Reduction of inflammation and pyrexia in the rat by oral administration of SDZ 224-015, an inhibitor of the interleukin-1 beta converting enzyme.

1. The aim of this study was to determine whether a synthetic inhibitor of the interleukin-1 beta converting enzyme (ICE) displays oral activity in models of inflammation. 2. To this end, the ICE inhibitor, SDZ 224-015, was examined in rat paw oedema, pyrexia and nociception tests. 3. SDZ 224-015 (0.3-300 micrograms kg-1) potently reduced carrageenin-induced paw oedema, with an oral ED50 of approximately 25 micrograms kg-1. This effect was independent of endogenous glucocorticoid, as shown by retention of activity upon adrenalectomy. 4. Pyrexia induced by lipopolysaccharide (0.1 mg kg-1 s.c.) or by interleukin-1 beta (100 ng i.v.) was also reduced, over a similar dose-range to oedema (oral ED50s 11 micrograms kg-1 and 4 micrograms kg-1 respectively). 5. SDZ 224-015 (0.2-5 mg kg-1, p.o.) displayed analgesic activity in the Randall-Selitto yeast-inflamed paw pressure test, significant at a dose of 1 mg kg-1, p.o. 6. Thus, SDZ 224-015 has potent oral activity in several acute models for inflammation, suggesting that ICE inhibitors may constitute a novel type of anti-inflammatory agent.

Induction of swelling, synovial hyperplasia and cartilage proteoglycan loss upon intra-articular injection of transforming growth factor beta-2 in the rabbit.

Transforming growth factor beta (TGF-beta) is a multifunctional homodimeric polypeptide with potent actions upon many target cells, including those of mesenchymal and haemopoietic lineage. The recent reports of high levels of the cytokine in rheumatoid synovium and synovial fluid, prompted this study into the effect of intra-articular injection of TGF beta-2 into rabbit knee-joints. Four daily injections of 1 microgram caused swelling, probably as a consequence of prostaglandin E2 production, synovial fibroblastic hyperplasia and a striking loss of femoral condyle proteoglycan. Using the polymerase chain reaction, no evidence could be obtained for the induction of interleukin-1 alpha gene expression in either synovial tissue or synovial fluid cells. These findings suggest that the TGF-beta present in the rheumatoid joint may contribute directly to the pathogenesis of rheumatoid arthritis.

Induction of neutrophil-mediated cartilage degradation by interleukin-8.

Neutrophil influx into the inflamed joint is a characteristic feature of disease flares in patients with rheumatoid arthritis. Recently, a protein produced by monocytes and fibroblasts that has chemoattractive/activating properties for neutrophils has been identified and characterized. This protein has been called interleukin-8 (IL-8). In this study, we cocultured neutrophils with 35S-sulfate-labeled cartilage and found that the addition of recombinant human IL-8 (rHuIL-8) caused rapid, neutrophil-mediated cartilage degradation that was the result of induction of neutrophil degranulation by the cytokine. With 10(-7)M rHuIL-8, 23% of the radiolabel was released into the culture medium in 4 hours, compared with a 9% release without the factor. At concentrations of up to 10(-6)M, rHuIL-8 had no direct effect upon cartilage breakdown. These findings indicate that IL-8 may participate in the pathogenesis of rheumatoid arthritis through the induction of neutrophil-mediated cartilage damage.

Contrasting modulation by transforming growth factor-beta-1 of insulin-like growth factor-I production in osteoblasts and chondrocytes.

The importance of locally produced insulin-like growth factor-I (IGF-I) in connective tissues has recently been recognized. It has been postulated that the action of anabolic hormones on bone may be mediated through local IGF-I release. However, whether IGF-I can also be modulated by other locally acting cytokines has not been addressed. Transforming growth factor-beta (TGF beta) is a polypeptide thought to be involved in the regulation of tissue growth and repair. Although the occurrence of TGF beta is ubiquitous, particularly high amounts are found in bone and cartilage. In this study the effect of TGF beta-1 on immunoreactive IGF-I production by osteoblasts and chondrocytes was investigated and compared to that of PTH on osteoblasts or basic fibroblast growth factor (bFGF) on chondrocytes. Both TGF beta-1 and PTH stimulated IGF-I release from osteoblasts, which was further enhanced when both were consecutively present. Contrastingly, although bFGF stimulated IGF-I release by chondrocytes, TGF beta-1 was inhibitory and also blunted the effect of bFGF when both were present concurrently. These findings demonstrate that the regulation of local IGF-I production in bone and cartilage may differ and illustrate the complex nature of local cytokine interactions.

Impairment of macrophage colony-stimulating factor production and lack of resident bone marrow macrophages in the osteopetrotic op/op mouse.

