Intestinal lymph-borne factors induce lung release of inflammatory mediators and expression of adhesion molecules after an intestinal ischemic insult.

BACKGROUND: Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. MATERIAL AND METHODS: Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. RESULTS: Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1ß, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB(4) and TXB(2) were found to be significantly increased. CONCLUSIONS: These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R.

Mycobacterium bovis bacillus Calmette-Guérin killed by extended freeze-drying targets plasmacytoid dendritic cells to regulate lung inflammation.

We have previously shown that bacillus Calmette-Guérin (BCG) inactivated by extended freeze-drying (EFD) reduces airway hyperresponsiveness, whereas live and heat-killed BCG fail to do so. However, the cells involved in the protective effect and the signaling and transcriptional networks that could reprogram T cell commitment after EFD BCG treatment remained to be elucidated. We investigated whether EFD BCG targets plasmacytoid dendritic cells (pDCs) potentially involved in the polarization of regulatory T cells (Tregs) and the transcriptional factors that regulate allergic inflammation. OVA-sensitized mice were s.c. injected with EFD, live, or heat-killed BCG. We analyzed after the injection of the various BCG preparations: 1) pDCs recruited in the draining lymph nodes (day 4); 2) transcription factors involved in inflammation and T cell commitment in spleen and lungs after OVA challenge (day 28). Airway hyperresponsiveness and transcription factors were determined after in vivo depletion of pDCs or Tregs in EFD BCG-treated and OVA-challenged mice. EFD BCG reduced inflammation via the recruitment of pDCs polarizing the differentiation of naive CD4+ T lymphocytes into Tregs. In vivo, pDC or Treg depletion at the time of EFD BCG treatment abrogated the protection against inflammation. EFD BCG treatment upregulated Forkhead-winged helix transcription factor (Treg signature) and downregulated GATA-3 and RORgammat (Th2 and Th17 signatures) more efficiently than live and heat-killed BCG. Moreover, only EFD BCG enhanced peroxisome proliferator-activated receptor gamma expression and blocked NF-kappaB activation, cyclooxygenase expression, and p38 MAPK phosphorylation. EFD BCG reduced allergic inflammation by recruiting pDCs that promoted Tregs; EFD BCG acted as a peroxisome proliferator-activated receptor gamma agonist and thus could be used in asthma and other inflammatory diseases.

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.

Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway.

Innate immune responses against microorganisms may be mediated by Toll-like receptors (TLRs). Intestinal ischemia-reperfusion (i-I/R) leads to the translocation of bacteria and/or bacterial products such as endotoxin, which activate TLRs leading to acute intestinal and lung injury and inflammation observed upon gut trauma. Here, we investigated the role of TLR activation by using mice deficient for the common TLR adaptor protein myeloid differentiation factor 88 (MyD88) on local and remote inflammation following intestinal ischemia. Balb/c and MyD88(-/-) mice were subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). Acute neutrophil recruitment into the intestinal wall and the lung was significantly diminished in MyD88(-/-) after i-I/R, which was confirmed microscopically. Diminished neutrophil recruitment was accompanied with reduced concentration of TNF-alpha and IL-1beta level. Furthermore, diminished microvascular leak and bacteremia were associated with enhanced survival of MyD88(-/-) mice. However, neither TNF-alpha nor IL-1beta neutralization prevented neutrophil recruitment into the lung but attenuated intestinal inflammation upon i-I/R. In conclusion, our data demonstrate that disruption of the TLR/MyD88 pathway in mice attenuates acute intestinal and lung injury, inflammation, and endothelial damage allowing enhanced survival.

Reduced allergic lung inflammation in rats following formaldehyde exposure: long-term effects on multiple effector systems.

