Background/Purpose:   Current understanding of pathogenesis suggests that RA is mainly a Th1 mediated process that promotes robust inflammatory cytokine production by “classically” activated macrophages.  This is in contrast to Th2 diseases such as parasitic infection or allergy that balance potential catastrophic tissue destruction from large worm invasion, or chronic inflammatory response to ubiquitous proteins, by skewing macrophages to an alternatively activated state. Alternatively activated cells appear to be generated from local macrophage proliferation in pure Th2 environments.  However, in conditions with concomitant Th1 stimuli, recruited monocytes can be skewed toward alternative activation as well.  It is unknown what role alternative macrophages may play in RA.  It seems reasonable to assume that their presence would be favorable given their “anti-inflammatory” properties.  However, they theoretically have the potential to hinder response to, and clearance of, a yet unknown foreign antigen.  Given that alternative macrophages display distinct proliferating capabilities, it seems reasonable to suspect that proteins controlling the cell cycle may be involved in their regulation.  One such protein, a cyclin dependent kinase inhibitor, p21, is decreased in RA patient synovium, and is associated with worse serum transfer-induced arthritis (STIA) compared to wild type (WT) controls.

Methods:   In vitro studies were done with bone marrow derived macrophages (BMDMs) and thioglycollate induced peritoneal macrophages (PMs).  Cells were treated with stimuli for classical, alternative, and regulatory macrophage differentiation.  Supernatants were analyzed for production of cytokines by ELISA, and for nitric oxide (NO) by colorimetry.  Quantigene assays were performed for various genetic markers of macrophage phenotype.  Mice were also injected IP with thioglycollate and IL-4, with or without BRDU added 3 hours prior to harvest.   Peritoneal cells were assessed by flow cytometry.  Finally, mice were treated with IV IL-4 + KBxN serum and observed for arthritis. 

Results:   In vitro analysis of skewed BMDMs and PMs revealed increased production of nitric oxide in p21-/- cells stimulated with interferon gamma + lipopolysaccharide compared to B6 control cells.  Conversely, p21-/- BMDMs and PMs stimulated by IL-4 had less relm alpha mRNA, a marker of alternative activation.  In vivo studies were concordant in that p21-/- mice treated with thioglycollate + IL-4 had less relm alpha protein expression by flow cytometry.  Intriguingly, there was increased local proliferation of p21-/- PMs as measured by 3 hr BRDU incorporation.  However, recently recruited p21-/- monocytes showed significantly less proliferation compared to B6.  Finally, IV IL-4 significantly dampened STIA in B6 mice whereas arthritis in p21-/- mice was not attenuated.

Conclusion:   p21 may promote homeostasis during inflammatory conditions by potentiating recruited monocytes and possibly local macrophage differentiation into alternatively activated states.  This appears to be independent of its role as a cell-cycle inhibitor because monocytes recruited to inflammatory sites have less proliferation in the absence of p21.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/p21-promotes-inflammatory-arthritis-resolution-by-facilitating-alternative-activation-of-macrophages/