Evidence against the existence of a membrane form of murine IL-1 alpha

Previous studies have demonstrated that paraformaldehyde-treated macrophages possess IL-1 alpha activity in a variety of bioassay systems. However, no definitive biochemical data in support of the membrane IL-1 alpha concept has been reported. The purpose of the present study was to determine if the biologic activity associated with treated cells is due to a membrane form of IL-1 alpha or alternatively, to the leakage of IL-1 alpha. If the former case was true, then the exposed membrane IL-1 alpha should bind anti-IL-1 alpha antibodies or be cleaved by mild trypsin treatment. In both instances, IL-1 alpha activity should be lost when measured in a subsequent IL-1 bioassay. Our results indicate that pulsing paraformaldehyde-treated normal or cell line macrophages with anti-IL-1 alpha antibodies or treating the cells with trypsin did not affect the ability of the treated cells to function in a murine thymocyte proliferation assay. Furthermore, the standard short term treatment of cells with paraformaldehyde (15 min) did not prevent the leakage of IL-1 alpha from the cells or the processing of the precursor forms of the protein. When cells were treated with paraformaldehyde for 2 h, they no longer released IL-1 alpha or possessed thymocyte stimulatory activity. We also found that short term glutaraldehyde treatment of macrophages completely blocked the release of IL-1 alpha from cells as well as the appearance of cell-associated IL-1 alpha activity. Our results support the conclusion that the stimulatory activity of paraformaldehyde-treated macrophages is not due to a membrane form of IL-1 alpha but is, in fact, due to the continuous release of IL-1 alpha from the cells.     

Involvement of a calpain-like protease in the processing of the murine interleukin 1 alpha precursor

Interleukin (IL) 1 alpha is synthesized as a 33-kDa precursor that is enzymatically cleaved to the 15-17-kDa forms that are found in the culture supernatants of activated macrophages. We have explored the possibility that calcium might enhance IL-1 processing and secretion via the stimulation of a calcium-dependent protease. We have found that lysates prepared from human peripheral blood monocytes, the human histiocytic lymphoma cell line U937, and the murine macrophage cell line P388D1 contain a calcium-dependent IL-1 alpha processing activity that cleaves the IL-1 alpha precursor to its mature form. Although NIH 3T3 mouse fibroblast cell lysates also contain IL-1 processing activity, lysates from the murine thymoma EL-4, the human epidermoid cell line HEp-2, and the human foreskin fibroblast line FS-4 lack this activity. IL-1 processing activity is inhibited by leupeptin and exhibits a molecular mass of 80-110 kDa. The processing activity is also inhibited by a monoclonal antibody directed against calpain type I. These results indicate that the processing of the IL-1 alpha precursor is mediated, at least in part, by a member of the calpain family of proteases. Mixing experiments revealed that lysates from EL-4 or HEp-2 cells contain an inhibitor(s) of the calpain-like protease in macrophage extracts. It is, therefore, likely that many non-macrophage cell types are unable to process the IL-1 alpha precursor because the calpain present in these cells is only weakly active due to the presence of a specific inhibitor(s) such as calpastatin.     

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.     

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.     

Studies on the synthesis and secretion of interleukin 1. I. A 33,000 molecular weight precursor for interleukin 1

Previous studies have suggested that murine interleukin 1 (IL 1) may be synthesized as a high m.w. precursor. Using specific antibodies against murine IL 1, we have analyzed the primary form of IL 1 synthesized by normal peritoneal macrophages and P388D1 cell line macrophages, and in vitro using poly (A)+ RNA from stimulated normal and cell line macrophages. In all cases, the labeled protein immunoprecipitated with the anti-IL 1 antibodies exhibited a m.w. of 33,000 on SDS gels. This 33,000 m.w. protein was not an aggregate of low m.w. IL 1. Addition of excess purified low m.w. IL 1 completely blocked the immunoprecipitation of the 33,000 m.w. protein. When cells were pulsed with [35S]methionine for 1 to 5 hr and then incubated in medium containing unlabeled methionine for 19 hr, labeled low m.w. IL 1 was detected in the culture fluid. If cells were pulsed with [35S]methionine to label the 33,000 m.w. protein and then incubated in the presence of a maximally effective concentration of the protein synthesis inhibitor, cycloheximide, the low m.w. IL 1 was still found in the culture fluid. Our results indicate that IL 1 is synthesized as a 33,000 m.w. precursor that is converted to the low m.w. form that is found in the culture fluid of stimulated murine macrophages.     

Membrane IL-1: IL-1 alpha precursor binds to the plasma membrane via a lectin-like interaction

Although biologically active IL-1 associated with plasma membrane has been demonstrated in both mouse and man, a biochemical mechanism for membrane anchoring has not been described. We have analyzed the nature of membrane IL-1 in stimulated murine macrophages. We show that it consists of an IL-1 alpha precursor bound to the plasma membrane via a lectin-like interaction that is specifically dissociated with D-mannose. The dissociated IL-1 was detected as both a biological activity and, by immunoprecipitation and SDS-PAGE, as a 33 kDa IL-1 alpha precursor. Treatment of macrophages with D-mannose before fixation depleted detectable IL-1 biological activity associated with the membrane. Pro-IL-1 alpha was glycosylated in these cells, as shown by incorporation of D-[14C]mannose; thus it is likely that a cell surface lectin binds pro-IL-1 via these carbohydrate residues. In addition to anchoring IL-1 precursor to the plasma membrane, this lectin-like interaction may also be important in the overall regulation of IL-1 release.     

