A synthetic peptide metalloproteinase inhibitor,but not Timp,prevents the breakdown of proteoglycan within articular cartilagein vitro

IL 1-stimulated pig articular cartilage fragments were cultured in the presence and absence of various metalloproteinase inhibitors. Tissue inhibitor of metalloproteinases (TIMP) was unable to stop the release of proteoglycan from the cartilage. Incubation of cartilage with a potent synthetic metalloproteinase inhibitor inhibited the release of proteoglycan in a dose-dependent fashion. The results suggest that low-M _r metalloproteinase inhibitors may have therapeutic potential in limiting connective tissue breakdown in conditions such as rheumatoid arthritis.     

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1α than to Il-1β. Treatment with Il-1α or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1β caused much less severe changes. Some variation in cellular response to Il-1α may result from the heterogeneity previously reported among articular chondrocytes.     

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes.

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.     

Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1beta (IL-1beta) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1beta induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1beta-treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1beta, thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.     

Mechanism of in vitro antitumor effects of interleukin 1 (IL 1)

In vitro studies suggest that purified IL 1 beta derived from normal human peripheral blood monocytes and human myelomonocytic cell line THP-1 cell supernatants was capable of modest augmentation of NK activity of purified LGL and of promoting monocyte cytotoxicity for the human melanoma A375 target cells. In addition, purified IL 1 beta also has direct cytostatic and cytocidal effects for A375 cells. A375 melanoma cells were cloned to obtain a homogeneous population of IL 1 receptor-bearing target cells. Recombinant human IL 1 alpha inhibited the proliferation of these cells within 48-72 h in a dose-dependent manner. Similar doses of recombinant IL 1 alpha exhibited inhibitory effects on the ornithine decarboxylase (ODC) activity of A375 cells by 6-24 h. Putrescine, a nontoxic product of the ODC pathway, could prevent the cytostatic effect of recombinant IL 1 alpha on these tumor target cells. This observation indicates that inhibition of the ODC pathway is causally related to the antiproliferative effect of IL 1 on these tumor cells.     

Effect of cytokines and anti-arthritic drugs on glycosaminoglycan synthesis by bovine articular chondrocytes

The effect of cytokines (interleukin-1, interleukin-1, and tumor necrosis factor ) and several antiarthritic drugs on glycosaminoglycan synthesis and secretion into medium by bovine articular chondrocytes was examined. Sulfated glycosaminoglycans (S-GAG) were measured by a modified 1,9-dimethylmethylene blue (DMB) dye binding assay. Hyaluronate (HA) was measured by an inhibition ELISA based on specific binding to a proteoglycan. All three cytokines caused a dose-dependent decrease in S-GAG production and a dose-dependent increase in HA production. Non-steroidal antiinflammatory drugs (NSAIDs, indomethacin, naproxen, and piroxicam, 1M) could not reverse the effect of IL-1 on inhibiting S-GAG and stimulating HA synthesis. The anti-inflammatory steroid (dexamethasone, 1M) depressed HA synthesis by 50–70% in the absence or presence of IL-1. Dexamethasone depressed S-GAG synthesis by 20–30% in the absence or presence of IL-1. Therefore, none of the tested anti-rheumatic drugs reversed the cytokine mediated changes in glycosaminoglycan synthesis by bovine chondrocytes.     

Effects of hexosamines and omega-3/omega-6 fatty acids on pH regulation by interleukin 1-treated isolated bovine articular chondrocytes

Previous work has shown that interleukin 1 (IL-1) increases the activity of acid extruders in articular chondrocytes, while the H⁺-adenosine triphosphatase (ATPase) inhibitor bafilomycin can prevent aggrecanase-mediated cartilage degradation. The H⁺ transport induced by IL-1 may therefore be required for proteinase activity. In the present study, the effects of hexosamines and fish oils on H⁺-ATPase activity have been characterised for isolated bovine articular chondrocytes. Cells isolated in the presence of IL-1 were acidified, and the fraction of acid extrusion mediated by Na⁺–H⁺ exchange and an H⁺-ATPase were determined using specific inhibitors. Exposure to IL-1 significantly enhanced both components of acid extrusion. Co-incubation with glucosamine or mannosamine attenuated the H⁺-ATPase fraction of efflux. The addition of glucosamine at 9 h after exposure to IL-1—when H⁺-ATPase activation is already apparent—was also able to abolish H⁺-ATPase activity, implying that hexosamines do not exert effects at the level of protein synthesis. Co-incubation with the glucose transport inhibitor phloretin elicited similar effects to the hexosamines, suggesting that modulation of adenosine triphosphate levels may underlie their effects on H⁺-ATPase function. The omega-3 fish oil linolenic acid but not the omega-6 fish oil linoleic acid reduced H⁺-ATPase activity to levels seen in IL-1-untreated cells, although total efflux remained elevated, as a result of an enhanced H⁺ leak. These observations support a model whereby IL-1 stimulates an H⁺-ATPase-dependent system, possibly involved in aggrecanase activation, which appears to be one of the target mechanisms interrupted by dietary supplements reported to have symptom-modifying effects on osteoarthritis.     

