Inhaled porcine pancreatic elastase causes bronchoconstriction via a bradykinin-mediated mechanism.

Neutrophil elastase has been linked to inflammatory lung diseases such as chronic obstructive pulmonary disease, adult respiratory distress syndrome, emphysema, and cystic fibrosis. In guinea pigs, aerosol challenge with human neutrophil elastase causes bronchoconstriction, but the mechanism by which this occurs is not completely understood. Our laboratory previously showed that human neutrophil elastase releases tissue kallikrein (TK) from cultured tracheal gland cells. TK has been identified as the major kininogenase of the airway and cleaves both high- and low-molecular weight kininogen to yield lysyl-bradykinin. Because inhaled bradykinin causes bronchoconstriction and airway hyperresponsiveness in asthmatic patients and allergic sheep, we hypothesized that elastase-induced bronchoconstriction could be mediated by bradykinin. To test this hypothesis, we measured lung resistance (RL) in sheep before and after inhalation of porcine pancreatic elastase (PPE) alone and after pretreatment with a bradykinin B(2) antagonist (NPC-567), the specific human elastase inhibitor ICI 200,355, the histamine H(1)-antagonist diphenhydramine hydrochloride, the cysteinyl leukotriene 1 receptor antagonist montelukast, or the cyclooxygenase inhibitor indomethacin. Inhaled PPE (125-1,000 microg) caused a dose-dependent increase in RL. Aerosol challenge with a single 500 microg dose of PPE increased RL by 132 +/- 8% over baseline. This response was blocked by pretreatment with NPC-567 and ICI-200,355 (n = 6; P < 0.001), whereas treatment with diphenhydramine hydrochloride, montelukast, or indomethacin failed to block the PPE-induced bronchoconstriction. Consistent with pharmacological data, TK activity in bronchial lavage fluid increased 134 +/- 57% over baseline (n = 5; P < 0.02). We conclude that, in sheep, PPE-induced bronchoconstriction is in part mediated by the generation of bradykinin. Our findings suggest that elastase-kinin interactions may contribute to changes in bronchial tone during inflammatory diseases of the airways.

A small-molecule, tight-binding inhibitor of the integrin alpha(4)beta(1) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep.

The leukocyte integrin very late antigen-4 (alpha(4)beta(1), CD49d/CD29) is an adhesion receptor that plays an important role in allergic inflammation and contributes to antigen-induced late responses (LAR) and airway hyperresponsiveness (AHR). In this study, we show that single doses of a new small-molecule, tight-binding inhibitor of alpha(4), BIO-1211, whether given by aerosol or intravenously, either before or 1.5 h after antigen challenge blocks allergen- induced LAR and post-antigen-induced AHR in allergic sheep. Multiple treatments with doses of BIO-1211 that were ineffective when given singly, were protective. BIO-1211 also provided dose-dependent inhibition of the early airway response (EAR) to antigen. In conjunction with the functional protection against the antigen-induced LAR and AHR, sheep treated with BIO-1211 before challenge showed significantly reduced: (1) numbers of eosinophils in bronchoalveolar lavage (BAL), (2) BAL levels of the inflammatory marker tissue kallikrein, and (3) numbers of inflammatory cells (lymphocytes, eosinophils, metachromatic staining cells, and neutrophils) in bronchial biopsies obtained after challenge when compared with corresponding biopsies after vehicle treatment. More importantly, we show for the first time that an inhibitor of alpha(4) was able to reverse post-antigen-induced AHR, thereby decreasing the time of recovery from the normal period of > 9 d to 3 d. Our results show that effective inhibition of antigen-induced airway responses can be achieved with single doses of a potent small-molecule inhibitor of alpha(4) and that such agents may be used therapeutically, as well as prophylactically, to alleviate allergen- induced inflammatory events. These data provide further support and extend the evidence for the role of alpha(4) integrins in the pathophysiologic events that follow airway antigen challenge.

