Chemokines and Cell Trafficking. John W Steinke, Ph.D. University of Virginia Charlottesville, VA

Transcription

1 Chemokines and Cell Trafficking John W Steinke, Ph.D. University of Virginia Charlottesville, VA

2 Disclosure Research Grant/Co-investigator: National Institutes of Health (NIH) Research Grant: Genentech Royalties/Licensing: immortalized cell line

3 Learning objectives Upon completion of this session, participants should be able to: Discuss rationale for chemokine nomenclature Indicate complexity of chemokine signaling pathways Identify families of molecules involved in adhesion and cell trafficking Outline process of cell trafficking and movement across endothelial surfaces Identify diseases associated with defects in chemokine receptors and cell trafficking Discuss current approaches of chemokine modulation in therapeutic development

4 What is a chemokine? -small peptides (8-10 kda) that are produced by a variety of cell types originally described as mediators of cell migration -48 members have been described that belong to one of four structural families in humans that are defined by the relative position of their cysteine residues -chemokines bind to at least 20 different receptors -chemokine receptors are a sub-group of the G-proteincoupled receptors characterized by a seven -helical transmembrane domain

5 Nomenclature

6 Chemokine families C chemokine subfamily (2 members) N C C CC chemokine subfamily (27 members) N CC C CXC chemokine subfamily (18 members) N CXC C CX3C chemokine subfamily (1 member) N CX3C C

7 Chemokine structure Nickel et al. JACI 1999;104:723-42

8 Chemokine nomenclature C chemokine subfamily XCL1- Lymphotactin XCL2- SCM-1 CC chemokine subfamily CCL2- MCP-1 CCL3- MIP-1 CCL5- RANTES CCL11- Eotaxin CCL17- TARC CCL22- MDC CXC chemokine subfamily CXCL8- IL-8 CXCL9- Mig CXCL10- IP-10 CXCL11- I-TAC CX3C chemokine subfamily CX3CL1- Fractalkine New additions CCL3L1 CCL4L1 For a complete list see S Commins, L Borish and J W Steinke Cytokines, interferons, and chemokines JACI 2010;125:S53-S72

9 CXC chemokine sub-nomenclature ELR vs non-elr (Glu-Leu-Arg) ELR is conserved motif immediately preceding the first cysteine amino acid ELR chemokines- angiogenic and act mainly through CXCR2 Include: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 Non-ELR chemokines- angiostatic and act mainly through CXCR3B and are induced by interferons Include: CXCL4, CXCL9, CXCL10, CXCL11

10 Common chemokines and basic function Chemokine CCL2 (MCP-1) CCL3 (MIP-1 ) CCL4 (MIP-1 ) CCL5 (RANTES) CCL11 (eotaxin) CCL17 (TARC) CCL19 (ELC) CCL21 (SLC) CCL22 (MDC) Function induces transendothelial migration of monocytes migration of T cells, NK cells, monocytes and immature dendritic cells migration of T cells, NK cells, monocytes and immature dendritic cells trafficking and homing of T cells and monocytes eosinophil chemotaxis migration of dendritic cells and Th2 cells induces migration of dendritic cells from periphery to draining lymph nodes induces migration of dendritic cells from periphery to draining lymph nodes migration of dendritic cells and Th2 cells

11 Common chemokines and basic function Chemokine CXCL1 (Gro- ) CXCL8 (IL-8) CXCL10 (IP-10) CXCL12 (SDF- 1 / ) CX3CL1 (fractalkine) Function enhances endothelial and epithelial cell wound healing neutrophil chemotaxis T cell chemoattractant induced by interferons induces migration of bone marrowderived progenitor cells acts as adhesion molecule and chemoattractant for monocytes and T cells

