Background/Purpose: Inflammatory arthritis, including rheumatoid arthritis, is characterized by neutrophil (PMN) recruitment into the joint in a highly regulated process controlled by chemoattractants (CAs). Four different chemoattractant receptors (CAR), BLT1, CCR1, CXCR2 and C5aR, are required and collaborate to mediate PMN recruitment into the joint in immune-complex (IC) induced arthritis. However, the precise role for each CA in the process of PMN recruitment into the joint in vivo remains unclear. This process begins with the capture of free-flowing leukocytes to the vessel wall, followed by rolling and then arrest on the vessel wall, release from adhesion and then crawling in all directions on the vessel to locate a receptive location for transendothelial migration (TEM) into the joint.

Methods: Multiphoton intravital imaging (MP-IVM) was performed to analyze the migratory behavior of wild-type (WT) and CAR-deficient (KO) PMNs in the joint of WT and CAR-KO-LysM-GFP mice, in which endogenous PMNs and macrophages express GFP, on day 7 after K/BxN serum transfer (KST). We analyzed the ability of WT and CAR-KO PMNs to enter the joint in short term adoptive transfer assays on day 7 after KST. We also analyzed joint tissue for the expression of C5a using immunohistochemistry and synovial fluid (SF) for the levels of CKs by ELISA. We analyzed the expression of CARs by flow cytometry on PMNs isolated from the bone marrow (BM), blood and SF. Lastly, we generated mixed bone marrow chimeric (BMC) mice with WT and CAR-KO BM cells transferred into lethally irradiated WT mice to analyze PMN recruitment into the joint through the entire course of arthritis development. Finally, the effect of CXCR2 ligands on SF PMN survival was evaluated.

Results: In the inflamed joint of WT- LysM-GFP mice, abundant PMN adhesion and TEM was observed. However, PMN adhesion and TEM were not observed in the joint of C5aR-KO-LysM-GFP mouse. PMN adhesion was observed in the joints of BLT1-KO-, CCR1-KO- and CXCR2-KO-LysM-GFP mice but PMN TEM was either not observed or was markedly diminished. Short term adoptive transfer assays demonstrated a decrease in the adhesion and TEM of C5aR-KO PMNs, whereas BLT1-KO, CCR1-KO and CXCR2-KO PMNs were able to adhere to the endothelium but had a decrease in TEM into the inflamed joint. 40% adoptively transferred C5aR-KO PMNs that arrested on the endothelium detached, while 20% of CCR1-KO PMNs that crawled on the endothelium detached. WT arrested and crawling PMNs were not observed to detach from the endothelium. C5a deposition was observed on the endothelium and cartilage in arthritic joints. C5a was detected in the joint on day 1, whereas the other CAs were detected at later time points. In mixed BMC mice with arthritis, CXCR2-KO PMNs were not observed in joint and CXCR2 ligands prevented SF neutrophils from undergoing apoptosis.

Conclusion: Our data demonstrate that C5a is the critical initiator of PMN adhesion on joint endothelium and is required to initiate joint inflammation in IC-induced arthritis. In addition, we have found that CCR1 contributes to the interaction of PMNs with the joint endothelium and that PMNs self-propagate their recruitment and survival via CXCR2 in the joint space.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/complement-c5a-receptor-is-the-key-initiator-of-neutrophil-adhesion-and-inflammation-in-immune-complex-induced-arthritis/