The lactoperoxidase system functions in bacterial clearance of airways.

Airway mucus is a complex mixture of secretory products that provides a multifaceted defense against pulmonary infection. Mucus contains antimicrobial peptides (e.g., defensins) and enzymes (e.g., lysozyme) although the contribution of these to airway sterility has not been tested in vivo. We have previously shown that an enzymatically active, heme-containing peroxidase comprises 1% of the soluble protein in sheep airway secretions, and it has been hypothesized that this airway peroxidase may function as a biocidal system. In this study, we show that sheep airway peroxidase is identical to milk lactoperoxidase (LPO) and that sheep airway secretions contain thiocyanate (SCN(-)) at concentrations necessary and sufficient for a functional peroxidase system that can protect against infection. We also show that airway LPO, like milk LPO, produces the biocidal compound hypothiocyanite (OSCN(-)) in vitro. Finally, we show that in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways. The data suggest that the LPO system is a major contributor to airway defenses. This discovery may have significant implications for chronic airway colonization seen in respiratory diseases such as cystic fibrosis.

Bronchial tissue kallikrein activity is regulated by hyaluronic acid binding.

Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses. To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation. Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml). The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor. Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule. Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC. Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl. Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity. Finally, TK activity bound to HA-Sepharose and could be eluted with HA. These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA. This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen. These events could be important in the regulation of kinin-mediated airway inflammation.

Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep.

Antigen challenge can elicit an allergic inflammatory response in the airways that involves eosinophils, basophils, and neutrophils and that is expressed physiologically as a late airway response (LAR) and airway hyperresponsiveness (AHR). Although previous studies have suggested that E-selectin participates in these allergic airway responses, there is little information concerning the role of L-selectin. To address this question, we examined the effects of administering an L-selectin-specific monoclonal antibody, DU1-29, as well as three small molecule selectin binding inhibitors, on the development of early airway responses (EAR), LAR and AHR in allergic sheep undergoing airway challenge with Ascaris suum antigen. Sheep treated with aerosol DU1-29 before antigen challenge had a significantly reduced LAR and did not develop postchallenge AHR. No protective effect was seen when sheep were treated with a nonspecific control monoclonal antibody. Treatment with DU1-29 also reduced the severity of the EAR to antigen. Similar results were obtained with each of the three small molecule selectin inhibitors at doses that depended on their L-, but not necessarily E-selectin inhibitory capacity. The inhibition of the EAR with one of the inhibitors, TBC-1269, was associated with a reduction in histamine release. Likewise, treatment with TBC-1269 reduced the number of neutrophils recovered in bronchoalveolar lavage (BAL) during the time of LAR and AHR. TBC-1269, given 90 min after antigen challenge also blocked the LAR and the AHR, but this protection was lost if the treatment was withheld until 4 h after challenge, a result consistent with the proposed time course of L-selectin involvement in leukocyte trafficking. These are the first data indicating that L-selectin may have a unique cellular function that modulates allergen-induced pulmonary responses.

Mechanism of pyocyanin- and 1-hydroxyphenazine-induced lung neutrophilia in sheep airways.

Pyocyanin (Pyo) and 1-hydroxyphenazine (1-HP) are extracellular products of Pseudomonas aeruginosa. To test whether these products were capable of producing an inflammatory response in the airways, combinations of Pyo and 1-HP at concentrations of 10(-4) and 10(-5) M were instilled into sheep airways, and indexes of inflammation were assessed by bronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazines caused a significant dose-dependent increase in the number of cells and neutrophils recovered by BAL. Control challenges produced no such changes. The lung neutrophilia was accompanied by an increased concentration of albumin in BAL. The increases in BAL neutrophils and albumin could be blocked by treating the sheep with the 5-lipoxygenase inhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophils when tested in vitro, but when alveolar macrophages (AM) were cultured in vitro in the presence of both Pyo and 1-HP (1 microM), the supernatants caused neutrophil chemotaxis. Analysis of AM culture supernatants incubated with the combination of pigments showed significant increases in leukotriene B4 and interleukin-8, and blocking these mediators separately or together reduced AM supernatant-induced neutrophil chemotaxis. We conclude that local instillation of Pyo and 1-HP can initiate an inflammatory response in the airways of sheep in vivo. This effect can be explained, in part, by the release of chemotactic factors produced by AM.