A potent adjuvant monophosphoryl lipid A triggers various immune responses, but not secretion of IL-1beta or activation of caspase-1

Lipid A, the membrane anchor portion of LPS, is responsible for the endotoxin activity of LPS and induces many inflammatory responses in macrophages. Monophosphoryl lipid A (MPL), a lipid A derivative lacking a phosphate residue, induces potent immune responses with low toxicity. To elucidate the mechanism underlying the low toxicity of MPL, we examined the effects of MPL on the secretion of proinflammatory cytokines by mouse peritoneal macrophages, a murine macrophage-like cell line (RAW 264.7), and a human macrophage-like cell line (THP-1). MPL enhanced the secretion of TNF-alpha, but not that of IL-1beta, whereas Escherichia coli-type lipid A (natural source-derived and chemically synthesized lipid A) enhanced the secretion of both cytokines. Although MPL enhanced the levels of IL-1beta mRNA and IL-1beta precursor protein to levels similar to those induced by lipid A, IL-1beta precursor processing in MPL-treated cells was much lower than that in E. coli-type lipid A-treated ones. Moreover, MPL, unlike E. coli-type lipid A, failed to induce activation of caspase-1, which catalyzes IL-1beta precursor processing. These results suggest that an immune response without activation of caspase-1 or secretion of IL-1beta results in the low toxicity of this adjuvant.     

Ca2+ stores and Ca2+ entry differentially contribute to the release of IL-1 beta and IL-1 alpha from murine macrophages

Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1 alpha and IL-1 beta) present in the cytosol of activated monocytes/macrophages. IL-1 beta is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1 beta and IL-1 alpha via a common pathway, dependent upon the release of Ca(2+) from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca(2+) alone do not promote IL-1 secretion because a concomitant efflux of K(+) through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1 alpha, independent of P2X7 receptor activation, but dependent upon Ca(2+) influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation.     

Soluble murine IL-1 receptor type I induces release of constitutive IL-1 alpha

IL-1 alpha and IL-1 beta are proinflammatory cytokines involved in the pathogenesis of many infectious and noninfectious inflammatory diseases. To reduce IL-1 toxicity, extracellular domains of the soluble (s) IL-1R are shed from cell membranes and prevent triggering of cell-bound receptors. We investigated to what extent murine sIL-1RI can neutralize the IL-1 produced by LPS-stimulated macrophages. When mouse peritoneal macrophages were incubated with LPS, addition of sIL-1RI significantly inhibited the bioactivity of IL-1. Stimulation of cells with sIL-1RI alone induced no bioactive IL-1. When immunoreactive cytokine concentrations were measured with specific radioimmunoassays, sIL-1RI alone appeared to induce a significant release of IL-1 alpha in a concentration-dependent manner. This effect was independent of new protein synthesis. The production of IL-1 beta or TNF-alpha was not influenced by sIL-1RI. There was no interference of sIL-1RI with the IL-1 alpha radioimmunoassay. In mice, an i.v. injection of sIL-RI alone induced a rapid release of IL-1 alpha, but not of TNF-alpha or IL-1 beta. Treatment of mice with sIL-1RI improved the survival during a lethal infection with Candida albicans. In conclusion, sIL-1RI induces a rapid release of IL-1 alpha from cells, as well as into the systemic circulation. Although this IL-1 alpha may be inactivated in circulation by the same sIL-1RI, this phenomenon probably has immunostimulatory effects at local levels where the sIL-1RI-induced IL-1 alpha acts in a paracrine or autocrine manner.     

Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Partial characterisation of the high and low molecular weight forms of P388D1-derived interleukin 1

The murine macrophage-derived cell line P388D₁ secretes the lymphokine inter-leukin 1 (IL-1) when stimulated by a variety of agents. When stimulated by bacterial lipopolysaccharide (LPS) the cells release IL-1 in both high and low molecular weight (m.w.) forms. The proportion of high m.w. IL-1 is reduced when IL-1-containing supernatants are concentrated by ammonium sulfate precipitation subsequent to hollow-fiber filtration. The high m.w. form can be converted to the low m.w. form by proteolysis, reduction and alkylation, or chromatography in a dissociating solvent. The low m.w. form remains as such, even when reconcentrated in fetal calf serum-containing medium. The high m.w. form thus likely consists of a complex between low m.w. IL-1 and another protein secreted by the P388D₁ cell line.     

Inhibition of apoptosis by calcium ionophores in IL-3-dependent bone marrow cells is dependent upon production of IL-4+.