Clinical and experimental evidences show that formaldehyde (FA) exposure has an irritant effect on the upper airways. As being an indoor and outdoor pollutant, FA is known to be a causal factor of occupational asthma. This study aimed to investigate the repercussion of FA exposure on the course of a lung allergic process triggered by an antigen unrelated to FA. For this purpose, male Wistar rats were subjected to FA inhalation for 3 consecutive days (1%, 90-min daily), subsequently sensitized with ovalbumin (OVA)-alum via the intraperitoneal route, and 2 weeks later challenged with aerosolized OVA. The OVA challenge in rats after FA inhalation (FA/OVA group) evoked a low-intensity lung inflammation as indicated by the reduced enumerated number of inflammatory cells in bronchoalveolar lavage as compared to FA-untreated allergic rats (OVA/OVA group). Treatment with FA also reduced the number of bone marrow cells and blood leukocytes in sensitized animals challenged with OVA, which suggests that the effects of FA had not been only localized to the airways. As indicated by passive cutaneous anaphylactic reaction, FA treatment did not impair the anti-OVA IgE synthesis, but reduced the magnitude of OVA challenge-induced mast cell degranulation. Moreover, FA treatment was associated to a diminished lung expression of PECAM-1 (platelet-endothelial cell adhesion molecule 1) in lung endothelial cells after OVA challenge and an exacerbated release of nitrites by BAL-cultured cells. Keeping in mind that rats subjected solely to either FA or OVA challenge were able to significantly increase the cell influx into lung, our study shows that FA inhalation triggers long-lasting effects that affect multiple mediator systems associated to OVA-induced allergic lung such as the reduction of mast cells activation, PECAM-1 expression and exacerbation of NO generation, thereby contributing to the decrease of cell recruitment after the OVA challenge. In conclusion, repeated expositions to air-borne FA may impair the lung cell recruitment after an allergic stimulus, thereby leading to a non-responsive condition against inflammatory stimuli likely those where mast cells are involved.

Cells isolated from bone-marrow and lungs of allergic BALB/C mice and cultured in the presence of IL-5 are respectively resistant and susceptible to apoptosis induced by dexamethasone.

We have previously reported that, in IL-5-stimulated bone-marrow cultures, dexamethasone upregulates eosinophil differentiation and protects developing eosinophils from apoptosis induced by a variety of agents. Recently developed procedures for the isolation of hemopoietic cells from allergic murine lungs have enabled us to evaluate how these cells respond to dexamethasone in IL-5-stimulated cultures, when compared with bone-marrow-derived cells isolated from the same donors, and whether differences in response patterns were linked to apoptosis. Ovalbumin challenge of sensitized mice increased significantly the numbers of mature leukocytes as well as hemopoietic cells recovered from digested lung fragments, relative to saline-challenged, sensitized controls. Both mature eosinophils and cells capable of differentiating into eosinophils in the presence of IL-5 were present in lungs from sensitized mice 24 h after airway challenge. Dexamethasone strongly inhibited eosinophil differentiation in IL-5-stimulated cultures of lung hemopoietic cells. By contrast, dexamethasone enhanced eosinophil differentiation in cultures of allergic bone-marrow cells, in identical conditions. Hemopoietic cells from lungs and bone-marrow were respectively susceptible and resistant to induction of apoptosis by dexamethasone. The dexamethasone-sensitive step was the response to IL-5 in culture, while accumulation of IL-5 responsive cells in allergen-challenged lungs was dexamethasone-resistant. Cells from lungs and bone-marrow, cultured for 3 days with IL-5 in the absence of dexamethasone, did not respond to a subsequent exposure to dexamethasone in the presence of IL-5. These findings confirm that IL-5-responsive hemopoietic cells found in challenged, sensitized murine lungs differ from those in bone-marrow, with respect to the cellular responses induced by dexamethasone, including apoptosis.

Allergenic sensitization prevents upregulation of haemopoiesis by cyclo-oxygenase inhibitors in mice.

1. We evaluated whether immunization affects bone-marrow responses to indomethacin, because allergenic sensitization and challenge upregulate responses to haemopoietic cytokines (including IL-5-driven eosinopoiesis) in murine bone-marrow, while indomethacin upregulates haemopoiesis and protects bone-marrow from radiation damage. 2. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized mice, with or without intranasal challenge. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with indomethacin (10(-7) - 10(-11) M) or aspirin (10(-7) - 10(-8) M). Total myeloid colony numbers and numbers of eosinophil-peroxidase-positive cells were determined at day 7. 3. In naïve BALB/c mice, indomethacin (10(-7) - 10(-9) M) increased GM-CSF-stimulated myeloid colony formation (P=0.003 and P=0.009, respectively). In contrast, it had no effect on bone-marrow of ovalbumin-sensitized and challenged mice. Indomethacin (10(-7) - 10(-9) M) also increased eosinophil precursor responses to IL-5 in bone-marrow of naïve (P<0.001 and P=0.002 respectively), but not sensitized-challenged mice. Aspirin (10(-7) M) had similar effects, equally abolished by sensitization. Enhancement of haemopoiesis by indomethacin required adherent cells from naïve bone-marrow. Nonadherent cells responded to IL-5 but not to indomethacin. Indomethacin was effective on bone-marrow from sham-sensitized, ovalbumin-challenged, but not from sensitized, saline-challenged mice. Plasma transfer from immune mice abolished eosinophil precursor responses to indomethacin in bone-marrow of naïve recipients. This was not prevented by previous removal of antibody from immune plasma. 4. COX inhibitors enhance haemopoiesis in naïve but not allergic mice. Responsiveness to indomethacin can be abolished either by active sensitization or by immune plasma transfer. Specific antibody is not involved.

