Prostaglandin E2 stimulates the production of interleukin-6 by neonatal mouse parietal bones

The pleiotropic cytokine interleukin-6 (IL-6) is thought to be involved in bone homeostasis. A number of bone resorbing agents have been shown to induce the release of IL-6 from bone. We wished to determine whether prostaglandin E2 (PGE2), which is a mediator of bone resorption, can elicit the production of IL-6. IL-6 was measured by the proliferative response of B9 hybridoma cells and could be completely neutralised by an anti-IL-6 antibody. Parietal bones from neonatal mice were maintained in culture in the presence of indomethacin (10(-6) M) with or without PGE2. The time course and dose-response to PGE2 of IL-6 production were determined. After 6 h in culture, 10(-8) M PGE2 produced significantly more IL-6 than the controls (P < 0.005). PGE2 (10(-6) M) stimulated the production of a mean of 12.8 ng/ml IL-6 over 6 h. Preincubating bones with indomethacin for 20 h prior to a 6 h culture with indomethacin led to a lowering of the production of IL-6 (mean 1.8 ng/ml) compared to bones cultured without the preincubation period (5.8 ng/ml). When the indomethacin preincubation period was used, a significant increase in IL-6 production was found with 10(-9) M PGE2 (P < 0.005), and 10(-6) M PGE2 caused the production of 39.9 ng/ml IL-6 over 6 h. Stripping endocranial and ectocranial membranes from bones demonstrated the membranes to be the major site of IL-6 production. However, intact bones were required for maximal stimulated IL-6 production.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNgamma) 100 u ml(-1), interleukin-1beta (IL-1beta) 1 u ml(-1) and lipopolysaccharide (LPS) 10 microg ml(-1) induced nitrite formation which could be inhibited by the competitive NOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME). IL-1beta alone (1-50 u ml(-1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNgamma 100 u ml(-1), IL-1beta 1 u ml(-1) and LPS 10 microg ml(-1) to induce both the iNOS and COX-2 pathways, and IL-1beta 3 u ml(-1) to induce COX-2 without iNOS activity. 3. Cells treated with IFNgamma 100 u ml(-1), IL-1beta I u ml(-1) and LPS 10 microg ml(-1) for 48 h either alone, or with the addition of L-NAME (0 to 10(-2) M), demonstrated inhibition by L-NAME of PGE2 (3.61 +/- 0.55 to 0.51 +/- 0.04 pg/l0(4) cells; P<0.001) and nitrite (34.33 +/- 8.07 to 0 pmol/10(4) cells; P<0.001) production. Restoration of the PGE2 response (0.187 +/- 0.053 to 15.46 +/- 2.59 pg/10(4) cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10(-6) M, but with the addition of the NOS substrate L-arginine (0 to 10(-5) M). 4. Cells incubated with IL-1beta 3 u ml(-1) for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10(-4) M), demonstrated increased PGE2 formation (1.23 +/- 0.03 to 2.92 +/- 0.19 pg/10(4) cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 microM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1beta 3 u ml(-1) for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10(-3) M) also demonstrated an increased PGE2 response (2.56 +/- 0.21 to 4.53 +/- 0.64 pg/10(4) cells; P<0.05). 5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.     

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma

Endothelins (ETs) are potent bronchoconstrictor agents postulated to contribute to the pathophysiology of asthma and other respiratory disorders. An increase in both the expression and release of immunoreactive (ir) ETs was reported in bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic patients. We investigated whether dexamethasone (DEX), a potent anti-inflammatory and anti-asthmatic drug, regulates the basal and stimulated release of ETs from guinea-pig cultured tracheal epithelial cells. These airway epithelial cells spontaneously release ET-1 over 24 h. When incubated in the presence of 10(-7) and 10(-6) M DEX for 24 h, basal production of ET-1 decreased by 32 and 29%. Lipopolysaccharide (LPS; 1, 5, 10 micrograms/mL), tumor necrosis factor-alpha (TNF alpha; 5, 10 ng/mL), and interleukin-1 beta (IL-1 beta; 1, 5 ng/mL) significantly increased the basal release of ET-1 after 24 h. When these cells were pretreated with DEX (10(-7) M) for a 24-h period, then incubated in the presence of LPS (10 micrograms/mL), TNF alpha (10 ng/mL), or IL-1 beta (1 ng/mL) for another 24 h, the stimulated release of ET-1 was inhibited by 48, 31, and 38%, respectively. At 10(-6) M, DEX decreased the stimulated release by 45, 37, and 46%, respectively. The present results show that DEX can regulate the basal release and inhibit the pro-inflammatory cytokine-stimulated production of ET-1 from guinea-pig cultured tracheal epithelial cells. They suggest that the beneficial effect of glucocorticoids in asthma may be related to the inhibition of ET synthesis.     

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.     

Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.     

Contribution of IL-1 beta to the enhancement of Campylobacter rectus lipopolysaccharide-stimulated PGE2 production in old gingival fibroblasts in vitro

Campylobacter rectus is associated with adult periodontitis. We previously reported that C. rectus lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in old cells of human gingival fibroblasts (HGFs) is higher than that in young cells. The present study examined whether an enhancement of C. rectus LPS-stimulated interleukin (IL)-1 beta production in old HGFs contributed to the increased production of PGE2. LPS was prepared from C. rectus ATCC33238. HGFs were established from healthy gingiva in three patients, aged 10-12 years. Cellular aging in culture was determined with increasing doubling. The cultured cells were treated with LPS (0.01-10 micrograms/ml), and the amount of IL-1 beta in the medium was measured after a 24 h incubation. The LPS-stimulated IL-1 beta production in each old cell (corresponding to 57-67% of complete life-span) was increased (1.6-2.6 times) compared to that in the young cells (corresponding to 17-20% of the life-span). The IL-1 beta mRNA synthesis in the presence of LPS in the old cells was higher than that in the young cells. The enhancement of LPS-stimulated PGE2 production was inhibited by anti-IL-1 beta antibody and by IL-1 receptor antagonist. These findings suggest that the greater ability of old cells to produce PGE2 in response to C. rectus LPS is due to their greater level of IL-1 beta.     

Effect of PDE4 inhibitors on zymosan-induced IL-8 release from human neutrophils: synergism with prostanoids and salbutamol

1. The activation of neutrophils with particulate stimuli such as zymosan induces the generation of the C-X-C chemokine interleukin (IL)-8. There is evidence that neutrophil derived IL-8 plays an important role in human diseases such as the adult respiratory distress syndrome. In the present study, we examined the effects of cyclic AMP elevating agents on the ability of human neutrophils to generate IL-8 in response to zymosan particles. 2. The PDE4 inhibitor rolipram had limited effect on zymosan-induced IL-8 generation. In contrast, the PDE4 inhibitors RP 73401 and SB 207499 concentration-dependently suppressed IL-8 generation. The potency of these inhibitors was RP 73401 > SB 207499 > rolipram which is correlated with their rank order of potency at inhibiting the catalytic site of purified neutrophil PDE4. Pretreatment of neutrophils with the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast had no effect on IL-8 generation. 3. The prostanoids prostaglandin E1 (PGE1) and PGE2 inhibited zymosan-induced IL-8 release from neutrophils in a dose-dependent manner, in response to 10(-5) M PGE1 and PGE2 inhibiting IL-8 generation by 89% and 75%, respectively. Similarly, the beta2-adrenoceptor agonist salbutamol also inhibited IL-8 generation, but it was less effective than the prostanoids. 4. Significant synergism between prostanoids or salbutamol and the PDE4 inhibitors to inhibit IL-8 generation was observed. In contrast, there was no significant synergism between PGE2 and the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast. 5. In order to evaluate the potential role of protein kinase A in mediating the inhibitory effects of cyclic AMP-elevating agents, we used the protein kinase A inhibitors, H 89 and KT 5720. Pretreatment of neutrophils with these drugs completely reversed the inhibitory effects of a combination treatment with rolipram and PGE2 on zymosan-induced IL-8 release. 6. Microscopic examination revealed that most neutrophils contained one or more zymosan particles and that combination treatment with rolipram and PGE2 noticeably reduced the number of ingested particles. Moreover, there was a significant reduction in the percentage of neutrophils which ingested three or more zymosan particles. 7. Thus, our results demonstrate that cyclic AMP-elevating agents modulate the ability of neutrophils to generate IL-8 in response to a particulate stimulus. However, these agents also modulate the ability of neutrophils to phagocytose zymosan particles. Whether this effect will translate into inhibition of the ability of neutrophils to deal with infectious agents needs to be investigated further.     

