPS, LTA) Acetylated low-density lipoproteins (LDL) Found on all phagocytes MARCO (macrophage receptor with collagenous struction) binds bacterial cell walls but not yeast Phagocytose apoptotic cells new factor MFG-E8 (released from activated macrophages and binds to apoptotic cells via phosphatidylserine) There are 6 members lipoteichoic acid = LTA There are 6 members lipoteichoic acid = LTA

19. Secreted PRRs activate the Complement (C�) System Complement system is activated by innate immunity Recognition by Complement receptors (CR) CR1, CR2, CR3, CR4, C5a, C3a Comprised of plasma proteins that when activated forms a triggered enzyme cascade Zymogens � activated by the cleavage of other proteases Precursor enzymes Function Facilitates the uptake & destruction of pathogens by phagocytes Induces an inflammatory responses Named Complement because it augmented the opsonization of pathogens by antibodies. Now it is clear it evolved as part of the innate immune system.Named Complement because it augmented the opsonization of pathogens by antibodies. Now it is clear it evolved as part of the innate immune system.

20. Activation of C� System The secreted PRRs activate C� through the classical and MB-Lectin pathway (pathogen dependent) -Alternate pathway does not depend on a pathogen binding protein (spontaneous cleavage of C3) Called Collectins (MBL & SP-A & SP-D) -Recognize the order of mostly CHO residues on microbes and not the host This pathway is important in early childhood and essentially act in lieu of antibodies The secreted PRRs activate C� through the classical and MB-Lectin pathway (pathogen dependent) -Alternate pathway does not depend on a pathogen binding protein (spontaneous cleavage of C3) Called Collectins (MBL & SP-A & SP-D) -Recognize the order of mostly CHO residues on microbes and not the host This pathway is important in early childhood and essentially act in lieu of antibodies

21. Secreted Pattern Recognition Molecules -pentraxins Acute-phase proteins are produced by liver cells in response to cytokines released by macrophages in the presence of bacteria. They include serum amyloid protein (SAP) (in mice but not humans), C-reactive protein (CRP), fibrinogen, and mannan- binding lectin (MBL). SAP and CRP are homologous in structure; both are pentraxins, forming five-membered discs, as shown for SAP (photograph on the right). CRP binds phosphorylcholine on certain bacterial and fungal surfaces but does not recognize it in the form in which it is found in host cell membranes. It both acts as an opsonin in its own right and activates the classical complement pathway by binding C1q to augment opsonization. MBL is a member of the collectin family, which includes C1q, which it resembles in its structure. We have already seen how MBL activates complement (see Section 2-7) and how it binds to pathogen surfaces (see Fig. 2.28). Like CRP, MBL can act as an opsonin in its own right, in addition to activating complement. SP-A and SP-D are surfactants A and D, both of which are collectins that coat bacterial surfaces, facilitating their phagocytosis. Photograph courtesy of J. Emsley. Photo from Nature 1994, 367:338-345. � 1994 Macmillan Magazines Limited. -pentraxins Acute-phase proteins are produced by liver cells in response to cytokines released by macrophages in the presence of bacteria. They include serum amyloid protein (SAP) (in mice but not humans), C-reactive protein (CRP), fibrinogen, and mannan- binding lectin (MBL). SAP and CRP are homologous in structure; both are pentraxins, forming five-membered discs, as shown for SAP (photograph on the right). CRP binds phosphorylcholine on certain bacterial and fungal surfaces but does not recognize it in the form in which it is found in host cell membranes. It both acts as an opsonin in its own right and activates the classical complement pathway by binding C1q to augment opsonization. MBL is a member of the collectin family, which includes C1q, which it resembles in its structure. We have already seen how MBL activates complement (see Section 2-7) and how it binds to pathogen surfaces (see Fig. 2.28). Like CRP, MBL can act as an opsonin in its own right, in addition to activating complement. SP-A and SP-D are surfactants A and D, both of which are collectins that coat bacterial surfaces, facilitating their phagocytosis. Photograph courtesy of J. Emsley. Photo from Nature 1994, 367:338-345. � 1994 Macmillan Magazines Limited.

