Monoclonal antibodies to murine IL-1 alpha. Production, characterization, and inhibition of membrane-associated IL-1 activity

We report the production of hamster anti-murine IL-1 alpha mAb and analysis of their specificity and suitability for use in murine IL-1 immunologic and biologic assays. The mAb bound to murine (Mu) rIL-1 alpha 3 but not rMuIL-1 beta as assessed by both direct ELISA and immunoprecipitation. They also inhibited the biologic activity of MuIL-1 alpha but not the activity of rMuIL-1 beta as measured in a T cell co-stimulator assay. These IL-1 alpha specific mAb only partially inhibited the co-stimulator activity found in macrophage culture supernatants but completely inhibited the co-stimulator activity of fixed peritoneal exudate cells. The data indicate that the species of IL-1 associated with murine macrophage membranes shares at least two epitopes with IL-1 alpha and probably represents a product of the IL-1 alpha gene. These reagents will be valuable for quantitative assessment of specific IL-1 proteins on cell surfaces, in culture supernatants, and in cell lysates. They will also be useful both in vitro and in vivo for determining the relative roles of the different IL-1 species in the development of biologic responses.     

IL-1 beta is secreted by activated murine macrophages as biologically inactive precursor

IL-1 alpha and IL-beta are distinct cytokines, produced by activated macrophages. The temporal sequence in the processing and secretion as well as the mechanism(s) by which IL-1 is secreted from the cells remain undefined. Here we have studied the production of IL-1 from murine macrophages after stimulation with LPS or Listeria monocytogenes by two distinct methods: i) immunoprecipitation of radio-labeled IL-1 peptides from culture supernatants, and ii) determination of IL-1 activity by neutralization with monospecific antisera to either form of IL-1. We confirmed that precursor and mature forms of both IL-1 alpha and IL-1 beta can be detected in the culture supernatants after stimulation of the macrophages with 10 to 20 micrograms LPS/ml but, in addition, we report the novel finding that IL-1 beta is exclusively secreted in its unprocessed precursor form after stimulation of the cells with either 0.5 to 1 microgram LPS/ml or with L. monocytogenes. Exposure of the cells to increasing amounts of LPS led to the appearance of a 20-kDa IL-1 beta peptide in the culture supernatants concomitant with the release of a processing activity for the IL-1 beta precursor. These data therefore suggest that, in a first step, IL-1 beta is secreted as an unprocessed precursor protein that in a second, postsecretory step is cleaved by a LPS-inducible protease, thus generating the 20-kDa IL-1 beta peptide. The latter represents the biologically active IL-1 beta inasmuch as the generation of IL-1 beta activity in the culture supernatants strictly correlated with the appearance of the 20-kDa IL-1 beta peptide.     

The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat alpha4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/alpha4 integrin pathway.     

A novel monoclonal antibody against murine IL-2 receptor beta-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells

A mAb specific for the murine IL-2R beta-chain (IL-2R beta) was produced by immunizing a rat with a rat transfectant cell line expressing a large number of cDNA-encoded murine IL-2R beta. The mAb, designated TM-beta 1, is specifically reactive with the murine IL-2R beta cDNA-transfectant but not with the recipient cell, and immunoprecipitates murine IL-2R beta of Mr 75 to 85 kDa. TM-beta 1 mAb completely abolished the high affinity IL-2 binding by inhibiting the ligand binding to IL-2R beta. Murine IL-2R beta was found to be constitutively expressed on a subpopulation of CD8+ T cells and almost all NK1.1+ NK cells in the spleen, whereas TM-beta 1 mAb inhibited the proliferation of spleen cells induced by 1 nM of IL-2. Interestingly, EL-4 cells that express murine IL-2R beta as detected by TM-beta 1 mAb can bind neither human nor murine IL-2 under the intermediate affinity conditions, although cDNA-directed human IL-2R beta expressed in the same EL-4 cells has been previously shown to manifest the intermediate affinity IL-2 binding. These results may imply that functional expression of IL-2R beta is differentially regulated between humans and mice. Finally, our neutralizing anti-IL-2R beta mAb TM-beta 1 will be useful not only for various in vitro studies but also for in vivo studies to directly investigate the possible involvement of the IL-2/IL-2R pathway in the generation and differentiation of T lymphocytes and NK cells.     

IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells

Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.     

