INTRODUCTION

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Exercise-induced asthma (EIA) is characterized by transient airway obstruction that typically occurs after hyperventilation with cold dry air. This hyperventilation-induced bronchoconstriction (HIB) usually peaks within 2-10 min after exercise ceases and spontaneously recovers 30-60 min thereafter. Inhalation of large volumes of cold dry air is believed to stimulate a cascade of events that result in airway narrowing in most asthmatic and in some normal individuals (1). The exact mechanisms involved are unknown. Anderson and coworkers suggested that hyperventilation-induced airway hyperosmolality stimulates mast cell degranulation, which in turn results in airway smooth muscle constriction (4). Airway surface fluid (ASF) osmolality does increase during hyperventilation, and correlates with the magnitude of obstruction that develops in a canine model of EIA (5). Changes in ASF osmolality in canine peripheral airways are associated with bronchial mucosal injury (6), mast cell degranulation (7, 8), and the elaboration of bronchoactive mediators (9); all of which are consistent with Anderson's original hypothesis.

Inhaled heparin inhibits HIB in human subjects (12, 13). It also attenuates antigen-induced bronchoconstriction and airway hyperresponsiveness in sheep (14). Although heparin attenuates methacholine-induced bronchoconstriction in humans with asthma (15), it does not alter airway reactivity to histamine in either asthmatic subjects or sheep (12, 13, 16). In addition to its anticoagulant properties, glycosaminoglycan heparin may modulate T cell function (17), neutrophil chemotaxis (18), complement activation (19), and smooth muscle growth (20). Heparin acts as a specific blocker of inositol triphosphate (IP₃)-mediated calcium release in various cell types (21, 22), and inhibits rat peritoneal mast cell degranulation in vitro (14) and anti-immunoglobulin E-induced histamine release from isolated human uterine mast cells (23). Thus, Ahmed and coworkers suggested that heparin may attenuate antigen-induced bronchoconstriction and hyperreactivity via the inhibition of IP₃-dependent mast cell mediator release (24).

Numerous animal and human studies using pharmacological interventions and bronchoalveolar lavage fluid (BALF) analyses suggest that a variety of eicosanoids contribute to the development of HIB (9, 25). The purpose of this study was to determine if inhaled heparin inhibits HIB in canine peripheral airways, and if its mode of action involves the inhibition of hyperventilation-induced mediator production and release. Specifically, we tested the hypothesis that heparin interferes with either the production or release of peptidoleukotrienes and prostanoids. In doing so, we examined the effect of treatment time and strength of stimulus on the efficacy of heparin in inhibiting HIB, and the effect of heparin on the concentration of eicosanoid mediators and cell profiles recovered in BALF immediately after hyperventilation with dry air.

	METHODS

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Experimental Technique

Dogs were handled and maintained in accordance with the Policy and Procedures Manual published by the Johns Hopkins University School of Hygiene and Public Health's Animal Care and Use Committee.

Anesthesia and instrumentation. Colony-bred male mongrel dogs (21.5 ± 1.3 kg, n = 6) were anesthetized with intravenous sodium thiopental (25 mg/kg). Intravenous fentanyl citrate (25 µg) was administered every 15 min and thiopental (50 mg) was supplemented as needed to maintain anesthesia. The depth of anesthesia was assessed by heart rate, blood pressure, canthal reflex, and presence of spontaneous movements and breathing. Dogs were intubated and mechanically ventilated (17 ml/kg) with room air; CO₂ was continuously monitored (LB-2; Beckman, Anaheim, CA) and maintained at ~ 4.5% by adjusting respirator frequency. Heart rate (HR) and mean arterial pressure () were recorded with a noninvasive blood pressure and heart rate monitor (Accutorr IA; Datascope, Paramus, NJ). Body temperature was monitored with a telethermometer and rectal probe (Yellow Spring Instrument, Yellow Spring, OH) and maintained with a warming pad.