Bronchopulmonary hyperreactivity and lung eosinophil sequestration but not their migration to the alveolar compartment are independent of interleukin-5 in allergic mice.

IL-5 is present in the lung and in the circulation following allergenic challenges in humans and in animals, but its role in bronchopulmonary hyperreactivity (BHR) and lung and bronchoalveolar lavage fluid (BALF) eosinophilia remains unclear. Because compartmentalization of IL-5 is recognized, the anti-IL-5 monoclonal antibody TRFK-5 or its isotype control GL113 were delivered selectively intranasally (i.n.) and/or intravenously (i.v.) before the prior i.n. challenge with 10 mug OVA in BALB/c and BP2 "Biozzi" mice immunized according to optimized protocols with read-outs taken 24 h later. IL-5 in the BALF was suppressed by i.n. TRFK-5, whereas its production persisted in the serum. Conversely, i.v. TRFK-5 suppressed IL-5 in the serum but not in the BALF. IL-5 was suppressed in conditioned medium from lung explants from mice treated with i.n. TRFK-5, which did not affect the other Th2 cytokines, IL-4 and IL-13. IL-5 is thus present in the alveolar, pulmonary and circulatory compartments following an i.n. allergenic challenge. When specific anti-IL-5 antibodies were delivered by the same i.n. route, BALF eosinophilia was markedly reduced, whereas BHR and lung eosinophil sequestration persisted totally or mostly, in both strains. The passage of eosinophils from lungs to alveoli depends on IL-5 released into the BALF, but not into circulation, whereas their lung sequestration and BHR are mostly IL-5-independent. IL-5 alone does not account for the complexities of BHR or of eosinophil tissue trapping, and lung-targeted immunobiologicals should be delivered into the appropriate compartment in order to assess the role of specific mediators in experimental airways/lung allergy.

Effects of fractions of traffic particulate matter on TH2-cytokines, IgE levels, and bronchial hyperresponsiveness in mice.

In recent decades an increased prevalence of allergic conditions has been observed in developed countries. Although lifestyles, exposure to infection, and diet are all likely important factors, many studies have also shown a strong link between industrialization and allergy. The aim of this study was to investigate which extract fractions from traffic particulate matter (TPM, collected in a tunnel in Prague) have the greatest impact on different inflammatory and immunological parameters, such as cytokine production, levels of immunoglobulin E (IgE) antibodies, and bronchial hyperresponsiveness (BHR) in mice, when the extracts are used together with birch pollen for immunization. BP2 mice were immunized with birch pollen and different fractions of TPM (fractions 1-8). They were provoked intranasally with a mixture of pollen and TPM or pollen alone before they were challenged with methacholine. The BHR was evaluated in a whole-body plethysmograph. Th2 cytokines and fibronectin concentrations were measured, and differential cell counts were performed in the bronchioalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titers. The highest titers of IgE and the highest BHR were found in the positive control mice (immunized and provoked with a mixture of pollen and TPM), followed by mice immunized with pollen and fraction 2 (which contains organic acids). Fraction 2 also induced the highest number of eosinophils and increased levels of interleukin 5 (IL-5) in the BAL fluid. The highest levels of IL-5, in BAL fluid and sera, were obtained in mice immunized with fraction 6 (moderately polar compounds), a somewhat surprising result since those mice did not produce any IgE, did not have any eosinophils in their BAL, or showed almost no BHR. Our data demonstrate that fractions 2 (organic acids) and 7 (highly polar compounds) seem to contain potential adjuvants stimulating the IL-5 production, the IgE synthesis, the eosinophil recruitment, and the bronchial hyperreactivity. Further characterization at the molecular level is now necessary to be able to identify the exact nature of those potential adjuvants. This will be of help in the future to improve the quality of the urban air aerosol.

Enhancement of methacholine-evoked tracheal contraction induced by bacterial lipopolysaccharides depends on epithelium and tumor necrosis factor.