22. Secreted Pattern Recognition Molecules 1. Collectins Recognizes microbial carbohydrates (CRD domain) Effector function mediated by collagenous domain Mannan-binding lectin (MBL) Recognizes patterns of mannose & fucose residues in a certain spatial orientation unique to microbes Initiates the lectin pathway of C� cleaving C2 & C4 Can function as an opsonin Binds a receptor on phagocytes (C1qRp) Surfactant proteins (SP-A / SP-D) lung Collectin: a family of structurally related, calcium-dependent sugar-binding proteins or lectins containing collagen-like sequences.Collectin: a family of structurally related, calcium-dependent sugar-binding proteins or lectins containing collagen-like sequences.

23. Collectins Structure is conserved and similar to other proteins with similar function: Some Complement proteins & Mannose Binding Protein Binds to bacteria, fungi & viruses Function by binding microbes and are important for mediating phagocytosis of alveolar macrophages SP = surfactant protein MBP � mannose binding proteinSP = surfactant protein MBP � mannose binding protein

24. Secreted Pattern Recognition Molecules � Cont. 2. Pentraxin Members include Serum amyloid protein (SAP) C-reactive protein (CRP) Recognize phosphorylcholines on microbes Functions as an opsonins Binds to C1q & activate classical C� pathway

25. Secreted Pattern Recognition Molecules � Cont. 3. Lipid Transferases LPS binding protein (LBP) Opsonin Bactericidal permeability increasing protein (BPI) Bactericidal protein 4. Peptidoglycan recognition proteins (PGRS) Recognizes peptidoglycans in evolutionarily distant organisms 4 human PGRS Function is unknown One has bactericidal effects Triggers a serine protease cascade in insects ? Complement cascade ? Less investigated proteinsLess investigated proteins

26. Inflammatory Response Hallmarks of an Inflammatory Response Redness Heat Swelling Pain Hallmarks of an Inflammatory Response Redness Heat Swelling Pain

27. Inflammatory Response Inflammatory response also releases cytokines that act on the hepatocytes of the liver but these cytokines can also stimulate other outcomes. IL-1, IL-6, and TNF-a activate hepatocytes to synthesize acute-phase proteins, and bone marrow endothelium to release neutrophils. The acute-phase proteins act as opsonins, while the disposal of opsonized pathogens is augmented by enhanced recruitment of neutrophils from the bone marrow. IL-1, IL-6, and TNF-a are also endogenous pyrogens, raising body temperature, which is believed to help eliminate infections. A major effect of these cytokines is to act on the hypothalamus, altering the body�s temperature regulation, and on muscle and fat cells, altering energy mobilization to increase the body temperature. At elevated temperatures, bacterial and viral replication are decreased, while the adaptive immune response operates more efficiently. Inflammatory response also releases cytokines that act on the hepatocytes of the liver but these cytokines can also stimulate other outcomes. IL-1, IL-6, and TNF-a activate hepatocytes to synthesize acute-phase proteins, and bone marrow endothelium to release neutrophils. The acute-phase proteins act as opsonins, while the disposal of opsonized pathogens is augmented by enhanced recruitment of neutrophils from the bone marrow. IL-1, IL-6, and TNF-a are also endogenous pyrogens, raising body temperature, which is believed to help eliminate infections. A major effect of these cytokines is to act on the hypothalamus, altering the body�s temperature regulation, and on muscle and fat cells, altering energy mobilization to increase the body temperature. At elevated temperatures, bacterial and viral replication are decreased, while the adaptive immune response operates more efficiently.

28. Leukocyte Adhesion One of the functions of the inflammatory response is to allow leukocytes to enter tissues from the circulation. Although the endothelial cells become leaky � leukocytes must enter via this 4-step adhesion cascade and each step depends on certain components.One of the functions of the inflammatory response is to allow leukocytes to enter tissues from the circulation. Although the endothelial cells become leaky � leukocytes must enter via this 4-step adhesion cascade and each step depends on certain components.

