Endotoxin-induced lethality in neonatal mice is counteracted by interleukin-10 (IL-10) and exacerbated by anti-IL-10.

The lethal effects occurring in neonatal (<24-h-old) BALB/c mice after challenge with 25 mg of lipopolysaccharide (LPS) per kg of body weight were significantly counteracted by pretreatment with recombinant interleukin-10 (rIL-10; 25 or 50 ng/mouse). Concordantly, blockage of endogenous IL-10 with the SXC1 monoclonal antibody increased LPS-induced mortality. Both IL-10 and SXC1 modulated the release of tumor necrosis factor alpha (TNF-alpha) so that, relative to controls, peak TNF-alpha values after LPS challenge were decreased by rIL-10 and increased by anti-IL-10.     

Lipopolysaccharide-induced production of interleukin-10 is promoted by the serine/threonine kinase Akt

The bacterial endotoxin lipopolysaccharide (LPS), is a potent inducer of the inflammatory response. Previous studies demonstrated that LPS-induced toxicity is reversed upon FcgammaR clustering by IgG immune complexes (IC) through upregulation of the anti-inflammatory cytokine IL-10. The PI3K-Akt pathway is also reported to reverse LPS-induced inflammation. In this study, we have examined the role of Akt in LPS-induced IL-10 production. First, we compared Akt activation in macrophages stimulated with either LPS alone, or with a combination of LPS and ICs. Our experiments revealed that while Akt was activated under both conditions, the level of activation was significantly higher in cells stimulated with LPS and ICs, suggesting that Akt may be involved in IC-induced upregulation of IL-10 production. Using several independent models we have then tested the notion that enhanced Akt activation may lead to enhanced LPS-induced IL-10 production. Over-expression of constitutively active Myr-Akt in the mouse macrophage cell line Raw 264.7 led to significant increase in IL-10 production in response to LPS. In addition, down-regulation of Akt by siRNA resulted in a decrease in LPS-induced IL-10 production. Peritoneal macrophages from transgenic mice with macrophage-specific expression of Myr-Akt produced significantly higher levels of IL-10 when stimulated with LPS, compared to their wild-type counterparts. Consistent with this observation, serum levels of IL-10, post-LPS challenge, was higher in the Myr-Akt transgenic mice compared to the wild-type mice. Taken together, these data demonstrate that Akt plays a critical role in LPS-induced production of IL-10.     

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2–10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0·01 to 100 μg/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

Leukocyte-derived IL-10 reduces subepithelial fibrosis associated with chronically inhaled endotoxin

Endotoxin (LPS), a Gram-negative cell wall component, has potent proinflammatory properties. Acute LPS exposure causes airway inflammation; chronic exposure causes airway hyperreactivity and remodeling. IL-10 is an important antiinflammatory cytokine, which is decreased in patients with airway disease, such as asthma and cystic fibrosis. To examine the physiologic and therapeutic role of IL-10 in acute and chronic LPS-induced airway disease. Mice were exposed to aerosolized LPS once or daily for 4 wk. Endpoints were airway inflammation, airway reactivity to methacholine, extracellular matrix protein expression, and histologic analysis. IL-10-deficient mice developed significantly enhanced airway cellularity and remodeling when compared with C57BL/6 mice after chronic LPS inhalation. However they demonstrated less airway hyperreactivity associated with higher inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), and lung lavage fluid nitrite levels. In a bone marrow transplantation model, the IL-10 antiinflammatory effect was dependent on the hematopoietic but not on the parenchymal IL-10 expression. Induced epithelial human IL-10 expression protected from the LPS effects and led to decreased collagen production. IL-10 attenuates chronic LPS-induced airway inflammation and remodeling. Physiologically, the antiinflammatory effect of IL-10 is mediated by hematopoietic cells. Therapeutically, adenovirus-driven expression of human IL-10 in airway epithelia is sufficient for its protective effect on inflammation and remodeling. The role of IL-10 on airway hyperreactivity is complex: IL-10 deficiency protects against LPS-induced hyperreactivity, and is associated with higher eNOS, iNOS, and airway nitrate levels.     

Interleukin-10 controls interferon-γ and tumor necrosis factor production during experimental endotoxemia

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-γ is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-γ synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-γ release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-γ levels. The ability of rIL-10 to inhibit LPS-induced IFN-γ synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 μg LPS resulted in a threefold decrease in peak IFN-γ serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2h before LPS challenge resulted in a marked increase in both TNF and IFN-γ serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 μg LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.     

