Arsenic trioxide reduces 2,4,6-trinitrobenzene sulfonic acid-induced murine colitis via nuclear factor-κB down-regulation and caspase-3 activation.

Arsenic trioxide, As(2)O(3), already used in human anti-cancer therapy, is also an efficient agent against the autoimmune and inflammatory diseases developed in MRL/lpr mice. Inflammatory bowel diseases (IBDs), notably Crohn's disease, which remain without efficient treatment, display autoimmune and inflammatory components. We, therefore, hypothesized that As(2)O( 3) may be active on IBDs. Using the 2,4,6-trinitrobenzene sulfonic acid-induced murine model of colitis, we demonstrate that As(2)O(3) used either in a preventive or a curative mode markedly reduced the induced colitis as assessed by macroscopic and microscopic scores, leading to prolonged mice survival. In addition, As(2)O(3) was able to inhibit NF-κB expression and DNA-binding in colon extracts leading to decreased cytokine gene expression (i.e. tumor necrosis factor-α, interleukin(IL)-1ß, IL-12, IL-17, IL-18, and IL-23). Interestingly, As(2)O(3) also reduced keratinocyte-derived chemokine (KC), inducible nitric oxide synthase (iNOS) mRNA levels, and myeloperoxidase (MPO) protein expression suggesting an impairment of neutrophils. This was associated with a marked increase of procaspase-3 and induced caspase-3 activation. This caspase-3 co-localized with MPO in the remaining neutrophils suggesting that As(2)O( 3) might have eliminated inflamed cells probably by inducing their apoptosis. These results assessed the potent anti-inflammatory effect of As(2)O( 3), that targets both NF-κB and caspase-3 pathways, and suggests a therapeutic potential for Crohn's disease and other severe IBDs.

IL-8 is a key chemokine regulating neutrophil recruitment in a new mouse model of Shigella-induced colitis.

The lack of a mouse model of acute rectocolitis mimicking human bacillary dysentery in the presence of invasive Shigella is a major handicap to study the pathogenesis of the disease and to develop a Shigella vaccine. The inability of the mouse intestinal mucosa to elicit an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes (PMN) may be due to a defect in epithelial invasion, in the sensing of invading bacteria, or in the effector mechanisms that recruit the PMN infiltrate. We demonstrate that the BALB/cJ mouse colonic epithelium not only can be invaded by Shigella, but also elicits an inflammatory infiltrate that, however, lacks PMN. This observation points to a major defect of mice in effector mechanisms, particularly the lack of expression of the CXC chemokine, IL-8. Indeed, this work demonstrates that the delivery of recombinant human IL-8, together with Shigella infection of the colonic epithelial surface, causes an acute colitis characterized by a strong PMN infiltrate that, by all criteria, including transcription profiles of key mediators of the innate/inflammatory response and histopathological lesions, mimics bacillary dysentery. This is a major step forward in the development of a murine model of bacillary dysentery.

A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma.

Mucus hypersecretion is a crucial feature of pulmonary diseases such as asthma, chronic bronchitis and cystic fibrosis. Despite much research, there is still no effective therapy for this condition. Recently, we showed that the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is required for mucus secretion by human bronchial epithelial cells in culture. Having synthesized a peptide corresponding to the N-terminal domain of MARCKS, we now show that the intratracheal instillation of this peptide blocks mucus hypersecretion in a mouse model of asthma. A missense peptide with the same amino acid composition has no effect. Based on quantitative histochemical analysis of the mouse airways, the peptide seems to act by blocking mucus release from goblet cells, possibly by inhibiting the attachment of MARCKS to membranes of intracellular mucin granules. These results support a pivotal role for MARCKS protein, specifically its N-terminal region, in modulating this secretory process in mammalian airways. Intratracheal administration of this MARCKS-related peptide could therapeutically reduce mucus secretion in the airways of human patients with asthma, chronic bronchitis and cystic fibrosis.

Leukotrienes mediate part of Ova-induced lung effects in mice via EGFR.

Antigen induces murine bronchial hyperreactivity (BHR), inflammation, mucus accumulation, and airway remodeling. To investigate whether leukotrienes (LT) mediate the effects of antigen [ovalbumin (Ova)], we studied 5-lipoxygenase (5-LO) expression in immunized BP2 mice and blocked LT synthesis with the 5-LO inhibitor zileuton or antagonized their effects with receptor antagonists [cysteinyl leukotriene (Cys-LT)-ra MK-571, LY-171883; LTB4-ra PH-163]. Cys-LT content increased in the bronchoalveolar lavage fluid (BALF) as early as 15 min after the intratracheal instillation of Ova. Zileuton inhibited LT release in the BALF and eosinophil recruitment in the lungs, and dose dependently reduced BHR, mucus accumulation, and remodeling, as did the LT-ra. Thus LT, released just after antigen challenge, might constitute the first step in accounting for the effects of Ova. Because mucus accumulation is regulated via the EGF receptor (EGFR), which is also implicated in the effects of LT, we studied this pathway with AG-1478, an EGFR tyrosine kinase inhibitor given at 0.5, 4, and 20 mg/kg. AG-1478 inhibited BHR, inflammation, and lung remodeling induced by Ova or by molecules themselves generated by Ova, such as LT, IL-13, and monocyte chemoattractant protein-1, which promote identical effects, suggesting the involvement of the EGFR pathway in the asthma-like syndrome observed.