The intracellular precursor of IL-1 beta is associated with microtubules in activated U937 cells

Treatment of the human histiocytic leukemia cell line U937 with PMA and LPS results in the induction of expression of IL-1. Rabbit polyclonal antibodies raised against mature rIL-1 have been used to investigate the intracellular location of the IL-1 beta precursor in U937 cells. The pattern of indirect immunofluorescence staining seen with these antibodies overlaps substantially with that seen by using a mAb raised against beta-tubulin. Depolymerization of tubulin by incubation of the cells at 0 degrees C before fixation results in the disruption of the pattern of IL-1 staining. IL-1 beta precursor in extracts of activated U937 cells is shown to co-cycle with exogenously added tubulin through two rounds of in vitro depolymerization/polymerization. In addition, immunoprecipitation of IL-1 from cell extracts at 30 degrees C but not at 0 degrees C results in co-precipitation of tubulin. Thus the IL-1 beta precursor is shown in vivo and in vitro to associate with the microtubules of the cytoskeleton.     

Detection of IL-1 alpha and IL-1 beta in the supernatants of paraformaldehyde-treated human monocytes. Evidence against a membrane form of IL-1

The concept of a membrane form of IL-1 arose from the observation that paraformaldehyde-treated macrophages display IL-1 bioactivity. Thus far, the biochemical characterization of a membrane form of the molecule has not been reported. In a recent publication we demonstrated that murine IL-1 alpha can be detected in the supernatants of paraformaldehyde-treated macrophages. These data indicate that the phenomenon of membrane IL-1 may result from leakage of IL-1 from inadequately fixed cells. In the current report we have extended our studies toward the examination of human IL-1 alpha and IL-1 beta. IL-1 activity can be detected in the supernatants of paraformaldehyde-treated human monocytes. Although anti-IL-1 alpha, but not anti-IL-1 beta, antibodies can efficiently block the IL-1 bioactivity, both IL-1 alpha and IL-1 beta can be found by immunoprecipitation in the supernatants of the fixed monocytes. IL-1 alpha is efficiently processed to the low m.w. form, whereas IL-1 beta remains predominantly as the inactive, precursor molecule. IL-1 is not found in the supernatants of monocyte membrane preparations, demonstrating that the leakage of IL-1 is from an intracellular, rather than membrane-bound source.     

A monoclonal antibody to the IL-1 beta peptide 163-171 blocks adjuvanticity but not pyrogenicity of IL-1 beta in vivo

The synthetic fragment VQGEESNDK, corresponding to the amino acid sequence in position 163-171 of human IL-1 beta, possesses the immunostimulatory but not the pyrogenic activity of the mature IL-1 beta polypeptide in vivo. To assess the relevance of this domain of IL-1 beta for its biologic activities, a mAb was raised against the synthetic peptide 163-171. The mAb Vhp20 could effectively recognize human rIL-1 beta in RIA and immunoblotting. In vivo, the mAb Vhp20 was able to selectively inhibit the immunostimulatory activity of IL-1 beta, but it could not affect the fever-inducing capacity of IL-1 beta. It is proposed that functional domains could be identified in the human IL-1 beta protein and that the fragment in position 163-171 is of major importance for the adjuvant capacity of the entire molecule, but irrelevant to its pyrogenic activity.     

Articular chondrocytes secrete IL-1, express membrane IL-1, and have IL-1 inhibitory activity.

