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Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis Richard C. Chou, Nancy D. Kim, Christian D. Sadik, Edward Seung, Yinan Lan, Michael H. Byrne, Bodduluri Haribabu, Yoichiro Iwakura, Andrew D. Luster Introduction • Leukocyte recruitment into tissue compartments is a tightly regulated process central to all inflammatory responses • Chemoattractants cooperate to control leukocyte migration by activating a family of related chemoattractant G protein-coupled receptors (GPCRs) • The K/BxN model of arthritis bears important clinical and histopathologic similarities to human rheumatoid arthritis, with synovial pannus formation and bone and cartilage erosions Introduction Reference : Current Protocols in Immunology Introduction • The K/BxN Arthritis Model • Mice expressing both the T cell receptor (TCR) transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) develop severe inflammatory arthritis serum from these mice causes a similar arthritis in a wide range of mouse strains, due to autoantibodies recognizing glucose-6-phosphate isomerase (GPI) Antibodies against GPI selectively deposit in the joints as immune complexes and fix complement lipid chemoattractant leukotriene B4 (LTB4) and its high-affinity receptor BLT1 are absolutely required for the development of arthritis in the K/BxN serum transfer model Introduction Reference : Immunologic Research 2005;32/1–3:5–13 Hypothesis The LTB4-BLT1 pathway delivers neutrophils expressing IL-1 and chemokines into the joint, which in turn amplifies and sustains neutrophil recruitment and inflammation via both neutrophil-derived and IL-1inducible chemokines. Result Exogenous IL-1β Restores Arthritis Susceptibility to Ltb4r1-/- Mice IL-1 was markedly decreased in the joints of arthritis-resistant Ltb4r1-/- mice (Kim et al., 2006) Exogenous murine recombinant IL-1β (mIL-1β) could restore ? i.p. on days 0, 1, 2 CP : BSA Injection of mIL-1β in addition to K/BxN serum induced polyarthritis in Ltb4r1-/- mice similar to ageand sex-matched C57Bl/6 wild-type (WT) control mice that received K/BxN serum The joints of WT, mIL-1β Ltb4r1-/- ->inflammation with leukocytic infiltrates, synovial hypertrophy, and joint erosion mIL-1β induced chemokine expression in the synovium of Ltb4r1-/- mice recruits Ltb4r1-/- PMNs into the joint in a BLT1-independent manner? Synovial tissue chemokine and cytokine levels Synovial fluid leukocyte chemokine receptor levels on day 4 after the injection of arthritogenic serum Ltb4r1-/- mice : attenuated induction of chemokines, IL-1β, and TNF-α mIL-1β -> Ltb4r1-/- mice : upregulated CCL4 (MIP-1β), CXCL2 (MIP-2), and CXCL1 (KC) : low level of IL-1β and TNF-α IL-1β acts downstream of BLT1 signaling to induce chemokine production in synovial tissues during inflammatory arthritis and recruits PMNs via their specific chemokine receptors. Result Neutrophil-Derived IL-1 Is Sufficient to Restore Arthritis Susceptibility in Ltb4r1-/- Mice PMNs were the source of IL-1β? BM PMNs from either WT or IL-1α/β-deficient mice were adoptively transferred into Ltb4r1-/- mice A,B. neutrophil-derived IL-1 is sufficient to restore disease in Ltb4r1-/- mice Synovial tissue chemokine and cytokine levels ->4 days after serum transfer C. inflammation with leukocytic infiltrates Result Activated PMNs Produce IL-1β murine PMNs produce IL-1β in the course of K/BxN arthritis? immunoblot analysis qPCR A,B. PMNs are probably the main cellular source of IL-1β in the synovial fluid ELISA D. supernatants of purified BM & SF PMNs from K/BxN transgenic mice incubated ex vivo for 21 hr dilution effect ELISA ELISA endogenously produced LTB4 had no apparent autocrine or paracrine effect on IL-1β production in SF PMNs ex vivo ELISA G. CP-105,696 -> BLT1-specific antagonist qPCR H, I. the effect of LTB4 on IL-1β mRNA and protein release in WT BM PMNs ELISA =>LTB4 is not the stimulus for neutrophil