An intracellular monocyte derived protein possessing interleukin 1 (IL-1) activity has been compared with the secreted from of IL-1. Our results indicated that this intracellular IL-1 had an apparent size greater than that of extracellular IL-1. Data are presented to show that a rabbit heterotypic antiserum prepared against epitopes on the secreted form of human IL-1 blocked the biologic activity of intracellular IL-1 in a dose dependent manner. Intracellular IL-1 had a mass of 30-40 kDa as determined by gel filtration using sephacryl-200 gel.
Interleukin-1 (IL-1) is a polypeptide that is produced during infection, injury, or immunologic challenge. There are two molecular forms, IL-1-beta and IL-1-alpha, and despite only a 26% amino acid homology, both forms induce a wide variety of biological changes. These include systemic effects such as fever, sleep, ACTH release, and increased sodium excretion. In vitro, IL-1 activates T and B lymphocytes and induces a variety of lymphokines, interferons, and other cytokines, particularly tumor necrosis factor, for the induction of inflammatory changes such as prostaglandin synthesis, activation of endothelial cells, and bone resorption. Despite the broad range of tissue targets, IL-1 receptors have been described primarily on lymphocyte lines and fibroblasts. A feature of these studies is the low numbers of receptor sites and a relatively low-affinity binding. There is also evidence for the existence of a second class of high-affinity receptors. Molecular weights of IL-1 receptors vary with the cell source; these have been demonstrated with molecular weight of 80, 70, and 60 kDa. In general, there is a discrepancy between receptor number and affinity and biological responses to IL-1. One explanation for the mechanism of action of IL-1, particularly on T cells, is the requirement for cross-linking of two membrane proteins. In some cells, the binding of IL-1 to putative receptors may be fast and transient, accounting for activation of intracellular responses without a measureable biological response (such as increased DNA synthesis). IL-1 activation of cells is an important biological response, and its mechanism remains in an unexplored domain.
In healthy cartilage, there is a balance between anabolic and catabolic activities of chondrocytes that maintains the functional integrity of the extracellular matrix. However, during osteoarthritis (OA), chondrocytes become more catabolically active and express increased levels of matrix degrading enzymes, such as MMPs and ADAMTSs. Increased MMP and ADAMTS activity results in a net loss of the extracellular matrix and therefore leads to cartilage damage. Previously, we found that the anti-inflammatory cytokine Interleukin 37 (IL37) is able to counter-regulate the catabolic status of chondrocytes by reducing the IL1β-driven expression of pro-inflammatory cytokines and catabolic enzymes.
Objectives
The goal of this study was to investigate, in human OA cartilage explants, the effect of IL37 on sulfated glycosaminoglycans (GAG) content and synthesis of extracellular matrix molecules and cartilage degrading enzymes to investigate its therapeutic potential in OA.
Methods
Human cartilage was obtained from eighteen OA patients undergoing total knee or hip arthroplasty. Biopsy punches of 4 mm in diameter were made to equalize explant size. After culturing overnight, explants were incubated for 48 h with three doses (1, 10 or 100 ng/ml) of recombinant-human IL37 (rhIL37). In the supernatant of the explant cultures, GAG release was measured with the DMB assay and levels of the ARGS neoepitope, which is one of the products of aggrecan degradation by ADAMTS5, were detected using Western Blot. Furthermore, gene and protein expression of extracellular matrix molecules and cartilage degrading enzymes were measured. Nitric oxide (NO), an important effector molecule that may suppress cartilage matrix synthesis, levels were measured in the supernatant of the explants culture using Griess reagens.
Results
Adding rhIL37 (100 ng/ml) to OA cartilage explants caused a highly significant reduction in GAG release to the supernatant of, on average, 32% in eighteen donors (Figure 1). Gene expression of the matrix molecules aggrecan and collagen type II was not affected, indicating that this effect of rhIL37 was not due to a loss of aggrecan synthesis. Another mechanism to prevent GAG release in cartilage is via inhibition of NO synthesis, but NO levels in the supernatant were comparable between rhIL37 treated groups and the control group. In contrast, after addition of rhIL37, ARGS neoeptiope levels, which reflect ADAMTS5 activity, were dose-dependently down regulated in the supernatant. Furthermore, protein analysis of the explants showed that rhIL37 reduced ADAMTS5 levels. These data indicate that IL37 interferes with the amount active matrix degrading enzymes in the cartilage matrix. However, gene expression of ADAMTS5 was not affected by rhIL37, indicating that the effect of IL37 on ADAMTS5 is post translational.
Conclusions
Our data show that rhIL37 reduces GAG release by OA cartilage explants. The mechanism behind this protective effect of IL37 probably runs via a reduction in ADAMTS5 abundance in the cartilage matrix, which is the main aggrecanase involved in OA This effect of IL37 on ADAMTS5 is probably post translational. Our data indicate that IL37 can maintain cartilage matrix integrity under OA conditions and is able to reduce the severity of cartilage destruction during OA.
Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis.
To improve our understanding of the metabolic role of cytokines in protein wasting, we estimated the rates of protein synthesis and degradation in muscle and liver tissues in intact rats treated with several doses of recombinant IL 1 and/or tumor necrosis factor (TNF)/cachectin. Protein breakdown in muscle and liver were derived in vivo from the relationship between [14C]leucine distribution and tissue dilution in reference to circulating leucine. Synthesis was derived from the relationship between [14C]leucine appearance in the protein-bound and free-tissue leucine pools. To specifically relate changes in leucine tracer metabolism to protein dynamics, we separately measured the effect of these cytokines on blood flow to different tissues. The increase in dilution of the tissue-free [14C]leucine by TNF and TNF/IL 1 mixture, but not by IL 1 alone, could not be explained by a hemodynamic effect of these cytokines. Rather, this finding indicated that muscle proteolysis is enhanced by TNF and synergistically augmented by the addition of IL 1. Compatible with these data was the finding that more prolonged infusions of recombinant TNF/cachectin and the combination with IL 1 increased urinary nitrogen excretion. Changes in [14C]leucine dilution in the liver were less pronounced than those in skeletal muscle and consistent with net anabolic effect of TNF on liver protein. We conclude that rats exposed systemically to sublethal doses of TNF respond with increasing muscle and decreasing liver proteolysis, similar to that observed in inflammation and in cancer.
When infused into the lateral cerebral ventricles of rabbits, human endogenous pyrogen (EP) preparations induced dose-dependent increases in slow-wave sleep concomitant with increasing body temperature. Heating EP to 70 degrees C destroyed its sleep-promoting and pyrogenic activity. Anisomycin (an antipyretic) prevented EP from increasing body temperature without affecting its sleep-promoting activity. Intravenous injection of EP induced fever and transient increases in slow-wave sleep but failed to induce prolonged increases in slow-wave sleep. We conclude that the somnogenic activity of EP is not secondary to its pyrogenic activity.
Abstract Cytokines are polypeptide mediators which act as communication signals among cells of the immune system as well as among other cells and tissues in the body. They are a heterogeneous and complex group and include interferons, tumor necrosis factor and chemokines. They play a key role in homeostasis and in host defence and are involved in such inflammatory and autoimmune diseases such as rheumatoid arthritis as well as infectious diseases such as HIV infection and septic shock. Modulation of the production and action of cytokines, as well as their exploitation as therapeutic agents, has been the object of intense and competitive research. This book overviews the field of cytokine research and describes the various approaches that have been taken to develop the pharmacology of these novel mediators. The pharmacology of cytokines is an exploding area which is entering the clinical arena. The book, in the framework of the immunobiology of cytokines, examines the interactions with the cytokine system of a variety of compounds ranging from simple synthetic chemicals to biotechnological products. In addition to examining individual agents and approaches, the book examines the pathophysiology of individual body systems and analysis specific contexts for the pathophysiology of these mediators as well as pharmacological approaches for their control.
