Biomedical subjects

S B Mizel

Publications and source records attributed to S B Mizel.

At least 19 recordsLinked to original sources

PubMed2001

Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin.

Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-alpha and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-alpha production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-alpha. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.

Animals↗

PubMed2000

mRNA and protein stability regulate the differential expression of pro- and anti-inflammatory genes in endotoxin-tolerant THP-1 cells.

The products of proinflammatory genes such as interleukin-1beta (IL-1beta) and cyclooxygenase-2 (COX-2) initiate many of the events associated with sepsis. Transcription of these genes is subsequently down-regulated, whereas expression of anti-inflammatory genes such as secretory interleukin-1 receptor antagonist (sIL-1 RA) is maintained. Differential expression is associated with endotoxin tolerance, a cellular phenomenon common to sepsis and characterized by reduced proinflammatory gene expression after repeated exposure to lipopolysaccharide. As a model for endotoxin tolerance, we examined the expression of COX-2 and sIL-1 RA in a human promonocyte cell line, THP-1. We observed a 5-fold decrease in COX-2 protein in endotoxin-tolerant cells relative to control cells. In contrast, sIL-1 RA protein increased 5-fold in control and tolerant cells and remained elevated. Decreased COX-2 production is due to repressed transcription and not enhanced mRNA degradation. In addition, COX-2 protein is turned over rapidly. Transcription of sIL-1 RA is also repressed during tolerance. However, sIL-1 RA mRNA is degraded more slowly than COX-2 mRNA, allowing continued synthesis of sIL-1 RA protein that is very stable. These results indicate that differential expression during endotoxin tolerance occurs by transcriptional repression of COX-2 and by protein and mRNA stabilization of sIL-1 RA.

Cell Line↗

PubMed2000

High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation.

Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176-5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-alpha production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-alpha production (EC(50)) was obtained with 1.5 x 10(-11) M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-alpha-inducing activity of the protein. Although less active than the wild-type protein, a Salmonella flagellin mutant composed of only the central hypervariable region retained substantial TNF-alpha-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-alpha-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

Cells, Cultured↗

PubMed2000

Proteasome-mediated regulation of interleukin-1beta turnover and export in human monocytes.

Interleukin-1beta is a secreted protein that accumulates in the cytosol as an inactive precursor (pIL-1beta) before processing and release of biologically active protein. To understand the impact of this property on IL-1beta production, we examined the intracellular stability of pIL-1beta in lipopolysaccharide (LPS)-stimulated human monocytes. Precursor IL-1beta was degraded with a relatively short half-life of 2.5 h in the promonocytic cell line, THP-1, and in primary monocytes. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) stabilized pIL-1beta levels in THP-1 cells, suggesting that degradation was proteasome-mediated, but this inhibitor was toxic for primary monocytes, causing release of pIL-1beta as well as the cytoplasmic enzyme, lactate dehydrogenase (LDH) into supernatants. In contrast, clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome, caused a dose-dependent stabilization of intracellular pIL-1beta, and this led to a corresponding increase in mIL-1beta and pIL-1beta but not LDH release into culture supernatants. Therefore, by regulating intracellular levels of precursor IL-1beta, the proteasome plays an important and previously unrecognized role in controlling the amount of biologically active IL-1beta that is exported by activated monocytes.

Acrylates↗

PubMed1999

Induction of cytokine synthesis by flagella from gram-negative bacteria may be dependent on the activation or differentiation state of human monocytes.

