Endogenous nitric oxide protects against T cell-dependent lethality during graft-versus-host disease and idiopathic pneumonia syndrome

The pathogenesis of idiopathic pneumonia syndrome (IPS), a noninfectious pulmonary complication of allogeneic bone marrow transplantation (BMT), has not been fully elucidated. However, several contributing factors have been proposed, including lung injury caused by reactive oxygen and nitrogen intermediates during preconditioning and development of graft-vs-host disease (GVHD). Studies on the role of reactive oxygen and nitrogen intermediates in IPS have yielded conflicting results. We have described a murine model of IPS, in which the onset of lung inflammation was delayed by several weeks relative to GVHD. This study evaluated whether the delay in onset of IPS was due to slow turnover of NO-producing, immunosuppressive alveolar macrophages (AM) following BMT. The results indicated that AM were immunosuppressive due to synthesis of NO. However, NO production and immunosuppressive activity by AM did not decline after BMT, but rather remained elevated throughout the 12-wk development of GVHD and IPS. In a 14-day model of IPS, continuous inhibition of NO with aminoguanidine (AG) reduced signs of IPS/GVHD, but also led to higher mortality. When AG treatment was initiated after onset of IPS/GVHD, rapid mortality occurred that depended on the severity of IPS/GVHD. AG-enhanced mortality was not due to inhibition of marrow engraftment, elevated serum TNF-alpha, liver injury, or hypertensive responses. In contrast, T cells were involved, because depletion of CD4(+) lymphocytes 24 h before AG treatment prevented mortality. Thus, NO production following allogeneic BMT affords a protective effect that helps down-regulate injury caused by T cells during GVHD and IPS.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Blockade of CXCR3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication after allogeneic stem cell transplantation (allo-SCT) that responds poorly to standard immunosuppressive therapy. The pathophysiology of IPS involves the secretion of inflammatory cytokines including IFN-gamma and TNF-alpha along with the recruitment of donor T cells to the lung. CXCR3 is a chemokine receptor that is expressed on activated Th1/Tc1 T cell subsets and the expression of its ligands CXCL9 (monokine induced by IFN-gamma (Mig)) and CXCL10 (IFN-gamma-inducible protein 10 (IP-10)) can be induced in a variety of cell types by IFN-gamma alone or in combination with TNF-alpha. We used a lethally irradiated murine SCT model (B6 --> bm1) to evaluate the role of CXCR3 receptor:ligand interactions in the development of IPS. We found that Mig and IP-10 protein levels were significantly elevated in the bronchoalveolar lavage fluid of allo-SCT recipients compared with syngeneic controls and correlated with the infiltration of IFN-gamma-secreting CXCR3(+) donor T cells into the lung. The in vivo neutralization of either Mig or IP-10 significantly reduced the severity of IPS compared with control-treated animals, and an additive effect was observed when both ligands were blocked simultaneously. Complementary experiments using CXCR3(-/-) mice as SCT donors also resulted in a significant decrease in IPS. These data demonstrate that interactions involving CXCR3 and its primary ligands Mig and IP-10 significantly contribute to donor T cell recruitment to the lung after allo-SCT. Therefore, approaches focusing on the abrogation of these interactions may prove successful in preventing or treating lung injury that occurs in this setting.     

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.     

Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation

Bone marrow transplantation (BMT) is an important therapeutic option for a variety of malignant and nonmalignant disorders. Unfortunately, BMT recipients are at increased risk of infection, and in particular, pulmonary complications occur frequently. Although the risk of infection is greatest during the neutropenic period immediately following transplant, patients are still vulnerable to pulmonary infections even after neutrophil engraftment. We evaluated the risk of infection in this postengraftment period by using a well-established mouse BMT model. Seven days after syngeneic BMT, B6D2F(1) mice are no longer neutropenic, and by 3 wk, they demonstrate complete reconstitution of the peripheral blood. However, these mice remain more susceptible throughout 8 wk to infection after intratracheal administration of Pseudomonas aeruginosa; increased mortality in the P. aeruginosa-infected BMT mice correlates with increased bacterial burden in the lungs as well as increased systemic dissemination. This heightened susceptibility to infection was not secondary to a defect in inflammatory cell recruitment to the lung. The inability to clear P. aeruginosa in the lung correlated with reduced phagocytosis of the bacteria by alveolar macrophages (AMs), but not neutrophils, decreased production of TNF-alpha by AMs, and decreased levels of TNF-alpha and IFN-gamma in the bronchoalveolar lavage fluid following infection. Expression of the beta(2) integrins CD11a and CD11c was reduced on AMs from BMT mice compared with wild-type mice. Thus, despite restoration of peripheral blood count, phagocytic defects in the AMs of BMT mice persist and may contribute to the increased risk of infection seen in the postengraftment period.     

