The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties.

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.

A base substitution in the interleukin-10 (IL-10) promoter between Sp1 and ets-1 binding sites is not associated with variation of IL-10 levels.

Interleukin 10 (IL-10) may play an important anti-inflammatory and immunoregulatory role in asthma. In this study, we investigated the role of a C to A substitution at position -627 of the IL-10 promoter, located in a necessary transcriptional region, which contains a number of putative transcriptional binding sites. The -627 nucleotide position is itself flanked by Sp-1 and ets-1 binding sites. We studied the allele frequency in 53 unrelated subjects from an admixed Caucasian, Asian and Pacific Islander group with personal or family histories of asthma. The frequency of homozygous C/C, heterozygous C/A, and homozygous A/A alleles at position -627 was 0.28, 0.44 and 0.28, respectively. In vitro assays indicated no differences between the C/C and A/A forms in binding transcriptional factors, especially Sp-1 factor, or in promoter activity. Moreover, in this selected population, there was no association between the C to A substitution and serum IL-10 levels. The mean level of IL-10 serum was determined to be 3.87 +/- 1.23 pg/ml in subjects carrying the A/A genotype, 3.47 +/- 0.57 for C/C genotype and 3.13 +/- 0.41 for the heterozygous (C/A genotype). This requires confirmation by comparing to non-asthmatic subjects. We conclude that although the -627 A allele occurs frequently (50% of alleles) in this selected group, in vitro assays and serum IL-10 levels suggest that the -627C-->A substitution represents a silent or neutral variant in the IL-10 promoter.

Polymorphisms in the interleukin-4 receptor alpha chain: association with traits of allergy and asthma in an admixed population in Hawaii.

Single nucleotide polymorphisms (SNPs) in the gene encoding the interleukin-4 receptor alpha chain (IL-4R alpha) have been associated with IgE levels or clinical atopy in some populations. Two SNPs that encode S503P and Q576R in the intracytoplasmic domain of the receptor are associated with loss or gain of function, respectively. We investigated the frequency of these SNPs and their association with traits of allergic asthma in 36 unrelated subjects selected from a racially admixed, clinically ascertained study population with family histories of asthma. The frequency of the 1682 T to C substitution that encodes S503P was 0.11 (70 alleles analyzed, from 29 TT homozygotes and 6 TC heterozygotes). The frequency of the 1902 A to G substitution that encodes Q576R was 0.26 (68 alleles analyzed, from 20 AA homozygotes, 10 AG heterozygotes and 4 GG homozygotes). In this atopic admixed sample, no significant association was detected between the variant genotypes and serum IgE levels, percentage of eosinophils, skin test reactivity, diagnosis of asthma or methacholine reactivity. More conclusive findings await clinical characterization of non-atopic, non-asthmatic subjects as well as more efficient and extensive haplotyping.

Differential effects of inhaled nitric oxide and hyperoxia on pulmonary dysfunction in newborn guinea pigs.

This study tested the hypothesis that inhaled nitric oxide (NO) and combined NO and hyperoxia will result in less pulmonary dysfunction and delay onset of respiratory signs compared with hyperoxia-exposed newborn guinea pigs (GPs). GPs were exposed to room air (n = 14), 95% O(2) (n = 36), 20 parts per million (ppm) NO (n = 14), or combined 20 ppm NO and 95% O(2) (NO/O(2), n = 13) for up to 5 days. Data evaluated included latency interval for onset of respiratory distress, pressure volume curves, lung histology, and bronchoalveolar lavage (BAL) polymorphonuclear cells (PMNs), proteolytic activity, and total protein. NO-exposed GPs did not develop respiratory distress and had no evidence of pulmonary dysfunction. O(2)-exposed GPs developed respiratory distress after 1-5 days (median 4.0) vs. 3-5 days (median 5.0) for NO/O(2) exposure (P < 0.05). BAL from O(2)-exposed GPs showed increased PMNs compared with NO/O(2)-exposed GPs. O(2)- and NO/O(2)-exposed GPs had comparable reduced lung volumes, lung histology, and increased BAL proteinase activity and total protein. In summary 1) O(2) exposure resulted in multiple measures of pulmonary dysfunction in newborn GPs, 2) 5-day exposure to NO produced no noticeable respiratory effects and pulmonary dysfunction, and 3) short-term exposure (

Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo.

