Recombinant Human Interleukin-1 Beta Research Articles

High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris

As an important pro-inflammatory cytokine, interleukin-1beta (IL-1β) participates in a variety of physiological and pathological responses. In order to obtain higher yielded recombinant human interleukin-1 beta (rhIL-1β), we cloned hIL-1β cDNA sequences based on the coding sequence of human mature IL-1β. After recombinant pPICZαA/hIL-1β was separated and sequenced, we transformed recombinant pPICZαA/hIL-1β into Pichia pastoris GS115, SMD1168 and X-33 strain via electroporation. The results showed that recombinant pPICZαA/ hIL-1β had the highest expression level in X-33 Pichia pastoris. Subsequently, rhIL-1β was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identified by Western blot. Then the fermentation process was optimized to increase product yield. Under the fermentation conditions of the absorption value of fermentation liquor before induction of 600, oxygen concentration of 20%, methanol concentration of 0.25% with pH 5.0, the yield of rhIL-1β reached to 250 mg/L after 72 h induction at 26°C. After aqueous two-phase extraction combined with chromatography, the purity of rhIL-1β was 95% and the yield was up to 85%. The biological activity of rhIL-1β was detected by MTT assay, and the result showed that rhIL-1β significantly inhibited the growth and proliferation of B16 melanoma cells.

Influence of human recombinant interleukin-1beta on protective and immunogenic efficacy of live plague vaccine

Influence of human recombinant interleukin-1beta on protective and immunogenic efficacy of live plague vaccine

Inhibitory effects of rhIL-1 beta pretreatment on bleomycin-induced pneumonitis in mice

We studied the effects of recombinant human interleukin-1 beta (rhIL-1 beta) pretreatment on bleomycin (BLM)-induced pneumonitis in mice. Lung injury was dose-dependently induced by BLM (50 mg/kg, 100 mg/kg, 150 mg/kg i.v.). The mice were pretreated with IL-1 (1 microgram/mouse i.p.) at 0.5, 6, 12 or 24 hours before the administration of BLM. Wet lung weight, lung weight-to-body weight ratio and bronchoalveolar lavage cell findings were analyzed with respect to time, and lung specimens on day 28 after administration of BLM were histopathologically examined. When mice were pretreated with IL-1 at 0.5 hr or 6 hr before the administration of BLM, changes in all the parameters were significantly suppressed. The results indicate that IL-1 pretreatment protects mice from BLM-induced pneumonitis and its effects are time-dependent.

The effects of PDTC on interleukin-1β-induced nitric oxide production in chondrocytes

In order to find new drugs to inhibit nitric oxide (NO) production, the effects of pyrrolidine dithiocarbamate (PDTC), a nuclear factor-kappa B (NF-kappaB) inhibitor, on recombinant human interleukin-1beta (rhIL-1beta)-induced NO production in chondrocytes were investigated. Rat chondrocytes were isolated and cultured, divided into control, P0, P1, P2, P3 and P4 groups. The chondrocytes in the P0, P1, P2, P3 and P4 groups were treated with different concentrations of PDTC (0, 3, 10, 30, and 50 micromol/L respectively) for 1 h and then incubated with 5 U/mL rhIL-1beta for 24 h. NO assay kit and RT-PCR were used to detect the NO content and the iNOS mRNA expression in the chondrocytes. The expression level of iNOS mRNA in control, P0, P1, P2, P3 and P4 groups was 0.02+/-0.01, 1.24+/-0.13, 1.21+/-0.14, 0.61+/-0.11, 0.40+/-0.09, 0.21+/-0.06, and the relative content of NO was 15.8+/-2.7, 100+/-14.8, 92.6+/-9.3, 68.3+/-14.2, 27.5+/-9.8, 19.8+/-3.6, respectively. In the P0, P1, P2, P3 and P4 groups, the expression of iNOS mRNA and NO production were significantly increased as compared with those in the control group. As compared with the P0 group, the expression of iNOS mRNA and NO content in control group were lower. In the P2, P3 and P4 groups, PDTC could significantly inhibit the expression of iNOS and NO production induced by rhIL-1beta in a concentration-dependent manner. It is suggested that PDTC can inhibit NO production and iNOS mRNA expression induced by IL-1beta, which may provide an alternative method for the treatment of osteoarthritis.

