Biphasic production of IL-8 in lipopolysaccharide (LPS)-stimulated human whole blood. Separation of LPS- and cytokine-stimulated components using anti-tumor necrosis factor and anti-IL-1 antibodies.

TNF, IL-1, and IL-6 are integral components of the cytokine cascade released in the response to inflammatory stimuli such as LPS. IL-8 is produced both in response to LPS as well as TNF and IL-1. The early, local production of TNF and IL-1 may therefore contribute to the subsequent expression of IL-8. This hypothesis was tested using LPS-stimulated human whole blood as an ex vivo model of local cytokine production. The production of TNF, IL-1 alpha, IL-1 beta, IL-6, and IL-8 was found to be responsive to a wide range of LPS concentrations (0.1 ng/ml-10 micrograms/ml). These cytokines were first detected between 1 to 4 h post-LPS stimulation, and reached plateau levels after 6 to 12 h. IL-8, however, also displayed a secondary wave of production, with the levels again increasing between 12 to 24 h. The IL-8 present in the plasma after LPS stimulation was biologically active, as assessed by neutrophil chemotaxis. In further studies, addition of anti-TNF and anti-IL-1 neutralizing antibodies, alone and in combination, to LPS-stimulated blood resulted in nearly complete ablation of the secondary phase of IL-8 synthesis at both the levels of protein and mRNA, while leaving the first, LPS-mediated phase of IL-8 synthesis unaffected. This model of cytokine production in human whole blood may reflect the sequence of events in a localized environment of inflammation where both a primary stimulus and the induced early cytokine mediators may serve to elicit multiple, temporally distinct phases of IL-8 production.     

Interleukin-1 (IL-1) receptor blockade reduces endotoxin and Borrelia burgdorferi-stimulated IL-8 synthesis in human mononuclear cells.

Interleukin-1 (IL-1) is a potent stimulator of IL-8 production by fibroblasts and monocytes. In the present study, we asked how much of endotoxin (LPS)-induced IL-8 production by human peripheral blood mononuclear cells was due to IL-1 induced by LPS. Cells were stimulated with either IL-1 beta, LPS, or Borrelia burgdorferi, and total IL-8 was determined by a specific radioimmunoassay. The addition of saturating concentrations of IL-1 receptor antagonist protein (IRAP) reduced the IL-1 beta-, LPS-, and B. burgdorferi-induced IL-8 synthesis by 85, 50, and 40%, respectively. Increasing the concentration of LPS did not affect the reduction in IL-8 synthesis observed in the presence of IRAP. Significant inhibition of the IL-1 beta-induced IL-8 synthesis was observed when IRAP was added 60 or 90 min after IL-1 beta; similarly, IL-8 synthesis after LPS was also reduced by delayed addition of IRAP. These data suggest that the ameliorative effects of IL-1 receptor blockade in models of inflammation and infection may be due, in part, to suppression of IL-1-induced IL-8.     

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1beta and tumor necrosis factor alpha

The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1beta and TNFalpha on the production of IL-8. The stimulatory effect of BK on the IL-1beta- or TNFalpha-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1beta and TNFalpha on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1beta and TNFalpha increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1beta as well as the IL-1beta-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1beta- and TNFalpha-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.     

Differential regulation by IL-4 and IL-10 of radiation-induced IL-6 and IL-8 production and ICAM-1 expression by human endothelial cells.

Radiation exposure results in an inflammatory reaction with acute as well as subacute consequences. Leukocyte infiltration is one of the predominant early histological changes and involves both cytokines and adhesion molecules. Endothelial cells play a key role in this reaction. We have previously shown the increased production of interleukin 6 (IL-6) and IL-8 and the upregulation in intercellular adhesion molecule 1 (ICAM-1) expression by HUVEC following gamma ray exposure. In the present study, we used the cytokines IL-4 and IL-10 to regulate these radiation-induced manifestations. Human umbilical vascular endothelial cells (HUVEC) were treated with IL-4 and IL-10 (50 pg/ml) either before or after 10- Gy irradiation. Three and seven days after irradiation, IL-6 and IL-8 production by HUVEC (either treated or non-treated) was assessed by enzyme-linked immunosorbent assay (ELISA). Our results show that IL-4, when added after irradiation, reversed the radiation-induced increase in IL-8 production, although slightly increased IL-6 production. IL-10 decreased both IL-8 and IL-6 production when added after irradiation. ICAM-1 expression was evaluated 3 days after irradiation by flow cytometry. The radiation-induced upregulation in ICAM-1 expression remained unaffected by the use of IL-4. Altogether, our results show that radiation-induced endothelial cell activation may be ameliorated by IL-4 and/or IL-10, which is of significance in designing strategies for cytokine-mediated intervention and/or therapy of radiation damage.     

