Chemokines Dr. habil. Kőhidai, László Chemotaxis Research Group Dept. Genetics, Cell- & Immunobiology, Semmelweis University 2011.

Classification of chemokines

Characterization of IL-8 chemokine

Dimerization of chemokines The CXC chemokines have a six stranded beta sheet dimer and the CC chemokines have an end-on-end dimer.  It was first thought that dimers were necessary for function of chemokines but tests have proven that chemokines can function as monomers.  It is possible that dimerizartion is just more thermodynamically favored

Receptor-ligand interaction chemokine NH2- HOOC- GAG Signalling

Characterization of IL-8 chemokine = Neutrophil activator peptide - NAP2; = Melanoma growth stimulator activity - MGSA Target cells: neutrophils, macrophages, T-Ly, mast cells, endothel, keratinocyte Homodimer (Leu25 + CH3 - blocks) Receptors Chtx. IL-8R1 4000 +++ IL-8R2 1700 + (7 loop transmembrane domain, G-prot.) Neutrophiles - ic. Ca2+ release - chemotaxis - exocytosis - resp. burst - histamine release micro bl.vessels perm. incr.

Chemokine receptors

FUNCTIONAL RESPONSES RANTES receptor Ca2+ channel Cell membrane Chemotaxis Adh. mol. expr. Proliferation Cytokine release IL-2 rec. expr. prot. against HIV Uropodium formation FUNCTIONAL RESPONSES RANTES receptor Cell membrane Ca2+ channel

Extravasation and chemokines/receptors

- Chemokines and adhesion molecules - Extravasation - Chemokines and adhesion molecules - http://www.nature.com/nri/journal/v4/n2/fig_tab/nri1268_F6.html The picture represents the sequential steps of leukocyte extravasation. Tethering of the leukocyte on the surface of activated endothelium occurs through interactions between L-selectin and its endothelial ligands, as well as P-selectin glycoprotein ligand 1 (PSGL1)-mediated contacts. Tethering and rolling precede firm adhesion, which is mediated by interaction of leukocyte integrins very late antigen 4 (VLA4, 4 1) and leukocyte function-associated antigen 1 (LFA1, L 2) with endothelial vascular cell-adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1), respectively. The actin cytoskeleton has a dual regulatory role during this step (see text). Integrin-mediated cell adhesion is enhanced by interaction of G-protein-coupled receptors — for example, CXC-chemokine receptor 4 (CXCR4), the receptor for stromal cell-derived factor 1 (SDF1 ), also known as CXC-chemokine ligand 12) — with chemokines immobilized on glycosoaminoglycans (GAGs) in a mechanism of receptor cross talk. During this step, leukocyte polarization occurs, with clustering of adhesion molecules in the cellular uropod. Finally, leukocytes extravasate and enter the target organ — a process that involves integrins as well as junctional adhesion molecules (JAMs) expressed by endothelial cells. For simplicity, not all of the domains of ICAM1, ICAM3 and VCAM1 are represented; they contain five, five and seven immunoglobulin domains, respectively. MTOC, microtubule-organizing centre.

- Chemokines and adhesion molecules - Extravasation - Chemokines and adhesion molecules - After exposure to a chemoattractant source in a soluble or immobilized gradient, leukocytes develop front-to-back asymmetry, including extension of a broad, actin-based, flat lamellipodium regulated by localized activation of molecules that are involved in actin polymerization, such as the CDC42–WASP–ARP2/3, RAC–WAVE–ARP2/3 or RhoA–DIA1 pathways, guided by the local concentration of chemotactic receptors in lymphocytes. RhoA-regulated activation of myosin is represented by myosin IIA localization at the leading edge and myosin IIB localization at the rear part of the cell. The opposing pole (the uropod) is thinner, which sometimes protrudes over the plane of cell interaction with the substrate and is involved in cell–cell contacts. Actin regulates the formation of the uropod and clustering of adhesion molecules such as intercellular adhesion molecule 1 (ICAM1), ICAM3, P-selectin glycoprotein ligand 1 (PSGL1), CD43 or CD44, using ezrin–radixin–moesin (ERM) proteins as membrane-actin cytolinkers. Other cytoskeletal polymers such as microtubules and intermediate filaments are also juxtaposed in this structure. ARP2/3, actin-related protein 2/3; MTOC, microtubule-organizing centre; ROCK, Rho-associated, coiled-coil containing protein kinase; WASP, Wiskott-Aldrich syndrome protein; WAVE, WASP family verprolin-homologous protein.

