Background/Purpose:

In rheumatoid arthritis (RA), the influx of inflammatory cells as well as the aggressive proliferation of fibroblast-like synovial cells (FLS) outstrips the oxygen supply from blood vessels leading to joint hypoxia.  Macrophages accumulate in hypoxic disease sites including RA joints where they possess broad pro-inflammatory, destructive and remodeling potential leading to inflammation and joint destruction. Macrophages respond to hypoxia by up regulating the hypoxia inducible transcription factors– HIF-1 and -2 normally degraded in the presence of oxygen. This study will attempt to understand the relative contribution of HIF-1 and HIF-2 expressing macrophages in RA and the genes/mechanisms involved in their activation.

Methods:

We obtained arthroscopy sections from RA patients for which tissue oxygen levels had been measured. This consisted of a random sample of mild (~40mmHg), moderate (~15mmHg) & severe (~3mmHg) joint hypoxia. We also used samples from a second cohort of patients scored with mild or severe disease (based upon extent of synovitis and vascularity), a sub group of which were also receiving anti-TNF therapy. Sections were immunostained with anti-HIF 1 and 2 and co-localised with the pan-macrophage marker CD68 as well as other macrophage markers (Flt-1, CD147, CD206 and Tie2).

Results:

In patients with mildly hypoxic joints, macrophages (CD68⁺) predominately expressed HIF-1 (20%) and CD147 and were found in small clusters localised to the lining layer, whilst macrophages in patients with severely hypoxic joints were in greater numbers (73%), throughout the biopsy. These macrophages predominately expressed HIF-2⁺ (>75%), Flt-1, Tie2 and CD206. A similar pattern was observed in patients with severe disease where sections expressed more HIF-2⁺Flt-1⁺ macrophages compared to those with mild scores (15 cells per field of view compared to 5 for mild p<0.01). There was no significant difference in HIF-1 expression.  Interestingly, this HIF-2⁺ macrophage subpopulation was absent in patients who had been successfully treated with anti-TNF.

Conclusion:

In patients with severely hypoxic joints macrophage numbers were significantly greater than in patients with mild hypoxia and predominantly expressed HIF-2⁺, which activates genes associated with both inflammation and angiogenesis. These cells expressed M2-like macrophage markers including Flt-1, Tie2 & CD206, important in tissue remodeling and angiogenesis. We are currently investigating the gene expression profile of these subpopulations using laser capture micro-dissection and gene arrays and will further study their relevance in animal models of RA using transgenic mice with targeted deletions of HIF-1 or HIF-2 in myeloid cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophages-in-hypoxic-rheumatoid-joints-preferentially-express-hypoxia-inducible-transcription-factor-2/