Supplementary Materials Supporting Information supp_106_15_6232__index. by monocytes that were in vitro activated with lipopolysaccharide, intracellular IL-17 expression was induced by in vivo activated monocytes in a TNF– and IL-1-impartial fashion. No role for IL-6 or IL-23 production by Ketanserin manufacturer either in vitro or in vivo activated monocytes was found. Instead, in vivo activated monocytes promoted Th17 responses in a SLRR4A cell-contact dependent manner. We propose that, in humans, newly recruited memory CD4+ T cells can be induced to produce IL-17 in nonlymphoid inflamed tissue after cellCcell interactions with activated monocytes. Our data also suggest that different pathways may be utilized for the generation of Th17 responses in situ depending on the site or route of accessory cell activation. 0.05; Fig. 1). Blocking both IL-1 and TNF- led to a synergistic effect in reducing both the percentage of IL-17+ T cells (50 9%, = 6) and IL-17 secretion (78 7%, = 6) relative to control conditions (Fig. 1 and = 6), anti-TNF- (= 9), anti-IL-6 (= 6), anti-IL-23 (= 3), and anti-IL-1/TNF- (= 6) Abs. *, 0.05 between cultures in absence or presence of LPS; +, 0.05 between LPS treatment in absence or presence of blocking mAbs, using a nonparametric matched pairs test (Wilcoxon). (= 6) and secretion (ELISA, = 6). (= 3). In Vitro Activated Monocytes from RA Patients also Promote Th17 Responses in an IL-1 and TNF–Dependent Manner. Because Th17 cells and IL-17A have been proposed to contribute to RA pathogenesis (15), we examined the induction of monocyte-driven Th17 responses in RA patients (= 9) vs. HC (= 16). The magnitude of the Th17 response observed in RA and HC cocultures was comparable, indicating that CD4+ T cells from RA patients are not intrinsically different in terms of their ability to adopt a Th17 profile in the presence of resting or LPS-stimulated monocytes (Fig. 2shows that in RA patients, like HC, the increase in IL-17+ T cells and IL-17 production caused by the addition of in vitro LPS-activated monocytes could be blocked by adding neutralizing Abs to IL-1 and TNF-. To better characterize these IL-17+ cells, we investigated the coexpression of Th17-associated cytokines. IL-17A+ cells from both HC and RA patients predominantly coexpressed TNF- (HC 53 14 and RA 48 Ketanserin manufacturer 12%), whereas coexpression of IFN- (HC 23 5 and RA 19 4%), IL-21 (HC 19 5 and RA 20 8%), and IL-22 (HC 9 3 and 13 4%) was also observed, albeit at lower levels. Addition of LPS to these cultures led to a global increase in Th17-associated cytokine expression, but did not change the proportion of IL-17+ cells coexpressing TNF-, IFN-, IL-21, or IL-22 (Fig. 2 and = 16) or RA patients (RA, = 9) were isolated and cocultured with anti-CD3 mAb for 3 days in the presence or absence of LPS. Cells were stimulated and IL-17 expression and production measured as described for Fig. 1. (and = 8, RA = 10), TNF- (HC = 4, RA = 7), IL-21 (HC = 3, RA = 4), or IL-22 (HC = 3, RA = 3). Representative dot plots are shown in shows that CD14+ monocytes are present at significantly increased numbers in the RA joints, compared with PB. Also, these SF-derived monocytes were highly activated with significantly Ketanserin manufacturer increased expression of CD40, CD86, HLA-DR, and CD54, compared with their PB counterparts (= 8; Fig. 3= 9), and was mirrored by a significant increase in IL-17 levels in the supernatants of these cultures. Interestingly, the increase in IL-17 was not due to a general increase in T cell.