Elastase contributes to antigen-induced mucociliary dysfunction in ovine airways.

Antigen-induced bronchoconstriction is associated with impairment of mucociliary clearance with a time course that is consistent with the initial influx of neutrophils into the airway. In this study we tested the hypothesis that elastase released from activated neutrophils contributes to the acute (0 to 6-hr) antigen-induced mucociliary dysfunction. Tracheal mucous velocity CTMV), an index of mucociliary function, was measured with a roentgenographic technique before and serially after airway challenge with Ascaris suum antigen alone, or after pretreatment with aerosolized alpha1-protease inhibitor (alpha1-PI, 10 mg) or the specific neutrophil elastase inhibitor ICI 200,355 (10 mg). Antigen alone significantly decreased TMV. Treatment with either alpha1-PI or ICI 200,355, given either at 30 min before antigen challenge or 1 h after challenge, significantly attenuated the antigen-induced reduction in TMV at 6 h after challenge, whereas sheep treated with inactivated alpha1-PI were not protected from this antigen-induced event. Inhalation of ovine elastase (obtained from stimulated neutrophils) significantly decreased TMV, and this effect was also blocked by pretreatment with alpha1-PI. Both alpha1-PI and ICI 200,355 inhibited the activity of elastase obtained from stimulated ovine neutrophils. To verify that the neutrophil numbers and elastase activity increased in sheep airways after antigen challenge, nine animals underwent bronchoalveolar lavage (BAL) at 2 h and 4 h after instillation of A. summ antigen. Four hours after challenge, the number of neutrophils had increased by 50-fold, and free elastase activity in lavage fluid had increased. These data indicate that the antigen-induced impairment of mucociliary clearance is partly dependent on increased elastase activity, and that elastase inhibitors may be useful in protecting against mucociliary dysfunction.

Scavenging of reactive oxygen species by a glycolipid fraction of Mycobacterium avium serovar 2.

Previous experiments indicated that MIF-A3, a peptidoglycolipid extracted from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18), inhibits the killing of Candida albicans by activated bovine peripheral blood-derived macrophages and murine thioglycollate-elicited peritoneal macrophages in vitro. Subsequent in vitro data from our laboratory indicated that this reduction in killing may be related to the ability of MIF-A3 to scavenge reactive oxygen species (ROS). In this study we examined this hypothesis directly by determining if MIF-A3 reduced exogenous H2O2-induced candidacidal activity. When Candida albicans was incubated with H2O2 (4 mM) alone, colony-forming units/ml x 10(4) (CFU/ml) were 0.4 +/- 0.1 (mean +/- SE, n = 4) as compared to 11.3 +/- 2.0 CFU/ml in control (untreated) cultures (p < .05). The addition of catalase at concentrations > or = 6.8 U/ml, completely blocked the fungicidal effect of H2O2. However, reducing the amount of catalase from 6.8 U/ml to 3.4 U/ml resulted in a loss of scavenging activity, which was associated with a 50% increase in H2O2-mediated killing. Substituting MIF-A3 (400 micrograms/ml) for catalase, also reduced H2O2-induced fungicidal activity. In the absence of MIF-A3, H2O2 reduced Candida albicans to less than 10(3) CFU/ml. However, in the presence of MIF-A3 the CFU/ml of Candida albicans increased 7.5-fold. Based on concentration-response curves of H2O2 inhibition vs. increasing amounts of catalase we determined that the relative inhibitory capacity of the MIF-A3 (400 micrograms/ml) was approximately 1.0 U/ml "catalase equivalents." These findings provide direct evidence that MIF-A3 can scavenge H2O2, and reduce H2O2-induced killing of Candida albicans.

