1. Richard C. Chou, Nancy D. Kim, Christian D. Sadik, Edward Seung, Yinan Lan, Michael H. Byrne, Bodduluri Haribabu, Yoichiro Iwakura, Andrew D. Luster Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis

2. 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

3. Reference : Current Protocols in Immunology Introduction

4. 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 B 4 (LTB 4 ) and its high-affinity receptor BLT1 are absolutely required for the development of arthritis in the K/BxN serum transfer model Introduction The K/BxN Arthritis Model

5. Reference : Immunologic Research 2005;32/1–3:5–13 Introduction

6. LTB 4 -BLT1 pathwayneutrophilsIL-1 chemokines chemokines.  The LTB 4 -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-1-inducible chemokines. Hypothesis

7.  Result Exogenous IL-1β Restores Arthritis Susceptibility to Ltb4r1 -/- Mice

8. Exogenous murine recombinant IL-1β (mIL-1β) could restore ? IL-1 was markedly decreased in the joints of arthritis-resistant Ltb4r1 -/- mice (Kim et al., 2006) Injection of mIL-1β in addition to K/BxN serum induced polyarthritis in Ltb4r1 -/- mice similar to age- and sex-matched C57Bl/6 wild-type (WT) control mice that received K/BxN serum i.p. on days 0, 1, 2 CP : BSA

9. The joints of WT, mIL-1β Ltb4r1 -/- ->inflammation with leukocytic infiltrates, synovial hypertrophy, and joint erosion

10. mIL-1β induced chemokine expression in the synovium of Ltb4r1 -/- mice recruits Ltb4r1 -/- PMNs into the joint in a BLT1-independent manner? 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. Synovial tissue chemokine and cytokine levels Synovial fluid leukocyte chemokine receptor levels

11.  Result Neutrophil-Derived IL-1 Is Sufficient to Restore Arthritis Susceptibility in Ltb4r1 -/- Mice

12. 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 C. inflammation with leukocytic infiltrates Synovial tissue chemokine and cytokine levels ->4 days after serum transfer

13.  Result Activated PMNs Produce IL-1β

14. murine PMNs produce IL-1β in the course of K/BxN arthritis? qPCR A,B. PMNs are probably the main cellular source of IL-1β in the synovial fluid immunoblot analysis dilution effect D. supernatants of purified BM & SF PMNs from K/BxN transgenic mice incubated ex vivo for 21 hr ELISA G. CP-105,696 -> BLT1-specific antagonist  endogenously produced LTB 4 had no apparent autocrine or paracrine effect on IL-1β production in SF PMNs ex vivo ELISA

15. H, I. the effect of LTB 4 on IL-1β mRNA and protein release in WT BM PMNs qPCR ELISA =>LTB 4 is not the stimulus for neutrophil IL-1β production in the K/BxN model. 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), -> deficient in the Fc receptor gamma chain J,K. 1) ICs induce IL-1β release from PMNs via FcR activation 2) LTB 4 -BLT1 pathway does not play an indirect autocrine or paracrine role in IC-mediated IL- 1β production and release

16.  Result Chemokine Induction in Synovial Tissue and Infiltrating Synovial Fluid Leukocytes

17. 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) Preclinical early onset early peak established peak qPCR ELISA C. CXCL2 increased dramatically starting on day 3 and remained very high through day 10 PMNs themselves contributed to chemokine production in the joint? qPCR ELISA D, E. CXCL2 was the most abundant neutrophil-active chemokine ligand expressed in SF PMNs

18. 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

19.  Result IL-1β Stimulates Chemokine Production in Endothelial Cells, Fibroblast- like Synoviocytes, and Macrophages

20. 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 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. qPCR

21.  Result Chemokine Receptor Expression on Neutrophils Recruited into the Joint

22. the relationship between chemokine expression in the arthritic synovium and the expression of their specific receptors on leukocytes recruited into the joint? qPCR A,B. 1. CCR1, CXCR2, and CXCR4 were detectable all days 2. BLT1 was expressed early C-D. BLT1 and CXCR2 protein surface expression by flow cytometry analysis with specific mAbs on BM and SF PMNs All Subpopulation Lost  BLT1 is downregulated on PMNs found in the joint and PMNs expressing CXCR2 are preferentially recruited into the inflamed joint at later time points 8 days after serum transfer

23.  Result CCR1 and CXCR2 Are Critical Receptors Downstream of BLT1 for Neutrophil Recruitment

24. 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  Ccr1 -/- Cxcr2 -/- mice were remarkably completely resistant to the development of arthritis

25. 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

26. 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 Discussion