Background/Purpose

Macrophages in rheumatoid arthritis (RA) synovial tissue (ST) produce high levels of inflammatory cytokines/chemokines and exhibit enhanced differentiation into osteoclasts in the pannus, playing the pivotal role in promoting inflammation and joint destruction. Recent observations demonstrate that effective therapy employing a TNF inhibitor results in a selective reduction of sublining RA ST macrophages within 24 hours. However, neither ingress of monocytes into the tissue nor apoptosis of macrophages in the RA tissue accounted for the reduction of macrophages.  Therefore, employing a murine model, studies were performed to define the role of CCR7 expression to promote macrophage egress from inflamed joints as a potential mechanism for therapeutic response.   

Methods

CCR7 expression in RA synoival macrophages was determined by two color immunohistochemistry employing anti-CCR7 and anti-CD68, RT-PCR and immunoblotting.  A human TNF transgenic (hTNF-tg) mouse line which spontaneously develops arthritis was employed, and treated with infliximab, administered intraperitoneally (10mg/kg, 1-3 doses). The clinical severity of the arthritis was defined as the sum score of joint swelling, inflammation, deformity and grip strength. Ankle histology was performed. The immune cell phenotypes and apoptosis were determined by flow cytometry. Ankle cell migration was tracked by PKH26 intra-articular injection and cell identification by flow cytometry. 

Results

CCR7 was increased in macrophages in RA ST and synovial fluid.  CCR7 expression on normal human macrophages was significantly increased at the mRNA and protein levels following incubation with TNFa, Pam3 or LPS. As expected the hTNF-tg mice developed arthritis beginning at week 4, progressing through week 12. The hTNF-tg mice treated with infliximab for 72 or 168 hours demonstrated significant clinical improvement. Histologic analysis identified significant reduction of inflammation, bone erosion and pannus formation after 168 hours of therapy.  Flow cytometric analysis demonstrated that ST macrophages were significantly reduced at 24 hours, prior to clinical improvement, and 72 and 168 hours following the initiation of therapy.  In contrast, other cell types including granulocytes, B or T lymphocytes and dendritic cells were not consistently or not significantly reduced. No increase of macrophage apoptotosis or necrosis in ankle ST was observed following treatment. Futher, although there was a reduction of PKH26 labeled macrophages in the ankles following therapy, there was also a reduction of macrophages in the popliteal lymph nodes and no increase in the percentage of PKH26 labeled macrophages was detected.

Conclusion

These observations demonstrate increased CCR7 on macrophages in the RA joint and that CCR7 on macrophages was increased by inflammation.  Macrophages, but not other cell types, in the inflamed synovium of hTNF-Tg mice were reduced early prior to clinical or histologic improvement, but we have yet to document egress as the mechanism.  The role of CCR7 in the therapeutic reduction of macrophages is being pursued by crossing hTNF-Tg mice with those deficient in CCR7-/-.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reduced-macrophages-in-the-synovium-contribute-to-the-effective-treatment-of-spontanneous-arthritis-observeded-in-human-tnf-transgenic-mice/