Extracellular metabolites of Pseudomonas aeruginosa produce bronchoconstriction by different mechanisms.

Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites. In this study we investigated the mechanism(s) responsible for this bronchial effect. BS were obtained from 48 h cultures and sterilized by filtration. Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL). Aerosols of BS (3 ml total) immediately increased SRL (541%). Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect. BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H1 antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B2 receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol). The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 +/- 21% protection was seen with chlorpheniramine pretreatment. These findings are characteristic of a BK-mediated response. Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 +/- 7 to 304 +/- 55 pg/ml. Control (broth) challenges produced no such change. To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.

Eosinophils and T lymphocytes are thought to be involved in allergic airway inflammation. Both cells express the alpha 4 beta 1-integrin, very late antigen-4 (VLA-4, CD49d/CD29); alpha 4-integrins can promote cellular adhesion and activation. Therefore, we examined the in vivo effects of a blocking anti-alpha 4 monoclonal antibody, HP 1/2, on antigen-induced early and late bronchial responses, airway hyperresponsiveness, inflammatory cell influx, and peripheral leukocyte counts in allergic sheep. Sheep blood lymphocytes, monocytes, and eosinophils expressed alpha 4 and bound HP 1/2. In control sheep, Ascaris antigen challenge produced early and late increases in specific lung resistance of 380 +/- 42% and 175 +/- 16% over baseline immediately and 7 h after challenge, respectively, as well as airway hyperresponsiveness continuing for 14 d after antigen challenge. Treatment with HP 1/2 (1 mg/kg, i.v.) 30 min before antigen challenge did not affect the early increase in specific lung resistance but inhibited the late-phase increase at 5-8 h by 75% (P < 0.05) and inhibited the post-antigen-induced airway hyperresponsiveness at 1, 2, 7, and 14 d (P < 0.05, for each time). Intravenous HP 1/2 given 2 h after antigen challenge likewise blocked late-phase airway changes and postchallenge airway hyperresponsiveness. Airway administration of HP 1/2 (16-mg dose) was also effective in blocking these antigen-induced changes. Response to HP 1/2 was specific since an isotypic monoclonal antibody, 1E6, was ineffective by intravenous and aerosol administration. Inhibition of leukocyte recruitment did not totally account for the activity of anti-alpha 4 antibody since HP 1/2 neither diminished the eosinopenia or lymphopenia that followed antigen challenge nor consistently altered the composition of leukocytes recovered by bronchoalveolar lavage. Because airway administration of HP 1/2 was also active, HP 1/2 may have inhibited cell activation. Reduction of platelet-activating factor-induced eosinophil peroxidase release from HP 1/2-treated eosinophils supports such a mechanism. These findings indicate a role for alpha 4-integrins in processes that lead to airway late phase responses and persisting airway hyperresponsiveness after antigen challenge.

Lipid mediators contribute to oxygen-radical-induced airway responses in sheep.

