Protective effect of estradiol on acute lung inflammation induced by an intestinal ischemic insult is dependent on nitric oxide.

INTRODUCTION: It has been shown that the innate immune system mediates acute lung inflammation triggered by intestinal trauma. Sexual dimorphism modulates the profile of TH1 and TH2 lymphocytes, and accordingly sex hormones may modulate acute lung inflammation by intestinal ischemia/reperfusion (I/R). Studies indicate that female rats are relatively resistant to organ injury caused by hemorrhagic shock and that the gut of female is more resistant than that of the male to deleterious effects of ischemic injury. At the present study, we investigated the effect of estradiol (E(2)) on the lung inflammation after intestinal I/R and its interaction with the nitric oxide (NO) pathway. METHODS: Anesthetized female rats submitted or not to 7 days ovariectomy (OVx) were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2 h of reperfusion. Groups of rats were treated with E(2) (17ß-estradiol, 280 µg/kg, s.c.) 24 h before ischemia and/or with the nonselective NO synthase inhibitor L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) (5 mg/kg, i.v.). In a parallel set of experiments, the selective NO synthase inhibitor, aminoguanidine (50 mg/kg i.v.), was given 1 h before ischemia. In all groups, lung vascular permeability (LVP) was assessed using the Evans blue dye extravasation method, neutrophil recruitment to the tissues by the standard myeloperoxidase (MPO) method, and endothelial NO synthase (eNOS) protein expression by Western blot. RESULTS: In OVx rats, LVP and MPO were increased after intestinal I/R as compared with intact controls. Estradiol reverted the LVP, but not MPO. Aminoguanidine reduced LVP in OVx rats. The E(2) protective effect on LVP was abolished by L-NAME; moreover, an increase in LVP even when compared with OVx rats treated only with L-NAME was observed. In addition, lung eNOS protein expression was reduced in OVx-I/R rats in comparison to intact controls and the E(2) inhibited this effect. CONCLUSIONS: Estradiol treatment is able to reduce lung inflammation due to intestinal I/R, but with the concomitant blockade of NOS activity, this effect is abolished. Nitric oxide probably reduces the vascular deleterious effects of intestinal I/R, and E(2) pretreatment reduces lung inflammation after intestinal I/R and exerts these effects by modulating eNOS protein expression in the lungs.

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.

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.

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.

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.

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.

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.

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.

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.

Interference of methysergide, a specific 5-hydroxytryptamine receptor antagonist, with airway chronic allergic inflammation and remodelling in a murine model of asthma.

BACKGROUND: Airway remodelling encompasses the structural changes observed in asthmatic airways. Mast cells, through the release of histamine and 5-hydroxytryptamine (serotonin), are implicated in early asthmatic reactions, bronchoconstriction and mucosal oedema, and in the development of bronchial hyperresponsiveness. However, the association between serotonin and remodelling processes in murine model of airways inflammation remains to be elucidated. OBJECTIVE: As serotonin is released by murine mast cells upon antigen challenge, we tested the hypothesis of its involvement in the development of inflammatory and remodelling processes in a murine model of chronic airway inflammation following prolonged allergen challenge. Methods BALB/c mice were exposed to aerosolized ovalbumin for 20 min 2 days a week, for 4 consecutive weeks. Two hours before each challenge, they were treated with methysergide (intranasally, 40 microg/kg). Forty-eight hours after the last aerosol challenge, bronchoalveolar lavage (BAL) and lung tissue were collected for analysis. RESULTS: Methysergide inhibited the allergen-induced increase in airway eosinophilia, reduced T helper type 2 (Th2) cytokines in lung, spleen or thoracic lymph nodes, and specific IgE levels. The extravasation of plasma and fibronectin production in the lung, and collagen deposition in the lung were also inhibited after methysergide treatment. Although methysergide treatment induced an increase in IFN-gamma levels, experiments with neutralizing antibody suggest that this is not responsible for inhibition. In addition, instillation of serotonin to immunized mice induced eosinophil recruitment to BAL, Th2 cytokine production and fibronectin release in lung as well as collagen deposition. CONCLUSION: Serotonin may contribute to the development and maintenance of remodelling through the release of cytokines and of fibrogenic mediators. Serotonin should therefore be considered as relevant for the development and maintenance of airway remodelling.

