Interleukin-17 and lung host defense against Klebsiella pneumoniae infection.

Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.

Keratinocyte growth factor attenuates hydrostatic pulmonary edema in an isolated, perfused rat lung model.

Hydrostatic pulmonary edema is a common complication of congestive heart failure, resulting in substantial morbidity and mortality. Keratinocyte growth factor (KGF) is a mitogen for type II alveolar epithelial and microvascular cells. We utilized the isolated perfused rat lung model to produce hydrostatic pulmonary edema by varying the left atrial and pulmonary capillary pressure. Pretreatment with KGF attenuated hydrostatic edema formation. This was demonstrated by lower wet-to-dry lung weight ratios, histological evidence of less alveolar edema formation, and reduced alveolar accumulation of intravascularly administered FITC-labeled large-molecular-weight dextran in rats pretreated with KGF. Thus KGF attenuates injury in this ex vivo model of hydrostatic pulmonary edema via mechanisms that prevent increases in alveolar-capillary permeability.

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.

Regulation of tumor necrosis factor cytotoxicity by calcineurin.

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.

Keratinocyte growth factor prevents ventilator-induced lung injury in an ex vivo rat model.

Mechanical ventilation has been shown to produce lung injury characterized by noncardiogenic pulmonary edema. Keratinocyte growth factor (KGF) is a heparin-binding growth factor that causes alveolar type II pneumocyte hyperplasia. KGF pretreatment and the resultant pneumocyte hyperplasia reduce fluid flux in models of lung injury. We utilized the isolated perfused rat lung model to produce lung injury by varying tidal volume and the level of positive end-expiratory pressure during mechanical ventilation. Pretreatment with KGF attenuated ventilator-induced lung injury (VILI). This was demonstrated by lower wet-to-dry lung weight ratios and less lung water accumulation in the KGF group. Further, KGF prevented the decline in dynamic compliance and alveolar protein accumulation in VILI. KGF pretreatment reduced alveolar accumulation of intravascularly administered fluorescein isothiocyanate-labeled high-molecular-weight dextran. Thus, pretreatment with KFG attenuates injury in this ex vivo model of VILI via mechanisms that prevent increases in permeability.

Randomized trial of the use of heliox as a driving gas for updraft nebulization of bronchodilators in the emergent treatment of acute exacerbations of chronic obstructive pulmonary disease.

OBJECTIVE: To determine whether the bronchodilator effects of albuterol and ipratropium bromide are greater if updraft nebulization is driven by 80% helium and 20% oxygen (HELIOX) than if driven by compressed room air (AIR) during the treatment of an acute exacerbations of chronic obstructive pulmonary disease (COPD). SETTING: The emergency department of a 750-bed inner-city community hospital. METHODS: Over a 12-month period, a convenience sample of 50 normoxic patients presenting with signs and symptoms of an acute exacerbation of COPD were prospectively randomized to receive either HELIOX or AIR as the driving gas for updraft nebulization of a mixture of albuterol 2.5 mg and ipratropium bromide 0.5 mg. Additional aerosol treatments with albuterol 2.5 mg were given at 20, 40, and 120 mins after randomization using the assigned gas. Spirometry was obtained while breathing room air before the first treatment (baseline) and at 1 hr and 2 hrs after the initiation of treatment. The primary measure of efficacy was the change in percent of predicted forced expiratory volume in 1 sec (FEV1) over the treatment period. A secondary measure of efficacy was the change in percentage of predicted forced expiratory flow after 25% to 75% of vital capacity had been expelled (FEF25-75). RESULTS: Twenty-five patients were randomized to each treatment group. Three patients (1 HELIOX, 2 AIR) were unable to complete the study. The baseline FEV1was 44% (95% confidence interval, 35% to 52%) of predicted in the HELIOX group and 39 (31% to 46%) of predicted in the AIR group. There were no adverse outcomes observed in either the HELIOX group or the AIR group. There were no significant differences in the change of FEV1 between the two groups by either the 1 hr or 2 hr time point (1 hr, HELIOX + 10% [7% to 13%], AIR + 9% [5% to 13%]; 2 hr HELIOX + 10% [6% to 15%], AIR + 10% [6% to 14%]). The improvement in FEF25-75 was significantly greater in the HELIOX group than in the AIR group at both the 1 hr time point (HELIOX + 14% [7% to 22%] vs. AIR + 7% [3% to 10%], p = .05) and at the 2 hr time point (HELIOX + 15% [8% to 21%] vs. AIR + 7% [4% to 11%], p = .05). CONCLUSION: Use of HELIOX as a driving gas for the updraft nebulization of bronchodilators during the first 2 hrs of treatment of an acute COPD exacerbation failed to improve FEV1 faster than the use of AIR. The faster improvement in FEF25-75 during the first 2 hrs of treatment was small and of uncertain clinical significance.

Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.

Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.

The role of the inflammatory response in chronic bronchitis: therapeutic implications.