Measurement of peripheral airway resistance (Rp). A fiberoptic bronchoscope (5.5-mm o.d., BF type P 10; Olympus Corporation of America, New Hyde Park, NY) was inserted through an airtight portal in the endotracheal tube and wedged in a sublobar bronchus. Sublobar bronchus airway pressure (Pbr) was measured with a polyethylene 90 catheter that was threaded through the suction port of the bronchoscope and connected to a pressure transducer (Statham Gould, Oxnard, CA). Compressed, dry, room temperature, 5% CO₂ in air was delivered around the catheter and into the wedged sublobar segment at 200 ml/min. Pbr was measured by stopping the ventilator during exhalation, and allowing the unobstructed areas of the lung to equilibrate with atmospheric pressure at functional residual capacity. Under this condition, Pbr decays to a plateau at a pressure greater than the atmospheric pressure in the surrounding unobstructed lung, and Rp = Pbr/200 ml/min = Pbr/3.33 ml/s.

Airflow challenge. In the dry air challenge (DAC), bronchoconstriction was induced by temporarily increasing the flow rate of 5% CO₂ in dry air from 200 to 2,000 ml/min for either 2 min (DAC-2) or 5 min (DAC-5).

Pretreatment with heparin. Unfractionated heparin sodium derived from porcine intestinal mucosa (Elkins-Sinn, Cherry Hill, NJ) was prepared daily in bacteriostatic water (10,000 U/ml; Elkins-Sinn), aerosolized (Ultra Neb 100; DeVilbiss, Somerset, PA), and delivered into the wedged sublobar segment via the bronchoscope. Bacteriostatic water (BW) served as the vehicle control. Each sublobar segment (which is estimated to subtend ~ 5% of the lung in a 20-kg dog) was treated with 10,000 U per sublobar segment, a dose that is similar to that used in human studies (12, 13). Heparin and BW aerosols were delivered at 500 ml/min in 5% CO₂ and air.

Measurements of partial thromboplastin time, coagulation time, and bleeding time. Partial thromboplastin time, coagulation time, and bleeding time were analyzed according to standard methods, using a 5-ml sample of venous blood obtained before and after treatment with heparin.

Bronchoalveolar lavage, differential cell counts, and mediator analyses. Three 20-ml aliquots of warm (37° C) Hanks' buffered saline solution were infused through the bronchoscope into a wedged sublobar bronchus, and each aliquot was recovered by gentle aspiration. The recovered BALF samples were pooled and stored at 4° C until the conclusion of the experiment. The BALF was then centrifuged at 4° C for 10 min at 1,300 rpm. The cell pellet from a 5-ml sample was resuspended in 1 ml of supernatant and a hemocytometer was used to determine total cell number. Differential cell counts of macrophages, lymphocytes, neutrophils, eosinophils, and epithelial cells were done on cytocentrifuged BALF samples strained with a modified Wright- Giemsa stain. Trypan blue exclusion was used to evaluate cell viability.

BALF was concentrated with a Sep-Pak C₁₈ cartridge (Waters, Milford, MA), eluted in 4 ml of methanol, and stored at 70° C. Aliquots of the eluate were analyzed as previously described (11), using commercially available enzyme-linked immunosorbent assay (ELISA) kits for prostaglandin D₂ (PGD₂: Cayman Chemical, Ann Arbor, MI), prostaglandin F₂ (PGF₂), thromboxane B₂ (TxB₂), and leukotriene LTC₄-D₄-E₄ (LTC₄-E₄: Neogen, Lexington, KY).

Experimental Protocol

Effect of heparin on HIB resulting from a 2-min DAC. Two bronchoscopes were simultaneously wedged in contralateral sublobar segments in each dog. After recording baseline Rp, each sublobar segment was exposed to DAC-2. Rp was recorded at 0.5, 2, 5, 10, and 15 min after the DAC-2. After Rp recovered to baseline, one sublobar location was treated with heparin, the other with BW. Sixty minutes after aerosol treatment, DAC-2 was repeated in each segment.