30. Na�ve and Memory T Cells Travel in Different Paths Na�ve (have not seen their antigen) T cells travel in the blood and lymphatics Memory T cells (have been activated by their antigen) can also travel through tissues

31. Lymphocyte Trafficking Patternsof Na�ve T Cells Lymphocyte recirculation was first described by Gowans and Knight in 1964 (Proc. R. Soc. London Ser. B. 159:257). About 10 years ago (Butcher Cell 67:1033-1036, 1991) the adhesive and activation processes were shown to impart specificity using homing receptors/adhesion molecules and chemokines/chemokine receptors. Lymphocytes use homing receptors/adhesion molecules and chemokines/receptors to recirculate between blood and lymph nodes. Na�ve T cells recirculate via blood to peripheral lymph node via CD62L on lymphocytes and PNAd on high endothelial cells of lymph nodes. This interaction is mediated by selectins. Chemokines are small molecules (8-10K in size). The lymph node tissue produces CCL21 (SLC) or CCL19 (ELC). The lymphocytes utilize a G-protein coupled receptor on the endothelium as a which is called CCR7 and binds to both chemokines.Lymphocyte recirculation was first described by Gowans and Knight in 1964 (Proc. R. Soc. London Ser. B. 159:257). About 10 years ago (Butcher Cell 67:1033-1036, 1991) the adhesive and activation processes were shown to impart specificity using homing receptors/adhesion molecules and chemokines/chemokine receptors. Lymphocytes use homing receptors/adhesion molecules and chemokines/receptors to recirculate between blood and lymph nodes. Na�ve T cells recirculate via blood to peripheral lymph node via CD62L on lymphocytes and PNAd on high endothelial cells of lymph nodes. This interaction is mediated by selectins. Chemokines are small molecules (8-10K in size). The lymph node tissue produces CCL21 (SLC) or CCL19 (ELC). The lymphocytes utilize a G-protein coupled receptor on the endothelium as a which is called CCR7 and binds to both chemokines.

32. Lymphocyte Trafficking Patternsof Na�ve T Cells Lymphocyte recirculation was first described by Gowans and Knight in 1964 (Proc. R. Soc. London Ser. B. 159:257). About 10 years ago (Butcher Cell 67:1033-1036, 1991) the adhesive and activation processes were shown to impart specificity using homing receptors/adhesion molecules and chemokines/chemokine receptors. Lymphocytes use homing receptors/adhesion molecules and chemokines/receptors to recirculate between blood and lymph nodes. Na�ve T cells recirculate via blood to peripheral lymph node via CD62L on lymphocytes and PNAd on high endothelial cells of lymph nodes. This interaction is mediated by selectins. Chemokines are small molecules (8-10K in size). The lymph node tissue produces CCL21 (SLC) or CCL19 (ELC). The lymphocytes utilize a G-protein coupled receptor on the endothelium as a which is called CCR7 and binds to both chemokines.Lymphocyte recirculation was first described by Gowans and Knight in 1964 (Proc. R. Soc. London Ser. B. 159:257). About 10 years ago (Butcher Cell 67:1033-1036, 1991) the adhesive and activation processes were shown to impart specificity using homing receptors/adhesion molecules and chemokines/chemokine receptors. Lymphocytes use homing receptors/adhesion molecules and chemokines/receptors to recirculate between blood and lymph nodes. Na�ve T cells recirculate via blood to peripheral lymph node via CD62L on lymphocytes and PNAd on high endothelial cells of lymph nodes. This interaction is mediated by selectins. Chemokines are small molecules (8-10K in size). The lymph node tissue produces CCL21 (SLC) or CCL19 (ELC). The lymphocytes utilize a G-protein coupled receptor on the endothelium as a which is called CCR7 and binds to both chemokines.

