Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung.

Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing.

Peptidase modulation of vasoactive intestinal peptide pulmonary relaxation in tracheal superfused guinea pig lungs.

The effects of enzyme inhibitors on vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (PaO) and VIP-like immunoreactivity (VIP-LI) recovery were studied in isolated tracheal superfused guinea pig lungs. In the absence of inhibitors, VIP 0.38 (95% CI 0.33-0.54) nmol/kg animal, resulted in a 50% decrease in PaO and 33% of a 1 nmol/kg VIP dose was recovered as intact VIP. In the presence of two combinations of enzyme inhibitors, SCH 32615 (S, 10 microM) and aprotinin (A, 500 tyrpsin inhibitor units [TIU]/kg) or S and soybean trypsin inhibitor (T, 500 TIU/kg), VIP caused a significantly greater decrease in PaO and greater quantities of VIP were recovered from lung effluent (both P < 0.001). The addition of captopril, (3 microM), leupeptin (4 microM), or bestatin (1 microM) failed to further increase pulmonary relaxation or recovery of VIP-LI. When given singly, A, T, and S did not augment the effects or recovery of VIP. The efficacy of S (a specific inhibitor of neutral endopeptidase [NEP]) and A and T (serine protease inhibitors) thus implicated NEP and at least one serine protease as primary modulators of VIP activity in the guinea pig lung. We sought to corroborate this finding by characterizing the predominant amino acid sites at which VIP is hydrolized in the lung. When [mono(125I)iodo-Tyr10]VIP was offered to the lung, in the presence and absence of the active inhibitors, cleavage products consistent with activity by NEP and a tryptic enzyme were recovered. These data demonstrate that NEP and a peptidase with an inhibitor profile and cleavage pattern compatible with a tryptic enzyme inactivate VIP in a physiologically competitive manner.

Effects of chronic airway inflammation on the activity and enzymatic inactivation of neuropeptides in guinea pig lungs.

The effects of airway inflammation induced by chronic antigen exposure on substance P (SP)-induced increases and vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (Pao), and the recovery of intact and hydrolyzed radiopeptide were studied in tracheally perfused guinea pig lungs. SP (10(-6) mol/kg) induced a significantly greater increase in Pao in lungs from antigen-exposed (30 +/- 5 cm H2O) than saline-exposed animals (15 +/- 1 cm H2O, P < 0.05). Significantly more intact 3H-SP and significantly less 3H-SP 1-7, a neutral endopeptidase (NEP) hydrolysis product, were recovered from the lung effluent of antigen-exposed than saline-exposed animals (P < 0.05). Injection of VIP (10(-9) mol/kg) induced significantly more pulmonary relaxation in saline-exposed compared with antigen-exposed lungs (62 +/- 4%, P < 0.001). In contrast to effluent from saline-exposed animals, lung effluent from antigen-exposed lungs contained less intact VIP, increased amounts of a tryptic hydrolysis product, and no products consistent with the degradation of VIP by NEP. These data indicate that inflamed lungs are more sensitive to the contractile effects of SP because it is less efficiently degraded by NEP and are less sensitive to the relaxant effects of VIP because it is more efficiently degraded by a tryptic enzyme. Changes in airway protease activity occur with allergic inflammation and may contribute to airway hyperresponsiveness.

Interferon gamma induces prostaglandin G/H synthase-2 through an autocrine loop via the epidermal growth factor receptor in human bronchial epithelial cells.

