Hyaluronic acid blocks porcine pancreatic elastase (PPE)-induced bronchoconstriction in sheep.

We previously showed that inhaled porcine pancreatic elastase (PPE) causes bronchoconstriction in sheep via a bradykinin-mediated mechanism. Hyaluronic acid (HA), in vitro, binds and inactivates airway tissue kallikrein (TK), the enzyme responsible for kinin generation. Therefore, we hypothesized that in vivo, HA should prevent PPE-induced bronchoconstriction by binding and inactivating TK. To test this, we measured pulmonary resistance (RL) in allergic sheep before and after inhalation of PPE alone (500 microg) and after pretreatment with either inhaled HA at 70 kD, designated low molecular weight (LMW)-HA or 200 kD, designated high molecular weight (HMW)-HA at different concentrations. Inhaled PPE increased RL 147 +/- 8% over baseline values and this effect was associated with a 111 +/- 28% increase in bronchoalveolar lavage fluid (BALF) TK activity. HA blocked the PPE-induced bronchoconstriction and the increase in BALF TK activity in a dose- dependent and molecular weight-dependent fashion. HA alone had no effect on RL. Instillation of PPE in the lung increased kinin concentrations in BALF, a result consistent with the PPE-induced increase in BALF TK activity. Our findings show that HA blocks PPE-induced bronchoconstriction in a dose-dependent and molecular weight-dependent fashion by a mechanism that may, in part, be related to inhibition of TK activity and the formation of kinins.

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.

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.

The interaction of alpha 1-proteinase inhibitor and tissue kallikrein in controlling allergic ovine airway hyperresponsiveness.

We reported previously that the development of airway hyperresponsiveness (AHR) 24 h after antigen challenge in allergic sheep was associated with increased tissue kallikrein activity (TK) and decreased alpha-1-proteinase inhibitor (alpha 1-PI) activity in bronchoalveolar fluid (BAL). The inverse correlation between TK and alpha 1-PI in these experiments suggested that administration of alpha 1-PI might reduce TK activity and block AHR. To test this hypothesis, airway responsiveness, as determined by calculating the cumulative carbachol breath units (BU) that increased specific lung resistance by 400% (PC400), was measured before and 24 h after aerosol challenge with Ascaris suum antigen in seven sheep hypersensitive to this antigen. On the next day, 30 min before the 24 h PC400 measurement, the sheep were treated with either aerosol alpha 1-PI (Prolastin, 10 mg/5 ml) or denatured (DN) prolastin (10 mg/5 ml), which had only 10% of its original activity. BAL was also performed before and 24 h after challenge for the measurement of TK and alpha 1-PI activity. Treatment with DN-Prolastin at 24 h after antigen challenge did not block antigen-induced AHR: PC400 fell from a baseline (mean +/- SE) of 26.0 +/- 3.2 BU to 11.2 +/- 1.5 BU after challenge (p < 0.05). This AHR was associated with increased TK (363%, p < 0.05) and decreased alpha 1-PI activity (65%, p < 0.05). Prolastin treatment at 24 h blocked the AHR: PC400 was 21.0 +/- 2.8 before and 23.2 +/- 3.7 after challenge (p < 0.05 versus DN-Prolastin) and the changes in BAL TK (28% increase) and alpha 1-PI activities (15% increase) were not different from baseline (both p < 0.05 versus DN-Prolastin). There was a significant inverse correlation between alpha 1-PI activity and TK activity in BAL, as well as the changes between baseline and 24 h in alpha 1-PI activity and TK activity in BAL Pretreatment (30 min before antigen challenge) with Prolastin also protected against the antigen-induced AHR. The effect of Prolastin was also seen against aerosol challenge with high-molecular-weight kininogen (HMWK), a substrate of TK. HMWK caused bronchoconstriction which was blocked by Prolastin (p < 0.05), and the bradykinin B2 antagonist, NPC-567 (indicating that kinins were generated), but not DN-Prolastin or the elastase inhibitor, ICI 200, 355. Although the negative association between alpha 1-PI activity and TK activity identified in this study does not prove cause and effect, our findings do raise the possibility that in vivo alpha 1-PI may regulate TK activity and allergen-induced AHR.