Characterization and identification of interleukin 1 receptors on bovine neutrophils.

Interleukin 1 (IL-1) has been shown to modulate various functional activities of neutrophils, presumably by first binding to cell surface IL-1 receptors. In this study, we characterized and identified IL-1 receptors on bovine neutrophils using direct and competitive receptor binding studies and affinity cross-linking analysis. The results of direct binding studies demonstrated that bovine neutrophils bound 125I-labeled bovine IL-1 by a single affinity binding site (Kd = 4.6 nM, 5600 binding sites per cell). Competitive receptor binding studies demonstrated that unlabeled recombinant bovine IL-1 beta, murine IL-1 alpha, and human IL-1 beta competitively blocked neutrophil binding of 125I-labeled bovine IL-1 beta. In contrast, the IL-1 receptor antagonist (IL-1ra) demonstrated no detectable binding to bovine neutrophils as judged by direct and competitive receptor binding assays. Affinity cross-linking of 125I-labeled bovine IL-1 beta to neutrophils was used to identify cell surface IL-1 receptors. Two specific cross-linked products were observed with molecular sizes of 89 kd (a deduced receptor size of 71.5 kd) and more than 200 kd. The latter may indicate a complex of IL-1 receptor-associated signal-transducing components, IL-1 receptors, and IL-1. The presence of 100 nM unlabeled bovine, murine, or human IL-1 during the receptor binding and cross-linking reactions prevented the formation of cross-linked complexes of 125I-labeled bovine IL-1 beta and its receptor. In contrast, the IL-1ra failed to inhibit the formation of cross-linked complexes of 125I-labeled bovine IL-1 beta and its receptor.     

Induction of articular cartilage degradation by recombinant interleukin 1 alpha and 1 beta

The purpose of this study was to compare the effects of human recombinant interleukin 1, alpha and beta, on articular cartilage. The effects of rIL-1 alpha and rIL-1 beta on proteoglycan degradation and synthesis following treatment of bovine articular cartilage in serum-free organ culture were quantified. Purified human IL-1 and both rIL-1 alpha and rIL-1 beta induced a two-fold or greater increase in glycosaminoglycan (GAG) release from cultured articular cartilage. Levels or rIL-1 alpha as low as 15 pM induced increased proteoglycan degradation whereas identical levels of rIL-1 beta did not. Killing of the cartilage cells abolished induced GAG release by all forms of IL-1. Analysis of proteoglycan size following IL-1 treatment showed limited degradation of material released into the culture medium or remaining within cartilage. Both forms of recombinant IL-1 inhibited GAG synthesis when continually present in the culture medium. Actinomycin D and cycloheximide inhibited IL-1 dependent cartilage destruction whereas indomethacin did not.     

Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs

Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol. wt proteins. Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1 alpha and IL-1 beta exist as single genomic copies. In addition, bovine IL-1 beta mRNA is approx. 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.     

Effect of estradiol on interleukin 1 synthesis by macrophages

The effect of estradiol on interleukin 1 (IL-1) synthesis/secretion by rat peritoneal macrophages was investigated. Peritoneal adherent cells (PAC) from adult female rats secreted greater amounts of IL-1 spontaneously than those from age-matched male rats or prepubescent female rats. Ovariectomy led to reduced synthesis of IL-1 by PAC but estradiol replacement therapy of such rats effectively increased IL-1 synthesis. IL-1 secretion was also stimulated when PAC from male rats was incubated with estradiol. A combination of estradiol and LPS in vitro enhanced secretion of IL-1 by PAC even more than estradiol alone. These data provide new evidence suggesting that estradiol may play an important role in regulating synthesis of IL-1 by macrophages.     

Structure and regulation of articular cartilage proteoglycan expression]

Beyond aggrecan, the major proteoglycan present in articular cartilage that confers resistance to compressive load and viscoelasticity to the tissue, other proteoglycan families have been described in cartilage. Among them, decorin, biglycan and fibromodulin which belong to the small leucine-rich proteoglycans family bind to matrix components, specially to collagen fibrils and thus regulate fibrillogenesis in cartilage and matrix integrity. These small proteoglycans can also interact with TGF-beta and modulate its bioavailability and stability. The third family is composed by cell surface proteoglycans as syndecans, glypican-1 and betaglycan. These molecules interact with various components of cell environment (growth factors, proteases, matrix components, etc.) and mediate numerous cell functions. Some modifications of one of these proteoglycan expression occur during degenerative pathologies and may lead to alteration of the functional properties of the tissue as well as variations in growth factor bioavailability. These factors are involved in the attempt of cartilage repair initiated by chondrocytes in the early stages of osteoarthritis.     