12 Chemokine receptors Picture from Heise et al JBC 2000; 275:

13 Chemokine receptor nomenclature C chemokine subfamily XCR1 CXC chemokine subfamily CXCR1-7 CXCR3 and CXCR3b CC chemokine subfamily CCR1-10 CCL18-PITPNM3 CX3C chemokine subfamily CX3CR1 membrane-associated phosphatidylinositol transfer protein 3 3 Decoy chemokine receptors DARC-duffy antigen receptor for chemokines D6 (ACKR2) CCRL1 (formally known as CCX-CKR)

14 Chemokine receptors on common immune cells Cell type Chemokine receptor Naïve T cell Th1 Th2 Th17 Eosinophil Basophil Neutrophil CXCR4, CCR7 CCR5, CXCR3 CCR4, CCR8 CCR6 CCR3 CCR3 CXCR1, CXCR2

15 Chemokine signaling Ca 2+ channel Chemokine protein tyrosine kinases SrcTPK Ca 2+ PLC PIP2 Ras Raf RhoA Pyk-2 Shc PI3K p125 FAK ZAP70 STAT1/STAT3 MEK PKB DAG IP3 PLD MAPK PKC cpla2 ERK Chemotaxis Degranulation AA metabolites Proliferation Cellular metabolism Focal adhesion

16 Regulation of chemokine receptors Homologous desensitization: G protein-coupled receptor kinases selectively phosphorylate chemokine-occupied receptors, leading to endocytic uptake of chemokine-receptor complexes Heterologous desensitization: Non-G protein-coupled receptor kinases phosphorylate ligand- free (non-engaged) chemokine receptors, preventing future G protein coupling and receptor activation

17 Function

18 Inflammatory vs Homeostatic Inflammatory: Homeostatic: Chemokines that are induced by pathogens, cytokines or growth factors that recruit effector leukocytes to sites of infection, inflammation, tissue injury and tumors Chemokines that are expressed in the bone marrow and lymphoid tissues that are involved in hematopoiesis, immune surveillance and adaptive immune responses

19 Adhesion molecules in leukocyte interactions Selectins Selectins Name Tissue distribution Ligand A family of cell-surface adhesion molecules that bind to sugar moieties on specific glycoproteins with mucinlike features. P-selectin (PADGEM, CD62P) E-selectin (ELAM-1, CD62E) L-selectin (LAM-1, CD62L) Activated endothelium and platelets Activated endothelium Leukocytes Sialyl-Lewis X PSGL-1(CD162) Sialyl-Lewis X PSGL-1(CD162), CD15, ESL-1 Sialyl-Lewis X, MAdCAM-1, GlyCAM-1, CD34

20 Adhesion molecules in leukocyte interactions Integrins Integrins Name Tissue distribution Ligand Heterodimeric cell-surface proteins involved in cell-cell and cell-matrix interactions. Play important role in adhesive interactions between lymphocytes and antigen-presenting cells and in lymphocytes migration into tissues. 1 : 1 (VLA-1, CD49a/CD29) 2 : 1 (VLA-2, CD49b/CD29) 3 : 1 (VLA-3,CD49c/CD29) 4 : 1 (VLA-4, CD49d/CD29) 5 : 1 (VLA-5, CD49e/CD29) Activated T cells, fibroblasts, mesangium, hepatocytes, monocytes Activated T cells, endothelium, platelets, basophils, NK, B cells Glemeruli, thyroid, basement membrane Monocytes, mast cells, eosinpohils, T and B cells, basophils, NK, dendritic cells Lymphocytes, monocytes, basophils, mast cells, endothelium, epithelium Laminin, collagen Collagen, laminin Laminin, collagen, fibronectin CS-1 domain VCAM-1, MAdCAM-1 fibronectin, JAM-B fibronectin