Chronic bronchitis is diagnosed clinically by a chronic productive cough. As implied by the term "bronchitis," chronic airway inflammation is typically found in the central airways in patients with persistent cough and mucous hypersecretion. Although the exact pathogenesis of chronic bronchitis remains unclear, bacterial colonization and the resulting inflammatory response are thought to be of central importance. The generation of pro-inflammatory cytokines and chemotactic stimuli by the airway epithelium likely play central roles in propagating the inflammatory response in patients with chronic bronchitis. Further insights into the initiating events and underlying mechanisms that result in the clinical syndrome of chronic bronchitis will likely provide novel opportunities for therapeutic interventions.

Innate immunity and pulmonary host defense.

The lung, in order to facilitate gas exchange, represents the largest epithelial surface area of the body in contact with the external environment. As normal respiration occurs, the upper and lower airways are repeatedly exposed to a multitude of airborne particles and microorganisms. Since these agents are frequently deposited on the surface of the respiratory tract, an elaborate system of defense mechanisms is in place to maintain the sterility of the lung. Innate defenses are primarily responsible for the elimination of bacterial organisms from the alveolus. Early bacterial clearance is mediated by a dual phagocytic system involving both alveolar macrophages and polymorphonuclear leukocytes. The recruitment and activation of inflammatory cells at a site of infection involves the orchestrated expression of leukocyte and vascular adhesion molecules, as well as the establishment of chemotactic gradients via the generation of proinflammatory cytokines and chemokines. Immunologic manipulation of innate immunity may serve as an important adjuvant therapy in the treatment of both immunocompromised and immunocompetent patients with severe lung infections. As the complexities of the host-pathogen interaction are further dissected and elucidated, it is likely that the therapeutic benefits from these approaches will be realized.

Serine/threonine phosphorylation in cellular signaling for alveolar macrophage phagocytic response to endotoxin.

Protein serine/threonine (ser/thr) phosphorylation is an early signaling event in macrophage activation. We investigated the changes in stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats challenged with intratracheal endotoxin. Animals were sacrificed 90 min post intratracheal lipopolysaccharide (LPS, 100 microg/rat) challenge. AMs were incubated with or without phosphatase inhibitors at 37 degrees C for 30 min. Phagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs were determined. LPS challenge activated SAPK/JNK activity and enhanced phagocytosis of AMs without altering phosphatase activity in these cells. Inhibition of phosphatase 1 and 2A activity with okadaic acid and calyculin A exerted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concentration of 1 microM increased the mean channel fluorescence intensity (MCF) and the percentage of cells engaged in phagocytosis (percent phagocytosis) in AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 microM enhanced both the MCF and percent phagocytosis of AMs from LPS-challenged rats. Calyculin A at a concentration of 10 nM increased the MCF phagocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagocytosis of AMs in both saline and LPS groups. AM CD18 expression was not altered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (calyculin A) of AM CD18 expression. These results suggest that ser/thr phosphorylation and dephosphorylation participate in mediating the phagocytic response of AMs to LPS.

Frequency and prognostic significance of pericardial effusion in primary pulmonary hypertension. PPH Study Group. Primary pulmonary hypertension.

Pericardial effusion was noted in 43 of 79 patients (54%) with severe primary pulmonary hypertension. Larger effusion was associated with hemodynamic and echocardiographic evidence of right heart failure, impaired exercise tolerance, and a poor 1-year prognosis.

Granulocyte colony-stimulating factor modulates the pulmonary host response to endotoxin in the absence and presence of acute ethanol intoxication.

Alcohol impairs neutrophil function and predisposes the host to infectious complications. Granulocyte colony-stimulating factor (G-CSF) increases both the number and functional activities of neutrophils. This study investigated the effects of G-CSF on the pulmonary response to endotoxin in rats with or without acute ethanol intoxication. Acute ethanol intoxication inhibited tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 production in the lung and suppressed the recruitment of neutrophils into the lung. Ethanol also inhibited CD11b/c expression on recruited neutrophils and suppressed the phagocytic activity of circulating neutrophils. G-CSF pretreatment up-regulated CD11b/c expression on circulating polymorphonuclear leukocytes, augmented the recruitment of neutrophils into the lung, and enhanced the phagocytic activity of circulating and recruited neutrophils in both the absence and presence of acute ethanol intoxication. G-CSF inhibited MIP-2 but not TNF-alpha production in the lung. These data suggest that G-CSF may be useful in the prevention or treatment of infections in persons immunocompromised by alcohol.

Effects of ibuprofen on the physiology and survival of hypothermic sepsis. Ibuprofen in Sepsis Study Group.