Effect of heparin on HIB resulting from a 5-min DAC. Two weeks later, we repeated the protocol described above in the same sublobar segments, except this time DAC-5 was used instead of the DAC-2 described above.

Effect of heparin on BALF cell profiles and mediator release. A similar protocol using DAC-5 was repeated after another 2-wk rest period, except this time BAL was done immediately after the DAC.

Effect of heparin on histamine-induced bronchoconstriction. A similar protocol, in which histamine was used to induce bronchoconstriction before and 60 min after heparin and BW aerosol treatment, was done at least 2 wk after the last protocol.

Statistical Analyses

Rp, cell, and mediator data were analyzed by Friedman two-way analysis of variance in conjunction with a Student-Newman-Keuls test for the comparison of individual treatment means. Statistical significance was judged at p < 0.05. Data were expressed as means ± SE.

	RESULTS

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Effect of Heparin on HIB Resulting from a 2-min DAC:

Before treatment with heparin, baseline Rp was 0.823 ± 0.161 cm H₂O/ml/s (n = 6) and increased ~ 67% within 5 min after DAC-2 (Figure 1A). Aerosolized heparin transiently increased Rp from 0.97 ± 0.16 to 1.47 ± 0.19 cm H₂O/ml/s (n = 6, p = 0.001). Sixty minutes after heparin treatment Rp had returned to baseline (0.924 ± 0.142, p > 0.05) and a second consecutive DAC-2 increased Rp by only 23% (Figure 1A). Compared with the preheparin response, postheparin hyperventilation-induced changes in Rp were significantly reduced 2, 5, and 10 min after the second DAC. In contrast, treatment with BW transiently increased Rp from 0.82 ± 0.18 to 1.13 ± 0.18 cm H₂O/ml/s (n = 6, p > 0.05), but did not affect HIB (Figure 1B). Neither heart rate, blood pressure, nor body temperature was affected by either heparin (Table 1) or BW aerosol treatment.

View larger version (20K): [in this window] [in a new window]   Figure 1.   Hyperventilation-induced changes in peripheral airway resistance (Rp) before (open circles) and 60 min after (solid circles) treatment with either heparin (A) or bacteriostatic water (B). Values represent means ± SEM of n = 6 experiments. *p < 0.05 compared with baseline; p < 0.05 for before versus 60 min after inhaled heparin.

Effect of Heparin on HIB Resulting from a 5-min DAC

Before treatment with heparin baseline Rp was 0.693 ± 0.147 cm H₂O/ml/s (n = 6) and increased ~ 79% within 5 min after DAC-5. Sixty minutes after heparin treatment Rp had returned to baseline (0.777 ± 0.145, p > 0.05), the second DAC increased Rp by only ~ 29% (Figure 2A). Compared with the preheparin response, postheparin hyperventilation-induced changes in Rp were significantly reduced at 2 and 5 min after the second DAC. Heparin attenuated HIB induced by DAC-5 by ~ 51%, a reduction similar (~ 62%) to that seen when heparin was tested against the milder DAC-2 stimulus (Figure 2B).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Hyperventilation-induced changes in peripheral airway resistance (Rp) before (open circles) and 60 min after (closed circles) treatment with heparin. (A) Hyperventilation with 2,000 ml/min for 2 min (from Figure 1A), (B) hyperventilation with 2,000 ml/min for 5 min. Values represent means ± SEM of n = 6 experiments. *p < 0.05 compared with baseline; p < .05 for before versus 60 min after inhaled heparin.