The purpose of this study was to determine if the bronchoconstriction and airway hyperresponsiveness (AHR) resulting from aerosolized xanthine (x; 0.1%)-xanthine oxidase (xo; 4.1 U) and the subsequent production of oxygen radicals is mediated by the secondary generation of lipid mediators. In seven conscious sheep, specific lung resistance (SRL) was measured before and after x-xo challenge; approximately 30 min later when SRL had returned to baseline, airway responsiveness to carbachol was determined from dose-response curves by calculating the cumulative provocating dose of carbachol in breath units (BU, defined as one breath of a 1% wt/vol carbachol solution) that increased SRL 400% over baseline (PD400). Inhaled x-xo caused in immediate increase in SRL of 162 +/- 36% (mean +/- SE; p less than 0.05) over baseline and decreased PD400 from a baseline value of 32.5 +/- 5.0 to 16.6 +/- 1.7 BU (p less than 0.05). Pretreatment with the H2O2 scavenger, catalase (CAT,; 38 mg aerosol), methylprednisolone succinate (MS; 1 mg/kg given intravenously), the cyclooxygenase inhibitor, indomethacin (IND; 2 mg/kg given intravenously), and the PAF antagonist, WEB-2086 (3 mg/kg given intravenously) all attenuated the x-xo-induced increase in SRL (p less than 0.05); the leukotriene D4 antagonist, MK-571 (5 mg by aerosol) had no effect. All agents inhibited the x-xo-induced decrease in PD400: mean BUs were 27 after CAT, 32 after WEB-2086, 34 after IND, 31 after MS, and 25 after MK-571 (all p less than 0.05 versus x-xo alone).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of P. aeruginosa-derived bacterial products on tracheal ciliary function: role of O2 radicals.

The purpose of this investigation was to evaluate the effects of bacterial products derived from Pseudomonas aeruginosa on the function of airway cilia and to assess the role of phagocytes and oxygen radicals in the observed responses. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from normal sheep by brush biopsy (70% epithelial cells, 18% macrophages, 11% neutrophils). Baseline CBF ranged between 678 and 1,126 min-1. After 20 min of perfusion with the cell free supernatant of P. aeruginosa culture (mucoid strain), a concentration-dependent depression of CBF was observed with a 58% inhibition at a 1:1 dilution (P less than 0.05). The P. aeruginosa-derived products pyocyanin and 1-hydroxyphenazine also decreased CBF in a dose-related fashion. The cilion-inhibitory effects of the supernatant and bacterial products were markedly attenuated after centrifugation of the brush preparation (80% epithelial cells, 16.5% macrophages, 3.5% neutrophils). Glucose/glucose oxidase also caused a rapid, concentration-dependent cilioinhibition or ciliostasis. Catalase blocked or attenuated the ciliary effects of the supernatant, bacterial products and glucose/glucose oxidase. Thus bacterial products released from P. aeruginosa impaired ciliary activity by a pathway which involved neutrophils and was mediated by toxic oxygen radicals.

Heterologous transplantation of activated murine peritoneal macrophages inhibits gamete interaction in vivo: a paradigm for endometriosis-associated subfertility.

Macrophage hyperactivation has been postulated to be the pathologic aberration in patients suffering from endometriosis-associated subfertility. In this report an in vivo model for macrophage-mediated infertility is described. Populations of macrophages were obtained from an inbred strain of mice (Balb/C) as follows: (1) in vivo hyperactivated macrophages (harvested from donor mice treated with intraperitoneal thioglycolate); (2) hyperactivated macrophages deactivated ex vivo with the protein synthesis inhibitor emetine; and (3) basal state (nonactivated) macrophages obtained from untreated mice. Recipient mice underwent ovarian hyperstimulation with pregnant mare serum gonadotropins; 2 x 10(6) macrophages were transferred on the afternoon of stimulation day 3 before injection of human chorionic gonadotropin (hCG) and mating. Unfertilized oocytes and 4-cell embryos were counted on day hCG +2 as a reflection of reproductive performance. Heterologous transfer of in vivo hyperactivated macrophages, but not basal state macrophages, significantly inhibited fertilization. This effect was largely reversed by pretreatment with emetine. These experiments confirm the relevance of macrophage-mediated interference with early reproductive performance and provide a model for the development of alternative therapies (e.g., immunomodulation of the peritoneal fluid environment) for endometriosis-associated subfertility.

Cellular LTB4 production differs in allergic sheep with and without late airway responses.

