Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll‐like receptor (TLR) expression. TLR1–TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDCbut not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL‐8, IP‐10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN‐α and IL‐12 including bioactive IL‐12 p70. Previous incubation of PDC with IL‐3 decreased the amount of CpG‐induced IFN‐α and shifted the cytokine response in favor of IL‐12. CpG ODN‐activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN‐stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL‐12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activatedT cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell‐controlled restriction to foreign antigens is maintained.

SummaryThe receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding spedfically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs foUows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.T NF, a polypeptide cytokine, produced primarily by mononuclear phagocytes, plays a key role in the initiation of the inflammatory response. It has a variety of effects, of different, even contrasting nature (1, 2). Evidence that some of these effects can be detrimental to the host have attracted attention to the mechanisms that regulate TNF function. The intracellular signals for the response to TNF are provided by cell surface receptors, of two distinct molecular species, to which TNF binds at high affinity (3, 4). Both receptors for TNF exist also in soluble forms (5-8), apparently derived by proteolytic cleavage from the cell surface forms (9). These soluble TNF receptors (sTNF-Rs) 1 can compete for TNF with the cell surface receptors and thus block its activity (5-8). It was therefore suggested that they function as physiological attenuators of the activity of TNF, safeguarding against its potentially harmful effects. We report here that the sTNF-Rs affect TNF function also by stabilizing its activity, most likely by preventing dissociation of the homotrimeric TNF molecules (10-13) to inactive monomers. Materials and Methods TNF and Its Soluble Receptors.Recombinant human TNF-c~ (TNF; 6 x 107 U/mg of protein), produced by Genentech Co.1 Abbreviations used in this paper: B-CLL, B-chronic lymphocytic leukemia; sTNF-R, soluble tumor necrosis factor receptor.(San Francisco, CA), was kindly provided by Dr. G. Adolf, of the Boehringer Institute (Vienna, Austria). Radiolabeling of the TNF with mI was performed by the chloramine-T method, as previously described (14). Native TNF was produced by stimulation of human peripheral mononuclear phagocytes with bacterial lipopolysaccharide for 6 h, as described elsewhere (15), and used without further purification. The soluble forms of the type I (p55) and type II (p75) TNF receptors (sTNF-RI and sTNF-RII, previously called TBPI and TBPII [81) were isolated from normal urine by ligand (TNF) affinity purification followed by reversed-phase HPLC, as described before (8).Cells. ...

The oncogenic latent membrane protein 1 (LMP1) of the Epstein-Barr virus recruits tumor necrosis factor-receptor (TNFR)-associated factors (TRAFs), the TNFR-associated death domain protein (TRADD) and JAK3 to induce intracellular signaling pathways. LMP1 serves as the prototype of a TRADD-binding receptor that transforms cells but does not induce apoptosis. Here we show that TRAF6 critically mediates LMP1 signaling to p38 mitogen-activated protein kinase (MAPK) via a MAPK kinase 6-dependent pathway. In addition, NF-kappaB but not c-Jun N-terminal kinase 1 (JNK1) induction by LMP1 involves TRAF6. The PxQxT motif of the LMP1 C-terminal activator region 1 (CTAR1) and tyrosine 384 of CTAR2 together are essential for full p38 MAPK activation and for TRAF6 recruitment to the LMP1 signaling complex. Dominant-negative TRADD blocks p38 MAPK activation by LMP1. The data suggest that entry of TRAF6 into the LMP1 complex is mediated by TRADD and TRAF2. In TRAF6-knockout fibroblasts, significant induction of p38 MAPK by LMP1 is dependent on the ectopic expression of TRAF6. We describe a novel role of TRAF6 as an essential signaling mediator of a transforming oncogene, downstream of TRADD and TRAF2.

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Objective. Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis.Methods. We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF)