Release of interleukin-1 from human synovial tissue in vitro.

During the enzymatic disaggregation of human synovium, used in the process of isolating synoviocytes, a factor was liberated into the culture medium that exhibited the thymocyte mitogenic properties of interleukin-1. Like interleukin-1, this synovial-derived mitogen could be isolated using an affinity column of antihuman leukocytic pyrogen. By gel filtration and isoelectric focusing, the mitogen cofractionated with human monocyte-derived interleukin-1. Finally, the isolated mitogen was shown to exhibit other properties of interleukin-1: stimulation of the secretion of interleukin-2, enhancement of the titer of acute-phase proteins in vivo, and stimulation of the release of prostaglandin E2 from human synoviocyte cultures. These observations suggest that interleukin-1 can be derived from the human synovium.

The four biochemically distinct species of human interleukin 1 all exhibit similar biologic activities.

The supernatants of human monocytes incubated with endotoxin are able to stimulate the proliferation of murine thymocytes in the presence of PHA. This is known as LAF (lymphocyte activating factor) activity and is a characteristic activity of interleukin 1 (IL 1). The LAF activity can be resolved into four major fractions: a 15,000 dalton (pI 7), a 15,000 (pI 5.5), a 35,000 (pI 7), and a 35,000 (pI 5.5) fraction. To determine whether these four fractions shared the other biologic activities ascribed to IL 1, they were compared in a series of bioassays. When standardized with respect to their LAF activities, the four fractions did not differ significantly as mitogens for murine thymocytes, inducers of IL 2, murine or human B cell activators, human chondrocyte or synoviocyte stimulants, or inducers of acute phase proteins in vivo. On the other hand, the samples differed markedly as stimulators of porcine synoviocytes, with the 15,000 dalton (pI 5.5) fraction being the only strongly active fraction. These results are consistent with the hypothesis that all four LAF could be products of a single gene, although the porcine receptor may be able to distinguish between them. If this is the case, all four fractions can properly be termed IL 1.

Biochemical heterogeneity of human interleukin-1.

Human interleukin 1 (IL-1), a mediator of several host defense responses, is usually considered to have a molecular weight of 15,000 daltons and an isoelectric point near neutrality. However, during purification, significant IL-1-like activity is also found with apparent molecular weights of greater than 80,000, 50,000, and 35,000 daltons. The active species with the highest apparent molecular weight (greater than 80 kilodaltons) is not a strong comitogen for thymocytes but exhibits relatively strong direct mitogenic activity. By affinity By affinity chromatography, it was shown that the direct mitogen is probably a contaminant distinct from the IL-1. Significant IL-1 activity is also recovered at approximately 50,000 daltons, but upon rechromatography it dissociates, suggesting that it is an aggregate. In contrast, the species eluting at 35,000 daltons and 15,000 daltons are stable upon rechromatography even in 6 M urea. Upon isoelectric focusing, the 15,000 and the 35,000 dalton species show similar patterns, with a prominent peak of IL-1 activity near neutrality and multiple peaks in the range of pI 4.5-5.8. Therefore, it appears that human monocytes secrete at least four biochemically distinct and noninterconvertible proteins, all of which stimulate the proliferation of murine thymocytes in the presence of PHA. Although the IL-1 preparations described above were prepared by pooling IL-1-rich supernatants from 12 donors, the heterogeneity is not due to genetic diversity. A similar set of four molecules with IL-1 activity was derived from the leukocytes of a single patient with chronic myelomonocytic leukemia and from a single human placenta.

Contamination of commercial leukocyte-derived human interleukin 2 with interleukin 1.

Commercial preparations of human leukocyte-derived IL-2 were tested for the presence of contaminating IL-1 by affinity chromatography. Two samples from Electro-Nucleonics were found to contain significant amounts of IL-1, suggesting that IL-1 may contaminate preparations of leukocyte-derived IL-2.

In vitro activation of human chondrocytes and synoviocytes by a human interleukin-1-like factor.

Monocytes have been shown to secrete factors which stimulate the destruction of cartilage. Since one of the monocyte products, interleukin-1 (IL-1), has been shown to stimulate the release of collagenase and prostaglandin E from synoviocytes, we have investigated whether IL-1 is also responsible for chondrocyte activation. Purified preparations of IL-1 derived from human blood monocytes stimulated the production of prostaglandin E and plasminogen activator by human articular chondrocytes. After Sephadex G-75 chromatography, the lymphocyte-activating and the chondrocyte-activating activities of IL-1 eluted together in the regions corresponding to the void volume and to Kav = 0.2-0.3 (Mr 30,000-45,000) and Kav = 0.5-0.65 (Mr 12,000-17,000). The major peak of stimulating activity was the 12,000-17,000 dalton peak. Upon further analysis of the 12,000-17,000 dalton peak by isoelectric focusing, the major peak of lymphocyte-activating factor activity was recovered at a pI of 6.3 with a minor peak at 4.6-5.3. Similar patterns of activity were observed when the fractions were assayed for the stimulation of the production of prostaglandin E and plasminogen activator by human chondrocytes and of prostaglandin E by human synoviocytes. Treatment of the partially purified lymphocyte activating factor with phenylglyoxal reduced the thymocyte-stimulating activity 99% and the chondrocyte-stimulating activity 100%. These results suggest that IL-1 may stimulate the degradation of connective tissues during inflammation.

Stimulation by human interleukin 1 of cartilage breakdown and production of collagenase and proteoglycanase by human chondrocytes but not by human osteoblasts in vitro.

Human articular chondrocytes in culture synthesise collagenase and neutral proteoglycanase in response to addition of a 12-17 kDa protein produced by cultured human monocytes. This factor copurifies with interleukin 1, as assessed by lymphocyte activating factor activity, on gel filtration chromatography and isoelectric focusing. The interleukin 1 and chondrocyte-stimulating activities are destroyed by pretreatment of the material with phenylglyoxal. The same materials also promote the release of glycosaminoglycans from cultures of intact bovine nasal cartilage. The proteoglycanase activity release from chondrocytes appears to be a metalloproteinase because it is inhibited by EDTA and not by phenylmethylsulphonyl fluoride (PMSF), and because detection of its activity is dependent on the presence of 4-aminophenylmercuric acetate. Human osteoblast-like cells do not respond to this factor by increased proteinase production, but are stimulated to produce prostaglandins. These results suggest that interleukin 1 has activities upon non-immune cells which promote the degradation of connective tissue matrices. Human osteoblasts do not synthesise neutral collagen- and proteoglycan-degrading enzymes and thus are unlikely to be directly responsible for the matrix degradation which occurs during bone resorption.

An interleukin 1 like factor stimulates bone resorption in vitro.

Many activities are now ascribed to the monokine interleukin 1 including enhancement of immune responses, stimulation of thymocyte proliferation, activation of B cells, stimulation of proteinase and prostaglandin production by connective tissue cells, stimulation of the production of acute phase proteins, induction of fever and the induction of neutrophilia. These activities were thought to be due to various different factors, but are now considered probably due to very similar, if not identical, molecules. The term interleukin 1 (IL-1) was coined to describe the factor released by monocyte/macrophages which acts on T and B lymphocytes. Only after this definition had been accepted was it shown that target cells other than lymphocytes were affected by IL-1. Products of human blood monocytes (mononuclear cell factor, MCF) have been implicated in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and periodontal disease. Bone resorption is often a feature of such diseases, and monocytes are frequently found at sites of localized bone resorption. Preliminary experiments with monocyte-conditioned medium indicated that MCF could stimulate bone resorption. We therefore undertook this study to verify these observations and to determine whether purified IL-1 could stimulate connective tissue breakdown in vitro.

Isolation of an interleukin-1-like factor from human joint effusions.

Interleukin-1 (IL-1) is a macrophage derived mediator whose properties suggest that it could play a role in the pathology of arthritis. To test this hypothesis, joint fluids from patients with serveral different arthritides were tested. Small amounts of IL-1-like activity were recovered from many of these joint fluids after affinity chromatography over a column of rabbit anti-human IL-1. Positive fluids were obtained from patients with rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, osteoarthritis, gout, and traumatic arthritis. Upon gel filtration, the joint derived factor displayed a molecular weight distribution similar to that of IL-1 derived from human monocytes stimulated in vitro. These results suggest that IL-1 is present in joint effusions and, therefore, might contribute to joint destruction.