Leukotrienes mediate murine bronchopulmonary hyperreactivity, inflammation, and part of mucosal metaplasia and tissue injury induced by recombinant murine interleukin-13.

Interleukin (IL)-13 induces bronchopulmonary hyperreactivity (BHR), eosinophilic inflammation, and mucus accumulation in the murine airways. To investigate the potential role of leukotrienes (LT) in mediating these effects, we studied the ability of IL-13 to induce the expression of 5-lipoxygenase (5-LO), we compared the effects of IL-13 and of various leukotrienes on different biological parameters and the interference by the 5-LO inhibitor zileuton (orally, 50 mg/kg, 3 times a day for 3 days), and by some antagonists. The cysteinyl (Cys)-LTs LTC4, LTD4, LTE4, and LTB4, (1 microg/d for 3 d, instilled intratracheally) induced BHR, cell recruitment, fibroblast growth, and mucus production and release into the airways. After the intratracheal instillation of recombinant murine (rm) IL-13, Cys-LT increased in the bronchoalveolar lavage fluid (BALF) at 15 min, followed by lower amounts at 3-6 h. Zileuton inhibited LT production in the BALF, eosinophil and neutrophil sequestration in the lungs, and their passage into the BALF. Zileuton and the Cys-LT-receptor antagonist (ra) LY171883 or MK-571, or the LTB4-ra PH-163 (at 3-10, 5-15, and 10 mg/kg, respectively, administered intratracheally), inhibited BHR by recombinant murine IL-13. Airways mucus after recombinant murine IL-13-challenge was reduced by zileuton and by LY171883, MK-571, and PH-163. LT also induced the vascular endothelium remodelling and collagen deposition. Overall, our results demonstrate the major involvement of LT in the effects of IL-13 on the lung.

Leukotrienes, IL-13, and chemokines cooperate to induce BHR and mucus in allergic mouse lungs.

In mice, intratracheal challenges with antigen (ovalbumin) or recombinant murine interleukin-13 (IL-13) induce lung inflammation, bronchial hyperreactivity (BHR), and mucus accumulation as independent events (Singer M, Lefort J, and Vargaftig BB. Am J Respir Cell Mol Biol 26: 74-84, 2002), largely mediated by leukotrienes (LT). We previously showed that LTC(4) was released 15 min after ovalbumin, and we show that it induces the expression of monocyte chemoattractant proteins 1 and 5 and KC in the lungs, as well as IL-13 mRNA. Instilled intratracheally, these chemokines induced BHR and mucus accumulation, which were inhibited by the 5-lipoxygenase inhibitor zileuton and by the cysteinyl-LT receptor antagonist MK-571, suggesting mediation by cysteinyl-LT. Because these chemokines also induced release of LT into the bronchoalveolar lavage fluid and IL-13 into the lungs, we hypothesize that LT- and chemokine-based loops for positive-feedback regulations cooperate to maintain and amplify BHR and lung mucus accumulation after allergic challenge and in situations where IL-13, LT, or chemokines are generated.

Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury.

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A(2) (sPLA(2)-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA(2)-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA(2)-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 microg/kg) induced an increase in pulmonary expression of sPLA(2)-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA(2)-IIA than AM from saline-treated animals. This ex vivo sPLA(2)-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA(2)-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.

Granulocyte depletion and dexamethasone differentially modulate airways hyperreactivity, inflammation, mucus accumulation, and secretion induced by rmIL-13 or antigen.

The intratracheal administration of interleukin (IL)-13 to hyperresponsive BP2 mice induces bronchopulmonary hyperreactivity (BHR), eosinophilia, mucus and MUC5AC accumulation, similar to those observed after ovalbumin (Ova) treatment when mice are immunized. mRNAs for IL-4 peaked at 6 h after Ova challenge, then vaned, whereas IL-13 expression was stable for a longer period, suggesting different effects. Inhalation of aerosolized methacholine by immunized mice 72-96 h after Ova reduced epithelial mucus content, and enriched the bronchoalveolar lavage fluid (BALF) mucus. The role of granulocytes for mucus accumulation was studied using vinblastine or the antigranulocyte antibody RB6-8C5, which interfered to a limited extent only with allergen-induced mucus accumulation. By contrast, eosinophilic and neutrophilic inflammation, as well as BHR, were completely suppressed. Granulocytes are thus involved in Ova-induced BHR, whereas mucus accumulation and BHR are unrelated. Granulocytes seem to be more implicated in rmIL-13-induced mucus, which is reduced by the antigranulocyte antibody, whereas BHR is unaffected. The glucocorticosteroid dexamethasone reduced all the parameters evaluated after Ova or after rmIL-13. Because the effects of IL-13 are glucocorticoid-sensitive, they probably involve secondary mechanisms.