IL-1β In Supernatant Research Articles

Activation of inflammasome by attenuated Salmonella typhimurium in bacteria-mediated cancer therapy.

Escherichia coli and attenuated Salmonella both naturally accumulate in a tumor mass, yet have distinct therapeutic efficacy: the E. coli K-12 strain (MG1655) cannot induce as significant a tumor suppression as attenuated Salmonella typhimurium, despite similar levels of accumulation in the tumor. To elucidate the mechanism of the robust antitumor effect of S. typhimurium, the cytokine profiles elicited by bacterial colonization in tumors were analyzed. C57BL/6 mice bearing MC38 tumors were injected with Salmonella or MG1655 in the tail vein. Tumors were collected 3 days post-infection and homogenized. Inflammasome-related signals were measured by real-time PCR, ELISA and western blot analysis. Only attenuated Salmonella triggered significant levels of the inflammatory cytokine IL-1β in the tumor, whereas tumor growth was significantly suppressed. In addition, transcript levels of the core molecules of inflammasome signaling, IPAF, NLRP3 and P2X7, were significantly elevated only in Salmonella-treated tumors. Upon direct interaction between Salmonella and BMDM, BMDM expressed inflammasome-related proteins such as NLRP3, IPAF and caspase-1 p10, and secreted a significant amount of IL-1β in supernatants. Coincubation assays with BMDM and Salmonella-treated MC38 cells (damaged cancer cells) revealed secretion of IL-1β only when TLR4 and inflammasome were activated by both LPS and damaged cancer cells. ATP released from damaged cancer cells was also identified as a mechanism of NLRP3 activation. In conclusion, Salmonella activate the inflammasome pathway using damage signals released from cancer cells and through direct interaction with macrophages.

Paeoniflorin down-regulates ATP-induced inflammatory cytokine production and P2X7R expression on peripheral blood mononuclear cells from patients with primary Sjögren's syndrome.

This study determined the effects of paeoniflorin (PF) on the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2X7R) expressed on peripheral blood mononuclear cells (PBMCs) and production of ATP-induced pro-inflammatory cytokines released by PBMCs in patients with primary Sjögren's syndrome (pSS). The pharmacological functions and cytotoxic effects of PF were dose dependent in PBMCs from 20 newly diagnosed pSS patients and 20 normal individuals. The optimum dose of PF was 100μM. PF significantly down-regulated the production of interleukin (IL)-1β and IL-6 from pSS PBMCs, and significantly inhibited ATP-induced expression of P2X7R, that might contribute to reduced IL-1β and IL-6. mRNA and protein levels of P2X7R on pSS PBMCs were significantly higher than in normal individuals (p=0.03, p<0.001). When PBMCs from subjects were stimulated in vitro with ATP in the presence of PF, P2X7R mRNA and protein levels were decreased significantly (p<0.001, p<0.001, respectively versus ATP group) in the pSS. Supernatant IL-1β and IL-6 levels were significantly lower in the PF group compared with ATP group (p<0.001, p<0.001). We show for the first time that PF-mediated reduction of IL-1β and IL-6 was due in part to the reduced expression and activation of the ATP sensor P2X7R on pSS PBMCs, indicating that PF might be useful for the management of pSS via down-regulating P2X7R expression. Thus, PF may provide a new therapeutic approach to regulate P2X7R-mediated pathologic responses of pSS.

Cyr61 participates in the pathogenesis of rheumatoid arthritis by promoting proIL-1β production by fibroblast-like synoviocytes through an AKT-dependent NF-κB signaling pathway

IL-1β plays a major role in the development of rheumatoid arthritis (RA). We previously showed that Cyr61 participates in RA pathogenesis as a proinflammatory factor. Here, we found that the levels of IL-1β and Cyr61 were higher in RA SF than in osteoarthritis (OA) SF. IL-1β mRNA and proIL-1β protein levels were remarkably increased in Cyr61-stimulated FLS; however, IL-1β was hardly detectable in the supernatant. We also found that the level of adenosine triphosphate (ATP) in SF and ST was significantly increased in RA patients and that the level of IL-1β in supernatants from Cyr61-activated FLS increased significantly when we added exogenous ATP to the culture. Mechanistically, Cyr61 induced proIL-1β production in FLS via the AKT-dependent NF-κB signaling pathway, and ATP caused Cyr61-induced proIL-1β to generate IL-1β in a caspase-1-dependent manner. Our results reveal a novel role of Cyr61 in RA that involves the promotion of proIL-1β production in FLS.

Cell death of THP-1 induced by puried Rv3671c protein of tuberculosis and the detection of TNF-α and IL-1β in Mycobacterium tuberculosis

To assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis (M.tuberculosis). The gene encoding Rv3671c protein of M.tuberculosis was cloned into pET-28a vector and then expressed in Escherichia coli. The Rv3671c was purified with Ni-NTA affinity and ion exchange chromatography. The detection of protein concentration was by Lowry method.THP-1 cell was stimulated with Rv3671c protein and cells were analyzed by Hochest staining under fluorescence microscopy to assay cell death (apoptosis and necrosis). TNF-α and IL-1β were detected by ELISA at each stimulating time. The Rv3671c protein of M.tuberculosis was successfully expressed in Escherichia coli. The purity of recombinant Rv3671c protein was 95%, and the protein concentration was up to 0.4 mg/ml. The nucleus of THP-1 was isolated and necrosis-like under fluorescence when cells were stimulated by Rv3671c protein. The levels of TNF-α and IL-1β in supernatant were 19 000 and 16 500 pg/ml respectively, and were significantly higher than control cells with the levels of 2100 and 3800 pg/ml separately. The necrosis of THP-1 cells could be stimulated by Rv3671c protein of M.tuberculosis and it was probably associated with high cytokines TNF-α and IL-1β levels.

P044 Pollutant particulate matter acts on bronchial epithelial and dendritic cells to promote a pro-inflammatory environment

Introduction Airborne particulate matter (PM) from ambient pollution is believed to exacerbate asthmatic disease, although the mechanisms underpinning this are unclear. Dendritic cells (DC) orchestrate immune responses in the airways, and lie directly underneath human bronchial epithelial cells (HBEC) that line the airways. HBEC are the first cells to encounter PM entering the lungs. This study explores the relationship of DC and HBEC following exposure to foreign noxious material such as PM. Methods Primary PM samples were obtained from the KCL Environmental Research Group. Pollutants were extracted from filters set up within the London Low Emission Zone to monitor the association of PM composition with firework activity. NIST SRM1648a (standard reference material) was obtained from National Institute of Standards and Technology. Due to the limit of primary sample supply, NIST was used as a surrogate prototypical urban ambient pollution PM. Oxidative potentials of PM were quantified by depletion rate of 2 μg/ml ascorbic acid.Cytokines TNF-α, GM-CSF, IL-6, IL-8, IL-10 and IL-1β in supernatants were determined by Cytokine Bead Array (CBA). Viability was measured using propidium iodide and Annexin V staining. Myeloid dendritic cells (Cd11c+) were isolated from peripheral blood mononuclear cells. Dendritic cells were cultured alone, or together with epithelial cells. Supernatants are collected after 24 h. CD80 surface staining was analysed by flow cytometry. Results The capacity of individual primary PM extracts to stimulate IL-6, IL-8 and GM-CSF production by primary bronchial epithelial cells strongly correlate with the oxidative potential of individual PM extracts, as measured by ascorbic depletion rates. Furthermore, by inducing antioxidants using sulforaphane, stimulation of these cytokines by PM was suppressed due to enhanced protection against oxidative stress. Dendritic cells alone produce a similar pro-inflammatory response to a high oxidative potential PM. In co-culture, stimulation with PM greatly increases the cytokine production compared to individually cultured epithelial cells or mDCs. IL-10 and IL-1β, which were both non-detectable in epithelial cells are also increased in dendritic cells co-cultured with HBEC and stimulated with PM. Conclusion Oxidative stress has been identified as a major determinant of the capacity of PM to induce pro-inflammatory cytokines in primary human bronchial epithelial cells. Epithelial cells interact with dendritic cells in co-cultures stimulated with PM to greatly potentiate pro-inflammatory responses.

Interleukin-1β Inhibition Prevents Choroidal Neovascularization and Does Not Exacerbate Photoreceptor Degeneration

The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

Ethanol Changes Gene Expression of Transcription Factors and Cytokine Production of CD4+ T-Cell Subsets in PBMCs Stimulated With LPS