The induction of prostaglandin G/H synthase (PGHS; prostaglandin endoperoxide synthase, cyclooxygenase) by proinflammatory cytokines accounts, at least in part, for the altered eicosanoid biosynthesis in inflammatory diseases. In secondary cultures of normal human bronchial epithelial cells (NHBECs), interferon-gamma (IFN-gamma, 10 ng/ml for 24 h) increased the amount of prostaglandin E2 (PGE2) released in response to stimulation with exogenous arachidonic acid (5 microM). The enhanced production of PGE2 reflected the upregulation of PGHS-2 as indicated by enhanced expression of PGHS-2 RNA and increased recovery of PGHS-2 protein in NHBECs. IFN-gamma did not alter the production of PGE2 in A549 cells (a human lung adenocarcinoma cell line) or 6-keto-PGF1alpha in human umbilical vein endothelial cells (HUVECs), although prostaglandin release and/or the expression of PGHS-2 RNA in these cell lines was upregulated by other proinflammatory cytokines. Induction of PGHS-2 RNA in IFN-gamma-treated NHBECs, which peaked at 24 h, suggested the presence of an intermediary substance regulating the expression of PGHS-2. When the binding between the epidermal growth factor (EGF) receptor and its ligands was disrupted by a neutralizing antibody (LA-1), IFN-gamma failed to upregulate the release of PGE2 and the expression of PGHS-2 RNA in NHBECs. Furthermore, IFN-gamma induced the expression of RNAs for a number of ligands at the EGF receptor TGF-alpha; heparin-binding EGF-like growth factor (HB-EGF); and amphiregulin in NHBECs, and when administered exogenously, these ligands increased PGE2 release from NHBECs. Heparin at the concentration that neutralized the function of amphiregulin, or antibodies against TGFalpha or HB-EGF also reduced the release of PGE2 from IFN-gamma-stimulated NHBECs. These data are consistent with the presence of an autocrine growth factor/EGF receptor loop regulating PGHS-2 expression and PGE2 synthesis in bronchial epithelial cells.

Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.

Eotaxin is a potent and specific eosinophil chemoattractant that is mobilized in the respiratory epithelium after allergic stimulation. Pulmonary levels of eotaxin mRNA are known to increase after allergen exposure in sensitized animals. In this study we demonstrate that TNF alpha and IL-1beta induce the accumulation of eotaxin mRNA in the pulmonary epithelial cell lines A549 and BEAS 2B in a dose-dependent manner. Cytokine-induced A549 cell mRNA accumulation was maximal at 4 h and was significantly enhanced when the cells were costimulated with IFNgamma. TNFalpha- and IL-1beta-induced increases in eotaxin mRNA were diminished in a dose-dependent manner by the glucocorticoid dexamethasone and were augmented by the protein synthesis inhibitor cycloheximide. Cytokine-induced increases in eotaxin mRNA expression correlated with increased eotaxin protein production and secretion, and dexamethasone inhibition of cytokine-induced eotaxin mRNA augmentation was associated with diminished eotaxin protein secretion. These findings, together with the known kinetics of TNF alpha and IL-1beta mobilization in asthmatic airways and the potent eosinophil chemotactic effects of eotaxin, define a mechanism linking inflammatory cytokine mobilization to eosinophil recruitment that may be relevant to the pathogenesis of asthma.

Overexpression of eotaxin and the CCR3 receptor in human atherosclerosis: using genomic technology to identify a potential novel pathway of vascular inflammation.

BACKGROUND: Unstable atherosclerotic lesions typically have an abundant inflammatory cell infiltrate, including activated T cells, macrophages, and mast cells, which may decrease plaque stability. The pathophysiology of inflammatory cell recruitment and activation in the human atheroma is incompletely described. METHODS AND RESULTS: We hypothesized that differential gene expression with DNA microarray technology would identify new genes that may participate in vascular inflammation. RNA isolated from cultured human aortic smooth muscle cells treated with tumor necrosis factor-alpha (TNF-alpha) was examined with a DNA microarray with 8600 genes. This experiment and subsequent Northern analyses demonstrated marked increases in steady-state eotaxin mRNA (>20 fold), a chemokine initially described as a chemotactic factor for eosinophils. Because eosinophils are rarely present in human atherosclerosis, we then studied tissue samples from 7 normal and 14 atherosclerotic arteries. Immunohistochemical analysis demonstrated overexpression of eotaxin protein and its receptor, CCR3, in the human atheroma, with negligible expression in normal vessels. Eotaxin was predominantly located in smooth muscle cells. The CCR3 receptor was localized primarily to macrophage-rich regions as defined by immunopositivity for CD 68; a minority of mast cells also demonstrated immunopositivity for the CCR3 receptor. CONCLUSIONS: Eotaxin and its receptor, CCR3, are overexpressed in human atherosclerosis, suggesting that eotaxin participates in vascular inflammation. These data demonstrate how genomic differential expression technology can identify novel genes that may participate in the stability of atherosclerotic lesions.

