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

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

Inflammation is a reaction to injury or infection

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

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

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

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

LIPOPOLYSACCHARIDE lipid A

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)

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

TLR SPECIFICITY 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)

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 4.Many cytokines NFkB important in this activation *5. Macrophages activation of TLR4 increases TNF and IL-1 that activate endothelial cells.

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

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

Rolling Molecules Selectins and their ligands Integrins and their ligands

Endothelial moleculeLeukocyte Receptor P-Selectin Sialyl-Lewis X PSGL-1 E-SelectinSialyl-Lewis X GlyCam-1L-Selectin *VCAM-1  

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

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

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

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

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

Activation of the Leukocyte for killing and phagocytosis

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

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

Genetic DiseaseDefect Leukocyte adhesion deficiency 1  -chain of CD11/CD18 Leukocyte adhesion deficiency 2 Fucosyl transferase required for sialylated oligosaccharide synthesis Chronic granulomatous disease X-linked Autosomal recessive Decreased oxidative burst NADPH oxidase (membrane) NADPH oxidase (cytoplasm) Myeloperoxidase deficiencyAbsent MPO-H 2 O 2 system Chediak-Higashi syndrome Protein involved in organelle membrane docking and fusion

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

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

Macrophage Phenotypes and Cancer M1 M2 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