Regulation of monocyte IL-12 production: augmentation by lymphocyte contact and acute ethanol treatment, inhibition by elevated intracellular cAMP

IL-12, a monocyte-derived cytokine, is pivotal in activation of cellular immune response and inflammation. Both inflammatory response and cellular immunity are impaired by acute ethanol consumption. Here, we found that in vitro acute ethanol treatment (25-100 mM) results in a dose-dependent and significant increase of IL-12 in IFN-gamma (100 U/ml) plus Staphylococcal enterotoxin B (SEB; 1 microg/ml) stimulated monocytes and mononuclear cells but not in unstimulated cells from non-alcoholic blood donors. There was significantly greater IL-12 production in the MNC population compared to isolated Mphi (P < 0.001). Prevention of monocyte surface contact with either purified T lymphocytes or monocyte-depleted MNC resulted in a significant, 65+/-20%, decrease in IL-12 production regardless of IFN-gamma, SEB or ethanol stimulation suggesting that Mphi T-cell surface contact provides an additional signal for IL-12 production. In addition to cell surface contact, soluble mediators, particularly IL-10 and PGE2 may regulate IL-12 production. The cyclooxygenase inhibitor, Indomethacin (10(-6)M), augmented both IL-12 and IL-10 levels in isolated monocytes and mononuclear cells whether induced by medium, SEB or SEB plus 25 mM ethanol suggesting that regulation of IL-12 production via the cyclooxygenase pathway is independent of IL-10. Finally, elevation of intracellular cAMP levels by dbcAMP treatment consistently inhibited IL-12 as well as IL-10 production in monocytes induced by IFN-gamma or IFN-gamma plus 25 mM ethanol. These data suggest that augmentation of monocyte IL-12 by acute ethanol is not mediated via the cAMP pathway.     

Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13

Exposure of monocytes to pro-inflammatory cytokines or lipopolysaccharide (LPS) may induce synthesis and expression of tissue factor (TF). In this paper we have focused on the induction of TF-activity in human monocytes by the pro-inflammatory cytokines recombinant human interleukin 1 (rhIL-1 alpha) (rhIL-1 beta) (rhIL-6) and human tumour necrosis factor alpha (rhTNF-alpha), measured as procoagulant activity (PCA) in a microtitre plate-based clot assay. In addition we have studied the modulation of IL-1 alpha/beta induced TF-mRNA and PCA by rhIL-4, rhIL-10 and rhIL13. IL-1 alpha and IL-1 beta induced a concentration dependent increase in TF-activity. Neither IL-6 nor TNF-alpha gave rise to procoagulant activity at the concentrations tested (0.2-20 ng/ml). IL-4, IL-10 and IL-13, all effectively diminished IL-1 alpha/beta induced PCA, shown at the protein- and at the mRNA-level, while cell viability was unaffected. These results add to the previously demonstrated role of IL-4 and IL-10 as inhibitors of LPS-induced TF-activity, showing that these anti-inflammatory cytokines are not specific for LPS-activation but interfere with other stimulating substances such as IL-1, which may be involved in diseases where LPS is not present.     

Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p < 0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p < 0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.     

Bacterial lipopolysaccharide increases interleukin-6 and prostaglandin release in rat cortical type I astrocytes by different mechanisms: role of anti-inflammatory agents

LPS stimulated IL-6 release in a concentration-dependent manner from rat cortical type I astrocytes. This stimulatory action was completely abolished by Dexamethasone (DEX), but was not affected by indomethacin (IND), a 5-cyclooxigenase inhibitor. LPS-induced IL-6 release was partially inhibited by BW 4AC, a 5-lipoxygenase inhibitor. LPS concentration-dependently increased the release of PGE2 from type I astrocytes, an effect completely inhibited by IND. To rule out the possibility that DEX was inhibiting LPS-induced IL-6 release by blocking IL-6 gene expression, we tested the effect of DEX on interleukin 1beta(IL-1)-induced IL-6 release. DEX slightly inhibited IL-1-induced IL-6 release, while IL-1 releasing action on IL-6 was significantly reduced by IND. The involvement of nitric oxide (NO) generation on LPS-induced IL-6 release was also studied. We found that L-NO-arginine, an inhibitor of nitric oxide synthase, concentration-dependently reduced LPS-induced IL-6 release in astrocytes. In conclusion, we provide evidence that LPS action on IL-6 and PGE2 release can be ascribed to the activation of different transduction mechanisms, which can be pharmacologically dissected with the aid of anti-inflammatory drugs.     

