Human recombinant interleukin 1-mediated suppression of glycosaminoglycan synthesis in cultured rat costal chondrocytes

Effects of human recombinant interleukin 1 (IL-1) on the synthesis of glycosaminoglycan were examined with cultured rat costal chondrocytes. Incorporation of [35S]sulfate into glycosaminoglycan was strikingly diminished by the addition of IL-1 in a dose- and time- dependent manner. When the cells were cultured with 340 micrograms/ml of IL-1 for 72 hr, the synthesis of glycosaminoglycan was inhibited to 10% of the control. On the other hand, IL-1 had no effect on the morphology and proliferation of the chondrocytes. The suppression of glycosaminoglycan synthesis remained unchanged after the addition of indomethacin, indicating that the effect of IL-1 is independent of the enhanced synthesis of prostaglandins.     

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.     

Normal human neutrophils are a source of a specific interleukin 1 inhibitor

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.     

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).     

Human recombinant interleukin 1 beta has no effect on intracellular calcium or on functional responses of human neutrophils

The effect of human recombinant interleukin 1 beta (rIL 1 beta) on human neutrophils was examined. rIL 1 beta, even at concentrations of 100 ng/ml (100 half-maximal T cell stimulating U/ml) did not change significantly the intracellular free calcium concentration, [Ca++]i, whereas the control stimulus, fmet-leu-phe, significantly elevated [Ca++]i. rIL 1 beta also failed to stimulate production of superoxide, degranulation of lysosomal enzymes, phagocytosis of bacterial particles, chemotaxis, or chemokinesis of human neutrophils. This is substantial evidence that superphysiologic concentrations of interleukin 1 have no direct effect on [Ca++]i, as well as on functional responses of neutrophils.     

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.     

The stimulations of arachidonic acid metabolism by recombinant murine interleukin 1 and tumor promoters or 1-oleoyl-2-acetyl-glycerol are synergistic

Recombinant murine IL 1 stimulated arachidonic acid metabolism by rat liver cells (the C-9 cell line) and squirrel monkey smooth muscle cells, and in the presence of tumor promoters this stimulation was synergistic. In the rat liver cells that had been prelabeled with [3H]arachidonic acid, the release of 6-keto-PGF1 alpha and arachidonic acid also was stimulated by the IL 1, and this release was synergistic in the presence of TPA. 1-Oleoyl-2-acetyl-glycerol (OAG) stimulated prostaglandin production, and IL 1 synergized the prostaglandin production in the presence of OAG. OAG and TPA mimic the endogenous activator of protein kinase C, 1,2-diacylglycerol, and therefore IL 1 may amplify arachidonic acid metabolism during signal transmission processes.     

Effects of human recombinant tumor necrosis factor-alpha and interleukin 1 on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes

We examined effects of human rTNF alpha on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes. The effects of human recombinant IL-1 alpha and IL-1 beta were also given attention. rTNF alpha, as well as rIL-1 alpha and rIL-1 beta, decreased the incorporation of [35S]sulfate into glycosaminoglycan to about 10% of the levels in the control. The half-maximal doses of rTNF alpha, rIL-1 alpha or rIL-1 beta required for the suppression of glycosaminoglycan synthesis (by rTNF alpha, rIL-1 alpha, and rIL-1 beta) were 2 ng/ml, 30 ng/ml, or 5 ng/ml, respectively. rTNF alpha stimulated incorporation of [3H]thymidine in the chondrocytes in a dose- and time-dependent manner. DNA synthesis was increased to about threefold over the control cultures in the presence of 1 microgram/ml rTNF alpha for 72 hr. The stimulatory effect of rTNF alpha on DNA synthesis was observed in both subconfluent and confluent cultures, whereas rIL-1 alpha and rIL-1 beta had no stimulatory activity on DNA synthesis. The addition of rTNF alpha to the cultures of chondrocytes stimulated DNA synthesis, even in medium containing no fetal calf serum. The fetal calf serum acted synergistically with rTNF alpha in increasing DNA synthesis. We propose that both TNF and IL-1 may be involved in inflammatory diseases of cartilage, and that TNF alpha, but not IL-1, may have some physiologic growth factor function for chondrocytes.     

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.     

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.     

