IgG1-mediated Acute Pulmonary Hypersensitivity Response in the Guinea Pig Involvement of Specific Lipid Mediators1 •2
JOHN W. WATSON, MARYROSE CONKLYN, and HENRY J. SHOWELL
Introduction
Guinea pigs have long been used as a model of pulmonary immediate hypersensitivity reactions. These reactions are triggered by the interaction of antigen with cytophilic immunoglobulins attached to resident pulmonary cells, such as mast cells, and alveolar macrophages as well as circulating cells, such as basophils. The anaphylactic release of mediators subsequent to cross-linking of surface-bound antibody produces bronchoconstriction, pulmonary vascular leak, and mucus secretion which, in turn, results in airway obstruction, dyspnea, and death. Both IgG, and IgE immunoglobulins sensitize pulmonary tissue in this species. However, in vitro studies using sensitized pulmonary tissue indicate that the mediators released and sensitivity to pharmacologic agents may depend on the species of sensitizing antibody (1,2). In vivo studies using partially purified serum preparations to selectively sensitize guinea pigs with either IgG or IgE antibodies also indicate that there may be marked antibody-dependent differences in the sensitivity of the acute antigen-driven pulmonary hypersensitivity responses to various pharmacologic agents (3). We have isolated and purified an antiovalbumin (anti-OA) IgG l antibody from the serum of guinea pigs actively sensitized to ovalbumin. Specific in vitro antigen challenge of lung tissue derived from animals passively sensitized with this anti-Oe-IgG, has previously been shown to result in the release of LTB4 , LTD4 , and histamine (4). In this study, we have characterized the in vivo pharmacologic modulation of an acute pulmonary hypersensitivity response mediated by this antibody to determine the involvement, if any, of platelet-activating factor (PAF), LTD4 , and the cyclooxygenase products of arachidonic acid (AA)
SUMMARY We determined the pulmonary obstructive response to aerosolized antigen challenge, and Its sensitivity to antagonists of specific lipid mediators, In IgG 1 passively sensitized (lgG 1-PS) guinea pigs. Antlovalbumln (OA)-lgG1 was Isolated by affinity chromatography from serum derived from actively Immunized Hartley guinea pigs. Propranolol and pyrilamine pretreated, IgG1-PS guinea pigs were challenged with aerosolized antigen and pulmonary obstruction was quantified by measurements of excised lung gas volume (ELGV). ELGVIncreased between 150 and 1,035% In a doseproportional fashion with Increasing antigen exposure (0.001 to 0.1% nebulizer concentration). The leukotrlene antagonists ICI-204,219 and SKF-104,353 exhibited dose-proportional Inhibitions In antigen-Induced elevations In ELGV, Inhibiting up to 65 and 87% at the maximal concentrations examined. Similarly, the platelet-activating factor (PAF) antagonists WEB-2086 and L-659,989 Inhibited antigen-Induced elevations In ELGV, Inhibiting up to 94 and 59% at the maximal concentrations examined. In contrast, the cyclooxygenase (CO) Inhibitor plroxlcam significantly enhanced (p < 0.05)the OA-Induced elevations In ELGV. Aerosolized PAFchallenge produced dose-proportional elevations In ELGVthat were significantly Inhibited by the LTD. antagonist ICI-204,219(38 and 43% Inhibition) and the CO Inhibitor plroxlcam (62 and 48% Inhibition) In sensitized and nonsensltlzed animals, respectively. We hypothesize that IgG1-dependent airway obstruction Is mediated In part by LTD. produced In response to PAF generation. AM REV RESPIR DIS 1990; 142:1093-1098
metabolism. To this end, guinea pigs were passivelysensitized with purified homologous anti-OA-IgG h pretreated with pyrilamine and propranolol, and challenged with aerosolized OA antigen. The involvement of PAF, the leukotrienes, and the cyclooxygenase products of arachidonic acid metabolism in the resulting pulmonary obstructive response was inferred by treating these animals with specific antagonists and enzyme inhibitors before antigen challenge. Since our data indicated that both PAF antagonists and LTD4 antagonists could block 85 to 95070 of the pyrilamine-and propranololmodified IgGrmediated obstructive response, we also evaluated the pharmacology of aerosolized PAF-induced airway obstruction to determine if PAF produces airway obstruction by stimulating LTD4 synthesis. Methods Isolation of IgOl IgG l antibodies were raised in male Hartley guinea pigs, immunized according to the method of Regal (5). Blood was collected by cardiac puncture and the serum stored at