OBJECTIVES: The objective was to compare the clinical and physiologic characteristics of febrile septic patients with hypothermic septic patients; and to examine plasma levels of cytokines tumor necrosis factor alpha (TNF-alpha and interleukin 6 (IL-6) and the lipid mediators thromboxane B2 (TxB2) and prostacyclin in hypothermic septic patients in comparison with febrile patients. Most importantly, we wanted to report the effect of ibuprofen treatment on vital signs, organ failure, and mortality in hypothermic sepsis. SETTING: The study was performed in the intensive care units (ICUs) of seven clinical centers in the United States and Canada. PATIENTS: Four hundred fifty-five patients admitted to the ICU who met defined criteria for severe sepsis and were suspected of having a serious infection. INTERVENTION: Ibuprofen at a dose of 10 mg/kg (maximum 800 mg) was administered intravenously over 30 to 60 mins every 6 hrs for eight doses vs. placebo (glycine buffer vehicle). MEASUREMENTS AND MAIN RESULTS: Forty-four (10%) septic patients met criteria for hypothermia and 409 were febrile. The mortality rate was significantly higher in hypothermic patients, 70% vs. 35% for febrile patients. At study entry, urinary metabolites of TxB2, prostacyclin, and serum levels of TNF-alpha and IL-6 were significantly elevated in hypothermic patients compared with febrile patients. In hypothermic patients treated with ibuprofen, there was a trend toward an increased number of days free of major organ system failures and a significant reduction in the 30-day mortality rate from 90% (18/20 placebo-treated patients) to 54% (13/24 ibuprofen-treated patients). CONCLUSIONS: Hypothermic sepsis has an incidence of approximately 10% and an untreated mortality twice that of severe sepsis presenting with fever. When compared with febrile patients, the hypothermic group has an amplified response with respect to cytokines TNF-alpha and IL-6 and lipid mediators TxB2 and prostacyclin. Treatment with ibuprofen may decrease mortality in this select group of septic patients.

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells.

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.

Innate immunity, cytokines, and pulmonary host defense.

Effective host defense against bacterial infection is dependent on the activation and recruitment of phagocytic cells. The initiation, maintenance, and resolution of this inflammatory response in the setting of bacterial pneumonia is dependent on the expression of cytokines. As the complexities of the host-pathogen interaction are further dissected and unraveled, immunologic manipulation of cytokine expression will likely become an important adjuvant therapy in the treatment of serious lung infections.

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats.

Several methodologies have been developed to assess alveolocapillary membrane permeability in acute lung injury. The purpose of this study was to determine the reliability of FITC-dextran compared with radioactive tracers to assess lung permeability alterations. After intraperitoneal administration of alpha-naphthylthiourea (ANTU, 50 mg/kg) or DMSO-ANTU vehicle, the animals were euthanized and their lungs were studied in an isolated-lung preparation. FITC-dextran or radiolabeled tracers were added to the perfusate. At 2 h the bronchoalveolar lavage (BAL) fluid from the ANTU group showed a significantly greater amount of fluorescence in the supernatant after centrifugation of BAL fluid compared with the DMSO group. Consistent results were observed with the radioactive tracers: there was an increase in extravascular albumin space and extravascular lung water compared with the control group. No cleavage of the FITC from the dextran molecule was evident by chromatography comparing samples recovered from the BAL fluid to the pure FITC-dextran molecule. In conclusion, measurement of FITC-dextran in the supernatant of BAL fluid after intravascular administration is a reliable method of assessing lung permeability changes in vivo and ex vivo.

Pulmonary stress injury within physiological ranges of airway and vascular pressures.

PURPOSE: The aim of this study was to assess the respective role of a small elevation in pulmonary capillary pressure, airway pressure, or both on alveolar capillary barrier permeability in an isolated perfused rat lung model. MATERIALS AND METHODS: Four groups were studied with low or high airway pressure (LA: 10 mL/kg (tidal volume); HA: 20 mL/kg), low or high pulmonary artery pressure (LP: 9 mm Hg; HP: 12 mm Hg): LALP, HALP, LAHP, and HAHP. The lungs were ventilated and perfused ex vivo for 30 minutes. Quantification of fluorescein isothiocyanate-labeled (FITC) dextran in bronchoalveolar lavage (BAL) fluid and radiolabeled tracers assessed alveolar capillary barrier permeability. RESULTS: BALF FITC-dextran was similar in the three groups with either one or two low-pressure parameters (LALP, LAHP, HALP), but high amounts were found in the HAHP group (375.2 x 10(-6) mg/mL v, respectively, 21.4, 26.2, and 30 x 10(-6) mg/mL, P = .0001). These results were consistent with the albumin space and extravascular lung water: higher values only in the HAHP group statistically different from the other groups (P < .002). Interalveolar pore examined with scanning electron microscopy showed an increase in diameters between LALP and HAHP (P < .0001). CONCLUSIONS: We can conclude that elevation of either the pulmonary artery pressure from 8 to 11 mm Hg or the alveolar pressure from 10 to 15 mm Hg alone does not change the permeability of the alveolar capillary membrane; however, there is an additive effect of these pressures.

Acute ethanol intoxication inhibits neutrophil beta2-integrin expression in rats during endotoxemia.

The effects of acute ethanol intoxication on neutrophil [polymorphonuclear leukocyte (PMN)] adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol. Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. Control animals received an intraperitoneal injection of saline. Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 microg/rat in 2.5 ml of saline) or saline. Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 microg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion. Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats. Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs. Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats. Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication. Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs. G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis. The impairment of beta2-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection. G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.