Mouse calvaria-derived osteoblastlike cells have been shown to produce macrophage colony-stimulating factor (M-CSF). This factor may be involved in osteoclastogenesis and thus in bone resorption. In the present study we investigated whether the production of M-CSF was altered in the osteopetrotic mouse mutant strain op/op, characterized by a decrease in osteoclast number and an impairment of bone resorption. Whole calvariae and cells, as well as skin and lung fibroblasts, of the op/op mouse were found to produce no measurable M-CSF, in contrast to tissue and cells derived from normal littermates. M-CSF was identified by colony assay in semisolid media and by inhibition of the biologic activity with antiserum against M-CSF. Furthermore, the number of resident macrophages, identified by F4/80 antigen (F4/80 Ag) immunohistochemistry, was drastically decreased in bone and bone marrow of the op/op mouse, but in skin these cells were normal in number and morphology. These findings suggest that both M-CSF and resident macrophages play a role in the mechanism of bone resorption. The op/op mouse appears to be a valuable model to further investigate such a hypothesis.

Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1.

It has been observed that bone resorption in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF) is accompanied by an increase in osteoclast number. Because the osteoclast is of hemopoietic lineage, recruitment could be regulated by colony-stimulating factors, one of which may be macrophage colony-stimulating factor (M-CSF). In this study, we show that the constitutive release of M-CSF activity by the osteoblastic cell MC3T3-E1 is enhanced by the presence of recombinant IL 1 alpha, recombinant TNF alpha, or by the concurrent presence of purified transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). Increased release of CSF by the osteoblast in response to these agents may provide a signal for the growth and maturation of osteoclast precursors leading to subsequent bone resorption.

Effects of transforming growth factor type beta upon bone cell populations grown either in monolayer or semisolid medium.

Bone has been shown to store large amounts of transforming growth factor type beta (TGF beta) and this has recently been found to be synthesized by bone-forming cells. We report on studies undertaken to examine the effects of platelet-derived TGF beta on different bone cell populations, isolated from 1-day postnatal rat calvaria by sequential enzymatic digestion. In addition, we tried to determine which of these cell populations synthesize TGF beta. In this regard, evidence was collected to indicate that cell populations which were shown to be enriched with osteoblast-like cells synthesize TGF beta. Although the production of the factor appeared to be limited to a particular cell type, its action was found to be of a more general character, as all cell populations were found to respond to TGF beta. Contrary to earlier reports, TGF beta was shown to be inhibitory upon cell proliferation. In this context, growth of cells released during early digestions was reduced considerably more than growth of those released during late digestions. Studies on the effect upon protein synthesis revealed that TGF beta specifically inhibited collagen but not the synthesis of noncollagenous proteins. The synthesis of collagen was altered to a greater extent in cells isolated during late digestions than in cells of the early populations. Further information on the TGF beta-mediated effects on bone cell biology was provided by data showing that both alkaline phosphatase and cAMP production in response to PTH was greatly reduced by TGF beta. Finally, experiments performed to determine whether TGF beta induces any of the bone cell populations to acquire the transformed phenotype revealed that only populations previously shown to be enriched with osteoblast-like cells formed colonies in soft agarose.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of hemopoietic growth factors by bone tissue and bone cells in culture.

This study was carried out to determine whether bone might be a source of hemopoietic growth factors. Both neonatal murine calvaria and primary cultures of cells isolated from calvaria released, upon stimulation with lipopolysaccharide, an activity that stimulated the growth of the interleukin (IL) 3-dependent cell lines, 32D cl, 123, and NSF 60. Upon gel filtration, this activity eluted with a molecular weight of 30,000 kDa. Further characterization, however, revealed that the major activity in conditioned medium was not IL 3. Activity was absorbed by DEAE-Sephacel at low salt concentration, whereas IL 3 does not adhere. Furthermore, an IL 3-specific antiserum did not neutralize the activity from cells and only partly neutralized the activity generated by whole calvaria. After gel filtration, the 30-kDa activity stimulated the growth of very large colonies in semisolid medium consisting mainly of granulocytes with the remainder being macrophages. No colony types belonging to other hemopoietic lineages were found, indicating, again, that the activity was not identical to IL 3. Subsequently, conditioned medium was fractionated by hydrophobic chromatography on Phenyl-Sepharose CL-4B, yielding two peaks of activity. Neutralization of activity with antisera to granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL 3 and use of colony assays showed that medium conditioned by whole calvaria contained GM-CSF and granulocyte CSF (G-CSF) in similar amounts together with a little IL 3, and medium conditioned with calvaria cells contained GM-CSF and little G-CSF. We conclude that bone releases hemopoietic growth factors that could contribute both to hemopoiesis and to the recruitment of osteoclasts from progenitors resident in the adjacent marrow.

Murine osteoblastlike cells and the osteogenic cell MC3T3-E1 release a macrophage colony-stimulating activity in culture.

