Biomedical subjects

J Saklatvala

Publications and source records attributed to J Saklatvala.

At least 91 records · Page 5Linked to original sources

PubMed1988

Interleukin-1 alpha modulates collagen gene expression in cultured synovial cells.

The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level.

Bucladesine↗

PubMed1988

Studies on the murine interleukin-1 receptor.

The multiple biological actions of interleukin-1 (IL-1) on its diverse range of target tissues is consequent upon interaction of the cytokine with specific, high affinity cell surface receptors. In this report we describe the covalent crosslinking of natural porcine IL-1 and recombinant human IL-1 to intact cells of the murine EL-4 6.1 [NOB-1] thymoma subline, and the solubilization of functional receptors from these cells. Crosslinking studies revealed the existence of two polypeptides, of 100 Kda and 80 Kda, which are involved in IL-1 recognition. Chromatographic studies and ligand-blotting of the soluble receptor demonstrated that the smaller of these two polypeptides, which appears to be a glycoprotein, is capable of interaction with both forms of IL-1.

Animals↗

PubMed1987

Murine interleukin-1 receptor: differences in binding properties between fibroblastic and thymoma cells and evidence for a two-chain receptor model.

The concentration of porcine interleukin-1 beta (pIL1 beta) required to elicit half-maximal IL2 production from NOB-1, a subline of murine thymoma EL4, was 100-fold greater than for p1L alpha. In contrast, similar doses of each type of IL1 stimulated increased lactate production by Balb/C 3T3 fibroblasts. Receptor-bound 125I-IL 1 alpha was displaced with equal efficiency by both unlabelled forms from 3T3 cells, but a 20-fold lower affinity for p1L1 beta was observed using NOB-1. Crosslinking experiments suggested that the IL1 receptors on each line consisted of two polypeptides of 80 and 100 kDa. The results provide the first evidence for a multiple-component IL1 receptor within which IL1 alpha and IL1 beta may bind at different loci, and suggest the receptors may have evolved differently in the two lines.

Animals↗

PubMed1987

Studies on the fate of receptor-bound 125I-interleukin 1 beta in porcine synovial fibroblasts.

Binding of porcine interleukin 1, radiolabeled with Bolton-Hunter reagent (125I IL 1), to monolayers of porcine synovial fibroblasts (PSF) was found to be a temperature-dependent process. The rate of uptake and the amount of cell-associated ligand was higher at 37 degrees C than at 4 degrees C or 19 degrees C, and exceeded the apparent equilibrium binding capacity. The amount of bound 125I IL 1 that was removed by brief treatment with acidic buffers decreased from 80% at 4 degrees C to 35% for PSF incubated at 37 degrees C; this procedure was used to distinguish surface-bound from internalized ligand. In untreated PSF, surface binding was maximal at 1 hr and was maintained for at least 5 hr during which time the internal pool continued to increase. The lysosomotropic agent methylamine (20 mM) decreased surface binding by 50%; monensin (20 microM) decreased the rate and extent of internalization. Cycloheximide (10 micrograms/ml) did not affect ligand uptake, hence, continual expression of surface receptors could not be ascribed to their de novo synthesis. 40% of the radioactivity taken up by PSF during incubation at 37 degrees C subsequently appeared in the culture medium upon prolonged postincubation (5 hr) in the absence of added 125I IL 1: 60% of this fraction was trichloroacetic acid-soluble in untreated cultures, but the extent of degradation was halved by treatment with methylamine or monensin. Direct measurement of the rate of internalization of prebound 125I IL 1 was obtained by monitoring the formation of covalently cross-linked ligand-receptor complexes after warming PSF monolayers to 37 degrees C. By using gel electrophoresis we observed a decrease (t1/2 = 9 to 11 min) in labeling of the major cross-linkable species.

Animals↗

PubMed1987

Interleukin-1 beta and interleukin-1 alpha stimulate the plasminogen activator activity and prostaglandin E2 levels of human synovial cells.

Monocyte-macrophage polypeptides (monokines) cause synovial cells to increase the levels of putative mediators of destruction and inflammation. This interaction may account for some of the properties of rheumatoid pannus. We report here that samples of purified human interleukin-1 beta (IL-1 beta) and recombinant IL-1 alpha stimulate both the plasminogen activator activity and prostaglandin E2 levels of human synovial fibroblast-like cells. The same holds true for purified pig IL-1 (catabolin) and recombinant murine IL-1. The elevation in plasminogen activator activity was inhibited by indomethacin, and this suggests that endogenous prostanoids are important in the IL-1-mediated stimulation of proteinase activity.

Dinoprostone↗

PubMed1987

Interleukin 1: purification and biochemical aspects of its action on cartilage.