33. Lymphocyte Trafficking Patterns of Effector/Memory T Cells When lymphocytes become activated (effector cells) they can then enter tissues via a similar mechanism. For homing receptors/adhesion molecules, activated lymphocytes down regulate CD62L and up regulate PSGL-1 a ligand that is called P-selectin ligand-1. Also the lymphocytes express a4b1 & CD44 (integrins). The lymphocytes also down regulate CCL7 and upregulate CXCL3. CXCL3 is expressed on most Th1 cells. Inflamed tissues express the adhesion molecules CD106 (VACM-1) Hyaluronic acid and P-selection. The tissue secretes CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC).When lymphocytes become activated (effector cells) they can then enter tissues via a similar mechanism. For homing receptors/adhesion molecules, activated lymphocytes down regulate CD62L and up regulate PSGL-1 a ligand that is called P-selectin ligand-1. Also the lymphocytes express a4b1 & CD44 (integrins). The lymphocytes also down regulate CCL7 and upregulate CXCL3. CXCL3 is expressed on most Th1 cells. Inflamed tissues express the adhesion molecules CD106 (VACM-1) Hyaluronic acid and P-selection. The tissue secretes CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC).

36. Phagocytosis Phagocytosis Definition: uptake of large particles (>0.5 mm) Actin-dependent, clathrin-independent High rate & efficiency of internalization Professional phagocytic cells Macrophages Neutrophils These cells have phagocytic receptors External receptors FcR, CR3, Mannose receptor Internal receptors TLRs

37. Macrophages(MQ) Blood - Called monocytes (1-6% WBC) Tissues - Called macrophages mature form of monocytes normally found in tissues such as gastrointestinal tract, lung, liver and spleen Functions: Phagocytose and kills after bactericidal mechanisms are activated (T cells) Produce cytokines/chemokines (initiates inflammation) Is an antigen presenting cell (co-stim. Molecules) They can also phagocytize cells undergoing apoptosis. This is detected via specific receptors.They can also phagocytize cells undergoing apoptosis. This is detected via specific receptors.

38. Neutrophils(PMN) Present in blood (55-60% of WBC) Not normally present in tissues Short lifespan - 12 hours Functions: First at the site of infection/injury Ingest and kill microbes after bactericidal mechanisms are activated (binding to pathogen) Primary (azurophil )and secondary granules (specific)Primary (azurophil )and secondary granules (specific)

39. Phagocytosis (MQ & PMN) Active process initiated by binding to pathogen Pathogen is surrounded and then internalized Phagocytosis requires 1. Binding to particle / microbe 2. Actin-filament formation under particle. 3. Membrane extension and fusion behind particle 4. Fusion with lysosome (endocytic pathway).Phagocytosis requires 1. Binding to particle / microbe 2. Actin-filament formation under particle. 3. Membrane extension and fusion behind particle 4. Fusion with lysosome (endocytic pathway).

40. Signaling Interactions during Phagocytosis Many signaling pathways are activated during phagocytosis: internalization active microbial killing induce production of inflammatory cytokines and chemokines -Protein kinase C Inhibitors block formation of actin filaments beneath particle -PI-3 Kinase Inhibitors block membrane extension and fusion behind particles -Phospholipase C Activates protein kinase C -Rho GTPases Regulates actin cytokeleton Provides receptor specificity Microbes can attack this process to evade phagocytosis. Many signaling pathways are activated during phagocytosis: internalization active microbial killing induce production of inflammatory cytokines and chemokines -Protein kinase C Inhibitors block formation of actin filaments beneath particle -PI-3 Kinase Inhibitors block membrane extension and fusion behind particles -Phospholipase C Activates protein kinase C -Rho GTPases Regulates actin cytokeleton Provides receptor specificity Microbes can attack this process to evade phagocytosis.