Acute ethanol intoxication has the potential to alter immune reactivity by various pathways. The aim of this study was to investigate T-helper cell subsets transcription factors and cytokines in human peripheral blood mononuclear cells (PBMCs) following a single dose of lipopolysaccharide (LPS) with or without ethanol exposure. Human PBMCs were cultured in the presence of 100 mM ethanol and/or 100 ng/ml LPS for various time periods (1, 3, 8, and 24 hours) and analyzed for the kinetics of gene expression by quantitative real-time PCR of selected transcription factors (T-bet, GATA3, Foxp3, and RORγt) and cytokines (TNF-α, IL-6, IL-10, and IFN-γ). The proportion of Th17 and Treg cells was identified 24 hours after treatment with ethanol and LPS by multiparameter flow cytometry. Viability and amount of dead cells were analyzed after 24 and 48 hours by MTT assay and flow cytometry. Following LPS challenge, gene expression of Foxp3 increased, whereas RORγt decreased after 3 hours, GATA3 decreased within 1 hour, whereas expression of T-bet did not change at any time. Gene expression of TNF-α, interferon-γ (IFN-γ), and IL-6 peaked after 3 hours, expression of IL-10 peaked after 8 hours. Ethanol suppressed the LPS-induced gene expression of Foxp3, RORγt, and T-bet after 8 hours, expression of TNF-α and IFN-γ was also suppressed after 3 and 8 hours. Markers of inflammation including TNF-α and IL-1β in supernatant of PBMCs were significantly decreased, while levels of IL-10 and IL-6 remained unchanged following ethanol exposure. Furthermore, ethanol-treated cells alone or in combination with LPS had significantly fewer IL-17- and IFN-γ-secreting CD4+ T cells but constant proportion of Treg cells when compared to control cells. Proliferation and viability of the cells were not influenced under these conditions. Alcohol interferes with the kinetics of Foxp3, RORγt, and T-bet gene expression and the production of TNF-α and IL-1ß and influences the balance of Treg/Th17 cells following LPS exposure.

The Activation of STAT3 Contributes to the Effect of Autocrined G-CSF On the Proliferation of HL60 Cells Induced by ATRA.

Abstract 4565 BackgroundIn previous works, we detected several significant cytokines in the serum of patients with APL after treatment with ATRA, and the results revealed a close association of serum G-CSF and the number of promyelocytes or more mature granulocytes. Some of the results also indicate that ATRA in vitro has a conspicuous effect on the expression of some cytokines, such as IL-1β, IL-8, TNF, G-CSF, and so on. At least, it is showed that cytokines in serum or supernatant secreted by APL cells perhaps played an important role in the genesis of leukocytosis. However, the role of G-CSF in proliferation of APL cells has not yet been explained clearly. In the present study, we analyse the secretion and expression of cytokines in APL patients who has hyperleukocytosis, study the relationship between cytokines and leukocytosis, and detect the signal transduction pathway of cytokines. ObjectiveTo detect the signal pathway of IL-1β and G-CSF secreted by primary acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). MethodsPrimary APL cells from ATRA-treated patients were cultured in RPMI 1640, then the level of supernatant IL-1β and G-CSF were detected by ELISA assay. The expression of IL-1β or G-CSF mRNA was measured by RT-PCR, and the level of STAT1α and STAT3 evaluated by Western blot. ResultsTreated with ATRA, the peripheral blood leucocytes of patients with APL was obviously up-regulated, and peaked at 12 days averagely, of which mainly were myelocytes and metamyelocytes. The secretion of IL-1β or G-CSF in supernatant was up-regulated (P<0.05). RT-PCR assay confirmed that in the primary APL cells, with higher secretion of IL-1β and/or G-CSF, the expression of IL-1β and/or G-CSF was up-regulated, correspondingly. Cytokines and signal transduction factors analysis indicated that the expression of STAT1α protein increased in all the patients, irrelevant to the expression of IL-1β or G-CSF, but the increase of STAT3 expression was consistent with the expression and secretion of G-CSF. ConclusionThe higher expression of G-CSF in primary APL cells induced by ATRA contributes to the activation of STAT3. Disclosures:No relevant conflicts of interest to declare.

Changes in endocrine and immune responses of neonatal pigs exposed to a psychosocial stressor

The aim of the study was to determine the effects of a single social isolation (4 h) of piglets on immediate changes in stress hormones and immune responses at 7, 21 or 35 days of age. This social stressor caused an increase in plasma ACTH and cortisol concentrations and a decrease in plasma TNF-α. The percentage of CD8 + cells increased and the CD4 + cell percentage decreased, resulting in decreasing CD4 +/CD8 + ratio. The observed changes were consistent for all days studied. Further, the isolation treatment resulted in diminished LPS-stimulated IL-1β and IL-10 production in whole-blood cultures. In isolated piglets, positive correlations were estimated between changes in percentage of CD8 + cells and cortisol, and negative ones between changes in plasma TNF-α and culture supernatant IL-1β with ACTH and cortisol. The data suggest that psychosocial stress in neonatal pigs induced immune alterations to maintain adaptive stability, but reflecting also negative emotions experienced by this treatment.