21 Adhesion molecules in leukocyte interactions Integrins Heterodimeric cell-surface proteins involved in cell-cell and cell-matrix interactions. Play important role in adhesive interactions between lymphocytes and antigen-presenting cells and in lymphocytes migration into tissues. Integrins-cont Name 6 : 1 (VLA-6, CD49f/CD29) L : 2 (LFA-1, CD11a/CD18) M : 2 (Mac-1, CD11b/CD18) D : 2 ( D/CD18) 4 : 7 (CD49d/CD 7; Act-1) Tissue distribution Memory T, monocytes, platelets, endothelium Monocytes, macrophages, T cells, neutrophils, dendritic cells Monocytes, macrophages, neutrophils, eosinophils, basophils, NK cells Eosinophils, basophils, monocytes, lymphocytes Some memory T cells, eosinophils, basophils (but not PMN) Ligand Laminin ICAM-1, -2, -3, -4, -5, E-selectin, JAM-A ICAM-1, -2, ic3b, fibrinogen, JAM-C ICAM-3, VCAM-1 VCAM-1, MAdCAM-1 fibronectin CS-1 domain

22 Adhesion molecules in leukocyte interactions Immunoglobulin superfamily Immunoglobulin superfamily Name Tissue distribution Ligand A family of proteins in which all members have at least one immunoglobulin or immunoglobulinlike domain. These proteins are involved in antigen recognition and cell-cell interaction within the immune system. ICAM-1 (CD54) ICAM-2 (CD102) ICAM-3 (CD50) ICAM-4 (CD242) Activated endothelium, fibroblasts, dendritic cells, epithelium Endothelium, dendritic cells, basophils, mast cells, monocytes Leukocytes, langerhans cells, endothelium Erythrocytes LFA-1, Mac-1, fibrinogen, rhinovirus LFA-1, Mß2 (CR3) LFA-1, CD18 LFA-1, Mac-1, CD18, CD49 b,d,e

23 Adhesion molecules in leukocyte interactions Immunoglobulin superfamily-cont Immunoglobulin superfamily Name Tissue distribution Ligand A family of proteins in which all members have at least one immunoglobulin or immunoglobulinlike domain. These proteins are involved in antigen recognition and cell-cell interaction within the immune system. VCAM-1 (CD106) PECAM (CD31) MAdCAM-1 NCAM (CD56) Activated endothelium, fibroblasts, dendritic cells, respiratory epithelium Activated leukocytes, platelets, endothelial cell-cell junctions Mucosal endothelium Neural cells, NK, T cell subsets VLA-4, 4/ß7 (act- 1; LPAM-1), Dß2 CD31,CD38 4/ß7 NCAM, heparin sulfate

24 Inflammatory cell migration +Eotaxi n,rant ES Granule Proteins Inflammation LTC 4 O 2

25 Rolling Shear stress sialyl-lewis X CXCL8 CXCR1 receptor E-selectin

26 Rolling-a little more complicated PSGL-1 ESL-1 ESL-1 PSGL-1 E-selectin P-selectin Adapted from Hidalgo, A. et al. Immunity 2007;26:477-89

27 Adhesion and spreading ICAM-1 Load-bearing bond Signaling bond Forming bond LFA-1

28 Adhesion- a closer look ESL-1 CD44 ESL-1 PSGL-1 E-selectin P-selectin CD44 L-selectin Adapted from Hidalgo, A. et al. Immunity 2007;26:477-89

29 Transmigration (diapedesis)

30 Cell-to-cell tight junctions Proteases Reactive oxygen species Mac-1 JAM-C VLA-4 JAM-B LFA-1 JAM-A PECAM-1 PECAM-1 PECAM-1 PECAM-1 CD99 CD99 CD99 CD99 P -catenin VE-cadherin VE-cadherin -catenin P

31 Transmigration (diapedesis)

32 Migration

33 Various stages eosinophil cell migration Borrowed from DiScipo, R.G. et al. A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo J Immunol 1999;162:

34 Exception-effector T cells ICAM-1 LFA-1

35 Physiological consequence of leukocyte extravasation Migration of T lymphocytes for immune surveillance Recruitment of activated lymphocytes and granulocytes during the acute and chronic inflammatory responses Homing and mobilization of hematopoietic progenitor cells