Immunosuppressive effect of mesenchymal stem cells on lung and gut CD8+ T cells in lipopolysaccharide‐induced acute lung injury in mice

ObjectivesAcute lung injury (ALI) not only affects pulmonary function but also leads to intestinal dysfunction, which in turn contributes to ALI. Mesenchymal stem cell (MSC) transplantation can be a potential strategy in the treatment of ALI. However, the mechanisms of synergistic regulatory effects by MSCs on the lung and intestine in ALI need more in‐depth study.Materials and methodsWe evaluated the therapeutic effects of MSCs on the murine model of lipopolysaccharide (LPS)‐induced ALI through survival rate, histopathology and bronchoalveolar lavage fluid. Metagenomic sequencing was performed to assess the gut microbiota. The levels of pulmonary and intestinal inflammation and immune response were assessed by analysing cytokine expression and flow cytometry.ResultsMesenchymal stem cells significantly improved the survival rate of mice with ALI, alleviated histopathological lung damage, improved intestinal barrier integrity, and reduced the levels of inflammatory cytokines in the lung and gut. Furthermore, MSCs inhibited the inflammatory response by decreasing the infiltration of CD8⁺ T cells in both small‐intestinal lymphocytes and Peyer''s patches. The gut bacterial community diversity was significantly altered by MSC transplantation. Furthermore, depletion of intestinal bacterial communities with antibiotics resulted in more severe lung and gut damages and mortality, while MSCs significantly alleviated lung injury due to their immunosuppressive effect.ConclusionsThe present research indicates that MSCs attenuate lung and gut injury partly via regulation of the immune response in the lungs and intestines and gut microbiota, providing new insights into the mechanisms underlying the therapeutic effects of MSC treatment for LPS‐induced ALI.     

Influence of DMT1 and iron status on inflammatory responses in the lung

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.     

Thalidomide reduces lipopolysaccharide/zymosan-induced acute lung injury in rats

Pharmacological therapies targeting fulminant lung inflammation in acute lung injury (ALI) need to be improved. We evaluated the effect of thalidomide, a chemical modulating both acute and chronic inflammation, on ALI induced by intravenous administration of lipopolysaccharide (LPS) and zymosan in male Sprague-Dawley rats. Injection of LPS and zymosan induced significant lung inflammation, as evidenced by increased neutrophil sequestration in lung tissue as well as enhanced nitric oxide metabolite (NO _x ^– ) production in the serum and bronchoalveolar lavage (BAL) fluid. Lactate dehydrogenase (LDH) activity and protein concentration in BAL fluid were significantly increased after administration of LPS and zymosan. Pulmonary microvascular permeability was determined using the Evans blue retention method, which showed a significant increase in microvascular permeability after LPS and zymosan administration, indicating the development of ALI. Animals that received thalidomide (100 mg/kg) 2 h prior to LPS injection had significantly reduced pulmonary NO _x ^– production, pulmonary microvascular permeability, and LDH activity and protein concentration in BAL fluid. We therefore conclude that thalidomide ameliorates lung inflammation and reduces ALI induced by combined LPS and zymosan administration in rats.     

Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice.

We examined the effect of lipopolysaccharide (LPS) treatment on the expression of manganese and copper/zinc superoxide dismutase (MnSOD and Cu/ZnSOD) mRNA and protein in resident peritoneal macrophages and lung endothelial cells derived from LPS-sensitive (LPS-s) and LPS-resistant (LPS-r) mice. Macrophages from both LPS-s and LPS-r mice treated with LPS for 24 h produced increased levels of MnSOD mRNA and protein. In contrast, levels of lung endothelial cell MnSOD mRNA and protein from LPS-s mice were increased by LPS treatment, while no increases in these parameters were observed in endothelial cells from LPS-r mice. Tumor necrosis factor-alpha (TNF alpha) treatment, however, did increase levels of MnSOD mRNA in both LPS-s and LPS-r endothelial cells to an equal extent. Both macrophage and endothelial cell Cu/ZnSOD mRNA and protein levels were not significantly affected by LPS treatment. These results demonstrate that the mutation that affects susceptibility to LPS in LPS-r mice exerts a differential influence on MnSOD inducibility in a cell specific manner.     

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Determinants of surfactant function in acute lung injury and early recovery

Relationships between lung function and surfactant function and composition were examined during the evolution of acute lung injury in guinea pigs. Lung mechanics and gas exchange were assessed 12, 24, or 48 h after exposure to nebulized lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) fluid was processed for phospholipid and protein contents and surfactant protein (SP) A and SP-B levels; surfactant function was measured by pulsating bubble surfactometry. Lung elastance, tissue resistance, and arterial-alveolar gradient were moderately elevated by 12 h after LPS exposure and continued to increase over the first 24 h but began to recover between 24 and 48 h. Similarly, the absolute amount of 30,000 g pelleted SP-A and SP-B, the phospholipid content of BAL fluid, and surfactant function declined over the first 24 h after exposure, with recovery between 24 and 48 h. BAL fluid total protein content increased steadily over the first 48 h after LPS nebulization. In this model of acute lung injury, the intra-alveolar repletion of surfactant components in early recovery led to improved surfactant function despite the presence of potentially inhibitory plasma proteins.     

Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome

The interaction of TNF-alpha with TNF receptor 1 (TNFR1) activates several signal transduction pathways that lead to apoptosis or NF-kappa B-dependent inflammation and immunity. We hypothesized that host TNFR1 expression contributes to noninfectious lung injury and inflammation commonly observed after bone marrow transplantation (BMT), termed idiopathic pneumonia syndrome (IPS). C57BL/6 TNFR1-sufficient (TNFR1(+/+)) and -deficient (TNFR1(-/-)) mice were total body irradiated with or without cyclophosphamide conditioning and were given bone marrow plus IPS-inducing donor spleen T cells from B10.BR wild-type mice. TNFR1(-/-) recipient mice exhibited improved early post-BMT survival associated with decreased permeability edema. In addition, the low lung compliance measured in anesthetized, ventilated TNFR1(+/+) mice on day 7 after BMT was restored to baseline during TNFR1 deficiency. Importantly, bronchoalveolar lavage fluid (BALF) inflammatory cells from TNFR1(-/-) vs. TNFR1(+/+) mice generated less nitric oxide (.NO) and nitrating species and exhibited suppressed programmed cell death as assessed using flow cytometry. However, cellular infiltration and levels of proinflammatory cytokines and chemokines were generally higher in BALF collected on day 7 after BMT from TNFR1(-/-) compared with TNFR1(+/+) recipient mice. Our results support a major role of host TNFR1 in regulation of .NO production and lung dysfunction after allogeneic BMT.     

Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection

The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6.     

Pneumocystis carinii infection sensitizes lung to radiation-induced injury after syngeneic marrow transplantation: role of CD4+ T cells

A murine model of bone marrow transplant (BMT)-related lung injury was developed to study how infection sensitizes lung to the damaging effects of total body irradiation (TBI) at infectious and TBI doses that individually do not cause injury. Mice infected with Pneumocystis carinii exhibited an asymptomatic, rapid, and transient influx of eosinophils and T cells in bronchoalveolar lavage fluid (BALF). In contrast, mice infected with P. carinii 7 days before receiving TBI and syngeneic BMT (P. carinii/TBI mice) exhibited severe pulmonary dysfunction, surfactant aggregate depletion, and surfactant activity reductions at 17 days post-BMT. BALF from P. carinii/TBI mice contained a disproportionate initial influx of CD4(+) T cells (CD4(+):CD8(+) ratio of 2.7) that correlated with progressive lung injury (from 8 to 17 days post-BMT). Levels of TNF-alpha in BALF were significantly increased in P. carinii/TBI mice compared with mice given either insult alone, with peak values found at 11 days post-BMT. In vivo depletion of CD4(+) T cells in P. carinii/TBI mice abrogated pulmonary dysfunction and reduced TNF-alpha levels in BALF, whereas depletion of CD8(+) T cells did not affect lung compliance or TNF-alpha. Lung injury was not attributable to direct P. carinii damage, since CD4-depleted P. carinii/TBI mice that exhibited no injury had higher average lung P. carinii burdens than either mice given P. carinii alone or undepleted P. carinii/TBI mice. Together, these results indicate that P. carinii infection can sensitize the lung to subsequent TBI-mediated lung injury via a process dependent on non-alloreactive CD4(+) T cells.     