-20 0 C. Antiovalbumin (OA)-IgG l was isolated by affinity chromatography using protein A-Sepharose CL-4B (Pharmacia Chemicals, Uppsala, Sweden) and OA-Sepharose 4B columns as follows. Antiserum (250to 300 ml) was applied to a protein A-Sepharose CL4B column (1.5 x 17ern) which flowed directly onto the OA-Sepharose 4B column (1.5 x 15 em), Both columns had been equilibrated with phosphate-buffered saline (PBS), pH 7.4. After washing with PBS, the columns were separated and independently eluted with 0.1 M glycine, pH = 3.0. The eluted protein fractions from the OA-Sepharose 4B column that consisted primarily of anti-OA IgG l were combined and dialyzed against citrate-phosphate buffer (CPB), pH = 7.4 (6). This sample was then reapplied to the protein ASepharose CL-4B column that had been (Received in original form June 19, 1989 and in revised form February 28, 1990) 1 From the Department of Immunology and Infectious Diseases,Central ResearchDivision, Pfizer Inc., Groton, Connecticut. 2 Correspondence and requests for reprints should be addressed to John W. Watson, Ph.D., Department of Immunology and Infectious Diseases, Central Research Division, Pfizer Inc., Groton, CT 06340.
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WATSON, CONKLYN, AND SHOWELL
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equilibrated in CPB, pH 7.4. The protein was then isolated by pH gradient using CPB. The anti-OA IgG. eluted between pH 6 and pH 5. The fractions containing anti-OA IgG. were combined and dialyzed against PBS. The purity of the preparation was determined by electrophoresis on an 8 to 25070 acrylamide gel using the Pharmacia Phast System. Specific activity was established by the ability to sensitize guinea pig tracheal rings with an ED so of 3.7 ± 0.5 (x ± SE) ug/ml.
Index of Airway Obstruction The volume of air trapped behind closed airways in excised guinea pig lungs was utilized as an index of a physiologic response to antigen and/or mediator challenge. In this species, excisedlung gas volume (ELGV) has been previously shown to increase in a dose-proportional manner with increasing aerosol concentrations of LTD.., methacholine, and histamine (7), and it has been shown to be highly correlated with measurements of pulmonary resistance and dynamic compliance (8). The rapidity with which ELGV increases following challenge, and its reversibility with isoproterenol infusion, are consistent with the hypothesis that smooth muscle constriction plays a role in mediator-driven elevations in ELGV (8). However, it is likely that ELGV could be influenced by any force that would contribute to the obstruction of airways at low lung volumes such as mucus secretion, peribronchial edema, a reduction in lung elasticity, or an alteration in airway surface forces. What is important in the context of this study is that ELGV is sensitive to LTD.., PAF, and histamine challenge and therefore can be used to indicate the presence of a physiologic concentration of these substances following antigen or mediator challenge. ELGV was determined by measuring the stable volume of air trapped within the excised lung at a transpulmonary pressure of zero em HzO. Immediately after death, the pleural space was opened and the lungs allowed to collapse to a stable volume. The lungs were surgically removed, the nonpulmonary tissues dissected away, then the trachea was shortened and tied just above the carina. The loss of trapped air from lungs treated in this manner is approximately 50J0/h and independent of the amount of gas within the lung (9). The volume of air within the lung was determined by measuring the negative force exerted by the lung when submerged in saline as described by Stengel and Silbaugh (9). Because the density of lung tissue is approximately equal to the density of saline, this force (in grams) is equal to the volume of air (in milliliters) trapped within the lung (Archimedes' principle). All volume measurements were completed within 5 min of excision. Volumes were normalized to animal weight to give excised lung gas volume in units of milliliters per kilogram. Aerosol Generation Aerosols were generated by a Pulmosonic model 25 nebulizer (Devilbiss Co., Somerset, PA) using 3 ml of test solution. This type of
nebulizer produces a 7 to 8 urn particle with a geometric standard deviation (SD) of 2 to 3. However, particle size may be altered before respiration as aerosols are recirculated in a closed loop system that consists of the nebulizer, a l-L expansion chamber, a circulating fan, and a 9-L exposure chamber. To determine the respirability of particles generated in this system, a group of five guinea pigs were exposed to aerosolized Evans blue dye for 10 min (nebulizer concentration 30 mg/ ml). Following death, their upper airways (nose to trachea) and lower airways (trachea and lungs) werehomogenized and dye extracted with formamide/acetonitrile. Spectrophotometric analysis of these supernatants indicated that, after substracting for background interference, the upper and lower airways contained an average of 2.5 and 32.0 f.1g Evans blue dye, respectively. Clearly, the particles generated by this system are preferentially distributed to the lung rather than to the upper airways.
Sensitizing Dose of IgG l Groups of guinea pigs were sensitized subcutaneously with varying doses of anti-OAIgG. (0.01, 0.03, 0.1, 0.3 mg/kg) 72 h before antigen challenge. Thirty minutes before challenge, guinea pigs weremedicated with pyrilamine maleate salt (5 mg/kg subcutaneously) and propranolol (5 mg/kg subcutaneously). During challenge, groups of three animals were allowed to breathe a 0.1810 ovalbumin aerosol (nebulizer concentration) for 5 min. Eight minutes after aerosol challenge, animals were killed with 130 mg sodium pentobarbital given intraperitoneally. ELGV was determined after death (""2 min after sodium pentobarbital). Antigen Dose-Response Curve Guinea pigs were sensitized with 0.375 mg/kg anti-OA-IgG. 72 h before antigen challenge. Thirty minutes before challenge, they were pretreated with propranolol (5 mg/kg subcutaneously) and pyrilamine maleate salt (5 mg/kg subcutaneously). Five animals were placed in the exposure chamber simultaneously and allowed to breathe antigen aerosol for 5 min. The aerosol antigen concentration was varied with the exposure group by stepwise increases in the nebulizer ovalbumin concentration. Eight minutes after challenge, animals were given 130mg Na pentobarbital intraperitoneally and ELGV determined following death. Pharmacology of Antigen-induced A irway Obstruction We chose a combination of anti-OA-IgG. (0.375 mg/kg) and aerosol antigen challenge (0.02 to 0.022%) that resulted in an average ELGV near 80870 of the maximal response (producing apnea and death). Guinea pigs were pretreated with pharmacologic agents at prechallenge time points that are consistent with published usage (10-13). Guinea pigs were also pretreated with propranolol (5 mg/kg subcutaneously) and pyrilamine male-
ate salt (5 mg/kg subcutaneously) 30 min before challenge. Control animals, orally dosed with vehicle, and test drug-pretreated animals (oral or aerosol dosing) werechallenged simultaneously in groups of four to six animals. During challenge, animals were placed in the aerosol exposure chamber and allowed to breathe ovalbumin aerosol for 5 min. Eight minutes after exposure, a time previously determined as the period of maximal bronchoconstriction, animals were killed with 130 mg sodium pentobarbital intraperitoneally and ELGV determined.
Aerosolized PAF Dose-Response Curve PAF was dissolved in normal saline containing 0.25 0J0 bovine serum albumin and stored on ice until it was added to the nebulizer. Normal, unsensitized guinea pigs were premedicated with propranolol (5 mg/kg subcutaneously for 30 min), placed in the aerosol chamber in groups of five, and allowed to breathe PAF aerosol for 5 min. The aerosol PAF concentration varied with the exposure group by stepwise increases in the nebulizer PAF concentration. Following challenge, guinea pigs were killed and the ELGV was determined. Pharmacology of Aerosolized PAF; LTD4 , or Methacholine-induced A irway Obstruction Animals were dosed with pharmacologic agents at pretreatment times consistent with previously published usage (10-13). Propranolol (5 mg/kg subcutaneously) pretreatment was given 30 min before challenge except in animals challenged with methacholine. During challenge, animals were placed in the exposure chamber and allowed to breathe PAF aerosol (300 ug/ml saline nebulizer concentration), LTD..aerosol (0.06 ug/ml), or methacholine Cl aerosol (0.02 mg/ml) for 5 min. Control animals, orally dosed with vehicle, and test drug-pretreated animals were challenged simultaneously in groups of four to six. Immediately after challenge, guinea pigs were killed and the ELGV was determined. Source of Reagents Ovalbumin (grade V), propranolol (DLpropranolol hydrochloride), and pyrilamine (maleate salt) were purchased from Sigma Chemical Company (S1. Louis, MO). WEB2086wasobtained from Boehringer Ingelheim (Ridgefield, CT). SKF-I04,353 was obtained from Smith, Kline and French Laboratories. L-659,989 was obtained from Merck, Sharp and Dohme Inc. (Rahway, NJ). Piroxicam and ICI-204,219 were synthesized by the medicinal chemistry division at Pfizer Inc. (Groton, CT). Statistical Significance Statistical significance was determined using Dunnett's t test for multiple comparisons with controls (14). Student's t test was utilized in cases where only two groups existed. Mean values were considered significantly different for p < 0.05.
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Results
The excised lung gas volume from normal control guinea pigs was 1.95 ± 0.47 ml/kg (x ± SD, n = 6), and this value was unchanged by sensitization with 0.375 mg/kg anti-OA-IgG t (1.86 ± 0.3; x ± SD, n = 5). Aerosol antigen chalenge of guinea pigs sensitized 72 h before challenge resulted in an elevation in the BLGV. Antigen-induced elevations in BLGV were associated with respiratory distress as the values for ELGV exceeded 18ml/kg. Deaths resulting from ventilatory insufficiency were associated with ELGV values above 20 ml/kg, The optimal dose of anti-Oa-Igfl, for sensitization was determined by varying the dose of IgG t , given72 h before challenge, with 0.1% ovalbumin aerosol. The threshold for sensitization with anti-OAIgG t was between 0.03 and 0.1 mg/kg, and the response plateaued above 0.1 mg/kg, Increasing levels of aerosolized antigen exposure produced a dose-proportional increase in ELGV (figure 1). Threshold pulmonary response as measured by ELGV occurred at nebulizer concentrations of 0.001 to 0.0030/0 ovalbumin. Maximal and lethal pulmonary responses to aerosolized antigen occurred at nebulizer concentrations of 0.03 to 0.1% ovalbumin. At the aerosolized antigen concentration used for the pharmacology experiments, 3 of 10animals died before being killed. The ELGV determined from these animals wereincluded in the control group's average ELGV. The elevations in ELGV produced by aerosol antigen challenge of anti-OAIgG t sensitized, propranolol, and pyrilamine-pretreated guinea pigs could be suppressed by bothleukotriene modulators and PAF antagonist. The leukotriene antagonists ICI-204,219 (ED so = 3.5 mg/kg given orally 2 h before challenge)
(figure 2A) and SKF-I04,353 (ED so = 0.37 mg/ml nebulizer concentration 5 min aerosol) (figure 2B), and the PAF antagonists WEB-2086 (ED so = 1mg/kg
given orally 45 min before challenge) (figure 3A) and L-659,989 (ED so = 7 mg/kg given orally 2 h before challenge) (figure 3B) each produced dose-proportional inhibitions of aerosolized antigen-induced elevations in ELGV. The cyclooxygenase inhibitor piroxicam had a tendency to exacerbate the antigen-induced elevations in ELGV,although this enhancement was not statistically significant at the usual antigen stimulus because this produced a near-maximal airway obstruction. Lowering the antigen concentration to 0.0120/0 uncovered a potent cyclooxygenase inhibitor enhancement (p < 0.05) of the antigen-induced airway obstruction where the ELGV after antigen challenge was 13.4 ± 1.6 (x ± SE, n = 8) ml/kg in control guinea pigs and 19.6 ± 1.4 (x ± SE, n = 8) ml/kg in guinea pigs pretreated with piroxicam (20 mg/kg given orally 2 h before challenge).
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before challenge) did not alter this response (EWV = 14.0 ± 0.8, n = 5). To determine if any of the antagonists or enzyme inhibitors exhibited nonspecific pulmonary effects at the dosages and exposure times utilized in our other protocols, we evaluated the effect of these compounds for which we had sufficient supplies on an aerosolized methacholine CI-induced elevation in ELGV (figure 5). A reduction in methacholine-induced elevations in ELGV could result from suppression of minute ventilation, nonspecific bronchodilatory activity, or muscarinic antagonism. Piroxicam, pyrilamine, WEB2086, and ICI-204,219 did not have a statistically significant effect on methacholine-induced bronchoconstriction.
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Inhalation of a PAF aerosol produced PAF (300 ug/ml nebulizer concentration) elevations in ELGV in propranolol-pre- did not elevate the ELGV above control treated guinea pigs, which were propor- . levels (control 2.3 ± 0.3 ml/kg, n = 5, tional to the nebulizer concentration of lyso-PAF 2.8 ± 0.3 ml/kg, n = 5). The PAP. In nonsensitized guinea pigs, the elevations in ELGV produced by PAF threshold for elevation of ELGV oc- challenge could be partially suppressed curred at 30 ug/ml, The nebulizer con- by a cyclooxygenase inhibitor or a leucentration of PAF producing a maximal kotriene antagonist (figure 4). The speresponse was 1,000 ug/ml, After chal- cific PAF antagonist WEB-2086 blocked lenge with aerosolized PAF (300 ug/ml 97.1 ± 3.5070 (x ± SE, n = 4) and 77.5 nebulizer concentration), the ELGV was ± 5.5070 (x ± SE, n = 4) of the PAFelevated to 15.8 ± 0.8 ml/kg (x ± SE, induced elevation in ELGV when nonn = 15)in nonsensitized animals and to sensitized animals were pretreated at 3 15.7 ± 0.9 ml/kg (x ± SE, n = 10) in and 0.3 mg/kg given orally 45 min besensitized animals. Challenge with lyso- fore challenge, respectively. The leukotriene antagonist ICI-204,219 (10mg/kg, given orally 2 h before challenge) inhibited 43.2 ± 5.0010 (x ± SE, n = 10, p < 100 0.01) and 38.3 ± 7.9% (x ± SE, n = 10, 90 p < 0.01) of the PAF-induced increases w 80 in EWV in nonsensitized and sensitized Z f 70 animals, respectively. The cyclooxygenase ~ 60 inhibitor piroxicam (20 mg/kg given orali 50 ly 2 h before challenge) inhibited 47.8 ± ~ 40 u 3.4070 (x ± SE, n = 5, p < 0.01) and 62.1 'S 30 ± 5.8% (x ± SE, n = 10, p < 0.01) of 20 the PAF-induced elevations in ELGV in 10 nonsensitized and sensitized animals, respectively. The HI antagonist pyrilaAEROSOLIZED PAF CHALLENGE mine did not produce a statistically sigFig. 4. Effect of several antagonists/inhibitors on aeronificant change in the PAF-induced elesolized PAF (300 I-&glml, 5 min)-induced elevations in vation in ELGV in either nonsensitized ELGV in sensitized (open bars) and nonsensitized (stipor sensitized animals. pled bars) guinea pigs. Control group n = 10sensitized, A 5-min challenge with aerosolized n = 15 nonsensitized. ICI-204,219 10 mglkg given orally 2 h before challenge, n = 10sensitized, n = 10 nonLTD4 (nebulizer concentration 0.06 J.Lg/ sensitized. Piroxicam 20 mglkg given orally 2 h, n = ml) produced an elevation of the EWV 10sensitized, n = 5 nonsensitized. Pyrilamine 5 mglkg to 14.0 ± 0.4 ml/kg (x ± SE, n = 5). subcutaneously 30 min, n = 10 sensitized, n = 4 nonPretreatment with the PAF antagonist sensitized. ** p < 0.01,NS = not statistically significant. Data expressed as x ± SE. WEB 2086 (10mg/kg given orally 45 min til
Discussion
L-659.989 mg/kg (p.o. 2 hrs)
Aerosolized OA challenge of guinea pigs passively sensitized with homologous antiovalbumin IgG., and pretreated with antihistamines and a ~-adrenergic blocker, produced dose-dependent increases in EWV. The PAF antagonists WEB-2086 (10, IS) and L-659,989 (12), as well as the LTD4 antagonists ICI-204,219 (13) and SKF-I04,353 (11), inhibited this response in a dose-proportional fashion (figures 2 and 3). These data are therefore consistent with the hypothesis that both PAF and LTD4 are released in IgG 1-mediated pulmonary hypersensitivity reactions. These studies were conducted under the protection of an HI antagonist because preliminary studies indicated that the IgG 1-mediated pulmonary hypersensitivity response is dominated by histamine, at least during and immediately af-
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ter antigen challenge. Similarly, animals were pretreated with propranolol because it significantly increased the sensitivity and maximal obstructive response to both antigen and mediator challenge. Therefore, although the data presented in figures 2 and 3 indicate that specific antagonists of LTD 4 and PAF block virtually all of the pulmonary obstructive response to antigen challenge, in reality this model has been configured to enhance the lipid mediator component of this response. Our observations that the histamineindependent airway obstructive response to aerosolized antigen challenge of IgG 1sensitized guinea pigs could be suppressed by LTD 4 antagonists are consistent with previous in vitro findings. Udem and coworkers (2) and Regal (1)observed leukotriene-associated bioactivity and leukotriene antagonist-suppressible