Effect of Heparin on BALF Cell Profiles and Mediator Release

The average volume of BALF recovered from wedged control, BW-treated, and heparin-treated sublobar segments was 37.4 ± 2.7, 38.8 ± 2.4, and 35 ± 3.5 ml, respectively. The BALF recovered from BW- and heparin-treated airways exposed to DAC contained a greater total number of cells than that recovered from the wedged control (16 ± 2.4, p < 0.05; Figure 3). DAC did not affect BALF lymphocyte and neutrophil cell counts. In contrast, when compared with unchallenged control airways, there was a marked increase in BALF macrophages, eosinophils, and epithelial cells recovered from heparin- and BW-treated airways that were exposed to DAC (p < 0.05). However, heparin treatment did not significantly reduce the number of any of these leukocytes. Although treatment with aerosolized heparin did not cause hematological abnormalities (Table 2), heparin increased the number of red blood cells (RBCs) in BALF recovered from airways exposed to DAC when compared with either control airways or BW-treated/DAC airways (p < 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Bronchoalveolar lavage fluid (BALF) cells per milliliter recovered from unchallenged wedged control airways (open columns) or dry air-challenged (2,000 ml/5 min) airways pretreated with either bacteriostatic water (hatched columns) or heparin (cross-hatched columns). Total = Total BALF nucleated cell concentrations; Mac = macrophages; Lym = lymphocytes; PMN = neutrophils; Eos = eosinophils; Epi = epithelial cells. Values represent means ± SEM of n = 6 experiments. *p < 0.05.

The effect of heparin on hyperventilation-induced eicosanoid mediator release is summarized in Figure 4. Hyperventilation significantly increased the concentrations of LTC₄-E₄, PGD₂, PGF₂, and TxB₂ in BALF recovered from BW-treated airways when compared with unchallenged control airways (p < 0.05). Pretreatment with aerosolized heparin significantly reduced the concentrations of LTC₄-E₄ and PGF₂ recovered in BALF, and abolished the hyperventilation-induced increases in PGD₂ and TxB₂ (Figure 4).

View larger version (26K): [in this window] [in a new window]   Figure 4.   Concentrations of eicosanoid mediators recovered in BALF from unchallenged wedged control airways (open columns) or dry air-challenged (2,000 ml/5 min) airways pretreated with either bacteriostatic water (hatched columns) or heparin (cross-hatched columns). LTC4-E4 = leukotriene C4/D4/E4; PGD2 = prostaglandin D2; PGF2 = prostaglandin F2; TxB2 = thromboxane B2. Values represent means ± SEM of n = 6 experiments. *p < 0.05.

Effect of Heparin on Histamine-induced Bronchoconstriction

Histamine aerosol increased Rp from 0.47 ± 0.17 cm H₂O/ml/s before to 0.52 ± 0.08 cm H₂O/ml/s (n = 3, p = 1.0) 60 min after treatment with heparin.

	DISCUSSION

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Inhalation of heparin attenuates HIB in the canine lung periphery by ~ 50-60% when compared with its vehicle control (Figure 1). The level of inhibition afforded by treatment with heparin is similar to that seen in subjects with asthma (12, 13), and compares favorably with other drugs previously used in our canine model. The efficacy of heparin is greater than that seen after treatment with either atropine (25) or methoxamine (28) (~ 25-30%); is similar to the effects of aminophylline (29), furosemide (30), indomethacin (9, 25), leukotriene antagonist MK-0591 (11), and noradrenaline (28) (~ 50-65%); and is less efficacious than salbutamol (~ 75-100%) (31, 32). The reduction in hyperventilation-induced airway obstruction caused by heparin is accompanied by a concomitant decrease in eicosanoid mediator concentrations recovered in post-DAC BALF (Figure 4). These results suggest that heparin inhibits HIB via the inhibition of either hyperventilation-induced eicosanoid production or release.

The commercial heparin used in this study contained benzyl alcohol as a preservative, which has been reported to relax canine airway smooth muscle in vitro (33). However, the use of bacteriostatic water (BW) as our vehicle control suggests that the concentration of benzyl alcohol used in commercial preparations of heparin does not alter HIB in canine peripheral airways (Figure 1). Similar findings were reported for humans (12, 13).

Numerous studies implicate mast cell-derived mediators in the pathogenesis of HIB (34). The use of a 5-lipoxygenase-activating protein antagonist confirmed that leukotrienes significantly contributed to the development of HIB in our canine model (11). However, heparin does not appear to act as a receptor antagonist (16, 23). The fact that no bronchodilating effect was seen in this (Figures 1 and 2) and other studies (12, 13) makes it unlikely that the protection provided by heparin against HIB results from any direct effect on baseline airway smooth muscle tone.