In vitro effects and possible clinical relevance of interleukin 3

Cytokines are mainly produced by monocytes and lymphocytes and influence a broad spectrum of physiological processes within the hematopoietic and the immune system. Clinical trials have mainly been initiated with a subgroup of cytokines namely the hematopoietic growth factors. Interleukin 3 (IL-3) was recently introduced into phase I and II trials. This factor was also termed "multipoietin" because of its ability to stimulate in vitro the hematopoietic stem cell and thus all three hematopoietic cell lines. The latest in vivo data, however, demonstrate that IL-3 stimulates leukopoiesis and megakaryopoieis with only marginal effects on erythropoiesis. In large clinical trials the therapeutic effect of this factor is investigated in patients with myelodysplastic syndromes. Further indications will be other primary bone marrow failures and aplasia caused by bone marrow transplantation, aggressive chemotherapy and radiation. Potential applications offered by combinations with other cytokines can hardly be foreseen.     

Age-related changes in the synthesis of matrix macromolecules by bovine articular cartilage

Calf and mature cow articular cartilage was labeled in vitro with [35S]SO4 and [3H]glycine and kinetics of incorporation of both isotopes by cartilage fragments was determined by scintillation spectroscopy. The cartilage fragments were then extracted in sequence with 4M GuHCl (Guanidium chloride) and pepsin. The pepsin digest was adjusted to 1.3 M NaCl and pepsin-solubilized collagen salted out. The 4M GuHCl extract, collagen and pepsin-resistent residue were then freeze-dried. The 4M GuHCl extract was further fractionated by DEAE (Diethylaminoethyl) 52 ion exchange chromatography to obtain protein and PG (Proteoglycan) fractions. The protein fraction was also characterised by SDS-PAGE and PG fraction by Sepharose C1-2B chromatography under associative conditions in the presence and absence of an exogenous HA (Hyaluronic acid). The GAG (Glycosaminoglycan) side chains of the PG samples were analysed by Sephadex G-200 column chromatography and their composition determined by paper chromatography after chondroitinase ABC digestion. Linear incorporation of both isotopes was observed from 1 to 18 hours of incubation and roughly equal amounts of [35S]SO4 counts were found on per cell bases in both cartilages although less [3H]glycine was incorporated by cow chondrocytes. It was also found that calf chondrocytes synthesize much greater proportion of the collagen whereas the cow cells synthesize PGs of smaller hydrodynamic sizes, bearing shorter GAG side chains that are enriched in KS (Keratan sulfate) and Ch-6S (Chondroitin-6 sulfate isomer). A failure of cow 35S-PGs monomers to interact with an exogenous HA in the presence of other extracted components was also demonstrated. The relevance of these findings for the mechanism of cartilage damage in aging and osteoarthritis is discussed.     

Immunorecognition of chondrocytes in articular cartilage activated by synovial interleukin 1.

A polyclonal antiserum which recognizes surface epitopes on IL1-activated pig chondrocytes has been used to immunolocalize chondrocytes responding to IL1 produced during co-culture of pig synovium and articular cartilage. Activation of the chondrocytes by the cytokine was restricted to the articular and subarticular region of the cartilage adjacent to the synovium. Chondrocyte activation was also seen when human rheumatoid synovium was co-cultured with the cartilage. The presence of IL1 in some synovial cells was confirmed by immunolocalization using antisera specific for IL1 alpha and IL1 beta.     

Effect of lipopolysaccharide on proteoglycan synthesis by adult human gingival fibroblasts in vitro.

The effect of lipopolysaccharide preparations from Salmonella enteritidis, Bacteroides gingivalis, and Actinobacillus actinomycetemcomitans on human gingival fibroblasts was studied. Lipopolysaccharide from all sources inhibited fibroblast proliferation in the concentration range of 0.5 to 50 micrograms/ml, with the lipopolysaccharide from A. actinomycetemcomitans having the strongest inhibitory effect. Assessment of the effect of lipopolysaccharide on gingival fibroblast metabolism indicated both total protein and proteoglycan synthesis to be inhibited with increasing concentrations of lipopolysaccharide. As for the antiproliferative effect, lipopolysaccharide from A. actinomycetemcomitans had the greatest inhibitory effect on cell synthetic activity. This inhibitory effect was determined by pulse-chase experiments to be a true depression in synthesis. Furthermore, the effect was independent of lipopolysaccharide-induced changes in cell proliferation and prostaglandin synthesis. This study confirmed the toxic effect of lipopolysaccharide on fibroblasts and, in particular, indicated that various lipopolysaccharide preparations vary in their potency to influence cell proliferation and extracellular matrix synthesis.