Effect of human intestinal macrophages on immunoglobulin production by human intestinal mononuclear cells isolated from patients with inflammatory bowel disease

The effect of macrophages on spontaneous immunoglobulin production by isolated human intestinal mononuclear cells (MNC) is unknown. Depletion of macrophages by adherence to fibronectin or by panning with macrophage-specific monoclonal antibody 3C10 lead to a significant reduction in IgA. IgG and IgM production by intestinal MNC from both normal (n = 10) and inflammatory bowel disease (IBD) (n = 13) mucosa. The reduction in immunoglobulin produced by macrophage-depleted intestinal MNC was greater in IBD patients than in normal controls. There was a significant correlation (r = 0.816, P less than 0.001) between the percentage of macrophages depleted by panning with 3C10 and the reduction in IgG produced by macrophage-depleted intestinal MNC. Addition of either fibronectin-adherent cells or the supernatant from these macrophage-enriched cells enhanced immunoglobulin production in a dose-dependent fashion. A greater increase in IgG production by macrophage-depleted cells was seen when cultured with supernatant from inflamed IBD mucosal cells, compared with that from normal mucosal cells. The soluble factor(s) responsible in the supernatant was acid and heat susceptible but was not affected by freezing and thawing. Addition of recombinant human interleukin-1 beta or human interferon-gamma to cell cultures did not influence immunoglobulin production. Thus, human intestinal macrophages enhance spontaneous immunoglobulin production by isolated intestinal MNC by secreting soluble factor(s) which remain to be fully characterized.

Mouse monoclonal anti–human thrombomodulin antibodies bind to and activate endothelial cells through NF‐κB activation in vitro

To clarify whether mouse monoclonal antibodies (mAb) against human thrombomodulin (TM), which react with human TM present on the endothelial cell (EC) surface, have anti-endothelial cell antibody (AECA) activity and influence antiinflammatory properties of human TM expressed on the EC surface in vitro. Three preparations of mouse mAb against human TM that react with different sites of the human TM epidermal growth factor-like domain were tested for their ability to 1) bind to ECs, 2) modulate cytokine secretion from ECs, EC adhesion molecule expression, and neutrophil adhesion to ECs, and 3) stimulate nuclear translocation of NF-kappaB through the degradation of cytoplasmic IkappaB in ECs. Recombinant human interleukin-1beta (IL-1beta) was used as a positive control, and mouse IgG1 and mouse IgG2a were used as negative controls. The 3 preparations of mouse mAb against human TM that bind to unfixed EC monolayers enhanced IL-6 and IL-8 secretion from ECs, up-regulated expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on EC monolayers, and enhanced neutrophil adhesion to ECs to a degree similar to that observed with IL-1beta stimulation, but they did not induce the secretion of tumor necrosis factor alpha or IL-1beta from ECs throughout the incubation period. The 3 preparations stimulated nuclear translocation of NF-kappaB through the degradation of cytoplasmic IkappaB. Mouse IgG1 and mouse IgG2a did not exhibit such effects. These results suggest the possibility that AECA can react with antigens such as TM that are present on the EC surface and activate ECs. Such events on ECs may lead to vascular inflammation and damage in patients with connective tissue diseases and vasculitis in which AECA are present.

Transiently enhanced LPS-induced fever following hyperthermic stress in rabbits

Hyperthermia has been shown to induce an enhanced febrile response to the bacterial-derived endotoxin lipopolysaccharide (LPS). The aim of the present study was to test the hypothesis that the enhanced LPS-induced fever seen in heat stressed (HS) animals is caused by leakage of intestinal bacterial LPS into the circulation. Male rabbits were rendered transiently hyperthermic (a maximum rectal temperature of 43 degrees C) and divided into three groups. They were then allowed to recover in a room at 24 degrees C for 1, 2 or 3 days post-HS. One day after injection with LPS, the post-HS rabbits exhibited significantly higher fevers than the controls, though this was not seen in rabbits at either 2 or 3 days post-HS. The plasma levels of endogenous LPS were significantly increased during the HS as compared to those seen in normothermic rabbits prior to HS. LPS fevers were not induced in these animals. One day post-HS, rabbits that had been pretreated with oral antibiotics exhibited significantly attenuated LPS levels. When challenged with human recombinant interleukin-1beta instead of LPS, the 1-day post-HS rabbits did not respond with enhanced fevers. The plasma levels of TNFalpha increased similarly during LPS-induced fevers in both the control and 1-day post-HS rabbits, while the plasma levels of corticosterone and the osmolality of the 1-day post-HS rabbits showed no significant differences to those seen prior to the HS. These results suggest that the enhanced fever in the 1-day post-HS rabbits is LPS specific, and may be caused by increased leakage of intestinal endotoxin into blood circulation.