Calcium ionophores inhibit apoptosis in the IL-3-dependent cell line BAF3 and maintain the cells in a viable noncycling state. In this report, an identical effect of ionophore was also demonstrated on the multipotent IL-3-dependent progenitor cell line FDCP-MIX and on the primary IL-3-dependent cell population that could be cultured from murine bone marrow. Inhibition of apoptosis required extracellular calcium and could be blocked by cyclosporin A. Nuclei from IL-3-dependent cells were found to lack a calcium-activatable nuclease that degrades chromatin in the linker region between nucleosomes, unlike the nuclei of lymphoid cells. The mechanism of action of calcium ionophore could be divided into two distinct steps. First, ionophore induced the production of a survival factor that stimulated DNA synthesis and was identified as IL-4. Second, ionophore inhibited the cell cycle of the various IL-3-dependent cells. IL-4 production could be inhibited by cyclosporin A and required extracellular calcium, whereas cell cycle arrest did not. This implied that factor production was the step that was necessary for inhibition of apoptosis and maintenance of cell viability. This was confirmed by the use of an anti-IL-4R antibody, which blocked the inhibition of apoptosis induced by calcium ionophores.     

Processing of interleukin-1 in cells of monocytic lineage is differentiation-dependent.

The interleukin-1 (IL-1) alpha and beta precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL-1 alpha and beta, we have investigated by immunoprecipitation the synthesis, their release and processing in a promyeloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes. We show that U937 monocytic cells, on induction with a tumor-promoting agent, synthesize and release into the culture medium proIL-1 beta but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL-1 alpha and beta without detectable processing of either cytokine. Processing and release of IL-1 alpha and beta by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL-1 alpha and beta show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL-1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17-kDa IL-1 alpha and beta.     

The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta

We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).     

The activation of human fibroblast prostaglandin E production by interleukin 1

We have examined the induction of prostaglandin E2 (PGE2) release from fibroblasts by human interleukin 1 (IL-1). A number of fibroblast cell lines appear to respond to IL-1 in a fashion similar to that seen with synovial fibroblast cultures. Using the Gin-1 primary fibroblast cell line, the earliest time where a significant increase in PGE2 release can be detected is 2 hr. Thereafter PGE2 appears to increase dramatically, with levels after 5 hr increased over 50-fold above baseline. IL-1 appears to directly induce the increase in PGE2 since removal of other proteins from culture medium does not affect induction. PGE2 induction by IL-1 also does not require cell proliferation. The induction appears to involve the synthesis of new protein since the enhanced release can be completely blocked by addition of actinomycin D or cycloheximide. Arachidonic acid mobilization in cells does not appear to be altered following IL-1 addition. However, the ability to convert arachidonic acid to PGE2 is increased following 5 hr of culture with IL-1. While increasing the release of PGE2, the addition of phorbol esters, alone or in combination with calcium ionophores, does not mimic the protein synthesis-dependent increase seen with IL-1. Taken together these results suggest that IL-1 induction of fibroblast PGE2 involves the synthesis of new protein or proteins involved in the conversion of free arachidonic acid to PGE2.     

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Generation of monoclonal antibodies to murine IL-1 beta and demonstration of IL-1 in vivo

The role of murine IL-1 beta in vitro and in vivo has not been defined. We describe here the production of neutralizing and immunoprecipitating mAb and polyclonal antibodies specific for murine IL-1 beta and their application to a characterization of the murine IL-1 beta protein. Immunization of either hamsters or rabbits with the recombinant mature form of murine IL-1 beta emulsified in CFA elicited antisera and hamster mAb that only recognized denatured IL-1 beta. In contrast, immunization with rIL-1 beta adsorbed to alum resulted in the generation of neutralizing and immunoprecipitating rabbit and hamster antisera and hamster mAb. All of the mAb recognize both the pro-form of IL-1 beta and the mature bioactive form produced by cultures of murine peritoneal macrophages. Using these antibodies, we demonstrate that approximately half of the IL-1 activity present in supernatants of LPS-treated cultured mouse macrophages is composed of IL-1 beta. Additionally, IL-1 beta as well as IL-1 alpha can be detected in the plasma of LPS-treated mice. These studies, therefore, demonstrate the production of IL-1 beta both in vitro and in vivo.     

Synergistic activation of granulocyte-macrophage colony-stimulating factor production by IL-1 and IL-2 in murine Th1 cells

Recent reports indicate that murine CD4+ Th1-type cloned T cells are insensitive to IL-1 because specific IL-1R are not detected on these cells and IL-1 does not modulate proliferative responses. However, we have determined that Th1 clones can respond to IL-1, because they function synergistically with IL-2 to induce granulocyte-macrophage-CSF secretion. This response to IL-1 plus IL-2 could be induced by IL-1 alpha or IL-1 beta and by membrane-bound IL-1 on macrophages. However, IL-1R could not be detected, and Th1 cells did not respond to IL-4 in the presence or absence of IL-1, as measured by either proliferation or granulocyte-macrophage-CSF production. Therefore, IL-1 functioned as a cofactor in Th1 cells stimulated with IL-2, but not with IL-4. A possible mechanism whereby IL-1 activates Th1 cells is discussed.