IL-1β production in the K/BxN model. qPCR ELISA J,K. 1) ICs induce IL-1β release from PMNs via FcR activation 2) LTB4-BLT1 pathway does not play an indirect autocrine or paracrine role in IC-mediated IL1β production and release -> deficient in the Fc receptor gamma chain Fc receptors are essential for the pathogenesis of serum transfer-induced K/BxN arthritis in which immune complexes deposit in the joints (Matsumoto et al., 2002), Result Chemokine Induction in Synovial Tissue and Infiltrating Synovial Fluid Leukocytes Neutrophil-Active Chemokine Expression Follows Temporal and Cell-Specific Patterns in Serum Transfer Arthritis CCR1 ligands -> CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CCL9 (MIP-1) CXCR2 ligands -> CXCL1 (KC), CXCL2 (MIP-2), CXCL5 (LIX), and CXCL7 (NAP-2) qPCR ELISA Preclinical early onset early peak established peak qPCR PMNs themselves contributed to chemokine production in the joint? qPCR ELISA C. CXCL2 increased dramatically starting on day 3 and remained very high through day 10 D, E. CXCL2 was the most abundant neutrophil-active chemokine ligand expressed in SF PMNs Immunohistochemical staining for CXCL1 and CXCL2 Arthritic ankle tissue sections 7 days after initial serum transfer. CXCL1 expression was seen primarily in the synovial tissue CXCL2 was primarily expressed by SF leukocytes other structural cells of the joint may also contribute to neutrophil recruitment by producing chemokines Result IL-1β Stimulates Chemokine Production in Endothelial Cells, Fibroblastlike Synoviocytes, and Macrophages IL-1 delivered to the joint by neutrophils could amplify neutrophil recruitment by inducing the release of neutrophil-active chemokines from synovial tissue cells? The pattern of chemokine production in fibroblast-like synoviocytes, endothelial cells, macrophages in response to IL-1β and TNF-α stimulation for 4 hr qPCR qPCR qPCR IL-1β and TNF-α significantly induced FLS, ECs, and MΦ to express neutrophil-active chemokines, neutrophils promote their own recruitment into the joint by releasing large amounts of CXCL2 and by releasing IL-1, which further amplifies neutrophil recruitment by inducing CXCL5 from FLS, CXCL1 from ECs, and CCL9 from MΦ. Result Chemokine Receptor Expression on Neutrophils Recruited into the Joint the relationship between chemokine expression in the arthritic synovium and the expression of their specific receptors on leukocytes recruited into the joint? qPCR qPCR A,B. 1. CCR1, CXCR2, and CXCR4 were detectable all days 2. BLT1 was expressed early 8 days after serum transfer All Subpopulation Lost All C-D. BLT1 and CXCR2 protein surface expression by flow cytometry analysis with specific mAbs on BM and SF PMNs BLT1 is downregulated on PMNs found in the joint and PMNs expressing CXCR2 are preferentially recruited into the inflamed joint at later time points Result CCR1 and CXCR2 Are Critical Receptors Downstream of BLT1 for Neutrophil Recruitment chemokines exert different roles in the recruitment of neutrophils during the initiation and progression of disease? using mice deficient in either CCR1 or CXCR2 marked delay severity never reached marked delay severity never reached Ccr1-/-Cxcr2-/- mice were remarkably completely resistant to the development of arthritis CCR1 and CXCR2 Mediate Early and Late Phases of Serum Transfer Arthritis Histological analysis and Histological scoring of Ccr1-/and Cxcr2-/- mice = Clinical measurements A critical and nonredundant role for CCR1 and CXCR2 in the recruitment of neutrophils and induction and progression of joint inflammation in this model of arthritis Discussion I. Neutrophils are a critical source of IL-1 in this arthritis model II. Immune complex activation of Fc receptors induces IL-1 production from neutrophils III. Sequential requirement for BLT1, CCR1, and CXCR2 for neutrophil recruitment IV. CCR1 and CXCR2 account for all of the neutrophil chemokine activity in model