An intensive communication intervention for the critically ill.

PURPOSE: We sought to determine the effects of a communication process that was designed to encourage the use of advanced supportive technology when it is of benefit, but to limit its burdens when it is ineffective. We compared usual care with a proactive, multidisciplinary method of communicating that prospectively identified for patients and families the criteria that would determine whether a care plan was effective at meeting the goals of the patient. This process allowed caregivers to be informed of patient preferences about continued advanced supportive technology when its continuation would result in a compromised functional outcome or death. MATERIALS AND METHODS: We performed a before-and-after study in 530 adult medical patients who were consecutively admitted to a university tertiary care hospital for intensive care. Multidisciplinary meetings were held within 72 hours of critical care admission. Patients, families, and the critical care team discussed the care plan and the patients' goals and expectations for the outcome of critical care. Clinical "milestones" indicative of recovery were identified with time frames for their occurrence. Follow-up meetings were held to discuss palliative care options when continued advanced supportive technology was not achieving the patient's goals. We measured length of stay, mortality, and provider team and family consensus in 134 patients before the intensive communication intervention and in 396 patients after the intervention. RESULTS: Intensive communication significantly reduced the median length of stay from 4 days (interquartile range, 2 to 11 days) to 3 days (2 to 6 days, P = 0.01 by survival analysis). This reduction remained significant after adjustment for acute physiology and chronic health evaluation (APACHE) 3 score [risk ratio (RR) = 0.81; 95% confidence interval (CI), 0.66 to 0.99; P = 0.04). Subgroup analysis revealed that this reduction occurred in our target group, patients with acuity scores in the highest quartile who died (RR = 0.60; 95% CI, 0.38 to 0.92; P = 0.02). The intervention, which allowed dying patients earlier access to palliative care, was not associated with increased mortality. CONCLUSIONS: Intensive communication was associated with a reduction in critical care use by patients who died. Our multidisciplinary process targeted advanced supportive technology to patients who survived and allowed the earlier withdrawal of advanced supportive technology when it was ineffective.

Urinary leukotriene E4 levels increase upon exposure to hypobaric hypoxia.

STUDY OBJECTIVE: To determine whether urinary leukotriene E4 (uLTE4) levels increase upon exposure to high altitude, and also to ascertain the relationship between uLTE4 levels and symptoms of acute mountain sickness (AMS). DESIGN: Prospective, unblinded, single-factor (altitude) experimental study. SETTINGS: US Army research laboratory facilities at sea level ([SL] 50 m), 1,830 m, and 4,300 m. PARTICIPANTS: Eight healthy male subjects ranging in age from 19 to 24 years. MEASUREMENTS: uLTE4 levels and symptoms of AMS were measured at just above SL (50 m), 3 1/2 days after being transported from SL to moderate altitude (MA) (1,830 m), and 1 1/2 days after ascent from 1,830 to 4,300 m (high altitude [HA]). Symptoms of AMS were assessed using standard indexes derived from the Environmental Symptoms Questionnaire weighted toward cerebral (AMS-C) and respiratory (AMS-R) manifestations. Oxygen saturation was measured noninvasively by pulse oximetry at SL and HA. RESULTS: The mean (+/-SEM) uLTE4 levels (pg/mg creatinine) were 67.9 (+/-13.2) at SL; 82.3 (+/-5.5) at MA; and 134.8 (+/-19.4) at HA (p < 0.05 comparing HA with SL and MA). CONCLUSIONS: We conclude that uLTE4 levels increase shortly after exposure to HA even after staging for 4 days at MA. Although this study does not clearly demonstrate a relationship between uLTE4 levels and symptoms of AMS, it supports the hypothesis that leukotrienes may be involved in the pathophysiologic state of AMS.