Surfactant protein A inhibits lipopolysaccharide-induced in vivo production of interleukin-10 by mononuclear phagocytes during lung inflammation

We previously demonstrated that resident alveolar macrophages from naive mice do not synthesize interleukin (IL)-10, whereas mononuclear phagocytes (MP) recruited during the lung inflammatory process are transiently competent for IL-10 production when exposed to lipopolysaccharide (LPS) in vitro. As surfactant protein A (SP-A), a member of the collectin family, inhibits LPS-induced in vitro IL-10 formation by bone marrow-derived macrophages, we studied its effect on MP under in vivo inflammatory conditions. When mice with LPS-induced inflamed lungs were given a second intranasal LPS administration, IL-10 concentration recovered in the bronchoalveolar lavage fluids varied as a function of the time interval between the two LPS doses. Thus, IL-10 concentration increased with the number of MP up to Day 3, and then decreased to undetectable values within 24 h, despite a continued increase in the number of MP. Analysis of IL-10 mRNA from purified MP indicated that gene expression correlated with the IL-10 level in the bronchoalveolar lavage fluid. In contrast to IL-10 production, SP-A concentrations during LPS-induced inflammation decreased with a nadir at Day 3, and then increased significantly within 24 h. Furthermore, intranasal administration of exogenous SP-A to mice with LPS-induced inflamed lungs led to a repression of the IL-10 production. In summary, this study demonstrates for the first time an in vivo inhibitory role of SP-A on the anti-inflammatory activity of MP, through inhibition of IL-10 production.     

Antiinflammatory properties of mycophenolate mofetil in murine endotoxemia: inhibition of TNF-alpha and upregulation of IL-10 release.

Mycophenolate mofetil (MMF) is a new immunosuppressive agent currently used in organ transplantation and under evaluation in immune-mediated inflammatory disorders such as rheumatoid arthritis. Although MMF was shown to inhibit purine nucleotide synthesis in lymphocytes, it is still unclear whether it might also exert direct antiinflammatory actions in vivo. To address this question, we evaluated the effects of MMF administration on the responses of mice to a single challenge with bacterial lipopolysaccharide (LPS). We observed that MMF treatment inhibits the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) upon LPS injection whereas it promotes IL-10 production. In parallel, MMF was found to protect mice from LPS-induced lethality. Inhibition of TNF-alpha release was also observed in IL-10-deficient mice indicating that it does not exclusively depend on the upregulation of IL-10 endogenous synthesis. In view of the differential effects of MMF on the LPS-induced production of TNF-alpha and NO on one hand and that of IL-10 on the other hand, we conclude that beside its immunosuppressive action at the lymphocyte level, MMF is also endowed with antiinflammatory properties.     

Effects of Lipopolysaccharide on Endothelial Cell Adhesion Molecule Expression in Interleukin-10 Deficient Mice

Interleukin (IL)-10 is known to inhibit the production of proinflammatory cytokines by macrophages suggesting that endogenous IL-10 may act as an anti-inflammatory agent. Because endothelial cell adhesion molecules (ECAMs) play a key role in the recruitment of leukocytes into tissue in response to an inflammatory stimulus (i.e., lipopolysaccharide (LPS)) and the following cytokine production, we wished to assess the importance of IL-10 as an endogenous modulator of ECAM expression using IL-10 deficient mice. Constitutive and LPS-stimulated expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin were measured in wild type C57BL/6 and IL-10 deficient mice with no signs of active enterocolitis, using the dual radiolabeled monoclonal antibody technique. We found that constitutive expression of these ECAMs did not differ between IL-10 deficient and WT mice for all organs tested. However, we demonstrated larger increments in LPS-induced expression of ICAM-1 and VCAM-1 in the vasculature of the small intestine in IL-10 deficient mice compared to WT mice. These findings suggest that endogenous IL-10 does not modulate constitutive or LPS-induced expression of ECAMs in most tissues, however it does appear to play an inhibitory role in LPS-stimulated expression of ICAM-1 and VCAM-1 in the intestinal vasculature.     