36 Moving past inflammation Additional roles for chemokines: Brain development-cxcl1(gro ), CXCL12(SDF-1), CCL3(MIP-1 ) Angiogenesis-CXCL5(ENA-78), CXCL8(IL-8) Wound healing- CCL2(MCP-1), CXCL8(IL-8) Hematopoiesis- CXCL12(SDF-1) Tumor immunity- CXCL4(PF-4), CXCL9(MIG), CXCL10(IP-10) Fibrosis- CCL2(MCP-1), CXCL8(IL-8) Cell survival/apoptosis- CXCL8(IL-8), CCL3(MIP-1 ) Embryonic development- CXCL12(SDF-1) Lymphocyte differentiation CCL11(eotaxin), CCL17(TARC) Organogenesis- CXCL12(SDF-1) Antigen recognition- CCL17(TARC), CCL22(MDC)

37 Production of CCL17 (TARC) and CCL22 (MDC) CCL17 is expressed in lymphoid tissue CCL22 is expressed in thymus, lung and spleen CCL17 and CCL22 both can be produced by activated myeloid DCs and B cells CCL17 and CCL22 both use the CCR4 receptor

38 Activities of CCL17 and CCL22 CCL17 and CCL22 in combination with CXCL12 promote platelet aggregation and adhesion molecule expression CCL22 production stimulated by prostaglandin D 2 and IL-9 in bronchial tissue leads to recruitment of eosinophils and Th2 cells to the lung CCL17 and CCL22 promote T cell-dc and T cell-b cell contact during antigen-driven immune activation

39 Production of CCL19 (ELC) and CCL21 (SLC) CCL19 and CCL21 are expressed by endothelium, stromal cells, and dendritic cells CCL19 and CCL21 both primarily use the CCR7 receptor, but can also use the CXCR3 receptor CCL19 and CCL21 both are targets for the decoy receptor CCRL1 (CCX-CKR)

40 Activities of CCL19 and CCL21 CCL19 and CCL21 together organize B and T cell areas of secondary lymphoid organs and aid in the recruitment and interaction of lymphocytes and APCs CCL21 is strongly expressed on high endothelial venules (HEVs) and is responsible for the recruitment of T cells and dendritic cells from the blood into the lymph nodes CCL19 and CCL21 promote tethering of APCs and T cells aiding in immune synapse formation Proliferation of a subset of NK cells is costimulated by CCL19 and CCL21

41 Disease

42 Chemokine/receptor defects and human disease CCR5/CCL3L1 HIV/AIDS CCR2/CXCR4/CXCL12 HIV/AIDS DARC P. vivax malaria CXCR4 WHIM s syndrome CXCL4 Heparin-induced thrombocytopenia CX3CR1, CCL2 Atheroslcerosis CCL2, 5, 7, 11/CXCL8 Asthma/Allergies CXCL12/CXCR4, R7 Cancer metastases CCL3L1/CXCL8/CCL2 SLE CCR5 West Nile Virus CCR5 Tickborne Encephalitis flavivirus infection CCR5/CCL2 HCV CCR5/CCL2/CCL4L1 Transplant rejection CCR5/CCR6 Rheumatoid arthritis CCL2/CCL3 Alzheimer s disease

43 WHIM s syndrome Inherited as an autosomal dominant allele of CXCR4 (carboxy terminus deletion) Non-CXCR4 mutations in GRK3 and ARRB2 that alter CXCR4 activity have been reported Less than 80 case reports Characteristics of disease Chronic neutropenia (<500 cells/ l) with bone marrow hypercellularity (myelokhathexis) Sinusitis, otitis, pneumonia and cellulitis common Warts (due to HPV infection later in life) Hypogammaglobulinemia Infections (HPV and bacterial) Often low or absent lymphocyte counts and low or absent IgG and/or IgA Early vaccine Ab response normal but lose protective Ab after 1 yr Treatment IVIG qmonthly may reduce incidence of infections in subjects with low IgG G-CSF or GM-CSF qdaily may normalize neutrophil counts Plerixator (CXCR4 antagonist) tested in 8 subjects increased