Human intestinal epithelial and smooth muscle cells are potent producers of IL-6.

BACKGROUND: Interleukin-6 (IL-6), a pluripotent cytokine, has traditionally been considered the product of proinflammatory cells. However, many other cell types have been shown to produce IL-6. Since intestinal inflammation is commonly associated with a vigorous systemic inflammatory response, we hypothesized that intestinal epithelial and smooth muscle cells might contribute to that response by producing IL-6. We therefore studied the capacity of differentiated human intestinal epithelial and smooth muscle cell lines to produce IL-6 in response to various proinflammatory stimuli. MATERIALS AND METHODS: CCL-241, a human intestinal epithelial cell line, and HISM, a human intestinal muscle cell line, were grown to confluency and then treated for 24 h with various concentrations of lipopolysaccharide, Clostridium difficile culture extract containing both toxin A and toxin B, recombinant human tumor necrosis factor-alpha (TNF-alpha), or recombinant human interleukin-1 beta (IL-1beta). Supernatants were then collected for IL-6 determination using an enzyme-linked immunosorbent assay. Cell numbers were determined using a Coulter counter. For comparison, parallel studies were performed using phorbol ester-primed U-937 and THP-1 human macrophage cell lines. RESULTS: Both human intestinal epithelial and smooth muscle cells produced IL-6 under basal conditions. In HISM cells, but not in CCL-241 cells, IL-6 release was increased slightly by treatment with C. difficile culture extract containing both toxin A and toxin B and with lipopolysaccharide. In both cell lines, IL-6 production was profoundly stimulated by treatment with IL-1beta and less so with TNF-alpha. Combinations of high-dose TNF-alpha and IL-1beta may have a slightly additive, but not synergistic, effect on IL-6 release. The amount of IL-6 produced by IL-1-stimulated intestinal cell lines was 70-fold higher than that produced by stimulated macrophage cell lines. CONCLUSIONS; Both intestinal epithelial and smooth muscle cells demonstrate the ability to release significant amounts of IL-6. The profound response to IL-1beta and TNF-alpha stimulation by both cell lines suggests that human intestinal parenchymal cells, influenced by paracrine mediators liberated from proinflammatory cells, might significantly contribute to the overall systemic inflammatory response by producing IL-6.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Normal and SV40 transfected human peritoneal mesothelial cells produce IL-6 and IL-8: implication for gynaecological disease.

High levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have been demonstrated in the peritoneal fluid of benign and malignant gynaecological disease. Peritoneal monocytes and macrophages, endometrial cells, endometrial and peritoneal stromal cells and tumour cells produce these cytokines in vitro. To investigate whether normal human peritoneal mesothelial cells (HPMC) produce IL-6 and IL-8, HPMC were isolated from omental biopsies. Primary HPMC (P-HPMC) were transfected with pSV3-neo encoding SV40 large T antigen (T-HPMC) to generate sufficient cells. T-HPMC preserved the characteristics of P-HPMC as assessed by phase contrast microscopy, electron microscopy, immunocytochemistry and flow cytometry (FACS) analysis. T-HPMC retained a stable phenotype up to passage 14-19, whereas P-HPMC proliferated poorly and became senescent by passage 4-6. T-HPMC and P-HPMC constitutively expressed IL-6 and IL-8 at both protein and mRNA level. IL-6 and IL-8 production was stimulated by recombinant human interleukin-1beta (hIL-1beta) or human tumour necrosis factor-alpha (hTNF-alpha) alone in a dose-dependent manner. Moreover, hIL-1beta or hTNF-alpha up-regulated IL-6 and IL-8 gene expression as determined by competitive PCR. In contrast, human interferon-gamma (hIFN-gamma) or lipopolysaccharide (LPS) showed no effect. These data indicate that (1) T-HPMC lines mimic the morphological and functional features of P-HPMC, (2) P-HPMC and T-HPMC constituitively produce IL-6 and IL-8, which is enhanced by hIL-1beta and hTNF-alpha and (3) HPMC in vivo may participate in the pathogenesis of benign and malignant gynaecological disease.