We have previously demonstrated that salmonellae, but not Escherichia coli or Yersinia enterocolitica, stimulates tumor necrosis factor alpha (TNFalpha) production in the human promonocytic cell line U38. Subsequent analysis revealed that the TNFalpha-inducing activity of salmonellae was associated with flagellin, a major component of flagella from gram-negative bacteria. In the present study, we have explored the basis for the apparent specificity of action of Salmonella flagella on TNFalpha expression in U38 cells and have extended this analysis to normal human peripheral blood mononuclear cells (PBMC). Flagella from the enteropathogenic E. coli strain E2348/69, Y. enterocolitica JB580, and Pseudomonas aeruginosa PAO1, which did not induce significant levels of TNFalpha production in U38 cells, were as potent as Salmonella flagella in terms of TNFalpha and interleukin 1beta activation in PBMC. However, TNFalpha production in U38 cells was greatly enhanced when these cells were stimulated with flagella from E. coli, Y. enterocolitica, and P. aeruginosa in the presence of a costimulant, phorbol 13-myristate acetate. These findings are consistent with the hypothesis that the activation or differentiation state of a monocyte may have a substantial effect on the cell's responsiveness to flagellum stimulation of cytokine synthesis. Furthermore, these results indicate that cytokine induction in monocytes may be a general property of flagella from gram-negative bacteria.

Cell Line↗

PubMed1998

Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line.

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-alpha) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoA transposon mutagenesis was used to identify the TNF-alpha-inducing factor. A mutant Salmonella strain which lacks the ability to induce TNF-alpha was isolated from a TnphoA library. Genetic analysis of this mutant demonstrated that the hns gene has been interrupted by transposon insertion. The hns gene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 is flhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutant Salmonella strains revealed a correlation between the ability to induce TNF-alpha and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutant Salmonella strains to upregulate TNF-alpha, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition, Salmonella FliC can confer the TNF-alpha-inducing phenotype on Escherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-alpha.

Cell Line↗

PubMed1997

Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.

Bacterial Proteins↗

PubMed1995

Differential sensitivity of interleukin-1 alpha and -beta precursor proteins to cleavage by calpain, a calcium-dependent protease.

In view of the observations that the calcium ionophores, A23187 and ionomycin, enhance the processing and secretion of interleukin-1 (IL-1 alpha) and IL-1 beta from macrophages, and IL-1 alpha processing is mediated by calpain, a calcium-dependent protease, we evaluated the possibility that calpain might also play a role in the processing of IL-1 beta. Whereas calpain-containing P388D1 macrophage lysates and purified calpain processed precursor IL-1 alpha to its mature 17-kDa form, precursor IL-1 beta was degraded by both sources of calpain. However, the activation of calpain in P388D1 cells that were transiently transfected with a cDNA expression vector encoding the precursor form of IL-1 beta did not result in the degradation of precursor IL-1 beta, but did result in the processing and secretion of IL-1 alpha, implying that precursor IL-1 beta is protected from calpain degradation in vivo. Furthermore, calpain did not enhance the processing of the IL-1 beta precursor by the IL-1 beta-converting enzyme. These results indicate that calpain is not involved in the processing of precursor IL-1 beta in vitro or in vivo. The IL-1 beta precursor may be protected from calpain degradation by a sequestering mechanism that involves a cytoplasmic factor(s) that reduces the sensitivity of IL-1 beta to attack by calpain or localizes IL-1 beta to a site that precludes any interaction with the protease. Although MDL 28,170, a calpain inhibitor, prevented the ionomycin-induced processing of precursor IL-1 alpha to the mature protein in P388D1 cells, it did not inhibit the ionomycin-induced secretion of the mature IL-1 alpha and -beta proteins expressed in these cells. These results indicate that a calcium-dependent factor other than calpain is involved in the secretion of the mature IL-1 proteins.

Animals↗

PubMed1995

Interleukin-1 beta secretion. A possible multistep process that is regulated in a cell type-specific manner.