44 Malarial resistance and DARC Defined as a minor blood group antigen that is expressed on Purkinje cells of the cerebellum, post-capillary venule endothelial cells, red blood cells and CD45RO+ T cells The molecule is a 7-transmembrane domain protein to which several members of the CC and CXC chemokine families can bind DARC is the receptor used by Plasmodium vivax to infect erythrocytes Most black Africans and 70% of African-Americans are resistant to vivax malaria due to a mutation in a GATA transcription-factor binding site in the promoter of the DARC gene that results in DARC being absent from the red blood cells

45 HIV entry through chemokine receptors HIV GP120 interacts with: CD4 co-stimulatory molecule chemokine receptor Relevant receptors Monocyte/macrophage: CCR5 T cell: CXCR4

46 Mechanism of entry Chinen J and Shearer WT JACI 2002;110:

47 HIV entry through chemokine receptors CCR5 32 receptor mutant Homozygotes- no infection Heterozygotes- slow disease progression CCL3L1 (CCR5 ligand) Low gene copy number is associated with lower chemokine concentrations and increased risk of HIV acquisition, higher viral loads and an accelerated rate of disease progression CCR2 V64I mutation associated with slow disease progression

48 Atherosclerosis-link to CX3CL1 and CX3CR1 CX3CL1 is a transmembrane glycoprotein with a chemokine domain on top of a mucin-like stalk that exists as a soluble or membrane-bound form Chemoattracts monocytes, NK cells and CD8+ T cells and induces cytotoxic granule release from NK cells Membrane-bound form mediates strong integrin-independent adhesion Picture from Rossi DL et al Genomics 1998; 47:163-70

49 Atherosclerosis-link to CX3CL1 and CX3CR1 Receptor expressed on the surface of neurons, activated vascular endothelium, NK cells and smooth muscle. CX3CL1 expression on endothelium stimulates P-selectin and ICAM-1 expression helping to initiate leukocyte aggregation. Both CX3CL1 and CX3CR1 are expressed in atherosclerotic plaques Two polymorphisms found in CX3CR1 are associated with disease T280M-protective by having lower affinity for CX3CL1 and preventing leukocyte recruitment into plaques V249I-associated with elevated risk of acute coronary syndrome and brain infarction

50 Metastases-chemokine link Metastatic propensity of tumors correlates with increased expression of CXCR4. Organs where metastatic cells home display peak levels of the CXCR4 ligand CXCL12. In contrast, expression of DARC is associated with a decrease in tumor-associated vasculature and reduction in metastatic potential. (leukemia, lung cancer, colorectal cancer) In animal models, inhibiting either CXCL12 or CXCR4 decreases metastases and tumor growth. CCX-CKR negative regulator of metastasis in breast cancer via a scavenger role. Low expression is correlated with poor survival in breast cancer patients.

51 Question #1 In humans, what is the smallest family of chemokines? A. CX3C B. C C. CC D. CXC

52 Question #2 Which chemokine receptor mutation has been linked to WHIM s disease? A. C3XCR1 A230M B. CXCR4 C. CCR2 D. CCR5 32

53 Question #3 Which of the following is a member of the immunoglobulin family and is involved in antigen recognition and cell-cell interactions? A. CD62L B. LFA-1 C. 1 7 D. ICAM-4

54 Question #4 Which chemokine receptor is used by Plasmodium vivax to infect cells leading to malaria? A. CCR2 B. CXCR3 C. DARC D. XCR2

55 Question #5 Chemokines are grouped based on A. Functionality B. Conserved cysteine residues C. Order in which they were identified D. Cellular expression patterns

56 Question #6 Homeostatic responses influenced by chemokines involve A. Recruitment of leukocytes to sites of infection B. Diapedesis C. Development of lymphoid structures D. Induction of cytokine synthesis by eosinophils