Two types of interleukin 1 (IL-1) have been identified and purified from pig leukocytes. These probably correspond to the two IL-1s identified in humans, rabbits, and rats. The IL-1s both cause cartilage and bone resorption and act on connective tissues cells via a common receptor. The cartilage proteoglycan degradation is due to limited proteolysis, but the enzymatic mechanism is not known. Tumor necrosis factor also has been shown to cause similar cartilage degradation and both cytokines inhibit resynthesis of proteoglycan. To date these are the only cytokines known to cause these effects on cartilage.

Animals↗

PubMed1986

Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts.

A monocyte-derived factor with IL-1-like properties has recently been shown to cause resorption of bone in organ culture. We have investigated the action of IL-1 on disaggregated populations of osteoclasts, incubated alone or in the presence of osteoblastic cells, in an attempt to identify the target cell for IL-1 in bone, and to elucidate the mechanism by which IL-1 induces osteoclastic resorption. Osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Under these conditions, the majority of osteoclasts form distinctive excavations in the bone surface within 24 h, the volume of which can be quantified by computer-assisted morphometric and stereophotogrammetic techniques. IL-1 had no effect on bone resorption by osteoclasts alone, but when incubated in the presence of calvarial cells or cloned osteosarcoma cells, it induced a 3.8 (+/- 0.38)-fold increase in osteoclastic bone resorption, with significant enhancement at concentrations of greater than or equal to 30 pg/ml. The osteoblastic populations themselves did not resorb bone. The mechanism by which osteoblastic cells stimulate osteoclasts did not appear to depend upon PG synthesis; nor could we detect a diffusible substance in the medium of stimulated cocultures. These results indicate that IL-1 stimulates bone resorption through a primary action on osteoblasts, which are induced by IL-1 to transmit a short-range signal that stimulates osteoclastic bone resorption.

Animals↗

PubMed1986

A common class of receptors for the two types of porcine interleukin-1 on articular chondrocytes.

IL1 causes chondrocyte-dependent degradation of the proteoglycans of the extracellular matrix of cartilage. The two types of porcine IL1 (pI5 and 8) were radiolabelled with 125I by use of the Bolton and Hunter reagent. Both labelled IL1s were fully active on cartilage. IL1/8 bound specifically to porcine articular chondrocytes. From Scatchard analysis there were calculated to be about 7,000 sites per cell with Kd 2.5 X 10(-10). All the bound radiolabelled ligand was displaceable with unlabelled IL1/8 or IL1/5 at similar concentrations. It was concluded that chondrocytes express a receptor site that reacts with either IL1.

Animals↗

PubMed1986

Serological characterization of acidic (pI 5.5) and neutral (pI 7.1) forms of 17kD and 35kD interleukin-1.

Human interleukin-1 (IL-1) was partially purified into the acidic and neutral 17,000 dalton forms and the acidic and neutral 35,000 dalton forms. Heterologous antiserum raised against purified human IL-1 (neutral, 17,000 daltons) neutralized only the neutral 35,000 dalton and 17,000 dalton isoelectric forms. Conversely, an antiserum raised against purified porcine acidic IL-1 (21,000 daltons) neutralized only the acidic 35,000 and 17,000 dalton forms. These results confirm earlier findings that the acidic and neutral species of IL-1 are not derived from the same gene. In addition, the results suggest that each isoelectric form of the 17,000 dalton IL-1 is probably derived directly from the corresponding 35,000 dalton IL-1.

Antigen-Antibody Complex↗

PubMed1986

Interleukin-1 inhibits the synthesis of collagen by fibroblasts.

Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.

Animals↗

PubMed1985

Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever.

Two forms of interleukin 1 (IL-1) were purified to homogeneity from the culture supernatants of pig buffy coat leukocytes stimulated with concanavalin A. The two proteins had identical Mr of 21,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but one, which had previously been purified as a cartilage-resorbing protein, had pI 5 (IL-1/5) and the other, pI 8.3 (IL-1/8). After initial gel filtration and separation by chromatofocusing IL-1/5 was purified by chromatography on hydroxyapatite and the ion exchangers, Mono S and Mono Q; IL-1/8 was purified by chromatography at pH 4.0 and pH 6.4 on Mono S. Purification was monitored by a cartilage proteoglycan release assay and both proteins had a final specific activity approximately 10(5) times that of the leukocyte culture medium. Medium conditioned by cells from 200 L of blood yielded approximately 15 micrograms of IL-1/5 and 50 micrograms IL-1/8. IL-1/8 augmented mouse thymocyte proliferation, stimulated synovial fibroblasts to produce prostaglandin E and latent collagenase, and was pyrogenic upon intracerebroventricular injection into rabbits. IL-1/5 has previously been shown to possess all these activities. An antiserum to each IL-1 was raised in rabbits. Each antiserum inhibited its respective IL-1 in a cartilage bioassay and stained it upon Western blotting. Neither antiserum inhibited or stained the other IL-1. We conclude that pig leukocytes make two immunologically distinct forms of IL-1 that have identical Mr, demonstrate the same range of biological activity, but differ in isoelectric point.

