Some reports have shown that IL-12 production by macrophages can be suppressed by TNF-

Some reports have shown that IL-12 production by macrophages can be suppressed by TNF-.11,30 As TGF- exposure of APCs was reported to increase their TNF- synthesis, we examined the possibility that reduced IL-12 secretion in these APCs is dependent on TNF-. To examine this possibility, we cultured thioglycolate-elicited PECs derived from either wild-type or TNF–deficient mice in the presence of antigen (OVA) and with or without TGF-. IL-12 secretion. Moreover, in the absence of TNF-R2, APCs exposed to TGF- failed to induce tolerance or regulatory cells known to participate in Eluxadoline this tolerance. Also, blocking of TNF-R1 signalling enhanced the ability of the APCs to secrete increased TGF- in response to TGF- exposure. Together our results support an anti-inflammatory role of TNF- in regulation of an immune response by TGF–treated APCs and suggest that TNF-R2 contributes significantly to this role. through regulation of macrophage CSF1R interleukin (IL)-12 production.11 Thus TNF- is also known for its biologically contrasting activity that negatively regulates inflammation. The activities of TNF- are mediated by two receptors, TNF-R1 (p55) and TNF-R2 (p75), which belong to the same family but are functionally distinct. Signalling through TNF-R1, which is considered the primary receptor, mediates the inflammatory effects of TNF-, while TNF-R2-mediated signals contribute to effects such as thymocyte proliferation, TNF–mediated skin necrosis and apoptosis of activated T cells.12C15 However, the two receptors are known to trigger overlapping intracellular signalling events.16,17 Therefore, regulatory or anti-inflammatory effects of TNF- have not yet been attributed to either one of these receptors. Previously it has been reported that transforming growth factor (TGF)–exposed antigen-presenting cells (APCs) acquire the ability to induce a form of peripheral tolerance that produces a deviation in the immune response away from a T helper type 1 (Th1) Eluxadoline response in an antigen-specific manner and results in a suppressed DTH response.18,19 This tolerance resembles that induced by eye-derived APCs that are exposed to TGF- in the ocular environment. The tolerogenic property of Eluxadoline TGF–exposed APCs was demonstrated to be dependent on TNF- as systemic administration of neutralizing anti-TNF- antibodies abrogated the tolerance.20 Although TGF–treated APCs have been shown to secrete increased levels of TNF-, it was not clear if this APC-derived TNF- was necessary to produce a deviation in the inflammatory immune response. Also, the mechanism by which TNF- contributes to suppression of DTH induced by TGF–exposed APCs remains unclear. When we compared the transcriptional programmes of TGF–exposed APCs and conventional APCs we noted increased message for TNF-R2 in the former cells.21 In this study we investigated further whether APC-derived TNF- is relevant in anti-inflammatory effects that lead to DTH suppression and if TNF-R2 plays a role in promoting such tolerogenic properties of these APCs. Our results indicate that indeed APC-derived TNF- is essential for the ability of APCs to produce a deviation in the immune response to achieve DTH suppression as well as to impair their secretion of IL-12, an important cytokine that supports the immune response during DTH. Similarly, TNF-R2 expressed by TGF–treated APCs is critical for their tolerogenic property. In the absence of this receptor, TGF–treated APCs fail to activate effectors with regulatory properties that are associated with their tolerance. Our observations suggest that TNF-R2 contributes significantly to the tolerance-inducing ability of TGF–treated APCs. Materials and methods Mice TNF-R2 (p75) KO, TNF- KO (C57BL/6 background, H-2b), C57BL/6-Tg(TcraTcrb)425Cbn/J [also known as OT-II C these are transgenic mice that express T-cell receptor (TCR) specific for chicken ovalbumin 323C339 in the context of I-Ab] and C57BL/6 (H-2b) mice, 6C8 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME). APCs Thioglycolate-elicited peritoneal exudate cells (PECs) were used as APCs. Cells were cultured in serum-free medium: RPMI-1640 (BioWhittaker, Walkersville, MD) containing 10 mm HEPES, 01 mm non-essential amino acids (NEAA), 1 mm sodium pyruvate, 2mm l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (BioWhittaker), 01% bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO) and ITS+ culture supplement [1 g/ml iron-free transferrin, 10 ng/ml linoleic acid, 03 ng/ml Na2Se and 02 g/ml Fe(NO3)3] (Sigma Chemical Co). PECs were obtained from mice that had received 2 ml of a 3% thioglycolate solution (Sigma Chemical Co) intraperitoneally (i.p.) 3 days earlier. In vitro treatments (2-5 103 cells/mouse), recipients were immunized subcutaneously into the nape of the neck with OVA/complete Freund’s adjuvant (CFA) (50 g). A week later these animals received an intradermal inoculation of OVA (200 g/20 l) into their right ear pinna. The left ear served as an untreated control. The thickness of both ears was measured immediately before and 24 hr after the OVA injection using a micrometer (Mitutoyo 227-101; MTI Corp., Paramus, NJ). The measurements.