We tested the hypothesis that allergic sheep that develop both early and late airway responses to inhaled Ascaris suum antigen (late responders) have an increased capacity to generate leukotrienes (LTs) compared with allergic sheep that show only early responses to inhaled antigen (acute responders). To test this hypothesis, we measured LTB4 production, in vitro, by granulocytes isolated from peripheral blood and by macrophages isolated from bronchoalveolar lavage (BAL) from both groups of sheep greater than or equal to 2 wk after the animal's last antigen challenge; LTB4 production by granulocytes isolated from BAL from both groups of sheep 6 and 48 h after local airway challenge with A. suum antigen was also measured. LTB4 production was induced by incubating cells (i.e., either granulocytes or macrophages) with calcium ionophore (A23187, 2 microM) and arachidonic acid (30 microM). LTB4 production was quantitated by high-performance liquid chromatography and verified by radioimmunoassay (RIA). On stimulation peripheral blood granulocytes from late responders (n = 7) produced (means +/- SD/10(6) cells) 13.3 +/- 5.2 ng LTB4 compared with 5.3 +/- 1.5 ng LTB4 (P less than 0.05) for acute responders (n = 7). This increased LTB4 production did not result from variations in granulocyte differential or cyclooxygenase activity (as indicated by RIA measurements of prostaglandin E2 production).(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-detectable effects of localized pneumonia on airway mucous cell populations: role of cyclooxygenase metabolites.

We examined the airway secretory apparatus of adult sheep with experimental pneumonia to look for morphologic and lectin-binding correlates of increased mucus production. The animals were inoculated in the right caudal lobar bronchus either with starch broth containing Pasteurella haemolytica (INF, n = 6), starch broth alone (SHAM, n = 6), or with P. haemolytica and subsequently treated (INF/T, n = 5) with 2 mg/kg indomethacin, subcutaneously three times daily for 6 days. In the INF and INF/T groups, a localized pneumonic infiltrate containing P. haemolytica organisms was present. The bronchi (18-23rd generation) adjacent to the pneumonic lesion had an increased gland volume fraction (6.3 +/- 3.7% in INF, 11.3 +/- 2.4% in INF/T, and 3.1 +/- 1.9% in SHAM, p less than 0.05 among the three). The mean population densities of BSA-reactive (identifying alpha-D-gal) cells were 41.9 +/- 2.7% in the INF, 40.1 +/- 5.6% in the INF/T, versus 14.3 +/- 1.5% in the SHAM group (p less than 0.05), while the corresponding values for PNA-reactive [identifying beta-D-gal(1----3)-D-galNAc] cells were 28.8 +/- 5.1%, 0%, and 0%, respectively. Nor morphologic abnormalities were seen in the trachea, but BSA staining was shifted to morphologically different mucous cells in the INF and INF/T. We conclude that in localized P. haemolytica pneumonia in sheep (1) there are morphologic changes of the airway secretory apparatus adjacent to the lesion, (2) the glycoconjugate profile of secretory cells adjacent to and remote from the lesion is altered, and (3) cyclooxygenase products influence the chemical composition of secretory cells.

Bacterial pneumonia stimulates macromolecule secretion and ion and water fluxes in sheep trachea.

In vivo instillation of Pasteurella haemolytica (greater than or equal to 10(7) colony-forming units/kg) into a lobar bronchus of sheep produced bacterial pneumonia by 7 days postinoculation. Infection was verified bacteriologically and histologically. Macromolecule secretion and ion and water fluxes were subsequently measured in tracheal tissues in vitro and were compared with values from sham-infected sheep. Macromolecules were radiolabeled with 35SO4 and [3H]threonine, and we measured the secretion of macromolecule-bound radiolabel onto the mucosa. Unidirectional fluxes of Cl-, Na+, and water were measured with radioactive tracers under open-circuit and short-circuit conditions. Lung infection increased basal secretion of bound 35SO4 (by 189%) and bound [3H]-threonine (by 110%). It significantly increased net Na+ absorption under open- and short-circuit conditions and induced open-circuit net absorption of Cl- and water (16 +/- 29 microliters X cm-2 X h-1). These changes were associated with specific recruitment of neutrophils and elevated levels of arachidonate metabolites (thromboxane B2 and leukotriene B4) in the airways. Thus the bacterial pneumonia-induced changes in tracheal mucus secretion may be the result of airway inflammation.