Previous work from our laboratory has shown that rabbit articular chondrocytes, like macrophages, produce reactive oxygen intermediates, express Ia antigen, and can mediate immunologic functions such as antigen presentation and induction of mixed and autologous lymphocyte reactions. We were interested in seeing if these cells could secrete interleukin-1 (IL-1) or express membrane form of IL-1 (mIL-1). Using the standard C3H/HeJ thymocyte assay, neither secreted IL-1 nor mIL-1 activity was detected in untreated or LPS-treated chondrocytes. However, the D10.G4.1 proliferation assay showed that chondrocytes, stimulated with LPS, secrete IL-1 and express the mIL-1 in a dose- and time-dependent manner. The IL-1 activity in LPS-stimulated chondrocyte supernatant and on fixed cells could be inhibited by anti-IL-1 antibodies. Sephadex G-75 chromatography of pooled, concentrated LPS culture supernatant resolved into two peaks of IL-1 activity at 13-17 and at 45-70 kDa, respectively. The bioactivity of chromatographic fractions were similar using both the thymocyte and D10.G4.1 bioassays. Western blot analysis of chondrocyte supernatant detects 17-kDa IL-1 beta; no processed 17-kDa IL-1 alpha was seen but IL-1 alpha-specific reactivity was observed at 64 kDa. Immunoblot analysis of chondrocyte lysates shows that cell-associated IL-1 is IL-1 alpha and is 37 kDa in size. PCR analysis shows the presence of mRNA for IL-1 beta and IL-1 alpha in LPS-treated cells; IL-1 beta mRNA was detected in untreated chondrocytes. The inability to detect IL-1 by the thymocyte assay is due to the presence of a chondrocyte inhibitor of IL-1 that can be demonstrated in cell sonicates, supernatants, and on paraformaldehyde-fixed chondrocytes. Chromatography of LPS-stimulated supernatant showed a peak of IL-1 inhibitory activity at 21-45 kDa. Chondrocytes which secrete IL-1 and express mIL-1 could play a critical role in maintaining chronic inflammation in rheumatoid arthritis. Therefore, the ability of chondrocytes to produce both IL-1 and an inhibitor to IL-1 is important in interpreting the mechanism of cartilage matrix maintenance and degradation.     

Characterization of murine IL-1 beta. Isolation, expression, and purification

One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library. We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli. rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis. Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity. The isoelectric point of rM IL-1 was found to be 8.85. The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein. Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM. Competition binding data suggested that murine and human IL-1 compete for a single class of receptor. Antisera were generated in rabbits against both murine and human IL-1. Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules. These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.     

Failure of IL-1 receptor antagonist and monoclonal anti-IL-1 receptor antibody to inhibit antigen-specific immune responses in vivo.

rIL-1R antagonist (rIL-1ra) and 35F5, a neutralizing mAb, have been shown to inhibit the ability of IL-1 alpha and IL-1 beta to bind to type I but not type II murine receptors. Additionally, IL-1ra and 35F5 inhibit a variety of inflammatory responses in vitro and in vivo. In the present report we have evaluated the activity of human IL-1ra and 35F5 in murine Ag-specific cell-mediated and humoral immune response models. Administration of IL-1ra, either twice daily or as a continuous infusion, did not inhibit the cytolytic T lymphocyte response to allogeneic splenocytes. The CTL response was also not inhibited by daily administration of 35F5. The delayed type hypersensitivity response to oxazolone was similarly refractory to administration of IL-1ra and 35F5. In the humoral immune response models, neither the splenic plaque response to SRBC nor the IgG or IgM response to TNP-keyhole limpet hemocyanin was inhibited by treatment with IL-1ra or 35F5. These data suggest that signaling through the type I IL-1R is not required for these Ag-specific immune responses.     

Differential effect of Cryptococcus neoformans on the production of IL-12p40 and IL-10 by murine macrophages stimulated with lipopolysaccharide and gamma interferon

In the present study, we examined the in vitro effect of Cryptococcus neoformans on the production of interleukin-12 (IL-12) and IL-10 by murine macrophages. At a dose of 1 x 10(5), 1 x 10(6) or 1 x 10(7) ml-1, a highly virulent strain of C. neoformans (strain YC-11) suppressed the production of IL-12p40 by a murine macrophage cell line, J774.1 stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma, while the production of IL-10 was not inhibited, but rather slightly augmented. The suppression of IL-12p40 production did not change by neutralizing anti-IL-10 mAb. A direct contact of C. neoformans with macrophages was largely involved in this inhibitory effect, since placement of a 0.45 micron pore membrane between the organism and macrophages prevented such effect. On the other hand, the culture supernatant of YC-11 did not inhibit macrophage IL-12p40 production when used at a lower dose, which contained an equivalent amount of capsular polysaccharide to that in the supernatant of YC-11 cultured at 1 x 10(5) or 1 x 10(6) ml-1, although it showed a small suppression at higher doses. Our results suggest that C. neoformans may suppress the induction of Th1 responses by inhibiting macrophage IL-12 production predominantly through a direct contact-dependent mechanism and to a lesser extent by a certain soluble factor(s) released from this microorganism.     