In a prior study, we found that the processed form of human interleukin-1 beta (mature IL-1 beta) is secreted to a significantly greater extent than the precursor form of the protein, indicating that the precursor domain acts in some manner to reduce the secretory potential of the protein. In view of this observation, we sought to define the sequence(s) in the IL-1 beta precursor that limit the secretion of the protein as well as the sequences in the mature protein that promote secretion. The P388D1 murine macrophage cell line and the Jurkat human T-cell line were transiently transfected with cDNA expression vectors encoding truncated forms of human precursor IL-1 beta proteins, lacking either the first 76, 94, 99, or 104 amino acids. The removal of increasing numbers of precursor amino acid residues resulted in a graded increase in the secretion of the truncated precursor IL-1 beta proteins from both cell lines. The minimal region of the precursor sequence required to inhibit the optimal secretion of IL-1 beta occurs between amino acids 100 and 104 for P388D1 cells and 95-99 for Jurkat cells. Deletion of the amino acids within these regions increased the secretion level of the truncated proteins to that of mature IL-1 beta. Mutagenesis of the mature IL-1 beta sequence revealed that a region of basic amino acids may play an important role in the optimal secretion of mature IL-1 beta in P388D1 cells, but not in Jurkat cells. Based on the differences in the structural requirements for IL-1 beta secretion in P388D1 and Jurkat cell lines, it is likely that the secretion of IL-1 beta may be subject to multiple levels of regulation that are differentially operative in different cell types.

Amino Acid Sequence↗

PubMed1995

Interleukin 1 activation of the AP-1 transcription complex in murine T cells is regulated at the level of Jun B protein accumulation.

We have examined the regulation of the AP-1 DNA transcription complex during T cell activation in response to interleukin 1 (IL-1) and phorbol ester (TPA) treatment of the IL-1 responsive murine thymoma T cell line, EL4 6.1 C 10. IL-1 synergistically enhances the stimulatory effect of TPA on AP-1-mediated gene expression in this cell line. To elucidate the mechanism(s) by which IL-1 enhances AP-1-mediated gene expression, we examined the effect of IL-1 on the synthesis and turnover of Jun B, the member of the jun gene family that is present in AP-1 complexes in EL4 cells. We found that IL-1 + TPA-treated cells contain significantly higher Jun B protein levels than cells treated with TPA alone. IL-1 promotes the prolonged accumulation of Jun B, whereas the cellular content of Jun B decreases dramatically after 6 hr in cells treated with only TPA. IL-1 enhancement of Jun B protein levels is not the result of a change in the turnover rate of the Jun B protein, but rather results from the maintenance of sufficient jun B mRNA to support continued accumulation of newly synthesized protein. In addition to Jun B, we found that the T cell AP-1 complex contains the Fra-1 protein, a member of the fos gene family. Although IL-1 dramatically increases Jun B accumulation, it does not enhance TPA-induced Fra-1 protein levels in EL4 cells. Thus, the stimulation of AP-1-mediated gene expression by IL-1 in EL4 cells is due to the promotion of Jun B protein accumulation that, in turn, facilitates Jun B heterodimerization with TPA-induced Fra-1 protein, thereby forming an active AP-1 complex.

Animals↗

PubMed1995

Regulation of macrophage activation and human immunodeficiency virus production by invasive Salmonella strains.

Salmonellae possess the ability to adhere to and invade macrophages and in so doing trigger a number of intracellular events that are associated with cellular activation. As an initial approach to defining the mechanisms by which invasive salmonellae alter macrophage function, we have explored the impact of Salmonella infection on the production of human immunodeficiency virus (HIV) in U1 cells, a promonocytic cell line latently infected with the virus. Infection of U1 cells with a pathogenic strain of Salmonella enteritidis resulted in a marked induction of macrophage activation and HIV production. The stimulatory effect of salmonellae was mediated by signals other than lipopolysaccharide. Salmonella mutants with specific defects in invasion or intracellular survival were markedly less effective in the induction of HIV production. In contrast to S. enteritidis, strains of Yersinia enterocolitica, Legionella pneumophila, and Escherichia coli did not induce HIV production. However, all of these bacteria induced comparable levels of gene expression mediated by the HIV long terminal repeat. The results of this study are consistent with the notion that invasive salmonellae possess the ability to activate the macrophage by at least one mechanism that is not shared with several other species of gram-negative bacteria. Furthermore, the expression of this unique property is maximal with Salmonella strains that are not only invasive but also capable of prolonged survival within the macrophage. Our results indicate that the U1 cell line may be a very useful model system with which to examine the biochemical pathways by which internalized salmonellae modulate the activation state of the macrophage.