Endotoxin and staphylococcus aureus enterotoxin a induce different patterns of cytokines

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-γ and TNF-β. LPS exhibited marked production of IL-1α, IL-1β, TNF-α, IL-6 and IL-8. After LPS stimulation IL-1α, IL-1β, TNF-α and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-β, IL-2, IFN-γ and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours.In contrast, peak production of IL-1α, IL-1β and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-β, TNF-α, IFN-γ and IL-2 was found with peak production 12–48 hours after initiation. Only low amounts of IL-6 were evident.The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections. The data may also imply that different immunomodulating approaches should be considered in life-threatening diseases with the two microbacterial agents.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines.

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-gamma and TNF-beta. LPS exhibited marked production of IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and IL-8. After LPS stimulation IL-1 alpha, IL-1 beta, TNF-alpha and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-beta, IL-2, IFN-gamma and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours. In contrast, peak production of IL-1 alpha, IL-1 beta and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-beta, TNF-alpha, IFN-gamma and IL-2 was found with peak production 12-48 hours after initiation. Only low amounts of IL-6 were evident. The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4)

The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Amnion cell biosynthesis of interleukin-8: regulation by inflammatory cytokines.

The cellular constituents of the placenta are important participants in the recruitment and trafficking of inflammatory cells within the placenta. In infection-induced labor, gestational tissues synthesize and release a variety of inflammatory cytokines whose effects include increased prostaglandin biosynthesis and the initiation of uterine contractions. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, has been recently described as being elevated in the amniotic fluid of mothers with chorioamnionitis. We investigated the biosynthesis of IL-8 by human amnion cells and its regulation by other inflammatory cytokines. Cultured amnion cells obtained from normal term placentae were found to produce IL-8 in response to pathophysiologic concentrations of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). Treatment of amnion cells stimulated by IL-1 beta with cycloheximide resulted in increased IL-8 production, while incubation of IL-1 beta treated amnion cells with actinomycin D resulted in a concentration-dependent decrease in detectable amounts of IL-8. Northern blot analysis of cultured amnion cells stimulated with IL-1 beta demonstrated a rapid increase in IL-8 mRNA which peaked at 2-4 hr. These in vitro results suggest inflammation of gestational tissues in vivo may result in locally produced IL-8 and, in association with other inflammatory mediators, may be important in the pathophysiology of infection-induced labor.     

Interleukin 8 production in whole blood assays: Is interleukin 10 responsible for the downregulation observed in sepsis?

Reduced cytokine production in ex vivo cultures has been regularly reported in patients suffering from sepsis syndrome. Using whole blood assays, we have now demonstrated that in sepsis patients, normal production of IL-8 was achieved with the higher concentration of lipopolysaccharide (LPS; 1 microg/ml) and with heat-killed streptococci, whereas the IL-8 production induced by lower LPS concentration (0.1 microg/ml) was significantly reduced as compared to healthy controls. In contrast, in patients undergoing cardiac surgery associated with cardio-pulmonary bypass, a group of patients with inflammation in the absence of infectious insult, none of the studied IL-8 productions were affected. Among the various anti-inflammatory cytokines known to regulate IL-8 production which we tested (i.e. IL-4, IL-10, IL-13, TGF-beta), IL-10 was the most active inhibitory cytokine in whole blood assays performed with blood samples from healthy subjects. However, its activity was not influenced by the amounts of LPS used. In addition, IL-10 also inhibited the heat-killed streptococci-induced IL-8 production and was the only cytokine to inhibit the release of IL-8 when TNF was added to LPS. It is worth noting that IL-13 which also inhibited the heat-killed streptococci-induced IL-8 production, failed to do so when the TNF production was analysed. Together, these data suggest that while circulating IL-10 in septic patients may be responsible for the hyporeactivity of circulating leukocytes, its presence is not sufficient to explain the observed dysregulation which occurs in septic patients.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

Endothelin-1 increases expression of cyclooxygenase-2 and production of interlukin-8 in hunan pulmonary epithelial cells

Inducible cyclooxygenase (COX-2) and inflammatory cytokines play important roles in inflammatory processes of chronic obstructive pulmonary disease (COPD). Endothelin-1 (ET-1) might be also involved in the pathophysilogical processes in COPD. In the present study, we determined whether ET-1 could regulate the expression of COX-2 and alter the production of interleukin-8 (IL-8) in human pulmonary epithelial cells (A549). Induced sputum samples were collected from 13 stable COPD patients and 14 healthy subjects. The COX-2 protein, ET-1, PGE(2) and IL-8 in these sputum samples were analyzed. A549 cells were incubated with ET-1 in the presence or absence of celecoxib, a selective COX-2 inhibitor. The expression of COX-2 protein in the cell and the amounts of PGE(2) and IL-8 in the medium were measured. The levels of COX-2 protein, ET-1, PGE(2) and IL-8 were significantly increased in induced sputum from COPD patients when compared to healthy subjects. ET-1 increased the expression of COX-2 protein, as well as the production of PGE(2) in A549 cells. Increased production of PGE(2) was inhibited by celecoxib. ET-1 also increased the production of IL-8. Interestingly, ET-1-induced production of IL-8 was also inhibited by celecoxib. These findings indicate that ET-1 plays important roles in regulating COX-2 expression and production of IL-8 in A549 cells. ET-1 mediated production of IL-8 is likely through a COX-2-dependent mechanism.