Leukocyte Endothelial Interactions

1. Leukocyte Endothelial Interactions Judith Berliner, Professor Departments of Biology and Medicine, UCLA

2. Leukocyte/endothelial interactions are a major event in the inflammatory process

3. Inflammation is a reaction to injury or infection

4. Inflammation and repair 1. Tissue exposed to infection, toxin, trauma 2. Damaged cells rapidly produce endothelial activators 3. In some cases, venules transiently increase permeability in response to activators 4. Plasma proteins enter the tissue and react with bacteria and toxins producing more activators

5. Inflammation and repair 5. Activated endothelial cells upregulate leukocyte adhesion molecules and chemotactic factors 6. Leukocytes bind to and enter the vessel wall 7. Acute: Neutrophils kill and macrophages engulf bacteria and toxins. Resolution 8. Chronic: macrophages are unable to remove source of injury. Granuloma

6. Inflammation and repair 9. Cytokines and growth factors produced by injured cells stimulate replication of nearby cells. Fibrosis 10. Angiogenesis occurs in response to additional growth factors. 11. Tissue architecture is restored

10. Activators of the endothelium to induce leukocyte EC interactions 1. Bacteria 2. Toxins 3. Autoantibodies 4. Oxidized Phospholipids 5. Advanced glycosylation end products 6. Cytokines: IL-1, TNF

11. OM peptidoglycan IM LIPOPOLYSACCHARIDE lipid A

12. INITIATION OF INFLAMMATION • RECOGNITION OF MICROBES BASED ON CONSERVED PATTERNS • LIPOPOLYSACCHARIDE (Gm-) • LIPOPEPTIDES (BACTERIA) • PEPTIDOGLYCAN (MOST BACTERIA) • FLAGELLA (MANY BACTERIA) • UNMETHYLATED CpG IN DNA • DOUBLE-STRANDED RNA (VIRAL)

13. TOLL-LIKE RECEPTORS single stranded RNA TLR8 Akira et al. Nature Rev Immun 2004

14. TLR1 Triacyl lipopeptides (Bacteria, mycobacteria) Soluble factors (Neisseria meningitidis) TLR2 Lipoprotein/lipopeptides (Various pathogens) Peptidoglycan (Gram-positive bacteria) Lipoteichoic acid (Gram-positive bacteria) Lipoarabinomannan (Mycobacteria) Glycolipids (Treponema maltophilum) Porins (Neisseria) Zymosan (Fungi) TLR3 Double-stranded RNA (Viruses) TLR4 Lipopolysaccharide (Gram-negative bacteria) Taxol (Plants) Fusion protein (Respiratory syncytial virus) Envelope protein (Mouse mammary-tumor virus) TLR5 Flagellin (Bacteria) TLR6 Diacyl lipopeptides (Mycoplasma) Lipoteichoic acid (Gram-positive bacteria) Zymosan (Fungi) TLR7 Imidazoquinoline (Synthetic) Single-stranded RNA (Viruses) TLR8 Imidazoquinoline (Synthetic) Single-stranded RNA (Viruses) TLR9 CpG-containing DNA (Bacteria and viruses) TLR SPECIFICITY -2004

15. Regulation of Endothelial Cell gene expression by TLR4 activation 1. P-Selectin and E-Selectin 2. MCP-1, IL-8, Platelet activating factor 3. ICAM-1, VCAM-1 • Many cytokines NFkB important in this activation *5. Macrophages activation of TLR4 increases TNF and IL-1 that activate endothelial cells.

16. Activators of the endothelium to induce leukocyte EC interactions 1. Bacteria 2. Toxins – Hydrogen Peroxide 3. Autoantibodies – Rheumatoid Arthritis 4. Oxidized Phospholipids - Atherosclerosis 5. Advanced glycosylation end products – Diabetes 6. Cytokines – TNF, IL-1

17. Activators of the endothelium to induce leukocyte EC interactions 1. Bacteria 2. Toxins – Hydrogen Peroxide,Silica 3. Autoantibodies – FC receptor 4. Oxidized Phospholipids – CD 36 5. Advanced glycosylation end products – RAGE 6. Cytokines – TNF, IL1 - TNF and IL-1 receptors

19. Rolling Molecules Selectins and their ligands Integrins and their ligands

21. SELECTINS • SELECTINS--LEUKOCYTE AND ENDOTHELIAL PROTEINS THAT MEDIATE CELL ADHESION TO CARBOHYDRATES • P-SELECTIN, ON ENDOTHELIA AND PLATELETS, UPON ACTIVATION RAPIDLY TRANSFERRED TO CELL MEMBRANE • L-SELECTIN, ON LEUKOCYTES, EXPRESSED CONSTITUTIVELY, BECOME MORE ADHESIVE IN STIMULATED LEUKOCYTES BUT SHED WITHIN MINUTES OF ACTIVATION • E-SELECTIN, ON ENDOTHELIA, INDUCED WITHIN HOURS BY INFLAMMATORY MEDIATORS

25. Regulation of Chemokines Levels and Activity Attachment to GAGs- localizes activity Cleavage by metalloproteinases Formation of homo and heterodimers m-RNA Degradation Protein Degradation

27. Effects of chemotactic factors on leukocyte activation 1. Chemotactic factors bind to GPCR 2. This leads to integrin activation 3. Integrin activation causes arrest followed by spreading and migration

30. Structure and Function of Integrins Integrins are heterodimers (formed by  and chains) receptors located on the surface of cells. The  subunit contains the RGD-binding site. Calcium is required for integrin binding activities. The cytoplasmic end is associated with cytoskeletal proteins. FAK

32. Leukocyte transmigration 1. Leukocyte migrates to EC junction 2. Leukocyte activates transient endothelial retraction 3. Leukocyte extends pseudopod into junctions 4. Leukocyte binds to homotypic molecules on EC and moves across monolayer 5. Leukocyte produces proteases and migrates across basement membrane in response to chemotactic factors

37. Activation of the Leukocyte for killing and phagocytosis

38. PHAGOCYTOSIS AND KILLING PHAGOCYTIC KILLING: -BY GRANULE CONTENTS -BY REACTIVE OXYGEN PRODUCTS EXTRACELLULAR KILLING

39. NEUTROPHILS: ARSENAL • REACTIVE OXYGEN PRODUCTS • NUTRIENT-BINDING PROTEINS • PROTEASES • LYSOZYME • MICROBICIDAL PEPTIDES • DEFENSINS • CATHELICIDINS

45. Diseases Associated with Inflammation and Angiogenesis Cancer Rheumatoid Arthritis Atherosclerosis Diabetic Retinopathy

47. Sources of Inflammatory Molecules in Tumors Extrinsic Pathway – Inflammation due to infection of cell injury Intrinsic Pathway – Oncogene Activation Both lead to increased macrophages in tumors

48. Macrophage Phenotypes and Cancer M1M2 Markers: IL12, TNF, IL6,ROS IL-4, IL-10, TGFB Functions: Attract lymphocytes Decrease lymphocyte entry Activate lymphocytes Decrease in lymphocyte activation Kill tumor cells Increase in angiogenesis Increase tumor cell growth