Mapping of biologically relevant sites on human IL-1 beta using monoclonal antibodies

mAb have been raised that recognize human IL-1 beta. Using overlapping peptide fragments expressed in yeast and bacteria, we have mapped the regions of the protein to which these antibodies bind. To assess the relevance of the different regions of IL-1 beta for the expression of its biologic activity, the ability of the antibodies to block IL-1 activity was assayed. Antibodies recognizing the regions 133-148 and 251-269 of human IL-1 beta could inhibit the activity of IL-1 beta, but not of IL-1 alpha, in two different biologic assays, the murine thymocyte proliferation and PGE2 release from human fibroblasts. Conversely, antibodies that recognize the region 218-243 have only a moderate inhibitory effect on the IL-1 beta biologic activity in both assays. Finally, an antibody mapping to the region 148-192 did not inhibit IL-1 beta activity either on thymocytes or on fibroblasts. It is suggested that IL-1 beta-induced cell activation involves different regions of the protein and that both N-terminal and C-terminal fragments are involved in the correct functioning of the IL-1 beta molecule.     

Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta.     

IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin.

Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability. The calcium ionophore A23187 and the detergent saponin caused complete release of nonprocessed 35-kDa pro-IL-1 beta and liberation into the extracellular medium of the cytoplasmic marker enzyme LDH and the lysosomal enzyme beta-N-acetylglucosaminidase. Hypotonic lysis resulted in the release of a 20-kDa IL-1 beta species that was distinct from the 17-kDa mature species. Importantly, incubation of the murine macrophages with the potassium/proton ionophore nigericin led to a quantitative conversion of pro-IL-1 beta to a 17-kDa species. The N-terminus of this nigericin-derived product possessed the amino acid sequence expected for mature biologically active IL-1 beta. Monensin, an ionophore similar to nigericin, did not induce release or proteolysis of IL-1 beta. Complete release of mature IL-1 beta required concentrations of nigericin in excess of 2 microM and a minimum of 10 min of treatment. Mature 17-kDa IL-1 beta was observed within the nigericin-treated cells before their lysis. Nigericin's effect was not limited to mouse peritoneal macrophages, inasmuch as the ionophore also induced release and proteolytic maturation of IL-1 beta produced by LPS-stimulated human peripheral blood monocytes. Treatment of macrophages with LPS and nigericin, therefore, results in a unique series of intracellular events that promote formation of mature 17-kDa IL-1 beta.     

IL-1beta-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells

Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a "tolerance-sparing" immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid DC with RAPA elicits the de novo production of IL-1beta by otherwise phenotypically immature DC. Interestingly, IL-1beta production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently identified receptor for IL-33, a cytokine favoring Th2 responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2-/- mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1beta-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1beta, RAPA-DC up-regulate ST2L and become refractory to proinflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.     

Alveolar macrophages differ from blood monocytes in human IL-1 beta release. Quantitation by enzyme-linked immunoassay

Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.     

Janus kinase 3 down-regulates lipopolysaccharide-induced IL-1 beta-converting enzyme activation by autocrine IL-10

ProIL-1 beta processing by IL-1 beta-converting enzyme (ICE) and the subsequent release of mature IL-1 beta are highly regulated events in the monocyte/macrophage response to pathogens. This process occurs in a controlled way through the activation of the constitutively expressed 45-kDa ICE precursor (proICE). To characterize the signaling pathways involved in ICE regulation in human monocytes/macrophages, we analyzed ICE activation in the presence of specific inhibitors of classic signaling pathways. Although LPS-induced ICE activity was not significantly affected by interruption of extracellular signal-regulated kinase, p38 kinase, or phosphoinositol 3-kinase, Janus kinase 3 (JAK3) inhibition produced a significant dose-dependent enhancement of LPS-induced ICE activity. Support for the inhibitory role of JAK3 was shown by the fact that IL-4 (which uses JAK1 and JAK3 signaling) suppressed LPS-induced ICE activity and by the finding that JAK3 knockout macrophages have increased LPS-induced ICE activation. To understand how JAK3 down-regulates LPS-induced ICE activity in monocytes, we hypothesized that JAK3 signaling enhances IL-10 production. In support of this model we show that LPS-induced IL-10 expression was synchronous with ICE deactivation, IL-4 induced the release of IL-10, exogenous IL-10 suppressed LPS-induced ICE activity, a neutralizing IL-10 Ab increased LPS-induced ICE activity, and, finally, JAK3 knockout macrophages displayed significantly reduced LPS-induced IL-10 production. These findings support a model in which JAK3 signaling enhances IL-10 production leading to down-regulation of ICE activation and suppression of IL-1 beta processing and release.