Protective effect of a single interleukin-12 (IL-12) predose against the toxicity of subsequent chronic IL-12 in mice: role of cytokines and glucocorticoids

The mechanisms of interleukin-12 (IL-12) toxicity were studied in mice using a schedule (murine rIL-12, 400 ng/mouse, intraperitoneally [IP] once daily for 5 days) that markedly reduced body weight and food intake. On day 5, IL-12-treated mice had elevated serum and spleen IFN-gamma and tumor necrosis factor (TNF). Serum sTNFR-P75 and corticosterone (CS) were also elevated. IL-12 toxicity was partially prevented by anti-IFN-gamma antibodies or dexamethasone (DEX). A pre-dose of IL-12 (200 ng/mouse on day -14) completely prevented the toxicity of subsequent IL-12. The IL-12 predose also inhibited IL-12-induced IFN-gamma levels, but did not modify IL-12-induced CS, TNF or sTNFR-P75. A protective effect was observed with a predose of lipopolysaccharide (LPS) or murine recombinant (r)IL-10. The protective effect of the IL-12 predose was reduced by coadministration of anti-IFN-gamma, but a predose of murine rIFN-gamma was not protective, suggesting that IFN-gamma is necessary but not sufficient for the protective effect of IL-12. The IL-12 predose specifically protected against IL-12 toxicity and did not modify LPS toxicity. These data indicate that IL-12 can induce tolerance to its own toxicity, probably through a downregulation of IL-12-induced IFN-gamma but independently of endogenous glucocorticoids. IFN-gamma, and possibly IL-10, might be important in this tolerance.     

Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO-), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-L-arginine methyl ester (L-NAME) blocked IL-8 release by approximately 90% in response to LPS (1 microg/ml), but did not affect the production of IL-1beta or TNF-alpha. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO- (2.5-80 microM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO- formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-gammaB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO-. Our study identifies ONOO- as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO- formation or scavenging ONOO- may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.     

Monocyte B7 and Sialyl Lewis X modulates the efficacy of IL-10 down-regulation of LPS-induced monocyte tissue factor in whole blood

Recent studies have investigated the use of anti-inflammatory cytokine, interleukin 10 (IL-10) to control the development of disseminated intravascular coagulation (DIC) in sepsis by down-regulation of monocyte tissue factor (MTF) induced by lipopolysaccharide (LPS) in the initial phase of the disease. In vitro and in vivo human studies have shown that a minimal (<1 h) delay in IL-10 treatment significantly reduces the cytokines ability to inhibit LPS-induced MTF expression and the end products of coagulation. In this whole blood in vitro study we investigated the role of lymphocyte and platelet interactions with monocytes to up-regulate MTF expression in the presence of IL-10 in the initial phase of exposure to LPS. Individual blockade of monocyte B7 or platelet P-selectin significantly (35%) reduced MTF expression (P<0.05). IL-10 showed a dose-dependent inhibition of LPS (0.1 microg/ml) induced MTF expression, with 56% inhibition at 1 ng/ml, maximizing at 5 ng/ml IL-10 (75%; P<0.05). Simultaneous exposure to LPS and IL-10 (1 ng/ml) or addition of IL-10 1 h after LPS, with individual B7 and P-selectin blockade significantly enhanced the inhibition of MTF expression by IL-10 (P<0.05). We conclude that the efficacy of IL-10 to control DIC could be enhanced by a simultaneous B7 and P-selectin blockade.     

Leukocyte migration across human peritoneal mesothelial cells is dependent on directed chemokine secretion and ICAM-1 expression