The prevalence of exercise-induced bronchospasm among US Army recruits and its effects on physical performance.

STUDY OBJECTIVES: To measure the prevalence of exercise-induced bronchospasm (EIB) and to determine its effect on the physical performance response to training in otherwise healthy young adults. DESIGN: Observational, retrospective study. SETTING: Fort Jackson, SC, May to July 1998. PARTICIPANTS: One hundred thirty-seven ethnically diverse US Army recruits undergoing an 8-week Army basic training course. MEASUREMENTS AND RESULTS: Subjects underwent exercise challenge testing at the end of basic training to evaluate for EIB (defined as a decrease in FEV(1) of > or = 15%, 1 or 10 min after running to peak oxygen uptake on a treadmill). Those subjects who were unable to run to peak oxygen uptake, or who were unable to perform two baseline FEV(1) maneuvers the results of which were within 5% of each other, were excluded from analysis. We measured peak oxygen uptake on a treadmill and the scores achieved on the components of the US Army physical fitness test (APFT). Of 137 subjects, 121 (58 men and 63 women) met our inclusion criteria. Eight subjects (7%) had EIB. Subjects who experienced EIB and unaffected control subjects both showed statistically significant gains in performance on the APFT events during basic training. At the end of basic training, peak oxygen uptake levels and APFT event scores were not significantly different between subjects with EIB and unaffected control subjects. CONCLUSIONS: Seven percent of the US Army recruits who were tested had EIB, but this did not hinder their physical performance gains during basic training. EIB per se should not be an absolute reason to exclude individuals from employment in jobs with heavy physical demands.

Hospitalizations for respiratory syncytial virus infection in Alaska Native children.

To characterize the epidemiology of Alaska Native children hospitalized for respiratory syncytial virus infections, we reviewed records of hospitalizations during the winter seasons of 1991 to 1992 and 1992 to 1993 at a hospital in Anchorage and a rural hospital in the Yukon Kuskokwim Delta (YKD) region of southwestern Alaska. The median age of hospitalization for respiratory syncytial virus infection was 2 months of age for YKD residents and 4.5 months for Anchorage residents. Sixteen percent of the hospitalized YKD children were less than 1 month of age, whereas the same was true for only 3% of the Anchorage children. Eight percent of the YKD patients required mechanical ventilation, whereas none of the Anchorage patients required ventilation. The median hospital stay was 4.8 days for YKD patients and 3.2 days for Anchorage patients. Hospitalization rates for infants less than 1 year of age were 33/1000 for Alaska Natives in Anchorage and 100/1000 for those in the YKD region. The extremely high hospitalization rate, especially among very young infants in the rural YKD region, points to a need for early preventive efforts.

Decline of Haemophilus influenzae type b disease in a region of high risk: impact of passive and active immunization.

Haemophilus influenzae type b (Hib) is a major cause of serious childhood bacterial infections. Before 1989 Alaska Native infants in the Yukon Kuskokwim Delta (YKD) had the highest recorded Hib disease rate, 2960:100,000 in children less than 1 year of age with 6 to 35 (mean, 13) cases/year between 1980 and 1988. In July, 1989, Alaska Area Native Health Service initiated a passive immunization project in the YKD using bacterial polysaccharide immunoglobulin (BPIG) administered at 3-month intervals to prevent Hib infections in infants less than 13 months of age. On January 1, 1991, after licensure of Hib conjugate vaccines for infants, the program was modified to a passive-active strategy using BPIG at birth and PedvaxHIB at 2, 4 and 12 months of age. Between July 1, 1989, and December 31, 1990, 80% of YKD children less than 1 year of age received at least 1 dose of BPIG. During this period there were 7 Hib cases in this age group, but only 1 of the cases had received any BPIG. Between January 1, 1991, and December 31, 1992, 4 Hib cases occurred in 2 YKD children. During the combined period, July 1, 1989, to December 31, 1992, the incidence of Hib disease for infants less than 1 year of age was 302:100,000. A dramatic decrease in Hib disease was observed in this high incidence region concurrent with implementation of passive and passive-active immunization strategies.