Animals↗

PubMed1985

Pig interleukin 1 (catabolin) is a potent stimulator of bone resorption in vitro.

A homogeneous form of pig interleukin 1 (catabolin) stimulates the resorption of mouse bones in culture. Concentrations as low as 25 pM are effective, demonstrating that it is more potent than 1,25-dihydroxy-vitamin D3 in our assay system. Catabolin was originally defined by its ability to stimulate glycosaminoglycan release from cartilage in culture and purified from pig mononuclear leucocyte supernatants. It also augments lectin-induced thymocyte proliferation, indicating that it is a form of pig interleukin 1. Bone resorbing factors are synthesized by other cell types, including fibroblasts and osteoblasts; we suggest that such cytokines are important in mediating the action of systemic hormones on bone.

Animals↗

PubMed1984

Pig catabolin is a form of interleukin 1. Cartilage and bone resorb, fibroblasts make prostaglandin and collagenase, and thymocyte proliferation is augmented in response to one protein.

Homogeneous catabolin from pig leucocytes induced proteoglycan breakdown, but not collagen breakdown, in explants of articular cartilage. It augmented lectin-induced proliferation of mouse thymocytes, stimulated production of prostaglandin E2 and collagenase by fibroblasts and chondrocytes, and increased Ca2+ release from mouse calvarial explants, all at concentrations down to 50 pM. In view of these effects it was concluded that pig catabolin is a form of interleukin 1.

Animals↗

PubMed1983

The cartilage-resorbing protein catabolin is made by synovial fibroblasts and its production is increased by phorbol myristate acetate.

Pig synovial fibroblasts were shown to produce a protein that caused live cartilage to resorb its proteoglycan matrix in vitro. Fibroblasts were obtained either from synovial tissue digest or by allowing them to grow out of explants. The population derived from the digests was homogeneous and free of macrophage-like cells after two passages, but was still producing the cartilage-resorbing protein after seven passages. The active protein was found to have Mr 20,000 on gell filtration, and pI 4.8 on isoelectric focussing in polyacrylamide gel. It was indistinguishable from a protein with the same activity from pig mononuclear leucocytes, which has been called catabolin. Production of the protein was increased if the synovial fibroblasts were cultured with the tumour promoter phorbol 12-myristate 13-acetate. Fibroblasts from other sources (joint capsule and peritoneum) also apparently made the protein. The possibility that catabolin is the same as interleukin-1 is discussed: if they are, then the results suggest that fibroblasts can make an interleukin-1-life protein.

Animals↗

PubMed1983

Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro.

Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.

Animals↗

PubMed1983

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro.

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.

Animals↗

PubMed1982

Lymphocytes induce resorption of cartilage by producing catabolin.

Mononuclear cells from pig blood, when cultured in the presence of lectins (concanavalin A or phytohaemagglutinin), release a factor that induces resorption of proteoglycan in explants of live bovine nasal cartilage. The factor had mol.wt. 20000 and pI 4.6-5.0, and was indistinguishable from the cartilage-catabolic protein catabolin isolated from culture medium of explants of pig synovial tissue. Since much of the catabolin from the mononuclear cells arose from the non-adherent population (98% lymphocytes) it was concluded that lymphocytes (and possibly monocytes) make catabolin.

Animals↗

PubMed1981

Characterization of catabolin, the major product of pig synovial tissue that induces resorption of cartilage proteoglycan in vitro.

1. Pig synovium in organ culture produces material which induces living cartilage to resorb its proteoglycan in vitro. 2. The bioassay for this material was to measure glycosaminoglycan released from explants of bovine nasal-septal cartilage cultured for 8 days. The performance of the assay was greatly improved by adding cortisol succinate (0.1mug/ml). This decreased the release of glycosaminoglycan from unstimulated cartilage without inhibiting its response to catabolic factors from the synovium. 3. By using this improved assay it was shown that 90% of the active materials in synovial culture medium were retained by dialysis membrane. 4. An active protein was partially purified from synovial culture medium by (NH(4))(2)SO(4) precipitation, ion-exchange chromatography, gel filtration and preparative isoelectric focusing. 5. This protein, called catabolin, had mol.wt. 17000 and pI4.6. 6. Synovial culture medium concentrated in dialysis tubing was subjected to gel chromatography and found to contain one major active component, which was eluted at the same position as the partially purified catabolin. 7. The synovial culture medium was not inactivated by heating (70 degrees C for 10min), nor were diluted preparations of partially purified catabolin, but concentrated crude preparations were thermolabile. 8. These results suggest that catabolin is the major substance produced by the synovial tissue in culture which induces resorption of proteoglycan of living cartilage in vitro. 9. Other cultured soft connective tissues produced catabolin-like activity. The example of sclera is shown, and production was inhibited by cortisol succinate (0.1mug/ml). 10. It is suggested that catabolin may be a general product of soft connective tissues in culture, and its physiological function may be to induce resorption of connective-tissue matrix after injury.

Animals↗