Escherichia coli↗

PubMed1993

Characterization of the structural requirements and cell type specificity of IL-1 alpha and IL-1 beta secretion.

The murine macrophage cell line, P388D1, was transfected with expression plasmids containing a cDNA for the precursor or mature form of human interleukin (IL)-1 alpha or -1 beta, and the extent of IL-1 protein secretion was analyzed. The secretion of IL-1 beta from cells transfected with a cDNA encoding the mature IL-1 beta was approximately 15-fold greater than from cells transfected with a precursor IL-1 beta cDNA. Although P388D1 cells transfected with precursor and mature IL-1 alpha cDNAs exhibited similar levels of secretion, the precursor IL-1 alpha cDNA-transfected cells secreted only the mature form of IL-1 alpha. Relatively high levels of IL-1 secretion were also observed in cultures of HeLa cells and the murine EL 4 and human Jurkat T cell lines that were transfected with a mature IL-1 alpha or -beta expression plasmid. Secretion was not observed when these cell lines were transfected with precursor IL-1 alpha or -beta expression plasmids. Secretion was also minimal when P388D1 cells were transfected with cDNAs encoding mature IL-1 beta proteins lacking the NH2- or COOH-terminal 20 amino acids. These results indicate that the mature forms of IL-1 proteins are the preferred substrates for secretion and that the maintenance of a specific conformation of these proteins may be required for optimal secretion.

Amino Acid Sequence↗

PubMed1992

Induction of AP-1 transcription factor components during T-cell activation by interleukin 1 and phorbol esters.

We have examined the effect of interleukin 1 (IL-1) and phorbol esters [12-O-tetradecanoylphorbol-13-acetate (TPA)] on the expression of various components of the AP-1 transcription factor complex during T-cell activation. We previously found that a chloramphenicol acetyltransferase reporter gene driven by the collagenase TPA responsive element was expressed upon stimulation of T-cells by TPA and that this expression was enhanced when IL-1 was added as a costimulant; IL-1 alone had no effect on TPA responsive element-chloramphenicol acetyltransferase expression. In this study, we have found that stimulation of T-cells by IL-1 and TPA is accompanied by activation of a subset of immediate early genes that comprise the AP-1 transcription factor complex. junB and fosB were rapidly induced following stimulation with TPA. Although the levels of other fos-related mRNAs were also elevated, their maximal induction was delayed by approximately 5 h. IL-1 alone had little or no effect, but enhanced TPA induced transcription and steady-state levels of these mRNAs. The expression of fos and jun during T-cell activation was accompanied by increased specific binding of JunB, FosB, and fos-related antigen containing complexes to the TPA responsive element. These findings indicate that the synergistic effect of IL-1 and TPA on AP-1 mediated gene expression is due, in part, to the ability of IL-1 to enhance the expression of genes encoding specific AP-1 transcription factor components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Activation of AP-1 by IL-1 and phorbol esters in T cells. Role of protein kinase A and protein phosphatases.