Steps of guiding and gradients

Chemokine producer cells

CXC kemokines

CC chemokines

Chemokine receptors and their ligand-specificity

Chemokine receptors and their ligand-specificity Receptor Ligand Target cell CXCR 1 IL-8, GRO-a, NAP-2, ENA-78 neutrophil, NK-cell 2 IL-8 neutrophil, NK-cell 3 IP-10, MIG, I-TAC monocyte, T-cell 4 SDF-1 monocyte, T-cell

Chemokine receptors and their ligand-specificity Receptor Ligand Target cell CCR 1 MIP-1a, RANTES, MIP-1b, monocyte, eosinophil MCP-1,-2,-3 2 MCP-1,-2,-3,-4,-5 monocyte 3 eotaxin, RANTES, MIP-1a, eosinophil, monocyte MIP-1b basophil 4 MCP-1, MIP-1a, RANTES monocyte 5 MIP-1a, MIP-1b, RANTES monocyte, T-cell 6 MIP-3b dendritic cell 7 MIP-3b act. T-cell 8 I-309 monocyte

Chemokine receptors and their ligand-specificity Receptor Ligand Target cell CX3CR 1 fractalkine monocyte, act. T- cell, NK-cell Receptor Ligand Target cell DARC 1 IL-8, GRO-a, NAP-2, RBC., endothel of (Duffy Ag) ENA-78,RANTES, post capillary venules MCP-1, MCP-3 Receptor Ligand Target cell Viral rec. US28 MIP-1a, RANTES, MCP ECRF-3 IL-8, GRO-a, NAP-2

Viral chemokine-binding proteins Virus modulation of the chemokine network. Depicted are the viral gene products (inner circle) that are chemokine receptor homologs (CXCRs or CCRs), chemokine homologs (CC or CXC), or secreted chemokine-binding proteins (CKBPs) that bear no homology to any known proteins. Host or viral ligands that bind viral chemokine receptors or viral CKBPs are illustrated on the outer circle. Similarly, host or viral receptors that are engaged by viral chemokines are also depicted on the outer circle. Ligands and receptors (outer circle) that are affected agonistically by the viral gene products are shown in blue; those affected antagonistically by viral gene products are shown in red. Those labeled in black text bind the viral gene product, but the interaction is uncharacterized. Note that some chemokines may activate a viral receptor positively but may be sequestered (e.g., hCMV US28). Inverse agonists are not shown. Virus abbreviations: CapV, capripoxvirus; FPV, fowlpox virus; EHV, HHV, and MHV, equine, human, and mouse herpesvirus, respectively; HSV, herpes saimiri virus; hCMV and mCMV, human and mouse cytomegalovirus, respectively; LSDV, lumpy skin disease virus; MDV, Marek’s disease virus; MC, molluscum contagiosum; SPV, swinepox virus; YLDV, Yaba-like disease virus. For a full review of the information depicted here, see refs [3 , 4 , 10 , 12 , 36373839 ]. ournal of Leukocyte Biology July 1, 2002 vol. 72 no. 1 24-34

Viral chemokine-binding proteins / chemokine receptors

Chemotactic potential of interleukines IL-2 IL-2 < IL-8 b chain is required for migratory effect LCF (lymphocyte chemoattractant factor) acts via IL-2 receptors BUT: - two LCF monomers - expression of CD4 are required IL-3 eosinophil = neutrophil IL-5 eosinophil > neutrophil IL-6 NK killer

Colony stimulating factors (~CSF) direct effect on „killing” mechanism Macrophage-CSF induced synthesis: granulocyte-CSF, g -interferon, TNF, IL-1 PGE2 inhibited mgration in neutrophils increased adhesion, margination phagocytosis, killing Granulocyte-CSF

CC chemokines Monocyta chemotactic protein (MCP) Macrophag inflammatory protein (MIP) Regulated upon activation, normal T cell expressed and secreted (RANTES) Homologies Structure: MCP1- MCP2 (62%) - MCP3 (71%) Effect: - pertussis toxin blocks MCP1 and MCP3 - cholera toxin blocks MCP2 - blocking Ser/Thr kinase influences mainly chemotaxis induced by MCP2 - MCP1 and MCP3 increases i.c. Ca2+ levels

Overlapping in ligands acting on CC-receptors

Tricks and mimics Acetylated peptide fragment from the ECM protein collagen mimics key sequences of IL-8. The acetylated tripeptide is acPGP, it induces neutrophil chemotaxis in vitro and emigration in vivo.

Regulation of integrines by chemokines

Significance in basic clinical aspects

Chemokines and tumors / metastasis Epithelial tumour cells produce inflammatory cytokines, such as tumour necrosis factor- (TNF- ), and chemokines, such as CCL2 and CXCL12. Leukocytes bearing the appropriate chemokine receptors are attracted to the malignant cells, and are themselves stimulated to produce more chemokines and inflammatory cytokines. Autocrine and paracrine networks are established that attract more leukocytes, especially T-helper 2 (TH2) lymphocytes, type-2 macrophages and pre-dendritic cells. The inflammatory leukocytes contribute to tumour growth and progression by producing proteases, angiogenic factors, growth factors and immunosuppressive cytokines. a | Cancer cells in a primary tumour have metastatic potential, but do not always express chemokine receptors. b | Some cancer cells acquire chemokine-receptor expression by gene mutation, gene fusion or local conditions, such as hypoxia. c | If local levels of the specific chemokine ligand are low, chemokine-receptor-expressing cancer cells can now respond to high levels of ligand at sites of metastasis and migrate towards the chemokine gradient. Alternatively, the acquisition of chemokine receptor might make tumour cells more likely to invade and spread. d | Chemokine ligand at the metastatic site can deliver anti-apoptotic and proliferative signals, and induce tumour-necrosis factor- . This cytokine can initiate a pro-tumour inflammatory network in the surrounding stroma. Hence, the chemokine ligand encourages the tumour cells to survive and grow. Nature Reviews Cancer 4, 540-550 (July 2004)

CC-receptors and HIV infection