BACKGROUND: Previous studies have demonstrated that induction of heat shock protein (HSP) 72 by whole-body hyperthermia reduces infarct size in an in vivo model of ischemia and reperfusion. Furthermore, hearts obtained from transgenic mice that overexpress HSP72 demonstrate improved functional recovery and decreased infarct size in vitro after global ischemia and reperfusion. METHODS AND RESULTS: To test the hypothesis that overexpression of HSP72 in transgenic mice reduces infarct size in vivo, transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Core body temperature was monitored with a rectal thermometer and maintained between 36.5 degrees C and 37.0 degrees C with a heating pad. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was smaller in [+]HSP72 mice compared with [-]HSP72 mice (12.7 +/- 2.8% [n = 7] versus 33.4 +/- 4.5% [n = 6], infarct size/risk area, respectively; P < .05; mean +/- SEM). CONCLUSIONS: Overexpression of HSP72 reduces infarct size in this in vivo transgenic mouse model of myocardial ischemia and reperfusion.

Substance P enhances electrical field stimulation-induced mast cell degranulation in rat trachea.

We previously demonstrated in an ex vivo rat tracheal model that chymotryptic activity is an index of mast cell degranulation and that substance P (SP) and electrical field stimulation (EFS) synergistically degranulate mucosal and connective tissue mast cells. In the current study, we found that the facilitatory effect of SP was apparent at concentrations as low as 10(-9) M. This effect was mimicked by 10(-7) M neurokinin A or by 10(-6) M capsaicin and was blocked by the NK1 receptor antagonist CP-96,345. SP + EFS-induced mast cell secretion was significantly attenuated by 10(-6) M tetrodotoxin. The response was also attenuated in tracheas from rats in which sensory nerves had been depleted by systemic pretreatment with capsaicin or in which sympathetic nerves had been depleted by systemic pretreatment with 6-hydroxy-dopamine. Atropine (10(-6) M) or indomethacin (10(-5) M) also attenuated SP + EFS-induced mast cell secretion. Our findings suggest the importance of a sensitizing rather than a direct stimulating effect of SP on mast cell degranulation. SP may increase the sensitivity of mast cells to EFS-discharged mediators or facilitate the release of mast cell-stimulating mediators from autonomic nerves.

Purification and characterization of dog mast cell protease-3, an oligomeric relative of tryptases.

The existence of a protein approximately 48% identical with mast cell tryptases was predicted previously from a dog mastocytoma cDNA. Antibodies raised against a peptide based on the deduced sequence suggested that the protein (dog mast cell protease-3, dMCP-3) is expressed in mast cells. In this report, characterization of the protein purified from mastocytomas reveals an N-glycosylated, high molecular weight, tryptic serine protease, which appears to be a tetramer of catalytic subunits, approximately half of which are linked by disulfide bonds. The oligomeric complex yields a single NH2-terminal sequence, which is identical with that predicted by dMCP-3 cDNA. This finding, and the lack of closely related genes on blots of genomic DNA, predict that each subunit is the product of one gene. Although dMCP-3 binds to heparin, it is active and stable at low ionic strength in heparin's absence. It resists inactivation by inhibitors in plasma but is sensitive to small inhibitors, e.g. leupeptin and bis(5-amidino-2-benzimidazolyl)methane (BABIM). dMCP-3 hydrolyzes extended peptidyl p-nitroanilides ending in basic residues, with P1 arginine preferred to lysine; it hydrolyzes the Arg18-Ser19 bond of calcitonin gene-related peptide but cleaves neither vasoactive intestinal peptide nor casein. These data suggest that dMCP-3 is a unique serine protease whose stability, formation of intersubunit disulfide bonds, inhibitor susceptibilities and substrate preferences differ from those of its closest relatives, the mast cell tryptases.

Multiple mechanisms for the effects of capsaicin, bradykinin and nicotine on CGRP release from tracheal afferent nerves: role of prostaglandins, sympathetic nerves and mast cells.

Application of capsaicin (CAP), bradykinin (BK) or nicotine (NIC) to intraluminally perfused rat tracheas induced an increase in calcitonin gene-related peptide (CGRP) levels in the perfusates. Depletion of sensory afferent CGRP with systemic CAP pretreatment resulted in a significant reduction of CGRP release evoked by CAP, BK or NIC. Chemical destruction of sympathetic nerve fibres by systemic pretreatment with 6-hydroxydopamine reduced CGRP release evoked by NIC, but did not alter the release produced by CAP or BK. Elimination of the tracheal mast cell population by pretreatment with compound 48/80 did not alter the effects of CAP, BK or NIC. CGRP release evoked by BK and NIC, but not CAP, was diminished by indomethacin, suggesting that cyclooxygenase products mediate the actions of BK and NIC. Prostaglandins, PGE1, PGE2, PGF2 alpha and PGI2, displayed stimulatory effects on CGRP release in the trachea. There are evidently multiple mechanisms mediating CGRP release from sensory terminals in rat trachea. It appears that CAP exerts a direct action on sensory nerves, while the effects of BK and NIC are mediated by PG synthesis. Sympathetic activation may be involved in NIC, but not BK, induced PG-mediated CGRP release.

Chymotryptic activity in perfusates of isolated rat trachea: correlation with mucosal and connective tissue mast cell secretion.

Serine proteinases participate in many inflammatory events in the airway. We therefore screened perfusates of isolated rat tracheas for tryptic, elastolytic, and chymotryptic serine proteinases. Only chymotryptic activity, indicated by hydrolysis of the synthetic substrate N-succinylalanylalanylprolylphenylalanyl p-nitroaniline (AAPF), was consistently detected in these perfusates. Basal levels of chymotryptic activity were not increased significantly by electrical field stimulation (EFS) (mean change +/- SEM: -0.05 +/- 0.05 m o.d. units, n = 4) or by 10(-7) M substance P (SP) (+0.04 +/- 0.02 m o.d. units, n = 14). However, the mean change after the stimuli were jointly administered (0.17 +/- 0.06 m o.d. units, n = 12) was significantly greater than control or after EFS (P = 0.01, one-way ANOVA). The SP + EFS-induced chymotryptic activity was inhibited by PMSF, soybean trypsin inhibitor, and chymostatin and was associated with an increase in histamine concentration and immunoreactivity to rat mast cell proteases (RMCP), indicating that the activity is due to mast cell degranulation. However, the activity was not significantly decreased by pretreating rats with systemic compound 48/80. SP + EFS-induced chymotryptic activity peaked rapidly and was associated with modest histamine release and an immediate peak in immunoreactivity to RMCP II, a marker of mucosal mast cells. Immunoreactivity to RMCP I, a marker of connective tissue mast cells, also increased after SP + EFS, but this immunoreactivity was either delayed or more sustained and did not coincide with the peak of chymotryptic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.

Degradation of airway neuropeptides by human lung tryptase.

Several lines of evidence suggest a possible role for mast cell proteases in modulating the biologic effects of neuropeptides. To explore the potential of such interactions in human airway, we examined the activity of human tryptase, the major secretory protease of human lung mast cells, against several neuropeptides with proposed regulatory functions in human airway. Using highly purified tryptase obtained from extracts of human lung, we determined the sites and rats of hydrolysis of vasoactive intestinal peptide (VIP), peptide histidine-methionine (PHM), calcitonin gene-related peptide (CGRP), and the tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Tryptase hydrolyzes VIP rapidly at several sites (Arg12, Arg14, Lys20, and Lys21) with an overall kcat/Km of 1.5 x 10(5) M-1 s-1 and hydrolyzes PHM primarily at a single site (Lys20) with a kcat/Km of 1.9 x 10(4) M-1 s-1. Tryptase also rapidly hydrolyzes CGRP at two sites (Arg18 and Lys24) with a kcat/Km of 2.7 x 10(5) M-1 s-1. The tachykinins are not hydrolyzed by tryptase. These observations raise the possibility that tryptase-mediated degradation of the bronchodilators VIP and PHM combined with exaggerated mast cell release of tryptase may contribute to the increase in bronchial responsiveness and the decrease in immunoreactive VIP in airway nerves associated with asthma. The favorable rates of hydrolysis of CGRP suggest that tryptase may also terminate the effects of CGRP on bronchial and vascular smooth muscle tone and permeability.

Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family.

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.

Protease inhibitors potentiate smooth muscle relaxation induced by vasoactive intestinal peptide in isolated human bronchi.

To examine the role of endogenous proteases in limiting the bronchodilating effects of vasoactive intestinal peptide (VIP) in human airway, we studied precontracted bronchial rings from five nonsmokers undergoing heart-lung transplantation for pulmonary hypertension, either primary or secondary to congenital heart disease. The protease inhibitors aprotinin, leupeptin, phosphoramidon, and soybean trypsin inhibitors significantly potentiated the bronchodilator response to VIP. Even in the presence of the four protease inhibitors, VIP-induced bronchodilation reversed spontaneously in some tissues. These studies show that degradation by endogenous airway proteases is an important determinant of the bronchodilating potency of VIP in isolated human airway.

Proteolytic inactivation of dog lung surfactant-associated proteins by neutrophil elastase.

The adsorption of pulmonary surfactant to an air/fluid interface is influenced by calcium-dependent interactions between its lipid and protein components. The latter include a glycoprotein of 28-36 kDa (SP-A) and two smaller hydrophobic proteins of 5-8 kDa (SP-B, SP-C). Neutrophil elastase and other proteolytic enzymes found in the alveolar washings in a variety of acute lung injuries may cleave the protein components of lung surfactant. To examine the hypothesis that free airspace elastolytic activity may thereby impair surfactant function, we analyzed the effect of neutrophil elastase on surfactant activity in vitro. The adsorption characteristics of dog surfactant and of complexes reassembled from purified surfactant components were examined after incubations with active or heat-inactivated neutrophil elastase. Surfactant preincubated with the active enzyme showed a marked concentration-dependent slowing of adsorption associated with proteolytic cleavage of SP-A. To determine whether elastase also decreases surface activity by affecting the hydrophobic proteins SP-B and SP-C, we studied the effect of incubating elastase with liposomes prepared from surfactant lipid fractions which contain SP-B and SP-C. The addition of intact SP-A to these liposomes incubated with inactive enzyme immediately enhanced adsorption speed. This enhancement was greatly attenuated in liposomes treated with active elastase, suggesting that one or both of the hydrophobic surfactant proteins had been affected by elastase. We conclude that proteolytic cleavage of surfactant proteins reduces adsorption speed in vitro and may disturb surfactant function in vivo.

Sulfur dioxide does not acutely increase nasal symptoms or nasal resistance in subjects with rhinitis or in subjects with bronchial responsiveness to sulfur dioxide.

We examined whether brief exposures to moderately high concentrations of sulfur dioxide (SO2) causes acute increases in nasal symptoms and nasal resistance in subjects with chronic rhinitis. We studied 19 subjects with allergic rhinitis and 3 subjects with chronic intermittent rhinorrhea, nasal congestion, and sneezing without any other manifestation of allergy. We found that the change in nasal resistance and symptoms caused by nasal inhalation of 4 ppm of SO2 for 10 min was no greater than the changes caused by nasal inhalation of conditioned room air. In a second set of experiments, we examined whether allergic subjects with demonstrable bronchomotor responsiveness to SO2 also had nasal responsiveness to the gas. We studied 8 subjects with a history of both asthma and allergic rhinitis. Each subject developed symptoms of dyspnea or wheezing and an increase in specific airway resistance of at least 8 L x cm H2O/L/s after breathing 1 or 2 ppm of SO2 by mouthpiece at 20 L/min, and did not develop these changes after breathing room air under the same conditions. No subject, however, developed more nasal symptoms or a greater increase in nasal airway resistance after tidally breathing SO2 through the nose than after breathing room air, even when the concentration of SO2 delivered to the nose was double the concentration delivered through the mouthpiece to the lower airways. We conclude that brief exposure to SO2 at a concentration of 4 ppm or less is unlikely to cause significant nasal dysfunction in most subjects with chronic rhinitis, and that in subjects with both allergic rhinitis and asthma, responsiveness to SO2 is not uniform throughout the respiratory tract.