We have examined the regulation of the AP-1 transcription complex in the IL-1-responsive murine T cell thymoma cell line EL-4 6.1 C10. Our results demonstrate that AP-1-mediated gene expression in T cells may be regulated by several signaling pathways and factors, including IL-1, protein kinase C, protein kinase A (PKA), and one or more serine/threonine-specific protein phosphatases. The activation of protein kinase C results in an increase in nuclear AP-1 DNA binding activity, as well as enhanced gene expression. IL-1 and agents that elevate intracellular cAMP levels do not, by themselves, induce AP-1 activation, but they synergize with phorbol esters. IL-1 and forskolin may enhance AP-1 function by different mechanisms, because forskolin enhanced gene expression without producing an increase in nuclear AP-1 DNA binding, whereas IL-1 increased AP-1-binding activity and gene expression. These observations, in conjunction with the lack of a demonstrable effect of IL-1 on cAMP production in EL-4 cells, are consistent with the view that IL-1 enhances AP-1 activation by a pathway that does not directly involve cAMP and PKA. However, the induction of AP-1 activity by IL-1 and phorbol esters is dependent upon the presence of PKA, as evidenced by the loss of AP-1 inducibility in cells transfected with a cDNA encoding protein kinase inhibitor, a specific inhibitor of PKA. The effect of protein kinase inhibitor on AP-1 activation in response to IL-1 and tetradecanoyl-phorbol-13-acetate was reversed in the presence of the serine/threonine protein phosphatase inhibitor okadaic acid. Thus, the level of AP-1 activity in T cells may be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases.

1-Methyl-3-isobutylxanthine↗

PubMed1991

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.

Animals↗

PubMed1991

Expression of interleukin-1 alpha and beta genes by human blood polymorphonuclear leukocytes.

Expression of IL-1 alpha and beta genes was studied in human blood PMN with close monitoring of the effects of contaminating mononuclear leukocytes (MNL). We provide evidence that PMN both transcribe and translate IL-1 alpha and beta genes after stimulation with LPS or IL-1 alpha. A combination of mouse thymocyte comitogen proliferation assay, ELISA, and immunocytochemistry was required to establish that IL-1 alpha and beta synthesis observed in preparations of PMN could not be accounted for by the low level of contaminating MNL. Synthesis of IL-1 beta in PMN exceeded that of IL-1 alpha, but little or no IL-1 alpha was released by PMN. Although increases in IL-1 mRNA after stimulation of PMN and MNL with LPS were similar, PMN were less efficient than MNL in translating IL-1 mRNA. In contrast, PMN and MNL IL-1 alpha and beta mRNAs were translated with equal efficiency in rabbit reticulocyte lysates, suggesting that synthesis of IL-1 in PMN is subject to some form of translational control. We conclude that PMN stimulated with LPS efficiently transcribe but inefficiently translate IL-1 genes relative to MNL. IL-1 beta transcription and translation predominates over that of IL-1 alpha, and IL-1 beta is the predominant IL-1 protein released by PMN. IL-1 can induce its own synthesis in PMN.

Base Sequence↗

PubMed1990

IL-1 secretion by macrophages. Enhancement of IL-1 secretion and processing by calcium ionophores.

In the present study we have demonstrated that the murine IL-1 alpha precursor lacks a cleavable signal sequence and does not undergo cotranslational translocation across microsomal membranes in vitro. Culture supernatants of the murine macrophage cell line, P388D, or from normal peritoneal macrophages collected within 0.5 to 3 h after stimulation contained the 33,000 m.w. precursor as the predominant form of IL-1 alpha. Over an 18-h period, the level of low m.w. IL-1 alpha increased as the secreted precursor was processed by extracellular and/or cell surface-associated proteolytic enzymes. The calcium ionophores A23187 and ionomycin were found to dramatically enhance the release and processing of murine and human IL-1. The rapid release of IL-1 in response to a change in the intracellular level of calcium does not appear to be caused by release of a membrane-bound form of the protein, nor is there evidence that IL-1 is packaged and released from cytoskeletal associated secretory granules. In marked contrast, calcium ionophores do not induce secretion of IL-1 from a nonmacrophage cell line that synthesizes but does not normally secrete IL-1. Our results suggest that activated macrophages possess a novel processing independent, possibly calcium-dependent, mechanism that allows for the release of the precursor forms of IL-1 alpha and IL-1 beta.

Animals↗

PubMed1990

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.

Cell Compartmentation↗