Respiratory transfer impedance between 8 and 384 Hz in guinea pigs before and after bronchial challenge.

We report a forced oscillatory technique for noninvasively measuring respiratory transfer impedance (Ztr) between 8 and 384 Hz in guinea pigs. This technique uses a device consisting of two chambers: one surrounding the animal's head that is used as a plethysmograph to measured flow through the airway opening and the other that surrounds the animal's body and is used to apply pressure oscillations to the body surface. Ztr was measured in spontaneously breathing awake guinea pigs and while the animals were anesthetized in normal and methacholine-challenged conditions. An eight-element model consisting of an airway compartment separated from a tissue compartment by a shunt gas compression compartment was fit to the data. Anesthesia increased central and peripheral airway resistance and bronchial airway wall compliance by 13, 31, and 44%, respectively, whereas it decreased tissue compliance by 37%. Compared with the unanesthetized condition, the methacholine challenge (20 micrograms/kg) resulted in an increase in central and peripheral airway resistance (69 and 319%, respectively) and a decrease in bronchial airway wall and tissue compliance (37 and 79%, respectively). This technique is capable of measuring Ztr in anesthetized and awake guinea pigs. Analysis of these data with this eight-element model provides reasonable estimates of airway and tissue parameters.

Substance P-induced histamine release in tracheally perfused guinea pig lungs.

The capacity of substance P (SP) and endogenously released tachykinins to liberate histamine was examined in isolated tracheally perfused guinea pig lungs. Increasing doses of tracheally injected SP were associated with the recovery of increasing amounts of histamine from lung effluent. The mechanism of SP-induced histamine liberation was explored in studies with neurokinin-(NK) receptor agonists and antagonists. Tracheal injection of either the NK1 agonist [Sar9,Met(O2)11]SP or the NK2 agonist [beta-Ala8]-neurokinin A-(4-10) was associated with a significant increase in histamine recovery from lung effluent. In addition, both the NK1 antagonist CP-99994 and the NK2 antagonist SR-48968 significantly inhibited SP-induced histamine release. These findings support the hypothesis that SP can liberate histamine from guinea pigs lungs by a mechanism that depends predominantly on NK1- and NK2-receptor activation. The liberation of endogenous tachykinins by acute tracheal injection of capsaicin was also associated with augmented histamine recovery, which was inhibited by combined NK1- and NK2-receptor blockade. Tracheal injection of SP was associated with an increase in the percentage of airway mast cells exhibiting histological evidence of degranulation. This study demonstrates that exogenous SP, as well as endogenous tachykinins released from capsaicin-sensitive neurons, can liberate histamine, most likely from airway mast cells, by a mechanism that depends predominantly on the activation of NK1 and NK2 receptors.

Angiotensin-converting enzyme genotype and physical performance during US Army basic training.

Prior studies have suggested that angiotensin I-converting enzyme (ACE) genotype correlates with superior physical performance in highly selected populations. This study assessed whether such an association exists in a heterogeneous population. Using polymerase chain reaction techniques, we determined the ACE genotypes (insertion/insertion, deletion/insertion, or deletion/deletion) of 62 male and 85 female US Army recruits. Before and after 8 wk of basic training, we determined peak oxygen uptake and performance on the Army Physical Fitness Test (APFT), which includes standardized measures of muscular endurance (sit-ups, push-ups) and a 2-mile run. Subjects of different ACE genotypes had similar peak oxygen uptakes and APFT scores, both before and after training. Subjects with genotype II had higher APFT scores than others, but the differences were not statistically significant. Furthermore, no ACE genotype group had a performance advantage in analyses that adjusted for baseline fitness. We conclude that ACE genotype does not have a strong effect on aerobic power or muscular endurance in healthy, young American adults drawn from an ethnically and geographically diverse population.

