Slit2 Regulates Hyaluronan & Cytokine Synthesis in Fibrocytes: Potential Relevance to Thyroid-Associated Ophthalmopathy

Abstract

Context: CD34+ fibrocytes have been implicated in development of thyroid-associated ophthalmopathy (TAO), a consequential autoimmune manifestation of Graves disease (GD). In TAO, CD34+ fibrocytes appear to masquerade as CD34+ orbital fibroblasts mixed with CD34- OF (collectively, GD-OF). Slit2, an axon guidance glycoprotein, is expressed by CD34- OF and attenuates GD-OF gene expression. Cardinal features of TAO include hyaluronan (HA) accumulation and cytokine-driven inflammation.

Objective: Compare expression of HA synthase isoenzymes (HAS1-3), UDP-glucose dehydrogenase (UGDH), synthesis of HA, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in fibrocytes and GD-OF. Determine whether Slit2 alters gene expression patterns.

Design/setting/participants: Patients with TAO and healthy donors were recruited from an academic practice.

Main outcome measures: Real-time polymerase chain reaction, HA, IL-6, and TNF-α immunoassays.

Results: HA synthesis and release from fibrocytes is substantially lower than in GD-OF. HAS1 expression dominates in fibrocytes while HAS2 in GD-OF. In contrast, HAS2 and UGDH expression dominate GD-OF and localize to CD34- OF. Recombinant human Slit2 (rhSlit2) substantially upregulates HA synthesis and HAS2 expression in fibrocytes but attenuates IL-6 and TNF-α production in these cells. In contrast, knocking down Slit2 in GD-OF reduces HA synthesis and HAS2 and UGDH expression while upregulating IL-6 and TNF-α.

Conclusion: The dramatic differences in HA, IL-6, and TNF-α production, and HAS and UGDH expression found in fibrocytes and GD-OF appear, at least in part, to be attributable to Slit2. These findings provide novel insight into the differences in gene expression exhibited by CD34+ fibrocytes and CD34+ OF and therefore reveal important aspects of disease pathogenesis.

Keywords: Graves disease; cytokine; fibroblasts; hyaluronan; ophthalmopathy; orbit.