Effect of atropine on tracheal mucociliary clearance and bacterial counts.

The purpose of this study was to determine if atropine, which has been shown to alter mucosal function, prolongs the persistence of inhaled bacteria in the trachea. In conscious sheep, bacterial counts in the trachea were determined by quantitative sterile brush cultures obtained before and serially after a controlled inhalation challenge with an aerosolized solution containing P. hemolytica (10(8) CFU X ml-1). The same animals were studied on two days, once without (control day) and once before and during intramuscular administration of 0.2 mg X kg-1 atropine sulfate at hourly intervals for up to 10 h (atropine day). On the control and atropine days, bacterial counts were zero before, and between 5 X 10(5) and 1.6 X 10(7) CFU X ml-1 immediately after inhalation of P. hemolytica. During the first 2 h after challenge, there was a similar semilogarithmic decline in bacterial counts on the control and atropine days despite the fact that mean tracheal mucociliary transport velocity remained unchanged on the control day, and ranged between 32% and 62% of baseline (p less than 0.05) during the 6-10 h post-drug observation period on the atropine day. However, the time to achieve sterility on the control day was less than or equal to 8 h in all animals, and greater than or equal to 8 h on the atropine day. We conclude that atropine prolongs the persistence of viable bacteria in the trachea. This effect of atropine may be related to an impairment of mucociliary clearance or to other alterations in mucosal function.

Excretion of cefamandole, cefazolin, and cephalothin into T-tube bile.

The biliary tract excretion of cefamandole, cefazolin, and cephalothin was measured in eight patients with T-tubes inserted into their common ducts after ductal exploration for biliary tract stones. Each patient received 1.0 g intravenously of each cephalosporin on 3 separate days; T-tube bile and serum were collected at selected time intervals thereafter. In seven patients, bile and urine were collected for 6 h after the administration of each drug. Mean peak levels of cefamandole, cefazolin, and cephalothin in bile were 352, 46, and 12 mug/ml, respectively. The respective mean peak serum levels were 55.0, 92.8, and 32.4 mug/ml. Despite the fact that peak serum levels of cefazolin were 1.5 times those of cefamandole, levels in bile of cefamandole were about 8 times those of cefazolin. Over a 6-h period, almost three times as much cefamandole was excreted into bile as was cefazolin. Therefore, in those patients with biliary tract sepsis, in whom a cephalosporin is indicated for therapy, cefamandole appears to be the drug of choice.

Effect of sodium cyanate upon the function of normal human polymorphonuclaer leukocytes.

Sodium cyanate, a drug that prevents sickling of hemoglobin S by virtue of its irreversible carbamylation of the N-terminal amino group of valine, was studied for its effect upon the function of normal human polymorphonuclear luekocytes. In concentrations of 500 and 100 mug/ml, sodium cyanate was found to inhibit killing by neutrophils of Staphylococcus epidermidis and Eschierichia coli but not of Streptococcus faecalis. Viability of cells and phagocytosis were not affected by cyanate; however, production of [14-C] carbon dioxide from [1-14-C] glucose and the iodination of 125-I during phagocytosis were significantly impaired. Cyanate is thought to inbibit the bacterixidal activity of neutrophils by interfering with the oxidative metabolism of gluxose via the hexose monophosphate shunt (theraby decreasing production of H-2-O-2) and by inbibiting iodination of ingested bacteria (either by competing with iodide as the oxidizable cofactor or by inhibiting myeloperoxidase). Since these effects of cyanate were all reversible by washing the nurtrophils free of the drug, it is unlikely that they are due to amino carbamylation.