Induction of IL-33 expression and activity in central nervous system glia

IL-33 is a novel member of the IL-1 cytokine family and a potent inducer of type 2 immunity, as mast cells and Th2 CD4+ T cells respond to IL-33 with the induction of type 2 cytokines such as IL-13. IL-33 mRNA levels are extremely high in the CNS, and CNS glia possess both subunits of the IL-33R, yet whether IL-33 is produced by and affects CNS glia has not been studied. Here, we demonstrate that pathogen-associated molecular patterns (PAMPs) significantly increase IL-33 mRNA and protein expression in CNS glia. Interestingly, IL-33 was localized to the nucleus of astrocytes. Further, CNS glial and astrocyte-enriched cultures treated with a PAMP followed by an ATP pulse had significantly higher levels of supernatant IL-1beta and IL-33 than cultures receiving any single treatment (PAMP or ATP). Supernatants from PAMP + ATP-treated glia induced the secretion of IL-6, IL-13, and MCP-1 from the MC/9 mast cell line in a manner similar to exogenous recombinant IL-33. Further, IL-33 levels and activity were increased in the brains of mice infected with the neurotropic virus Theiler's murine encephalomyelitis virus. IL-33 also had direct effects on CNS glia, as IL-33 induced various innate immune effectors in CNS glia, and this induction was greatly amplified by IL-33-stimulated mast cells. In conclusion, these results implicate IL-33-producing astrocytes as a potentially critical regulator of innate immune responses in the CNS.

The effect of lidocaine on neutrophil CD11b/CD18 and endothelial ICAM-1 expression and IL-1β concentrations induced by hypoxia-reoxygenation

Background: Lidocaine has actions potentially of benefit during ischaemia-reperfusion. Neutrophils and endothelial cells have an important role in ischaemia-reperfusion injury. Methods: Isolated human neutrophil CD11b and CD18, and human umbilical vein endothelial cell (HUVEC) ICAM-1 expression and supernatant IL-1β concentrations in response to hypoxia-reoxygenation were studied in the presence or absence of different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL−1). Adhesion molecule expression was quantified by flow cytometry and IL-1β concentrations by ELISA. Differences were assessed with analysis of variance and Student-Newman-Keuls as appropriate. Data are presented as mean ± SD. Results: Exposure to hypoxia-reoxygenation increased neutrophil CD11b (94.33 ± 40.65 vs. 34.32 ± 6.83 mean channel fluorescence (MCF), P = 0.02), CD18 (109.84 ± 35.44 vs. 59.05 ± 6.71 MCF, P = 0.03) and endothelial ICAM-1 (146.62 ± 16.78 vs. 47.29 ± 9.85 MCF, P < 0.001) expression compared to normoxia. Neutrophil CD18 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL−1) treated cells compared to control (71.07 ± 10.14 vs. 109.84 ± 35.44 MCF, P = 0.03). Endothelial ICAM-1 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL−1) treated cells compared to control (133.25 ± 16.05 vs. 146.62 ± 16.78 MCF, P = 0.03). Hypoxia-reoxygenation increased HUVEC supernatant IL-1β concentrations compared to normoxia (3.41 ± 0.36 vs. 2.65 ± 0.21 pg mL−1, P = 0.02). Endothelial supernatant IL-1β concentrations in lidocaine-treated HUVECs were similar to controls. Conclusions: Lidocaine at clinically relevant concentrations decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1β concentrations.

The effect of lidocaine on neutrophil CD11b/CD18 and endothelial ICAM-1 expression and IL-1β concentrations induced by hypoxia–reoxygenation

Lidocaine has actions potentially of benefit during ischaemia-reperfusion. Neutrophils and endothelial cells have an important role in ischaemia-reperfusion injury. Isolated human neutrophil CD11b and CD18, and human umbilical vein endothelial cell (HUVEC) ICAM-1 expression and supernatant IL-1beta concentrations in response to hypoxia-reoxygenation were studied in the presence or absence of different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)). Adhesion molecule expression was quantified by flow cytometry and IL- 1beta concentrations by ELISA. Differences were assessed with analysis of variance and Student-Newman-Keuls as appropriate. Data are presented as mean+/-SD. Exposure to hypoxia-reoxygenation increased neutrophil CD11b (94.33+/-40.65 vs. 34.32+/-6.83 mean channel fluorescence (MCF), P = 0.02), CD18 (109.84+/-35.44 vs. 59.05+/-6.71 MCF, P = 0.03) and endothelial ICAM-1 (146.62+/-16.78 vs. 47.29+/-9.85 MCF, P < 0.001) expression compared to normoxia. Neutrophil CD18 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (71.07+/-10.14 vs. 109.84+/-35.44 MCF, P = 0.03). Endothelial ICAM-1 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (133.25+/-16.05 vs. 146.62+/-16.78 MCF, P = 0.03). Hypoxia-reoxygenation increased HUVEC supernatant IL-1beta concentrations compared to normoxia (3.41+/-0.36 vs. 2.65+/-0.21 pg mL(-1), P = 0.02). Endothelial supernatant IL-1beta concentrations in lidocaine-treated HUVECs were similar to controls. Lidocaine at clinically relevant concentrations decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1beta concentrations.