The osteoclast may be of hematopoietic lineage and as such its development could be regulated by colony-stimulating factors. Since there is much interest as to whether osteoblasts influence bone resorption, we examined whether bone cells produce colony-stimulating activity. Both cells isolated from neonatal calvaria and the osteogenic cell MC3T3-E1 were found to constitutively release a colony-stimulating activity possessing characteristics of a macrophage colony-stimulating factor, as determined by basic biochemical purification and by identity of colonies induced in cultures of bone marrow cells. Release could be increased by the presence of the bone-resorbing agents lipopolysaccharide and 1,25 dihydroxyvitamin D3. We conclude that the osteoblast may contribute to both the processes of osteoclast formation and of hematopoiesis through the secretion of colony-stimulating activity into the adjacent bone marrow.

Transforming growth factor-beta reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture.

Transforming growth factor beta (TGF-beta) regulates cell growth and differentiation. Since it is abundant in bone, we have studied the effect of the polypeptide upon the growth and phenotypic expression of murine osteoblastic cells in monolayer culture. Its actions were compared to those of epidermal growth factor (EGF), another hormonally active polypeptide known to alter bone cell function. Picogram amounts of TGF-beta were found to inhibit the growth and phenotype (alkaline phosphatase and cAMP response to parathyroid hormone) of the clonal nontransformed MC3T3-E1 osteoblastic cell line. EGF also inhibited phenotypic expression, although at higher (nanogram) concentrations, but stimulated cell growth. The low concentration of TGF-beta required to inhibit growth and phenotype of osteoblastic cells together with its abundance in bone suggest that TGF-beta may be an important regulator of bone cell function.

Retinoids and synovial factor(s) stimulate the production of plasminogen activator by cultured human chondrocytes. A possible role for plasminogen activator in the resorption of cartilage in vitro.

Agents such as retinol, interleukin 1 and catabolin stimulate resorption of cultured cartilage. This process seems to be mediated by chondrocytes, but the mechanism by which breakdown occurs remains unknown. We have found that (10(-6)-10(-8) M) retinoic acid and (1 X 10(-6) M) retinol, in the presence or absence of a factor derived from cultured synovium (synovial factor), stimulate the degradation of fibrin by human chondrocytes in culture. Plasminogen was required for the enhancement of fibrinolysis, suggesting that the breakdown depended upon the production of plasminogen activators and subsequent liberation of plasmin. However, the chondrocytes did not release significant amounts of plasminogen activator, and the effects of the synovial factor and retinoids resulted from augmentation of the production or activity of enzymes which remained bound to the cell layer. The role of plasminogen in the resorption of cultured cartilage was also investigated. In the presence of plasminogen, (1 X 10(-8) M) retinoic acid or synovial factor stimulated the breakdown of cultured bovine nasal cartilage, but in the absence of plasminogen, the effect of synovial factor was abolished and that of retinoic acid reduced. However, in cultures containing both retinoic acid and synovial factor the resorption process was not affected by removal of plasminogen. Thus, the resorption of cartilage matrix in vitro may be partially mediated by plasminogen activators and plasmin.

Modulation of connective tissue metabolism by partially purified human interleukin 1.

We have investigated the relationship between the monokine interleukin 1 (IL-1) and the connective tissue-stimulating activities produced by monocytes such as mononuclear cell factor (MCF). Using almost exclusively human tissue we have monitored a wide range of MCF-like activities through the partial purification of IL-1 by gel filtration and isoelectric focusing. Activities measured include stimulation of chondrocytes to produce prostaglandins, plasminogen activator and proteoglycanase, enhancement of synovial cell proliferation, and stimulation of cartilage resorption, in addition to IL-1 (lymphocyte activating factor) activity. The activities described show the same molecular heterogeneity; the active material has similar potencies in the different systems, and removal of IL-1 activity by pretreatment with phenylglyoxal also results in loss of the connective tissue-stimulating activities. These results show that the factors responsible for this wide range of activities are very closely related to IL-1 and give further evidence in support of the possible involvement of IL-1 in the processes of joint destruction occurring in chronic inflammatory conditions such as rheumatoid arthritis.

Partial purification of a factor from human synovium that possesses interleukin 1, chondrocyte stimulating and catabolin-like activities.

Human synovial explants in culture release material that stimulates the production of prostaglandin E2 (PGE2) and several extracellular enzymes by human chondrocytes. Fractionation of conditioned medium by gel filtration revealed a protein of approx. 15 kDa, which in addition to stimulating production of PGE2 and plasminogen activator by human articular chondrocytes, possessed interleukin 1 activity and induced cartilage degradation. Further purification using iso-electric focussing again showed co-elution of these activities with a major pI of 6.9 and a minor pI of 5.1-5.3. This study indicated that human synovium releases a factor that is closely related to or identical with interleukin 1 and suggests that this protein may participate in cellular interactions that occur within the rheumatoid joint.