Inhaled bacterial lipopolysaccharides (LPSs) induce an acute tumour necrosis factor-alpha (TNF-α-) dependent inflammatory response in the murine airways mediated by Toll-like receptor 4 (TLR4) via the myeloid differentiation MyD88 adaptor protein pathway. However, the contractile response of the bronchial smooth muscle and the role of endogenous TNFα in this process have been elusive. We determined the in vivo respiratory pattern of C57BL/6 mice after intranasal LPS administration with or without the presence of increasing doses of methacholine (MCh). We found that LPS administration altered the basal and MCh-evoked respiratory pattern that peaked at 90 min and decreased thereafter in the next 48 h, reaching basal levels 7 days later. We investigated in controlled ex vivo condition the isometric contraction of isolated tracheal rings in response to MCh cholinergic stimulation. We observed that preincubation of the tracheal rings with LPS for 90 min enhanced the subsequent MCh-induced contractile response (hyperreactivity), which was prevented by prior neutralization of TNFα with a specific antibody. Furthermore, hyperreactivity induced by LPS depended on an intact epithelium, whereas hyperreactivity induced by TNFα was well maintained in the absence of epithelium. Finally, the enhanced contractile response to MCh induced by LPS when compared with control mice was not observed in tracheal rings from TLR4- or TNF- or TNF-receptor-deficient mice. We conclude that bacterial endotoxin-mediated hyperreactivity of isolated tracheal rings to MCh depends upon TLR4 integrity that signals the activation of epithelium, which release endogenous TNFα.

Tumor necrosis factor is not associated with intestinal ischemia/reperfusion-induced lung inflammation.

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: putative role of leukotriene B4 and nitric oxide.

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco's modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. Incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 microM) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with l-arginine (1 microM). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh.

Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation.

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days, and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route. One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB 4, TXB 2, IL-1 beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung. Vitamins C, E and apocynin reduced the levels of LTB 4 in BAL-cultured cells of the FA and FA/OVA groups, but increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1). In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability. Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned.

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome.

IL-2-induced vascular leak syndrome (VLS) is an important mechanism explaining the toxic effects of this cytokine and limiting its therapeutic use. We previously characterized a mouse model of IL-2-induced pulmonary VLS used to demonstrate that NK lymphocytes are involved in early/acute phase VLS (after one IL-2 injection). We also showed that NK cells and polymorphonuclear neutrophils (PMN) are involved in the late/chronic phase of the syndrome (after four daily IL-2 injections). In this study we use our mouse model to evaluate the role played by the IL-2 receptor (IL-2R) in VLS induction. Mouse and human IL-2R are different since the mouse IL-2Rbeta chain does not recognize IL-2. Here, we compare the acute and late VLS responses in human IL-2Rbeta transgenic and C57BL/6 wild type mice. Parameters linked to early phase VLS (bronchoconstriction and PMN mobilization) are enhanced in human IL-2Rbeta transgenic mice. By contrast, parameters used to measure late events (protein leakage and edema) are similar in human IL-2Rbeta transgenic mice and C57BL/6 wild type animals. However, after four IL-2 injections, the cellular content of the bronchoalveolar lavage fluids was different between the two types of animals. This study also characterizes a humanized animal model that could be further used to study human IL-2 activity and side effects in vivo.

Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory response to inhaled endotoxin.

Inhaled endotoxin induces an inflammatory response that contributes to the development and severity of asthma and other forms of airway disease. Here, we show that inhaled endotoxin-induced acute bronchoconstriction, TNF, IL-12p40, and KC production, protein leak, and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecule MyD88. Bronchoconstriction, inflammation, and protein leak are normal in Toll/IL-1R domain-containing adaptor inducing IFN-beta-deficient mice. MyD88 is involved in TLR, but also in IL-1R-associated kinase 1-mediated IL-1R and -18R signaling. We exclude a role for IL-1 and IL-18 pathways in this response, as IL-1R1 and caspase-1 (ICE)-deficient mice develop lung inflammation while TLR4-deficient mice are unresponsive to inhaled LPS. Significantly, using bone marrow chimera, we demonstrate that both hemopoietic and resident cells are necessary for a full MyD88-dependent response to inhaled endotoxin; bronchoconstriction depends on resident cells while cytokine secretion is mediated by hemopoietic cells.

Modulation of the induction of lung and airway allergy in the offspring of IFN-gamma-treated mother mice.

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-gamma reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-gamma was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-gamma-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-gamma-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-gamma than Nor/nor mice when stimulated with Con A. At the age of 6-7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-gamma, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-gamma during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.

Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNF-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome.

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 alpha,beta MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 alpha,beta MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.

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.

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.