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.

Role of mononuclear cells of IgA nephropathy on ICAM-1 expression in mesangial cells.

ObjectivesTo investigate the possible role of mononuclear cells and their products in the pathogenesis of IgA nephropathy, in vitro expression of ICAM-1 on cultured mouse mesangial cell(MC) was examined after stimulation with mononuclear cell culture supernatant from patients with IgA nephropathy.MethodsPeripheral blood mononuclear cells (PBMC) were isolated and cultured from 18 patients with primary IgA nephropathy, 8 normal controls and 5 patients with non-IgA nephropathy (FSGS 1, MGN 3, MPGN 1). ICAM-1 expression on cultured mouse MC by TNF-α, IL-1β and culture supernants of PBMC were analyzed using a cell ELISA method. The concentration of IL-1β and TNF-α in culture supernatants was measured by using a commercially available radioimmunoassay kit.ResultsAddition of human recombinant TNF-α induced an increased ICAM-1 expression in a dose-dependent manner. The expression of ICAM-1 was further increased after co-stimulation with TNF-α and IL-1β. Addition of PBMC culture supernatants into mouse MC induced significantly higher expression of ICAM-1 by supernatants from the patients with IgA nephropathy compared with that from normal controls.The concentration of TNF-α and IL-1β in supernatants from the patients with IgA nephropathy was significantly higher than that from those with non-IgA nephropathy.ConclusionTNF-α and IL-1 released from mononuclear cells induced the up-regulation of ICAM-1 expression and this may be related to the immune pathogenesis of IgA nephropathy.

Modulation of interleukin 1β production in macrophages stimulated with lipopolysaccharide by the protein kinase inhibitor staurosporine

Inhibitors of key components of intracellular signaling pathways were used to detect differences in the regulation of the production of interleukin 1β (IL-1β) and tumor necrosis factor α (TNFα) in macrophages activated with lipopolysaccharide (LPS). The protein kinase inhibitor staurosporine caused an increase in the level of IL-1β in supernatants of macrophages stimulated with LPS. Calphostin C and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) dihydrochloride, specific inhibitors of protein kinase C (PKC), also promoted enhancement of IL-1β secretion, but the effect was not as pronounced as that of staurosporine. In contrast, all three substances inhibited TNFα production. Measurement of IL-1β in lysates and supernatants of macrophage cultures indicated that staurosporine effected enhancement of the production of the cytokine in the cellular fraction, the greater portion of which was not secreted. Kinetics of accumulation of IL-1β mRNA and production of the cytokine during a 24 h period showed that enhanced production of IL-1β obtained 24 h after LPS stimulation of macrophages in the presence of staurosporine paralleled the increased levels in mRNA specific for the cytokine.

Analysis of ovine IL-1β production in vivo and in vitro by enzyme immunoassay and immunohistochemistry

A monoclonal antibody (mAb) specific for ovine IL-1β was produced and, in conjunction with a polyclonal rabbit antiserum, used to develop a sensitive enzyme immunoassay (EIA) for ovine interleukin 1β (IL-1β). The mAb neutralised the activity of recombinant ovine IL-1β (rOvIL-1β) and native OvIL-1 in an ovine thymocyte proliferation assay. However, it did not neutralise the biological activity of rOvIL-1β in the murine NOB1 CTLL assay. The mAb did not react with rOvIL-1α, IL-2, IL-4, IL-8, tumor necrosis factor-α, gamma-interferon or recombinant human IL-1β in indirect EIA. Immunohistological staining of activated alveolar macrophages and frozen lymph node sections demonstrated that the mAb detected IL-1β secreted by ovine macrophages (CD11c-positive). The EIA was highly sensitive, detecting less than 50 pg ml −1 of rOvIL-1β and low levels of native IL-1β in supernatants from lipopolysaccharide-stimulated macrophages. The EIA did not detect heat-inactivated IL-1β.