41. Killing Mechanisms Phagosome - membrane bounded vesicle that becomes acidified Lysozome - granules that contain products that damage or kill pathogens Enzymes Lysozyme - dissolves cell walls of some bacteria Acid hydrolases - digests bacteria Proteins Lactoferrin - binds Fe++ needed for bacterial growth Vitamin B12-binding protein Peptides Defensins and cationic proteins - direct antimicrobials

42. Killing Mechanisms - cont. Respiratory Burst Activated following phagocytosis Stimulated by PRR Requires increased oxygen consumption Produces substances that are directly toxic to the bacteria Oxygen-derived products O2-, H2O2 & Myeloperoxidase Nitrogen-derived products NO (nitrogen oxide) Produced by inducible NO synthase (iNOS) enzyme Enzyme is induced by cytokines (LT, TNFb)

43. NADPH Oxidase Fig. 18.3 The NADPH oxidase complex. NADPH is a large, multisubunit enzyme complex whose assembly in the phagocyte membrane is triggered by a phagocytic stimulus. For simplicity, only four major components are shown here: gp91phox and p21phox, which together form the membrane complex known as cytochrome b558, and p67phox and p47phox, which normally reside in the cytoplasm. On delivery of a phagocytic stimulus and even soluble stimuli including PMA and fMLP to the cell, p47phox becomes hyperphosphorylated and binds to p67phox and other cytosolic components, and these components migrate to the membrane to form the complete complex with cytochrome b558. Fig. 18.3 The NADPH oxidase complex. NADPH is a large, multisubunit enzyme complex whose assembly in the phagocyte membrane is triggered by a phagocytic stimulus. For simplicity, only four major components are shown here: gp91phox and p21phox, which together form the membrane complex known as cytochrome b558, and p67phox and p47phox, which normally reside in the cytoplasm. On delivery of a phagocytic stimulus and even soluble stimuli including PMA and fMLP to the cell, p47phox becomes hyperphosphorylated and binds to p67phox and other cytosolic components, and these components migrate to the membrane to form the complete complex with cytochrome b558.

44. Enzyme Reactions of Respiratory Burst Respiratory Burst NADPH NADP+ Superoxide + dismutase 2 O2 2 O- H2O2 Myeloperoxidase Enzyme which is stored in primary granules of PMN & MQ and uses the products of the respiratory burst. H2O2 + C1- Chloramines

45. Professional APC

46. Regulation of Adaptive Response Non adaptive recognition receptors called Pathogen Recognition Receptors (PRR). These recognize PAMPs or pathogen associated molecular patterns. This leads to activation of immune response. Receptors can exist on the surface of the cell or intracellular in endocytic compartments. Recognition is based on two principles 1. microbial non-self (PRR/PAMP) 2. Missing self -Inhibitory receptors (NK cells) NK cells will be discussed later and you have already discussed complement. However, we will touch on the receptors of the complement system that the innate immune system uses for recognition. -Factor H of Complement Recognition of a pathogen causes to signals to be released and this induces the development of T cells. co-stimulator signals cytokinesNon adaptive recognition receptors called Pathogen Recognition Receptors (PRR). These recognize PAMPs or pathogen associated molecular patterns. This leads to activation of immune response. Receptors can exist on the surface of the cell or intracellular in endocytic compartments. Recognition is based on two principles 1. microbial non-self (PRR/PAMP) 2. Missing self -Inhibitory receptors (NK cells) NK cells will be discussed later and you have already discussed complement. However, we will touch on the receptors of the complement system that the innate immune system uses for recognition. -Factor H of Complement Recognition of a pathogen causes to signals to be released and this induces the development of T cells. co-stimulator signals cytokines

47. T cells Recirculate to �Find� Antigen-loaded Dendritic cells

48. Mucosal Immunity � Reading Assignment Immunobiology (6th Edition) Janeway, Travers, Walpert and Capra Chapter 10 (p. 432-445). Neutra, MR et al Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu. Rev. Immunol. 14:275-300, 1996 Wright, JR. Immunoregulatory Functions of Surfactant Proteins. Nature Review Immunol. 5:58-68, 2005. Cheroutre, H. Start at the beginning: new perspectives on the biology of mucosal T cells. Annu. Rev. Immunol. 22:217-46, 2004. Weiner, H. Oral tolerance: immune mechanism and the generation of Th3-type TGF-beta-secreting regulatory cells. Microbes & Infection 3:947-954, 2001.