Inhibition of ATP-Binding Cassette Transporter Downregulates Interleukin-1β-Mediated Autocrine Activation of Human Dermal Fibroblasts

Fibroblasts constitute an important source of cytokines during inflammatory processes in the skin. Interleukin-1 is a potent, pleiotropic cytokine that is induced in activated human dermal fibroblasts. Interleukin-1 further induces many inflammatory mediators, including the chemokine interleukin-8. As fibroblasts express both interleukin-1 and the interleukin-1 receptor complex, the cellular response may be enhanced by autocrine activation. Interleukin-1alpha and interleukin-1beta lack a signal peptide and are translocated at the plasma membrane using an alternative secretory pathway, which may involve ATP-binding cassette transporter proteins. We hypothesize that inhibition of this pathway prevents secretion of interleukin-1, thereby downregulating interleukin-1-dependent autocrine induction of interleukin-8. We used the ATP-binding cassette 1 transporter inhibitor glybenclamide, which has been previously shown to block interleukin-1beta secretion in human monocytes. Using enzyme-linked immunosorbent assay, we assessed the effect of glybenclamide on interleukin-8 production in human dermal fibroblasts. In interleukin-1beta-transfected human dermal fibroblasts, interleukin-8 was induced through an autocrine activity of interleukin-1beta. Glybenclamide disabled this activation loop and significantly reduced interleukin-8. In human dermal fibroblasts that were stimulated with tumor necrosis factor alpha to reach high interleukin-1 expression levels, glybenclamide similarly suppressed interleukin-8. In contrast, glybenclamide did not affect interleukin-8 production in cells stimulated with interleukin-1 only. Glybenclamide did not affect caspase-1 in fibroblasts, which was expressed as an inactive precursor form, irrespective of treatments with tumor necrosis factor alpha and/or glybenclamide. Using overexpressing, interleukin-1-transfected COS-1 cells, inhibition of interleukin-1alpha and interleukin-1beta secretion was directly demonstrated on Western blots. These results are consistent with glybenclamide preventing externalization of interleukin-1 and subsequent autocrine induction of interleukin-8 in human dermal fibroblasts. Acting through such a mechanism, ATP-binding cassette transporter inhibitors may downregulate inflammation locally.

Prostaglandin E2 Regulates the Level and Stability of Cyclooxygenase-2 mRNA through Activation of p38 Mitogen-activated Protein Kinase in Interleukin-1β-treated Human Synovial Fibroblasts

The p38 MAPK mediates transcriptional and post-transcriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E(2) (PGE(2))-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1 beta-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE(2) release following a recombinant human (rh) IL-1 beta signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE(2) completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1 beta-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1 beta stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1 beta for 3-4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE(2), unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE(2) monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE(2). Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that IL-1 beta increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE(2)/p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE(2) release by rhIL-1 beta is primarily the result of PGE(2)-dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase.

Expression of macrophage migration inhibitory factor during Pseudomonas keratitis

Macrophage migration Inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine, and has been shown to play a role in the regulation of neutrophil chemokines and angiogenesis. Corneal epithelial and endothelial cells have been shown to express MIF. This study evaluated the expression of MIF during Pseudomonas keratitis in mice and in vitro using a corneal epithelial cell line. Three strains of P. aeruginosa, 6294 (invasive strain), 6206 (cytotoxic strain) and Paer1 (non-infectious strain) were used. Both cytotoxic and invasive strains were isolated from human corneal ulcers and the Paer1 strain was isolated from a non-infectious condition. Following challenge in mouse corneas or a corneal epithelial cell line, corneal homogenates or lysed corneal epithelial cells were used to isolate RNA. Migration inhibitory factor mRNA expression in the mouse cornea or human corneal epithelial cells was examined by reverse transcription-polymerase chain reaction analysis, and was found to be expressed as early as 4 h after the injury (scratch controls) or infection in the mouse corneas. Migration inhibitory factor mRNA in scratch controls and Paer1-inoculated corneas showed peak levels at 4 h post-challenge and this dropped by 24 h post-challenge. Corneas challenged with invasive and cytotoxic strains showed peak expression 24 h post-challenge. Migration inhibitory factor mRNA levels were significantly higher in invasive and cytotoxic strain inoculated corneas compared to Paer1 inoculated corneas. Challenging the corneal epithelial cell line with Pseudomonas 6294 and 6206 strains induced peak expression at 8 h and levels were decreased by 12 h. Epithelial cells inoculated with recombinant human interleukin-1beta protein induced very high levels of MIF mRNA at all time points compared to infected and control corneal epithelial cells. High expression of MIF in the infected corneas suggests that it may have a role in the pathogenesis of corneal disease induced by invasive and cytotoxic strains of P. aeruginosa.