Plasma eotaxin level and severity of asthma treated with corticosteroid.

Our understanding of asthma severity was advanced by the identification of biomarkers which account for differences in lung function impairment. We tried to examine the effects of corticosteroid treatment on known correlates of asthma severity including peripheral eosinophil counts, total IgE, IL-5, and eotaxin Levels in plasma. We compared these biomarkers among groups of stable asthmatics categorized by the dose of corticosteroid (N: steroid-free, n = 25; L: low-dose inhaled, n = 27; MH: medium or high-dose inhaled, n= 19; O: inhaled plus oral, n= 8). Next we compared these markers and peak expiratory flow rate (PEFR) in unstable asthmatics before and after treatment with steroids (n = 22). Eotaxin levels in the O group were higher than those in the N and MH groups (P < 0.05). Logistic regression analysis demonstrated that plasma eotaxin level was correlated with the severity of asthma defined by treatment intensity (P = 0.01) and % FEV1 (P = 0.04) while the other markers were not. Eotaxin levels did not change after steroid treatment in unstable patients, whereas eosinophil counts decreased in parallel with PEFR. Among biomarkers of asthma severity studied, plasma eotaxin level was not significantly affected by corticosteroid treatment, and was associated with the severity even in the presence of steroid therapy.

Glutathione prevents the early asthmatic reaction and airway hyperresponsiveness in guinea pigs.

The prevalence of asthma has increased worldwide. The reasons for this rise remain unclear. Oxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is the major representative of the class of nonprotein thiols and plays a pivotal role in a variety of enzymatic and nonenzymatic reactions that protect tissues against oxidative stress. In antioxidative reactions, GSH is converted into its oxidized form, glutathione disulfide (GSSG) that in its turn is enzymatically reduced into GSH to maintain a physiological redox balance. We used a guinea pig model of asthma to assess whether the early asthmatic reaction is associated with decreased lung levels of glutathione, and whether decreased glutathione is implicated in the increased airway smooth muscle reactivity that is associated with exposure of the lungs to allergen. Lung glutathione levels were decreased immediately after the onset of the early asthmatic reaction in vivo and associated with the release of 8-iso-PGF(2alpha), an indicator for oxidative stress. Glutathione ethylester, a glutathione precursor, blunted the airway obstruction during an early asthmatic reaction in a perfusion model and glutathione depletion rendered the airways hyperreactive. Glutathione ethyl ester in the buffer prevented this hyperreactivity. These results indicate that glutathione can modulate the early asthmatic reaction as well as the airway hyperresponsiveness.

Peptidase modulation of airway effects of neuropeptides.

SP and NKA are potent endogenous bronchoconstrictors, whereas VIP is a potent endogenous bronchodilator. There is abundant evidence that these neuropeptides are released in the lung in a variety of conditions and that they have the capacity to modulate the bronchoactivity of the same stimuli that release them. On many occasions, their bronchoactive effects are masked by their degradation at or near the site of their release. However, when the microenvironment is modified to decrease their cleavage, they can express enhanced physiologic effects. Although it appears that the human asthmatic lung may be an environment in which the effects of neuropeptides can be amplified, the role of neuropeptides in the pathogenesis of airway obstruction remains speculative.

Prostaglandin G/H synthase-2 is the constitutive and dominant isoform in cultured human lung epithelial cells.