Heparin may have multiple sites of action. It has multiple nonanticoagulant properties, which include modulation of various proteases, anticomplement activity (19), antiinflammatory action, and the potential to inhibit cell growth (37, 38). Heparin has been shown to bind to inositol triphosphate receptors and to inhibit the inositol triphosphate-induced release of calcium in various tissues including airway smooth muscle. Previous studies suggest that the antiallergic actions of heparin may be related to the inhibition of mediator release from mast cells (16, 23). Heparin inhibits histamine release in vitro (23), and prevents mast cell degranulation in mice (39). In addition, heparin attenuates HIB in subjects with asthma and acute antigen-induced bronchoconstriction in sheep without modifying the effects of histamine (12, 13, 16). These observations suggest that heparin inhibits allergen- and hyperventilation-induced airway responses by modifying mediator release derived from degranulated mast cells. The present study is consistent with this hypothesis and shows for the first time that inhaled heparin either inhibits or abolishes the release of eicosanoid mediators in vivo (Figure 4).

Hyperventilation-induced increases in LTC₄-E₄ and PGF₂ are significantly inhibited, and PGD₂ and TxB₂ remain at baseline levels, in heparin-treated/DAC airways (Figure 4). The marked effect of heparin on PGD₂, which is a relatively specific mast cell product (40), supports the notion that heparin inhibits hyperventilation-induced IP₃-dependent mast cell degranulation (7, 8). Although heparin may possess mast cell-"stabilizing" qualities similar to that reported for disodium cromoglycate (41), we cannot rule out the possibility that heparin directly interferes with eicosanoid metabolism.

Inhaled heparin tends to reduce the number of epithelial cells recovered in BALF after DAC when compared with BW-treated/DAC airways (Figure 3), but this effect is not significant. Thus, it is unclear if heparin inhibits HIB in part by protecting the bronchial mucosa from hyperventilation-induced injury (6, 7). Compared with BW, heparin did not have any effect on BALF inflammatory cell profiles in airways exposed to DAC. However, treatment with either heparin or BW 60 min before DAC increased macrophages, eosinophils, and the total number of cells recovered in BALF immediately after DAC. Because BALF cell profiles are normally either not affected or decreased immediately after DAC (9, 29), these data suggest that pretreatment with solutions containing benzyl alcohol initiates inflammatory cell infiltration. Our data reveal that leukocyte infiltration increased within 60 min after inhalation of heparin and bacteriostatic water, and this observation is consistent with in vitro and in vivo studies showing that heparin can act as a chemoattractant for monocytes and neutrophils (42, 43). Although heparin has been reported to inhibit airway inflammation in several animal models, all these studies examined cell infiltration 24 h after an airway challenge (44). This, in combination with our observation, suggests that the antiinflammatory activity of heparin is a time-dependent phenomenon.

Heparin did not prolong the partial thrombin time, bleeding time, or coagulation time during our experiments (Table 2). These data suggest that the inhibitory effect of heparin is unrelated to its anticoagulant activity. However, further study similar to that done in examining the effect of a nonanticoagulant fraction of heparin in sheep (47) would be necessary to confirm this. We did find that the number of red blood cells in BALF recovered from sublobar segments exposed to aerosolized heparin was significantly increased when compared with either their paired BW-treated airways or unchallenged control airways. Thus, although inhaled heparin did not affect systemic hematological activity, it does cause either local bronchovascular or pulmonary vascular hemorrhage.

In conclusion, our data provide direct evidence that inhaled heparin inhibits eicosanoid mediator production and release caused by hyperventilation with dry air, and significantly attenuates HIB. Although our data suggest that heparin inhibits mast cell degranulation, it also supports the hypothesis that heparin interferes in general with eicosanoid metabolism. This inhibitory action also may be derived in part from a tendency of aerosolized heparin to diminish hyperventilation-induced mucosal injury.