Ectopic lung transplantation induces the accumulation of eosinophil progenitors in the recipients' lungs through an allergen- and interleukin-5-dependent mechanism.

BACKGROUND: Airway challenge of ovalbumin-sensitized mice induces intrapulmonary accumulation of eosinophil progenitors. OBJECTIVE: To evaluate whether allergen-challenged lungs release factors promoting intrapulmonary accumulation of haemopoietic cells, and define the role of allergic lung injury, we developed a transplantation model. METHODS: Lung tissue from allergen-challenged, sensitized donors was ectopically grafted in syngeneic recipients, and haemopoietic progenitors inside the lungs of the recipients were quantified. RESULTS: In BALB/c mice, accumulation of progenitors occurred only when: (a) donors were sensitized and airway challenged with homologous allergen; (b) and recipients were sensitized. Grafts from the appropriate donors released biologically active IL-5, which was effective in sensitized recipients. The effect of the appropriate donor-recipient combination was prevented by neutralizing anti-IL-5 antibody. Grafts from unchallenged, sensitized donors synergized with recombinant IL-5 in sensitized recipients. Unlike BALB/c, grafts from naïve IL-5 transgenic CBA/Ca mice (whose lungs contained a large number of progenitors, independently of sensitization and challenge) were effective in non-transgenic, ovalbumin-sensitized recipients. CONCLUSION: This shows that: (a) intrapulmonary accumulation of progenitors is independent of immunological injury; (b) grafts systemically release IL-5, which is required for progenitor accumulation in the recipients' lungs; (c) and sensitization is required for full responsiveness to IL-5 and for generation of lung-derived signals that synergize with IL-5.

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.

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.

Prostaglandin E2 and dexamethasone regulate eosinophil differentiation and survival through a nitric oxide- and CD95-dependent pathway.

Apoptosis, involving both CD95/CD95L interactions and their modulation by nitric oxide (NO), is central to regulation of mature eosinophil numbers. However, its role in regulating eosinophil production from bone-marrow precursors is unknown. We examined the effects of prostaglandin E2 (PGE2) and dexamethasone on eosinophil differentiation and survival in murine bone-marrow cultures, and their relationship to: NO production as well as CD95/CD95L-dependent apoptosis. Bone-marrow cultures were established with IL-5, alone or in association with PGE2, dexamethasone or both. PGE2 (10(-7)M) inhibited eosinophil differentiation by selectively inducing apoptosis in developing eosinophils. Dexamethasone (10(-7)M) protected developing eosinophils from PGE2-induced apoptosis. Since dexamethasone prevents induction of nitric oxide synthase (NOS), we evaluated the role of NO in the effects of both PGE2 and dexamethasone. NO donors (SNAP and SNP) down-modulated eosinophil precursor responses to IL-5. SNAP induced apoptosis through a dexamethasone-resistant mechanism. The NOS inhibitors, Nomega-nitro-L-arginine and aminoguanidine, blocked the effects of PGE2 on developing eosinophils. PGE2 was ineffective in bone-marrow from knockout mice lacking inducible NOS. PGE2 up-regulated CD95 and CD95L expression in developing eosinophils. Neither PGE2 nor SNAP were effective in cultures from CD95L-deficient gld mice. These data suggest that PGE2 induces apoptosis in developing eosinophils through inducible NOS, leading to NO-dependent activation of the CD95L/CD95 pathway, while dexamethasone antagonizes the effects of PGE2 on the same targets.

Induction of bone-marrow eosinophilia in mice submitted to surgery is dependent on stress-induced secretion of glucocorticoids.

