The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll͞IL-1 receptor (TIR) domain, a motif that defines the IL-1͞Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NF B, and A52R potently blocked both IL-1-and TLR4-mediated NF B activation. MyD88 is a TIR domaincontaining adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domaindependent intracellular signaling.

Control of mitogen-activated protein kinase (MAPK)cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH 2 -terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK. Mitogen-activated protein kinase (MAPK)1 cascades control the activity of three sets of effector protein kinases (extracellular signal-regulated protein kinases (ERKs), Jun kinases (JNKs), and p38s). The central element in each MAPK pathway is a module of three protein kinases, MAPKK kinase, MAPKK, and MAPK (1). The three sets of effector MAPK differ in type of activating stimulus: JNKs and p38/HOG-1 primarily respond to stress (e.g. heat shock), and ERKs primarily respond to mitogens. However, a stimulus can activate more than one class of MAPK; the contribution of each pathway is cell typedependent, and MAPK pathways can both synergize and antagonize. This is caused in part by regulatory proteins influencing signaling by a range of mechanisms including scaffolding (e.g. JIP-1, STE5), regulating localization (e.g. Ksr), or recruitment to targets (e.g. 14-3-3 proteins) (2-4). Here we describe a novel family of MAPK control proteins, homologues of fly tribbles.Drosophila tribbles was shown to regulate String activity and hence mitosis during ventral furrow formation (5-8). A canine Trb-2-like protein has been described in the literature as a transiently expressed, mitogen induced, and highly labile cytoplasmic phosphoprotein, but its biological function was not characterized (9, 10). Rat Trb was shown to be rapidly upregulated during neuronal cell apoptosis (11). Recently Trb-3 has been reported to regulate Akt activation in liver by insulin (12) and regulate ATF4 activity (13,14). We show here that Trbs bind to MAPKK and regulate MAPK activation suggesting that Trb function may be broader than reported previously. , FLAG-MEK-1 (16), , and LHRE-TK-luc (18) were described earlier. V12 Ras was a gift of J. Downward. pAP-1 luc, pNFB luc, pFR luc, pFA-CHOP, pFA2-Elk-1, pMEKK-1 pMEK-1, and pMEK-3 were part of the PathDetect system (Stratagene). Quantitative real time-PCR was performed to characterize the expression profile of human tribbles genes by using the Human Rapid-Scan panel (Origene). MRNA levels are expressed as relative units normalized for glyceraldehyde-3-phosphate dehydrogenase expression. MATERIALS AND METHODS PlasmidsCell 85060701) and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin-streptomycin. Raw cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin-streptomycin. Cells (1.5 ϫ ...

Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.