Lipopolysaccharide induces biochemical alterations in chicks trained on the passive avoidance learning task

We have recently shown that activation of the immune system with lipopolysaccharide (LPS) results in memory-processing deficits for the passive avoidance learning task in the day-old chick. The current study examined two important issues in understanding the mechanisms underlying these memory deficits associated with immune system activation, namely, whether LPS (1) impairs Na(+)/K(+)-ATPase functioning and (2) increases corticosterone (CORT) concentrations in chicks trained on the task. As the effects of LPS on sickness behavior have only previously been characterized in older chickens, this study also tested whether LPS is able to produce similar alterations in day-old chicks. LPS decreased brain Na(+)/K(+)-ATPase activity and increased plasma concentrations of CORT in chicks trained on the passive avoidance learning task. These findings give an insight into some of the mechanisms that may be responsible for the LPS-induced memory-processing deficits. Consistent with previous research in older chickens, LPS increased body temperature in a dose-dependent manner, however, only the lowest dose of LPS tested significantly decreased food intake in the day-old chicks.     

Interferon-gamma (IFN-gamma) and prostaglandin E2 (PGE2) regulate differently IL-12 production in human intestinal lamina propria mononuclear cells (LPMC).

IL-12 modulates Th1 immune response during chronic colitis. Mechanisms regulating IL-12 synthesis in human intestine are poorly understood. The aim of this study was to investigate the effect of IFN-gamma and PGE2 on lipopolysaccharide (LPS)-stimulated LPMC IL-12 production. Normal LPMC cultures were run in the presence or absence of IFN-gamma and/or PGE2 before LPS stimulation. To examine the role of endogenous PGE2 on LPS-stimulated IL-12 release, LPMC cultures were added of indomethacin before LPS stimulation. IL-12, IL-10 and IL-8 were measured by ELISA. No IL-12 was detected in either unstimulated or LPS-stimulated LPMC cultures. In contrast, LPMC released IL-8 (650 +/- 125 pg/ml) and IL-10 (75 +/- 25 pg/ml) in response to LPS. Treatment of LPMC with IFN-gamma facilitated LPS-stimulated IL-12, whereas it completely abrogated IL-10 production. IL-12 release by LPMC stimulated with IFN-gamma and LPS was significantly inhibited by exogenous IL-10. The addition of PGE2 to IFN-gamma-treated LPMC cultures inhibited in a dose-dependent manner LPS-induced IL-12 secretion. Furthermore, IL-12 was detectable (85 +/- 25 pg/ml) in the supernatants of LPMC cultures treated with indomethacin and LPS. In contrast to the effect on IL-12, PGE2 significantly augmented LPS-stimulated LPMC IL-10 production. However, the inhibition of IL-12 by PGE2 was only partially reversed by anti-IL-10. In a simplified model of LPS tolerance, we finally showed that monocyte-derived macrophages exhibited reduced IL-12 production after repeat LPS stimulation. In these cell cultures, indomethacin abrogated the induction of LPS desensitization. IFN-gamma and PGE2 modulate differently the LPMC responsiveness to LPS in terms of IL-12 synthesis.     

Effect of protein kinase C inhibitor (H-7) and calmodulin antagonist (W-7) on pertussis toxin-induced IL-1 production by human adherent monocytes. Comparison with lipopolysaccharide as a stimulator of IL-1 production.

Human adherent monocytes stimulated with 1 microgram/ml pertussis toxin (PT) produced interleukin-1 (IL-1), as measured by thymocyte co-stimulation assay and enzyme-linked immunosorbent assay (ELISA), specific for IL-1 alpha and IL-1 beta. To clarify the role of protein kinase C (PKC) and calmodulin in IL-1 production, we investigated the effects of a PKC inhibitor, H-7, and a calmodulin antagonist, W-7 on PT- and lipopolysaccharide (LPS)-induced IL-1 production by monocytes. Addition of 10 microM and 20 microM H-7 to the culture medium markedly suppressed both PT- and LPS-induced IL-1 production. PT-induced IL-1 production was significantly suppressed by 5 microM and 10 microM W-7. However, LPS-induced IL-1 production was not suppressed by W-7 at the concentrations tested. When monocytes were labelled with Quin 2/AM, IL-1 production by monocytes stimulated with PT and LPS was markedly suppressed. These results indicate that different pathways are involved in the IL-1 production by PT and LPS; both calmodulin- and PKC-dependent processes are necessary for the IL-1 production induced by PT, whereas LPS-induced IL-1 production is dependent on the PKC. Inhibition of IL-1 production by interfering with intracellular Ca2+ trafficking in Quin 2/AM-loaded monocytes may be associated with the inhibition of PKC and calmodulin activity.     