Interleukin-1beta-induced prostaglandin E2 production in human myometrial cells: role of a pertussis toxin-sensitive component.

The objective of this study was to evaluate the possible role of pertussis toxin (PTX)-sensitive G-protein(s) in interleukin-1beta (IL-1) signaling in human myometrial cells (HMC). Primary cultures of HMC were stimulated with human recombinant IL-1 alone or in combination with PTX. Prostaglandin (PG) E2 in the medium was measured by radioimmunoassay, cyclooxygenase type 2 (Cox-2) and IkappaB by western analysis, and the activities of two members of the mitogen-activated protein kinase (MAPK) family of enzymes, ERK-2 and JNK, by the phosphorylation of appropriate substrates. IL-1 increased PGE2 output during an 18-hr long incubation by 21.7-fold (n = 5 experiments). This increase was inhibited by 57% after pretreatment overnight with PTX. IL-1-induced expression of Cox-2 protein was also suppressed to a similar degree in PTX-treated HMC cultures. Degradation of the nuclear factor kappa B (NF-kappaB)-inhibiting protein (IkappaB), a critical step in IL-1 signaling to the nucleus, was significantly inhibited by PTX, as was IL-1-induced activation of ERK-2 and JNK. It is suggested that the occupied IL-1 receptor-generated signal in HMC is transmitted by multiple pathways. One is coupled to a PTX-sensitive G-protein upstream from the MAPK phosphorylation cascade. This, in turn, may interact with another signaling pathway, the activation of NF-kappaB, via the phosphorylation of the IkappaB kinase complex.

Gonadotropin-releasing hormone messenger ribonucleic acid and protein expression in Vero cells.

Interleukin-1 (IL-1) is a major regulator of local cellular interactions during embryonic implantation. We hypothesized that gonadotropin-releasing hormone (GnRH) may also play a role in the embryonic/epithelial dialogue during early implantation. To examine this hypothesis, we examined the ability of IL-1 to regulate GnRH mRNA and protein expression in Vero cells. Viable Vero cells (1 x 10(5)/well) were cultured in multiple-well tissue culture plates for in vitro studies and in 4-well chamber slides for immunohistochemical study. Confluent Vero cells were cultured with increasing concentrations of recombinant human IL-1 beta for an additional 24 hr. Vero cell expression of GnRH and GnRH receptor mRNAs was measured with polymerase chain reaction (PCR) and nested PCR, respectively. GnRH protein expression was validated by immunohistochemistry study. The quantitative level of GnRH mRNA expression regulated by IL-1 beta in Vero cells was determined by quantitative competitive PCR (QC PCR) with standard curve methodology. RT-PCR revealed beta-actin, GnRH, and GnRH receptor mRNA expression in Vero cell cultures. Immunostaining confirmed the presence of GnRH protein in Vero cells. Quantitative PCR demonstrated IL-1 beta up-regulation of Vero cell GnRH mRNA expression (p < 0.05). These results suggest that Vero cell mRNA and protein expression of GnRH may play a substantial role in early embryo/epithelial dialogue during embryo coculture, with an embryotrophic effect due to expression of GnRH by Vero cells.

Cyclic tensile strain suppresses catabolic effects of interleukin-1beta in fibrochondrocytes from the temporomandibular joint.