1 We examined bone-marrow in mice receiving subcutaneous implants of heat-coagulated egg white, which are known to present chronic eosinophilic inflammation at the implant site. Egg white implants (EWIs) induced marked bone-marrow eosinophilia, and increased bone-marrow cell responses to granulocyte-macrophage colony-stimulating factor and interleukin-5 in culture. These effects were observed as early as 24 h and lasted for, at least, 30 days in implant recipients. 2 We found, however, that increased eosinophil production was also observed in control mice which underwent surgery but received no EWI (sham-implanted mice), up to 15 days post-surgery. As this suggests an important contribution of nonspecific stress mechanisms to eosinopoiesis, we further evaluated the role of stress hormones produced by the adrenal glands in the bone-marrow eosinophilia of sham-implanted mice. 3 Bone-marrow eosinophilia in mice undergoing surgery was dissociated from increases in other haemopoietic lineages. Surgery by itself increased circulating corticosterone levels by 24 h, and the increase was prevented by inhibition of adrenal glucocorticoid production by metyrapone. The effect of surgery on bone-marrow eosinophilia was prevented by pretreatment with both the glucocorticoid receptor antagonist, mifepristone, and metyrapone, and by surgical adrenalectomy. 4 By contrast, cathecolamine receptor antagonists (propranolol, prazosin and yohimbine) were ineffective, indicating that cathecolamine release from the adrenal glands was not responsible for the effects on bone-marrow. 5 These results highlight a critical role for stress-induced glucocorticoid hormones in selectively upregulating bone-marrow eosinopoiesis in mice submitted to surgery.

Arthropod-derived histamine-binding protein prevents murine allergic asthma.

Because histamine receptor type I blockade attenuates allergic asthma, we asked whether complete neutralization of histamine by an arthropod-derived, high affinity histamine-binding protein (EV131) would prevent allergic asthma. Intranasal administration of EV131 given before Ag challenge in immunized mice prevented airway hyperreactivity by 70%, and abrogated peribronchial inflammation, pulmonary eosinophilia, mucus hypersecretion, and IL-4 and IL-5 secretion. Saturation with histamine abrogated the inhibitory effect of EV131 on bronchial hyperreactivity. The inhibitory effect of EV131 on bronchial hyperreactivity was comparable to that of glucocorticosteroids. These results demonstrate that histamine is a critical mediator of allergic asthma. Therefore, complete neutralization of histamine, rather than specific histamine receptor blockade, may have a profound effect on allergic asthma.

NK cells and polymorphonuclear neutrophils are both critical for IL-2-induced pulmonary vascular leak syndrome.

The mechanism of IL-2-induced vascular leak syndrome (VLS) is still poorly understood. Cells of both innate and adaptive immune systems have been implicated, but no definitive conclusions have been reached concerning their respective roles. In this study we report a new mouse model of IL-2-induced pulmonary VLS used to obtain a detailed analysis of the early events (sequestration of polymorphonuclear neutrophils and bronchoconstriction) and late events (modifications in the cell and protein content of bronchoalveolar lavages, followed by edema) that characterize this lung injury. This model and knockout animals are used to reconsider the importance of the different leukocyte lineages in early and late events. Recombinase-activating gene 2(-/-) mice are used to demonstrate that adaptive lymphocytes, including NK T cells, are not required for pulmonary VLS induction. By contrast, results obtained with newly described recombinase-activating gene 2(-/-)/IL-15(-/-) mice indicate that NK cells play a key role in both early and late events. In parallel, polymorphonuclear neutrophil depletion is used to evaluate the contributions made by these cells to the late alterations occurring in the lung. Furthermore, when used in combination with inhibition of NO synthase, granulocyte depletion was completely effective in protecting mice from the late events of IL-2-induced pulmonary VLS. Together our results indicate that both NK and PMN cells play a central role in the late events of IL-2-induced VLS.