Roles of CC chemokine receptors (CCRs) on lipopolysaccharide-induced acute lung injury

The aim of the present study was to evaluate the effects of the CC chemokine receptor (CCR) 2b and CCR1 antagonist RS504393 as well as the roles of CCRs on lipopolysaccharide (LPS)-induced acute lung injury (ALI). In A549 cell line, treatment with RS504393 significantly inhibited the expression of CCR1, CCR2 and interleukin (IL)-8 after either LPS or tumor necrosis factor-α stimulation. An ALI model with intranasal LPS administration was used on C57BL/6J, CCR1, CCR2 and CCR3 knockout mice. Treatment with RS504393 had a noteworthy preventative effect on LPS-induced over-expression of IL-1β, plasminogen activator inhibitor and CCR2. In CCR1 and CCR2-deficient animals, LPS-induced less increase of lung weight, bronchoalveolar lavage (BAL) leukocytes and IL-6 compared to the C57BL/6J and CCR3 knockout mice. This was most prominent in the CCR2 knockout mice where no LPS-induced lung edema and no increase of IL-6 in BAL fluid occurred. Our results indicate that CCR2, and to some extent CCR1, play pivotal roles in the development of ALI.     

Differential regulation of monocytic tumor necrosis factor-α and interleukin-10 expression

Activation of human monocytes by bacterial endotoxin (LPS) results in an initial burst of inflammatory cytokines like tumor necrosis factor (TNF)-α which is followed by the secretion of anti-inflammatory mediators like interleukin (IL)-10. The signaling pathways in IL-10 induction are unknown. Here, we show that the regulation of IL-10 expression is more complex than that of TNF-α. LPS-induced TNF-α and IL-10 expression requires early activation of protein tyrosine kinases (PTK). Moreover, delayed addition of PTK inhibitors blocked IL-10, but not TNF-α, suggesting the impact of a late PTK activity. Two inducers of PTK activity are the downstream mediators of LPS activation, TNF-α and cyclic adenosine monophosphate (cAMP). Both mediators synergistically up-regulate IL-10 expression. Downstream of PTK activation, they use distinct pathways. TNF-α, but not cAMP-induced IL-10 gene expression was inhibited by pyrrolidine dithiocarbamate, suggesting the involvement of reactive oxygen species. Inhibition of protein kinase C (PKC) suppressed LPS-induced TNF-α and IL-10 expression as well, but, unlike TNF-α, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) did not induce IL-10 expression. Furthermore, PKC is not involved in late events of IL-10 activation, as delayed addition of PKC inhibitors did not suppress LPS-induced IL-10 expression and did not influence cAMP- or TNF-α-induced IL-10. The modulation of IL-10 expression by inflammatory mediators suggests a regulatory circuit of the inflammatory response.     

The intratracheal administration of endotoxin and cytokines. III. The interleukin-1 (IL-1) receptor antagonist inhibits endotoxin- and IL-1-induced acute inflammation.

Endotoxin, a lipopolysaccharide (LPS) component of gram-negative bacteria, induces alveolar macrophages to express interleukin-1 (IL-1). Lipopolysaccharide and IL-1 both cause severe acute neutrophilic inflammation in the lung after intratracheal injection, suggesting that LPS-induced IL-1 expression contributes to the pathogenesis of LPS-induced acute inflammation. In the present study, the role of IL-1 in LPS-induced acute pneumonia was investigated by quantitating the acute inflammation occurring at 6 hours after the intratracheal injection of LPS as compared to the same timepoint after the intratracheal coinjection of LPS and IL-1 receptor antagonist (IL-1ra). The IL-1ra was found to inhibit LPS-induced acute inflammation (P greater than 0.0001) as measured by the number of neutrophils recovered in bronchoalveolar lavage. The LPS-induced emigration of neutrophils was inhibited by as much as 45%. Recombinant IL-1 beta-induced neutrophil emigration into the lung was inhibited by 95% when IL-1ra was coinjected intratracheally with IL-1 beta. Coinjection of recombinant IL-1 beta and LPS increased the neutrophilic exodus as compared to the intratracheal injection of either agent alone. Intratracheal injection of LPS induces a progressive increase in IL-1ra mRNA expression in whole-lung RNA preparations, suggesting that endogenous IL-1ra may play an important role as a negative feedback mechanism to downregulate LPS initiated IL-1-mediated acute inflammation. In conclusion IL-1ra inhibits both LPS- and IL-1-induced neutrophilic inflammation and may therefore prove clinically useful as an anti-inflammatory agent for the therapy of either septic or aseptic IL-1-mediated acute inflammation.