To discern the effects of continuous passive motion on inflamed temporomandibular joints (TMJ). The effects of continuous passive motion on TMJ were simulated by exposing primary cultures of rabbit TMJ fibrochondrocyte monolayers to cyclic tensile strain (CTS) in the presence of recombinant human interleukin-1beta (rHuIL-1beta) in vitro. The messenger RNA (mRNA) induction of rHuIL-1beta response elements was examined by semiquantitative reverse transcriptase-polymerase chain reaction. The synthesis of nitric oxide was examined by Griess reaction, and the synthesis of prostaglandin E2 (PGE2) was examined by radioimmunoassay. The synthesis of proteins was examined by Western blot analysis of the cell extracts, and synthesis of proteoglycans via incorporation of 35S-sodium sulfate in the culture medium. Exposure of TMJ fibrochondrocytes to rHuIL-1beta resulted in the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), which were paralleled by NO and PGE2 production. Additionally, IL-1beta induced significant levels of collagenase (matrix metalloproteinase 1 [MMP-1]) within 4 hours, and this was sustained over a period of 48 hours. Concomitant application of CTS abrogated the catabolic effects of IL-1beta on TMJ chondrocytes by inhibiting iNOS, COX-2, and MMP-1 mRNA production and NO, PGE2, and MMP-1 synthesis. CTS also counteracted cartilage degradation by augmenting expression of mRNA for tissue inhibitor of metalloproteinases 2 that is inhibited by rHuIL-1beta. In parallel, CTS also counteracted rHuIL-1beta-induced suppression of proteoglycan synthesis. Nevertheless, the presence of an inflammatory signal was a prerequisite for the observed CTS actions, because fibrochondrocytes, when exposed to CTS alone, did not exhibit any of the effects described above. CTS acts as an effective antagonist of rHuIL-1beta by potentially diminishing its catabolic actions on TMJ fibrochondrocytes. Furthermore, CTS actions appear to involve disruption/regulation of signal transduction cascade of rHuIL-1beta upstream of mRNA transcription.

Influence of interleukin-1beta and hyaluronan on proteoglycan release from equine navicular hyaline cartilage and fibrocartilage

Proteoglycan (PG) release, in response to recombinant human interleukin-1beta (rh-IL-1beta), was measured in cartilage explants obtained from the equine distal sesamoid bone (navicular bone). Fibrocartilage from the surface of the navicular bone apposing the deep digital flexor tendon and hyaline cartilage from the surface of the navicular bone articulating with the middle phalanx were labelled with 35SO4. Hyaline cartilage from the distal metacarpus was used as a control tissue. Following radiolabel incorporation, the three cartilage types were treated with rh-IL-1beta (100 U/mL) in the presence of hyaluronan (0.2, 2, 20, 200 and 2000 microgram/mL). rh-IL-1beta-Induced PG release was measured by scintillation assay of PG-bound radiolabel. Increases in PG release of 94% (P < 0.01), 101% (P < 0.05) and 122% (P < 0.05), in response to rh-IL-1beta, were noted in fibrocartilage, navicular hyaline cartilage and metacarpal hyaline cartilage, respectively. Hyaluronan (0.2 microgram/mL) significantly reduced rh-IL-1beta-induced PG release in metacarpal hyaline cartilage (P < 0.01). In fibrocartilage and navicular hyaline cartilage, hyaluronan did not reduce PG release and at some concentrations appeared to increase PG release, although this was not statistically significant. These experiments show that (i) fibrocartilage and hyaline cartilage of the navicular bone release PGs in response to rh-IL-1beta, and (ii) hyaluronan does not prevent rh-IL-1beta-induced breakdown of navicular bone cartilage.

Interleukin-1 Beta Promotes Functional Recovery of Crushed Peripheral Nerve

This study was conducted to elucidate the role of the cytokine interleukin-1 beta on peripheral nerve recovery following crush injuries of two different magnitudes. Eighty-eight female rats were divided into four groups. A 5-mm segment of the right sciatic nerve was subjected to a 100-g crush load for 2 hours in the rats in Groups A1 and B1 or to a 15,000-g crush load for 10 minutes in the rats in Groups A2 and B2. The rats in Groups A1 and A2 received 10 microg/100 g body weight human recombinant interleukin-1 beta intraperitoneally 48, 24, and 1 hours before the nerve injury. The rats in Groups B1 and B2 were treated with an equal volume of normal saline solution with identical schedule guidelines. Walking-track tests (sciatic functional index) performed at intervals until 56 days after the crush and measurements of the contractile force of the extensor digitorum longus muscle made until 28 days were used to evaluate functional recovery of the nerve. During the second week after injury, the rats treated with interleukin-1 beta (A1) had an earlier recovery on the walking track than did those treated with saline solution (B1); this difference reached significance (p < 0.05) at day 11. Although Group A2 demonstrated a trend toward earlier recovery compared with Group B2, there was no significant difference between the two groups. After low or high-load crush injury, tetanic contractile forces were greater in the rats treated with human recombinant interleukin-1 beta than in those treated with saline solution. The results suggest that treatment with human recombinant interleukin-1 beta before crush injury can promote function in the peripheral nerve after the injury. However, the mechanisms that underlie the observed beneficial effects are not completely understood and only speculations can be made.

Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase.

1. Human myometrial smooth muscle cells (HMSMCs) in culture were exposed to recombinant human interleukin-1beta (IL-1beta, 10 ng ml-1) for 1 to 24 h. Cyclooxygenase-2 (COX-2) mRNA and protein were rapidly induced, with expression sustained at 24 h. 2. Cycloheximide (10 microg ml-1, 6 h) blocked IL-1beta-induced COX-2 protein expression and super-induced COX-2 mRNA expression. Induction of COX-2 mRNA and protein was blocked by dexamethasone (1 microm, 6 h). 3. IL-1beta-induced COX-2 expression was accompanied by a 3-fold increase of prostaglandin E2 release into the culture medium. 4. IL-1beta induced a transient (5-30 min) activation of p42/44 and p38 mitogen-activated protein kinase (MAPK) enzymes in HMSMCs. Activity of p38 MAPK was monitored by in-gel activity of its substrate MAP kinase-activated protein kinase-2 (MAPKAP kinase-2). Induction of MAPKAP kinase-2 activity was prevented by the p38 MAPK inhibitor SB 203580 (10 microm, 5-30 min). 5. COX-2 protein expression detected after 6 h IL-1beta stimulation was blocked by SB 203580 (10 microM). Exposure of HMSMCs to 10 ng ml-1 IL-1beta for only 30 min induced a level of COX-2 protein expression at 6 h culture similar to that detected in cells exposed to the cytokine for 6 h. 6. Exposure of cells to SB 203580 (10 microM during only the first 30 min of IL-1beta stimulation was effective in blocking COX-2 protein expression assayed after 6 h in culture. 7. This study has established that a transient activation of the p38 MAPK cascade is involved in IL-1beta-stimulated COX-2 expression in human myometrial smooth muscle cells. Induction of COX-2 by IL-1beta in HMSMCs provides support for the hypothesis that autocrine prostaglandin signalling in the myometrium, initiated by elevated intrauterine cytokine concentrations, plays a role in regulating myometrial contractility during labour.

Interleukin-1 beta promotes functional recovery of crushed peripheral nerve.

This study was conducted to elucidate the role of the cytokine interleukin-1 beta on peripheral nerve recovery following crush injuries of two different magnitudes. Eighty-eight female rats were divided into four groups. A 5-mm segment of the right sciatic nerve was subjected to a 100-g crush load for 2 hours in the rats in Groups A1 and B1 or to a 15,000-g crush load for 10 minutes in the rats in Groups A2 and B2. The rats in Groups A1 and A2 received 10 microg/100 g body weight human recombinant interleukin-1 beta intraperitoneally 48, 24, and 1 hours before the nerve injury. The rats in Groups B1 and B2 were treated with an equal volume of normal saline solution with identical schedule guidelines. Walking-track tests (sciatic functional index) performed at intervals until 56 days after the crush and measurements of the contractile force of the extensor digitorum longus muscle made until 28 days were used to evaluate functional recovery of the nerve. During the second week after injury, the rats treated with interleukin-1 beta (A1) had an earlier recovery on the walking track than did those treated with saline solution (B1); this difference reached significance (p < 0.05) at day 11. Although Group A2 demonstrated a trend toward earlier recovery compared with Group B2, there was no significant difference between the two groups. After low or high-load crush injury, tetanic contractile forces were greater in the rats treated with human recombinant interleukin-1 beta than in those treated with saline solution. The results suggest that treatment with human recombinant interleukin-1 beta before crush injury can promote function in the peripheral nerve after the injury. However, the mechanisms that underlie the observed beneficial effects are not completely understood and only speculations can be made.