These actions on neuronal guidance are mediated through Ca2+ dependent mechanisms (32). populace (GD-OF). In contrast to fibrocytes, GD-OF express vanishingly low levels of MHC-2, Tg, TSHR, and AIRE. Further, the amplitude of IL-6 induction by TSH in GD-OF is usually substantially lower. The molecular basis for this divergence between fibrocytes and CD34+ OF remains uncertain. Here we report that Slit2, an axon guidance glycoprotein, is usually constitutively expressed by the CD34? OF subset of ADP GD-OF. Culture medium conditioned by incubating with GD-OF and CD34? ADP OF (CM) substantially reduces levels of MHC-2, Tg, TSHR, and AIRE in fibrocytes. Expression can be restored by specifically depleting CM of Slit2. The effects of CD34? OF CM are mimicked by rhSlit2. TSH induces Slit2 levels in GD-OF by enhancing both Slit2 gene transcription and mRNA stability. These findings suggest that Slit2 represents a TSH-inducible factor within the TAO orbit that can modulate the inflammatory phenotype of CD34+ OF and therefore may determine the activity and severity of the disease. Introduction Fibrocytes derived from the monocyte lineage appear to play critical functions in tissue remodeling and wound healing (1C3). They display ADP a CD45+CD34+CXCR4+Col I+ phenotype, constitutively express high levels of major histocompatibility complex Class II (MHC-2), and several co-activation molecules through which they engage in complex bidirectional interactions with T cells (4, 5). They produce a diverse array of immunologically impactful molecules including cytokines (2, 6). Fibrocyte differentiation depends on T cells (4). Unexpectedly, fibrocytes express several autoantigens (7) including thyroid-specific proteins such as thyroglobulin (Tg), thyrotropin receptor (TSHR), sodium-iodide symporter, and thyroperoxidase (7C10). Expression of these thyroid proteins in fibrocytes is dependent on autoimmune regulator protein (AIRE) (10). Fibrocytes have recently been implicated in the pathogenesis of several autoimmune diseases, including rheumatoid arthritis (11), type I diabetes mellitus (12), and Graves disease (GD) (13, 14). In GD, orbital connective tissues surrounding the eye become infiltrated with T and B cells, mast cells and fibrocytes which appear to participate in the development of thyroid-associated ophthalmopathy (TAO). TAO remains a vexing, disfiguring, and potentially blinding condition without effective and safe medical therapies (15). A critical question remaining to be answered is why some patients with GD develop severe eye disease while others do not manifest any ocular abnormalities. By virtue of their display FHF4 of functional TSHR, fibrocytes can produce proinflammatory cytokines in response to both TSH and the pathogenic, TSHR-activating immunoglobulins (TSI) that drive hyperthyroidism of GD (13, 16, 17). Once in the TAO orbit, fibrocytes transition to CD34+ orbital fibroblasts (CD34+OF). There they coexist with residential CD34? OF in a mixed fibroblast populace (GD-OF). Yet GD-OF fail to express the high Tg and TSHR levels found in fibrocytes cultured from the circulation (8, 10). Vanishingly little is currently known about the factors generated within the human orbit that determine the behavior of infiltrating cells recruited from the bone marrow. We proffer that CD34? OF exert a modulating influence(s) on CD34+ OF, resulting in the dramatically lower expression levels of MHC-2, thyroid proteins, and AIRE, a phenomenon that has remained unexplained. Fibrocytes may represent a critical link between the autoimmunity occurring within the thyroid and orbit in GD yet the factor(s) regulating expression of thyroid-related proteins by these cells once they infiltrate the orbit remains unidentified. Slit2, an axonal guidance/repellent glycoprotein, constrains neuron migration and enforces midline integrity in the developing brain (18, 19). Three different mammalian orthologs of Slit have been identified (18). Members of the Slit family negatively influence leukocyte chemotaxis (20, 21) and mediate chemo-repulsion of diabetogenic T cells (22). The Slit2 pathway has been implicated in the regulation of angiogenesis and appears to alter the clinical behavior of human cancers (23). Its ADP expression correlates with tumor outcome (24). Slit2 acts through its cognate receptor, ROBO1, a surface transmembrane glycoprotein displayed on target cells (25). Recent evidence suggests that the Slit2/ROBO1 pathway plays a regulatory role in fibrocyte differentiation into lung fibroblasts and thus helps determine tissue remodeling in fibrotic lesions (26). Healthy lung tissues secrete Slit2 and attenuate the differentiation of fibrocytes while Slit2 expression is diminished in tissue surrounding lung fibrosis (26). Here.