Constitutive and PMA-induced interleukin-1 production by the human astrocytoma cell line T24

Astrocytes and glial cells of different species produce interleukin-1 (IL-1) in vitro. In all cases, however, the evidence relied on the detection of IL-1 activity in biological assays. In this report we describe a human astrocytoma cell line (T24) which produces IL-1 constitutively and upon induction with phorbol myristate acetate in vitro. The IL-1 activity was detected in the culture supernatant by a modified assay measuring IL-1-dependent induction of IL-2 production by EL-4 cells. The active molecule had a molecular weight of 17 kDa on gel filtration and an isoelectric point of 5.2. The activity was not neutralized by a goat antibody reacting against pI 7 IL-1. In contrast, rabbit antibody reacting against pI 5 and pI 7 IL-1 neutralized all the IL-1 activity. Cell-associated IL-1 activity was detected in the supernatant of freeze-thawed cell lysates. Serological evidence as well as isoelectric point determination further supported that the predominant form of IL-1 synthesized was of the pI 5 type, and immunoprecipitation of 35S-labeled cell lysate with monospecific polyclonal antibody to IL-1 alpha or IL-1 beta detected only IL-1 alpha precursor. However, Northern blot analysis of astrocytoma cells indicated that mRNA encoding for both IL-1 species were present. These results, therefore, provide unequivocal evidence that human astrocytoma cells synthesize both forms of IL-1 message and yet only activity corresponding to the pI 5 form is detectable inside and outside these cells, suggesting that the inactive pI 7 IL-1 precursor, if made, is not processed to the mature active 17-kDa form.     

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).     

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.     

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.     

Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules.

mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb.     

Phosphorylation of intracellular precursors of human IL-1

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.     

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.     

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.     

Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Possible role of calpain in normal processing of beta-amyloid precursor protein in human platelets

Abnormal proteolytic processing of beta-amyloid precursor protein (APP) underlies the formation of amyloid plaques in aging and Alzheimer's disease. The proteases involved in the process have not been identified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets generated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative alpha-secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal-dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous calpain. Finally, the secretion of APP from intact platelets was inhibited by cell-permeable calpain inhibitors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties.     

Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.     

Lysates of two established human tumor lines contain heparin-binding growth factors related to bovine acidic brain fibroblast growth factor

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNA's were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNA's for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.     

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.     

Effects of human anti-IL-1 alpha autoantibodies on receptor binding and biological activities of IL-1.

Immunoglobulin G (IgG) antibodies to interleukin 1 alpha (IL-1 alpha) are frequently found in the sera of healthy human individuals. The effects of these autoantibodies on receptor binding and biological activities of human IL-1 were tested. Using the murine T-lymphocyte line NOB-1, human thyrocytes and human foreskin fibroblasts, the antibodies competitively inhibited the biological activity of human recombinant IL-1 alpha (rIL-1 alpha). The degree of inhibition correlated with 125I-rIL-1 alpha binding to IgG in different immunoglobulin preparations and in individual sera. These antibodies also neutralized the IL-1 activity of isolated membrane fragments and lysates of human blood monocytes activated by lipopolysaccharide. In contrast, the supernatant IL-1 activity was not affected. Stronger inhibition of biological activity and cell binding of 125I-rIL-1 alpha was obtained with NOB-1 cells than with human thyrocytes. The antibodies failed to interfere with the biological activity of rIL-1 beta. It is concluded that IgG autoantibodies of IL-1 alpha in the sera of healthy humans selectively inhibit the biological activity of the soluble and membrane-associated forms of IL-1 alpha in vitro, and that the degree of biological inhibition afforded by these antibodies depends upon the target cell.     

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.     

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.