Adoptive transfer of acute lung injury

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.     

Exuberant inflammation in nicotinamide adenine dinucleotide phosphate-oxidase-deficient mice after allogeneic marrow transplantation

We have shown that NO and superoxide (O-*2)contribute to donor T cell-dependent lung dysfunction after bone marrow transplantation (BMT) in mice. We hypothesized that inhibiting superoxide production during inducible NO synthase induction would suppress oxidative/nitrative stress and result in less severe lung injury. Irradiated mice lacking the phagocytic NADPH-oxidase (phox(-/-)), a contributor to superoxide generation, were conditioned with cyclophosphamide and given donor bone marrow in the presence or absence of inflammation-inducing allogeneic spleen T cells. On day 7 after allogeneic BMT, survival, weight loss, and indices of lung injury between phox(-/-) and wild-type mice were not different. However, the majority of macrophages/monocytes from phox(-/-) mice given donor T cells produced fewer oxidants and contained less nitrotyrosine than cells obtained from T cell-recipient wild-type mice. Importantly, suppressed oxidative stress was associated with marked infiltration of the lungs with inflammatory cells and was accompanied by increased bronchoalveolar lavage fluid levels of the chemoattractants monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha and impaired clearance of recombinant mouse macrophage-inflammatory protein-1beta from the circulation. Furthermore, cultured macrophages/monocytes from NADPH-deficient mice produced 3-fold more TNF-alpha compared with equal number of cells from NADPH-sufficient mice. The high NO production was not modified during NADPH-oxidase deficiency. We conclude that phox(-/-) mice exhibit enhanced pulmonary influx of inflammatory cells after BMT. Although NO may contribute to increased production of TNF-alpha in phox(-/-) mice, the data suggest that NADPH-oxidase-derived oxidants have a role in limiting inflammation and preventing lung cellular infiltration after allogeneic transplantation.     

The essential role of the intestinal microbiota in facilitating acute inflammatory responses

The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-alpha, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-alpha and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-alpha or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their "no inflammation" phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.     

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.     

Identification with MRI of the pleura as a major site of the acute inflammatory effects induced by ovalbumin and endotoxin challenge in the airways of the rat

Inflammatory effects in the rat lung have been investigated, non-invasively by MRI, at early time points (3 and 6 h) after ovalbumin (OA) or endotoxin (LPS) challenges. Six hours after challenge with OA, a strong, even inflammatory signal was present around the periphery of the lung in a region corresponding to the pleura. Histological analysis confirmed the presence of marked edema associated with the pleural cavity of OA-treated animals. Lower levels of pleural edema were observed in MRI and histological evaluation of LPS-treated animals and no abnormality was observed in actively sensitized and na?ve, saline-treated groups. Diffuse edematous signals were detected in the lung 3 and 6 h after challenge with OA or LPS; the signal volumes were larger at both time points following OA instillation. Bronchoalveolar lavage (BAL) fluid analysis performed 6 h after challenge revealed increased levels of protein and greater cellular activation in OA- than in LPS-treated animals. Furthermore, increased levels of peribronchial edema were found by histology 6 h after OA. BAL fluid and histological assessments demonstrated that the inflammatory signals were due to edema and not mucus as no significant changes in BAL mucin concentrations or differences in goblet cells were identified between OA or LPS challenge and their respective vehicle groups. Our data show that MRI is able to detect, non-invasively, inflammatory signals in both the lung and the pleura in spontaneously breathing animals, highlighting its potential to study the consequences of pulmonary insults on both sites.