Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

The processing of interleukin-1 beta studied with antibodies raised against synthetic peptides from the precursor N-terminal region

The exact sequence of events during processing of human interleukin-1 beta (IL-1 beta) and the fate of the N-terminal region are unknown. We have used anti-peptide sera specific for the precursor and mature regions of IL-1 beta to study biosynthesis. These were raised against peptides corresponding to amino acids 1-15, 17-32, and 43-54 of the precursor and a peptide corresponding to the C-terminal 33 amino acids of mature human IL-1 beta. Antiserum to the mature region peptide immunoprecipitated the 35-kD precursor from cell lysates and 17-kD mature IL-1 beta and a 31-kD protein from the culture supernatants from radiolabeled human peripheral blood monocytes stimulated with lipopolysaccharide (LPS). Antisera to peptides from the precursor region also immunoprecipitated the 35-kD IL-1 beta precursor but not the 31-kD or 17-kD forms. Of the precursor-specific sera, only antiserum to amino acids 1-15 specifically recognized any other proteins; a peptide of 18 kD and a low molecular weight peptide, both of which accumulated in the medium. The 18-kD protein was not recognized by any of the other antisera and is unlikely to be the N-terminal region of the precursor removed during processing. Pulse-chase experiments indicated that the 31-kD protein could be a processing intermediate and also that it was itself an end product along with full-length precursor. Only 17-kD mature IL-1 beta had biological activity.     

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.     

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.     

Structure-function mapping of interleukin 1 precursors. Cleavage leads to a conformational change in the mature protein

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.     

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).     

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.     

Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells.

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.     

Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export

The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.     

ATP treatment of human monocytes promotes caspase-1 maturation and externalization

Mechanisms that regulate conversion of prointerleukin-1beta (pro-IL-1beta) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1beta, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1beta secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1beta. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1beta and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. On the other hand, anion transport inhibitors and alkylating agents arrest ATP-treated monocytes in a state where membrane latency is maintained. The data provided support the hypothesis that stimulus-coupled IL-1 post-translational processing involves a commitment to cell death.     

A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.     

Juxtacrine effects of IL-1 alpha precursor promote iNOS expression in vascular smooth muscle cells

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.     

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.     

Compartmentalization of mammalian proteins produced in Escherichia coli

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.     

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.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

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.     

Cloning of rabbit interleukin-1 beta: differential evolution of IL-1 alpha and IL-1 beta proteins

We have cloned the rabbit IL-1 beta cDNA, which encodes a 268 amino acid precursor similar in length to other sequenced IL-1 precursors. Comparison of all published IL-1 alpha and IL-1 beta sequences respectively indicates that the IL-1 alpha gene family is evolving faster than the IL-1 beta family, and that the two genes diverged approximately 270 million years ago. Surprisingly, there are differences in the regions preferentially conserved within the two families. The IL-1 alpha family is most conserved at the amino terminus whereas the IL-1 beta family is most conserved in the carboxy-terminal half. This is despite the fact that the carboxy-terminal half encodes the active portion of both molecules and would be expected to adopt a similar beta-sheet structure in IL-1 alpha as in the published X-ray structure of mature IL-1 beta. These findings suggest that differences in the function and properties of the IL-1 alpha and IL-1 beta precursor molecules may have been conserved. These differences may therefore provide an explanation for the existence of two IL-1 molecules.