Globule leukocytes and mast cells in the rat trachea: their number, distribution, and response to compound 48/80 and dexamethasone.

Globule leukocytes in the epithelium of the rat trachea may be counterparts of mucosal mast cells that are located in the gastrointestinal tract. If they are indeed similar to mucosal mast cells, globule leukocytes would be expected to decrease in number in rats treated with dexamethasone but not in rats treated with compound 48/80, an agent which causes non-antigenic degranulation of connective tissue mast cells. In this study, we determined the number and compared the distribution of globule leukocytes and connective tissue mast cells in the tracheas of pathogen-free rats. We then determined whether the number of these two types of cells changes in rats treated for 5 days with compound 48/80, dexamethasone, a combination of compound 48/80 and dexamethasone, or saline. We identified globule leukocytes and mast cells in whole mounts and histological sections of rat tracheas by using a histochemical reaction that demonstrates the chymotrypsin-like protease (chloroacetate esterase) present in mast cell granules. Using this method, we found that approximately 225,000 globule leukocytes were present in the epithelium of the trachea. These cells were most abundant in the rostral trachea. Rats treated with dexamethasone had a 91% reduction in the number of globule leukocytes with protease-containing granules, but rats treated with compound 48/80 had a normal number of these cells. We found some 55,000 connective tissue mast cells in the same tracheas. Mast cells were most abundant in the posterior membrane of the caudal trachea and in the lamina propria between cartilaginous rings. Rats treated with compound 48/80 had a 96% reduction in mast cells with protease-containing granules, but rats treated with dexamethasone had a normal complement of mast cells. We conclude that globule leukocytes are abundant in the tracheas of healthy rats, are similar in morphology and pharmacological responses to mucosal mast cells located in other organs of rats, and are more numerous than and have a different distribution than connective tissue mast cells. Globule leukocytes in the tracheal epithelium may have a role in respiratory defenses similar to that of mucosal mast cells in other organs.

Effect of eucapnic hypoxia on bronchomotor tone and on the bronchomotor response to dry air in asthmatic subjects.

Because hypoxia has been shown to cause bronchoconstriction and to potentiate bronchomotor responsiveness in animals, we investigated whether hypoxia has similar effects in subjects with asthma. We measured specific airway resistance (SRaw; the mean of 5 sequential readings taken 30 s apart) before and immediately after each of 15 asthmatic subjects breathed a mixture of 8% O2 in N2 until hemoglobin saturation (SaO2; by ear oximetry) fell to 80% or less for at least 2 min. We maintained end-tidal CO2 at resting levels, the temperature of the inspirate at 22.0 +/- 1.1 degrees C, and the dew point at 18.5 +/- 1.6 degrees C (mean +/- SD). The SaO2 fell to 70 +/- 8%; minute ventilation rose to 28.4 +/- 8.5 L/min, and heart rate rose by 27 +/- 6 beats/min. The SRaw did not increase significantly in the group (baseline SRaw, 6.61 +/- 2.36; posthypoxia SRaw, 6.69 +/- 2.21 L X cm H2O/L/s) or in any subject. To determine if hypoxia increases bronchomotor responsiveness, we also compared the responses to eucapnic hyperpnea with dry air and with dry gas mixtures of 7 to 10% O2 in N2 in a randomized, double-blind sequence in 9 of the subjects. We measured SRaw in each subject before and after stepwise increases in minute ventilation, for 3 min at each level, until SRaw doubled or until the subject's maximal voluntary ventilation was achieved. The SaO2 fell to 82% or less at each level of ventilation with the hypoxic gas mixture. The 2 stimulus-response curves thus obtained did not differ in any subject.(ABSTRACT TRUNCATED AT 250 WORDS)