UV-irradiated epidermal cells produce a specific inhibitor of interleukin 1 activity

UV irradiation of epidermal cells (EC) in vitro and in vivo leads to an enhanced synthesis of the immunostimulating cytokine interleukin 1 (IL 1). However, UV exposure in vivo also results in local as well as systemic immunosuppression. Therefore, it was tested whether UV-exposed murine EC in culture in addition to IL 1 release an inhibitor of IL 1 activity. Supernatants of UV-irradiated BALB/c EC and of a transformed keratinocyte cell line (Pam 212) were evaluated for their ability to suppress IL 1-mediated thymocyte proliferation. Crude supernatants derived from either UV-exposed or unirradiated EC did not interfere with IL 1 activity. When supernatants were subjected to HPLC gel filtration, fractions eluting at approximately 40 kD significantly blocked the activity of EC-derived IL 1 and murine recombinant IL 1. The release of this inhibitory cytokine (EC-derived contra-IL 1 [EC-contra-IL 1]) was confined to UV-exposed BALB/c or Pam 212 keratinocytes, since no inhibitory activity was detected in supernatants of unirradiated cells. EC-contra-IL 1 also blocked IL 1-induced fibroblast proliferation but did not suppress IL 2 or IL 3 activity. Moreover, EC-contra-IL 1 did not inhibit spontaneous proliferation of a variety of cell lines (Pam 212, P388D1, L 929, EL 4). With the use of chromatofocusing EC-contra-IL 1 exhibited a pI of 8.8, and upon reversed-phase chromatography it eluted within three distinct peaks. Therefore, murine UV-exposed EC, in addition to the production of immunoenhancing cytokines, also may release immunosuppressing mediators and thereby participate in UV-induced immunosuppression. These findings further support the notion that the epidermis may not only be considered as a simple barrier against harmful agents but represents an active element of the immune system.     

Human interleukin 1 is a cytocidal factor for several tumor cell lines

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.     

Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.     

Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme

Time-dependent secretion of immunoreactive-endothelin (IR-ET) from cultured porcine aortic endothelial cells was markedly suppressed by phosphoramidon is due to proteinase inhibitor. Analysis of the culture supernatant with or without phosphoramidon by reverse-phase high performance liquid chromatography confirmed that the suppression of IR-ET secretion by phosphoramidon is due to a decreae in secretion of endothelin-1-like materials. The secretion of the C-terminal fragment (CTF, 22-39)-like materials of big ET-1 was also decreased by phosphoramidon, whereas there was an increased secretion of big ET-1-like materials. These data strongly suggest that phosphoramidon suppresses the secretion of ET-1 from cultured endothelial cells by inhibiting the conversion of big ET-1 to ET-1. It is most likely that phosphoramidon-sensitive metalloproteinase is responsible for the processing of big ET-1 in vascular endothelial cells.     

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.     

Growth-promoting effect of recombinant interleukin 1 and tumor necrosis factor for a human astrocytoma cell line

IL 1 can exhibit dichotomous effects in the sense that it is cytotoxic for certain cells, although growth promoting for other cells. Because IL 1 is growth promoting for astrocytes, but cytotoxic for melanoma cells, the current investigation evaluated the effect of IL 1 upon astrocytomas. The human astrocytoma U373 was found to incorporate [3H]thymidine after exposure to recombinant human IL 1 alpha and IL 1 beta and murine IL 1 alpha. Surprisingly, U373 also incorporated [3H]thymidine after exposure to recombinant TNF. The response of the U373 to IL 1 may be used as a simple and sensitive assay for IL 1.     

Caveolae are a novel pathway for membrane-type 1 matrix metalloproteinase traffic in human endothelial cells

The extracellular matrix (ECM) distinctly modulates membrane type 1-matrix metalloproteinase (MT1-MMP) in human endothelial cells (ECs). Herein, ECM-dependent RhoA activation is shown to regulate MT1-MMP localization and activity as well as clathrin-independent internalization in confluent ECs. In this regard, caveolae are revealed as the major MT1-MMP endocytic pathway in human ECs. Thus, MT1-MMP is present at caveolae with caveolin-1 and both proteins together with alpha v beta 3 integrin colocalize at endothelial motility-associated extensions. Remarkably, caveolae traffic is required for proper MT1-MMP localization, activity, and function in migratory ECs as demonstrated by both treatment with caveolae-disrupting agents or selective targeting caveolin-1 expression by interference RNA. Thus, caveolae-mediated traffic constitutes a novel mechanism for MT1-MMP regulation in ECs during angiogenesis.     

The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes

Deficiency of ABCA1 causes high density lipoprotein deficiency and macrophage foam cell formation in Tangier disease. ABCA1 was also postulated to mediate the secretion of IL-1beta from monocytes and macrophages. We investigated the contribution of ABCA1 to IL-1beta secretion from human monocytes and macrophages of normal donors and Tangier disease patients. Neither an anti-ABCA1 antisense oligonucleotide nor ABCA1 deficiency interfered with LPS-induced secretion of IL-1beta from full blood or freshly isolated monocytes. By contrast, anti-ABCA1 antisense oligonucleotides decreased the LPS-induced secretion of IL-beta from macrophages by 30-50%. The secretion of the precursor pro-IL-1beta and TNFalpha was not inhibited. Compared to normal macrophages, LPS-stimulated Tangier disease macrophages secreted less IL-1beta relative to TNFalpha. Also the spontaneous secretion of IL-1beta by Tangier macrophages was lower than by control cells. We conclude that IL-1beta is secreted from monocytes by an ABCA1-independent pathway and from macrophages by ABCA1-dependent and -independent pathways.