Two isoforms of prostaglandin G/H synthase (PGHS; prostaglandin endoperoxide synthase, cyclooxygenase) have been identified; PGHS-1 is expressed constitutively in most tissues, whereas PGHS-2 is thought to be induced by various proinflammatory cytokines and growth factors. In this study, we determined which isoform of PGHS mRNA, protein, and activity was present constitutively in A549 (a human lung adenocarcinoma cell line) and in untransformed (normal human bronchial epithelial or NHBE) and transformed (16HBE4o-) human bronchial epithelial cells. Two PGHS-2-specific inhibitors, NS-398 and L-745, 337, blocked the release of prostaglandin E2 from A549 cells with mean inhibitory concentrations of 5 and 18 nM, respectively, but did not inhibit its release from human bronchial smooth muscle cells (BSMC) at a concentration of 10 microM. Northern and immunoblot analysis demonstrated that BSMC expressed PGHS-1 mRNA and protein constitutively, whereas epithelial cells expressed PGHS-2 mRNA and protein constitutively with either undetectable (A549, 16HBE4o-) or very low levels (NHBE) of PGHS-1. We conclude that PGHS-2 is the dominant PGHS isoform in unstimulated and stimulated lung epithelial cells in culture.

Endogenous nitric oxide and allergic bronchial hyperresponsiveness in guinea pigs.

To address the role of endogenous pulmonary nitric oxide (NO) in the modulation of airway tone, we investigated changes in expired NO levels, measured by chemiluminescence, and the effect of inhibition of NO synthase on inflammation-associated bronchial hyperresponsiveness in guinea pigs. Mixed expired gas NO levels were similar at baseline in antigen-exposed and unexposed animals and increased transiently to a similar degree during histamine-induced bronchoconstriction in both groups of animals [155 +/- 12% (15 +/- 1 to 23 +/- 4 ppb, P < 0.01) and 162 +/- 19% (16 +/- 2 to 25 +/- 3 ppb, P < 0.01) of baseline, respectively, after administration of 30 nmol/kg histamine]. Although inhibition of NO synthase with intravenous NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) enhanced bronchial responsiveness to histamine by 30 +/- 8% in unexposed animals (P < 0.05), L-NAME did not enhance histamine responsiveness in antigen-exposed animals exhibiting bronchial hyperresponsiveness 24 h after antigen exposure. Thus bronchial hyperresponsiveness induced by repeated pulmonary antigen exposure may be associated with a transient defect in NO-related homeostatic bronchodilator activity.

Exhaled nitric oxide following leukotriene E(4) and methacholine inhalation in patients with asthma.

Nitric oxide (NO) is a molecular gas that can be recovered in higher levels from the exhaled gas of subjects with asthma than from subjects without asthma. However, the precise mechanisms responsible of promoting increased fraction of expired nitric oxide (FE(NO)) in asthma are unknown. As leukotriene antagonism has been shown to reduce FE(NO) in patients with asthma, we hypothesized that leukotrienes mediate the increased FE(NO) encountered in this condition. Furthermore, because leukotriene antagonism stabilizes serum eosinophil markers during reductions in inhaled corticosteroid doses, and FE(NO) has been shown to correlate with sputum eosinophils in asthma, we reasoned that the effect of leukotrienes on FE(NO) might be mediated by eosinophils recruited to the airway by leukotrienes. To test this hypothesis, we performed methacholine and leukotriene (LT) E(4) bronchoprovocation challenges in 16 subjects with atopic asthma and measured FE(NO) and sputum differential counts before and after bronchoprovocation. We then compared FE(NO) in the seven subjects who developed increased sputum eosinophils following LTE(4) inhalation with values measured after methacholine inhalation in these seven subjects. Following LTE(4) inhalation, eosinophils rose from 4.01 +/- 0.89% pre-LTE(4) to 8.33 +/- 1.52% post-LTE(4). The mean change in sputum eosinophils from baseline after LTE(4) inhalation was larger than that after methacholine inhalation (+4.31 +/- 1.25% versus -1.14 +/- 0.93%). After LTE(4) inhalation, FE(NO) levels did not differ from prechallenge baseline or from levels following methacholine inhalation (ANOVA p > 0.05). These data indicate that neither LTE(4) nor recruitment of eosinophils into the airway by LTE(4) is a sufficient stimulus to acutely increase FE(NO) in subjects with asthma.