Transforming growth factor-β inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced ¹²⁵I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of M_r =116 and 80 kDa and a minor band of M_r =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Identification of disulfide-linked transforming growth factor-beta 1-specific binding proteins in rat glomeruli

We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type consists of a group of proteins that bind TGF-beta 1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions, four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000 and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the 90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000 and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta 1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously described type III TGF-beta receptor.     

A transforming growth factor beta (TGF-beta) receptor from human placenta exhibits a greater affinity for TGF-beta 2 than for TGF-beta 1

Affinity-labeling techniques have been used to identify three types of high-affinity receptors for transforming growth factor beta (TGF-beta) on the surface of many cells in culture. Here we demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-beta receptors. Using a chemical cross-linking technique with 125I-TGF-beta 1 and 125I-TGF-beta 2 and bis(sulfosuccinimidyl)suberate (BS3), we have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-beta receptor type III, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type III receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using 125I-TGF-beta 1 or 125I-TGF-beta 2 and varying amounts of competing unlabeled TGF-beta 1 or TGF-beta 2, revealed that both the placental type III proteoglycan and its core glycoprotein belong to a novel class of type III receptors that exhibit a greater affinity for TGF-beta 2 than for TGF-beta 1. This preferential binding of TGF-beta 2 to placental type III receptors suggests differential roles for TGF-beta 2 and TGF-beta 1 in placental function.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Further studies of the role of transforming growth factor-beta in human B cell function

This study was designed to address three specific questions in human B cells. First, to determine whether transforming growth factor-beta (TGF-beta)2 has similar biologic effects on B cell function as does TGF-beta 1. Second, to test the hypothesis that TGF-beta 1 is an autocrine growth and differentiation inhibitor. Finally, because multiple receptor species for TGF-beta have been identified on other cell types, to determine by chemical cross-linking and competitive binding studies the nature of the TGF-beta 1 R present on normal and transformed B cells. Exogenous TGF-beta 2 was found to be functionally similar to TGF-beta 1 in its inhibition of factor dependent normal B cell proliferation and Ig secretion. When an antibody, specific for the active form of TGF-beta 1, was added in conjunction with IL-2 to previously stimulated B cell cultures, there was a 14.4 +/- 4.2% increase in B cell proliferation, a 22 +/- 6% increase in IgG production, and a 33 +/- 8.6% increase in IgM production when compared to control cultures. Chemical cross-linking of 125I-TGF-beta 1 to normal B cell membranes identified two major cross-linked species of 65 and 90 kDa. A fivefold excess of unlabeled TGF-beta 1 competitively inhibited the detection of both of these bands while a 50-fold excess of unlabeled TGF-beta 2 did not inhibit the 90-kDa band and only partially inhibited (60%) of the 65-kDa band. Chemical cross-linking of 125I-TGF-beta 1 to transformed B cell membranes identified only a single band of 60 kDa. Scatchard plot analysis of 125I-TGF-beta 1 binding to normal B cells that was competitively inhibited with increasing concentrations of unlabeled TGF-beta 1 revealed both high and low affinity binding sites whereas analysis of 125I-TGF-beta 1 binding in the presence of increasing concentrations of unlabeled TGF-beta 2 revealed only low affinity sites. These findings demonstrate that TGF-beta 2 is as effective as TGF-beta 1 in inhibiting human B cell function, that small amounts of active TGF-beta 1 are present endogenously in in vitro cultures which partially inhibit B cell function, that two major TGF-beta 1 R cross-linked complexes of 65 and 90 kDa are present on normal B cells, and that transformation of B cells may be accompanied by changes in the TGF-beta 1 R.     

Contribution of interleukin-1 receptor accessory protein B to interleukin-1 actions in neuronal cells

Interleukin (IL)-1 is an important neuroimmunomodulator and a key mediator of inflammation during brain disorders. It acts on neuronal and glial cells via binding to the IL-1 type 1 receptor and IL-1 receptor accessory protein (IL-1RAcP). More recently, a neuronal-specific isoform of IL-1RAcP, named IL-1RAcPb, has been identified. Our aim was to determine the role of IL-1RAcPb in IL-1 actions in neuronal and glial cells, and to further explore the signaling mechanisms of IL-1 in neurons. We found that IL-1RAcPb deletion had no effect on IL-1α- and IL-1β-induced activation of the extracellular signal-regulated kinase 1/2 or IL-6 release in glial cultures, although IL-6 release in response to high IL-1α concentration (30 IU/ml) was significantly reduced. We identified the p38 kinase as a key signaling element in IL-1α- and IL-1β-induced IL-6 synthesis and release in neuronal cultures. IL-1RAcPb deletion had no effect on IL-1α- and IL-1β-induced IL-6 release in neurons, but significantly reduced IL-1α- but not IL-1β-induced p38 phosphorylation. Our data demonstrate that the p38 signaling pathway plays an important role in IL-1 actions in neurons, and that IL-1RAcP may regulate some, but not all, neuronal activities in response to IL-1α.     