BACKGROUND: Leukocyte migration into the peritoneal cavity is a diagnostic feature of peritonitis in patients treated with peritoneal dialysis (PD). While neutrophil (PMN) influx is characteristic of the acute phase of peritoneal infection, significant mononuclear cell (MNC) infiltration, occurs throughout the whole period of infection. Recent data suggests that human peritoneal mesothelial cell (HPMC) adhesion molecule expression and the synthesis of chemotactic cytokines may be important in the process. METHODS: In the present study we have examined, the regulation and directed secretion of chemokines (IL-8, MCP-1 and RANTES) and the basolateral to apical migration of unstimulated leukocytes across mesothelial cell monolayers using an in vitro model where HPMC were grown on the porous membrane of tissue culture inserts. Separate experiments have defined the importance of chemokine synthesis and ICAM-1 expression in the transmigration process. RESULTS: Apical stimulation of HPMC with IL-1 beta or TNF alpha resulted in a time and dose dependent up-regulation of IL-8, MCP-1 and RANTES mRNA expression and synthesis. This secretion was predominately into the apical compartment (> 85%) with all chemokines. Apical pre-stimulation of HPMC resulted in a dose- and time-dependent migration of both PMN and MNC across HPMC. Neutrophil migration was significantly reduced in the presence of appropriate concentrations of polyclonal IL-8 antibody (IL-1 beta (100 pg/ml) 153 +/- 12 versus anti-IL-8 (100 ng/ml) 71 +/- 7 (X 10(3)) PMN, N = 6, P < 0.02) and in the presence of anti-ICAM-1 F(ab)'2 fragments or soluble ICAM-1. Constitutive and cytokine stimulated mononuclear cell migration was significantly reduced in the simultaneous presence of polyclonal MCP-1 or RANTES antibody. CONCLUSIONS: These data demonstrate that HPMC synthesize IL-8, MCP-1 and RANTES in response to inflammatory cytokines. HPMC-derived C-x-C and C-C chemokines might contribute to the intra-peritoneal recruitment of leukocytes during peritoneal inflammation.     

The role of leukocyte emigration and IL-8 on the development of lipopolysaccharide-induced lung injury in rabbits

Leukocyte emigration and alveolar macrophage-derived cytokines may contribute to lung microvascular injury associated with adult respiratory distress syndrome. We have used mAbs against cell adhesion molecules on leukocytes (anti-CD18 and anti-CD49d) or against IL-8 to investigate these contributions. Intratracheal (i.t.) instillation of LPS (50 microg/kg) caused a significant increase in bronchoalveolar lavage polymorphonuclear leukocytes (PMNs) without an increase in mononuclear cells (MNCs) or an increase in lung permeability. Injection of LPS (10 microg/kg) i.v. at 24 h after i.t. LPS caused significant increases in bronchoalveolar lavage PMNs, MNCs, IL-8, and monocyte chemotactic protein-1, as well as increases in lung permeability. Rabbits that were administered i.t. LPS followed by i.v. LPS and treated with anti-CD18 mAb had a significantly lower lung permeability index and emigration of fewer PMNs but no change in MNC emigration compared with saline treatment. Anti-IL-8 mAb treatment resulted in a significantly lower lung permeability index with no change in PMN emigration compared with no treatment. These results suggest that PMN emigration is necessary but not sufficient for the development of LPS-induced lung injury, and that IL-8 plays a significant role in PMN-dependent lung injury, independent of PMN emigration.     

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.     

Modulatory effect of interleukin-10 on the production of platelet-activating factor and superoxide anions by human leucocytes

We observed that human monocytes (MO) and polymorphonuclear neutrophils (PMN) stimulated by lipopolysaccharide (LPS) produce platelet-activating factor (PAF) in a pattern characterized by an early and a delayed peak of synthesis. The early peak of PAF synthesis was due to a direct stimulation of these cells through mCD14 receptor as it was inhibited by anti-CD14 monoclonal antibody. The delayed and sustained peak of PAF synthesis was dependent on protein synthesis and cytokine production as shown by the inhibitory effect of cycloheximide on both MO and PMN, and of anti-tumour necrosis factor-alpha (anti-TNF-alpha) and of anti-interleukin-8 (anti-IL-8) neutralizing antibodies on MO and PMN respectively. IL-10 completely prevented this second, cytokine-dependent peak of PAF synthesis. In contrast, IL-10 markedly enhanced the first peak of PAF synthesis both in MO and PMN. Moreover, IL-10 was shown to modulate the production of superoxide anions (O2-) on both MO and PMN. As suggested by previous studies, IL-10 inhibited the delayed production of O2-. In the present study, we observed that IL-10 directly stimulated an early production of O2-. In addition, IL-10 enhanced the synthesis of O2- by MO and PMN challenged with LPS. The IL-10-induced O2- production was dependent, at least in part, from its effect on PAF synthesis, as it was inhibited by the PAF receptor antagonist WEB 2170. These results suggest that IL-10 may upregulate the early synthesis of PAF and O2- triggered by direct LPS stimulation, whereas it may downregulate the delayed production of these mediators.