Biological effects and binding properties of transforming growth factor-beta on human oral squamous cell carcinoma cells

The effects of transforming growth factor-beta (TGF-beta) on three human oral squamous cell carcinoma cell lines, HSC-2, HSC-3, and HSC-4, were investigated. Although these cell lines were equally sensitive to epidermal growth factor, responses to TGF-beta were variable. Dose-dependent inhibition of cell growth and [3H]thymidine incorporation of HSC-4 were observed by the addition of TGF-beta, whereas growth inhibitory effects on HSC-2 and HSC-3 were marginal. Moreover, treatment of HSC-4 with TGF-beta led to a more than 300-fold increase in fibronectin secretion into the medium. In contrast, TGF-beta did not increase the secretion of fibronectin on HSC-2 and HSC-3. Scatchard analysis of the binding of TGF-beta suggested that all squamous cell carcinoma cell lines have similar binding properties, with two classes of binding sites for TGF-beta. Affinity labeling of 125I-TGF-beta to cell surface receptors revealed the two major affinity crosslinked bands with Mr values of 65 kDa (type I) and 280 kDa (type III). A concomitant loss of 85 kDa band (type II) was observed in all squamous carcinoma cell lines examined. Although the proportions of type I and type III receptors were variable, the type I receptor, which is reported to be the main functional receptor in mediating the TGF-beta action, was commonly observed in these squamous cell carcinoma cell lines. These results indicate that the heterogeneity in response to TGF-beta between cell lines may be due to the difference in the signal transduction pathway of TGF-beta.     

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.     

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.     

Salidroside attenuates interleukin-1β-induced inflammation in human osteoarthritis chondrocytes

Salidroside, a bioactive constituent isolated from Rhodiola rosea, has been reported to have anti-inflammatory effects. However, the effects of salidroside on interleukin (IL)-1β-stimulated osteoarthritis (OA) chondrocytes remain to be elucidated. Thus, this study aimed to evaluate the anti-inflammatory effects of salidroside on IL-1β-stimulated human OA chondrocytes and explore its underlying mechanisms. Our results showed that salidroside significantly inhibited the production of nitric oxide and prostaglandin E-2, as well as suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1β-stimulated chondrocytes ( P < .05). In addition, salidroside also suppressed IL-1β-induced matrix metalloproteinases production in human OA chondrocytes ( P < .05). Furthermore, pretreatment with salidroside prevented IL-1β-induced NF-κB activation in OA chondrocytes ( P < .05). In conclusion, the current study demonstrated that salidroside inhibited the IL-1β-induced inflammatory response in OA chondrocytes via inhibition of NF-κB activation.     

Identification of IL-1 receptors on human monocytes

The expression and functional analysis of IL-1 beta R on human monocytes were investigated. Binding of 125I-IL-1 to human monocytes was found to be specific and saturable. Scatchard plot analysis revealed a single class of receptors with a binding constant of 600 pM and a receptor density of approximately 100 binding sites per cell. At 37 degrees C 54% of the labeled ligand was internalized over 2 h of incubation. Addition of 0.2% sodium azide to the cells reduced ligand internalization to 9% of total bound. Cross-linking studies revealed that the IL-1R in human monocytes had a Mr of 80 kDa. The addition of IL-1 to monocytes caused changes in membrane Ag expression as assessed by flow cytometric analysis. The results of this study identify IL-1 receptors on monocytes and suggest that IL-1 may act as an effector molecule for monocytes by enhancing expression of Ag correlated with cell differentiation and immune function.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

A biochemical and kinetic analysis of the interleukin-1 receptor. Evidence for differences in molecular properties of IL-1 receptors

This study describes the biochemical characterization and kinetic analysis of the interleukin-1 (IL-1) receptor in Raji human B-lymphoma and EL4 murine T-lymphoma cells. The internalization of 125I-IL-1 was studied in both cell types by an acid extraction technique which removes surface bound ligand. At 37 degrees C, binding to Raji IL-1 receptors was almost entirely cell surface (91%). EL4 cells, in contrast, internalized 59% of ligand at this temperature and this was almost totally inhibited by sodium azide. Receptor binding studies showed that the B-cells had a lower binding affinity but much higher receptor density per cell (KD = 2.1 nM, Ro = 7709) than the T-cells (KD = 0.4 nM, Ro = 241). The receptor binding affinity of two IL-1 analogs, Glu-4 and clone 18, was determined in competitive binding studies. In the B-cells the analogs had binding affinities of 25 and 90%, respectively, whereas in the T-cells the affinities were 0.2 and 200%, respectively. Chemical cross-linking studies showed that the IL-1 receptor in B-cells had a lower molecular weight than that in T-cells (68 kDa compared to 80 kDa). In summary these studies demonstrate that structural differences exist between IL-1 receptors in Raji and EL4 cells.     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.     

Transforming growth factor-beta is a potent inhibitor of IL-1 induced protease activity and cartilage proteoglycan degradation

Treatment of chondrocytes in culture with interleukin-1 results in the production of neutral proteases that cause the degradation of the large aggregating proteoglycan. TGF-beta is a pleiotropic growth factor that has been shown to induce differentiation of cartilage and, in some cases, was able to inhibit the IL-1-dependent processes. In this report, we examined whether TGF-beta could block the IL-1 induced catabolic effects on chondrocytes. After treatment with IL-1 beta (30 ng/ml), rabbit articular chondrocytes produced approximately 2 units of neutral protease activity. Under identical conditions, TGF-beta 1 alone did not induce any protease activity. However, a combination of IL-1 and TGF-beta resulted in a dramatic reduction in the level of protease activity. The inhibitory effect of TGF-beta was also observed at the level of proteoglycan incorporation into the extracellular matrix. The IL-1 treated chondrocytes failed to incorporate proteoglycans into their extracellular matrix. However, addition of TGF-beta in the presence of IL-1 resulted in partial reversal towards a normal extracellular matrix. These studies indicate that TGF-beta can block and at least partially inhibit the catabolic effects of IL-1 on chondrocytes.