bioactivity, following antigen challenge of pulmonary tissue derived from animals passively sensitized with purified IgG i More recently, Cheng and coworkers (4) have documented elevated levels of histamine, LTD4 , and LTB4 after antigen challenge of chopped lung from IgG 1-sensitized guinea pigs. To date, the role played by PAF in IgG Imediated pulmonary tissue responses has not been investigated. However, the presence of a PAF antagonist suppressible bronchoconstriction has been documented in both actively (10, 16) and passively (10, 17, 18) sensitized guinea pigs. Keep in mind that both the actively sensitized and the crude-serum sensitized animals used in previous studies possessed both IgE and IgG 1 homocytotropic antibodies and therefore did not demonstrate a PAF-antagonist suppressible pulmonary response to sensitization by a specific antibody as described here. The role, if any, played by PAF in IgE-mediated pulmonary hypersensitivity responses has yet to be established. In this study, pretreatment of IgG 1sensitized guinea pigs with the cyclooxygenase inhibitor piroxicam significantly enhanced the aerosolized antigeninduced airway obstruction. These data are consistent with previous studies using actively sensitized guinea pigs (19) and guinea pigs passively sensitized with either crude IgG 1 - or IgE-rich serum (3). Investigations into the mechanism of this enhancement have indicated that indomethacin increases histamine and leukotriene release while suppressing the release of PGEz-like material after antigen challenge of pulmonary tissue derived
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from actively sensitized guinea pigs (20, 21). Although leukotrienes clearly mediate airway obstruction in this model, the effect of PGEz has not been established. However, PGE z has been shown to reduce mediator release after antigen challenge of pulmonary tissue from actively sensitized guinea pigs (22), reduce mucus secretion in a number of species (23), and relax carbachol-contracted guinea pig tracheal preparations (24), all of which could reduce the pulmonary obstructive response to antigen challenge. Although our study does not address mechanism, our observations are consistent with a cyclooxygenase inhibitormediated enhancement of leukotriene formation/ release and/or inhibition of PGE z formation/release. Our observation that both PAF antagonists and LTD 4 antagonists each inhibit up to 85 to 95010 of the pyrilamine-modified IgG 1-mediated airway obstructive response, and literature reports documenting PAF-mediated leukotriene synthesis (25, 26) and Ll'Da-antagonist-suppressible PAF activity (27-31), led us to hypothesize that leukotrienes, synthesized subsequent to PAF generation, were responsible for the IgG 1-mediated airway obstruction. To clarify the role that PAF plays in this response, we challenged control and drug pretreated guinea pigs with aerosolized PAP. We found that aerosolized PAF - but not lyso-PAF -induced a dose-proportional airway obstruction. The obstructive response to PAF was partially blocked by the LTD 4 antagonist ICI-204,219 and the cylooxygenase inhibitor piroxicam, whereas the histamine antagonist pyrilamine did not have a significant effect (figure 4). Conversely, the airway obstructive response to aerosolized LTD 4 was unaffected by pretreatment with the PAF antagonist WEB-2086. Therefore, these data are consistent with the hypothesis that airway obstruction produced by aerosolized antigen challenge of IgG 1-sensitized guinea pigs is mediated in part by Ll'Ds-syntheslzed secondary to PAF synthesis in that a significant fraction of the obstructive response to aerosolized PAF can be inhibited by a LTD4 antagonist. However, these data are inconsistent with this hypothesis in that a significant fraction of the PAF-mediated airway obstruction could . be blocked by the cylooxygenase inhibitor piroxicam, which enhanced rather than reduced antigen-induced airway obstruction. One possible explanation for these data would invoke PAF, functioning as an intracellular messenger, to pro-
mote 5-lipoxygenase product formation within the cells that have been activated by antigen. However, when PAF is exogenously applied to respiratory epithelium, other cells, whose response to PAF includes both cyclooxygenase and 5-lipoxygenase product formation, may be involved in the pulmonary obstructive response. Our observations, in part, are consistent with a report by Abraham and coworkers (27), which demonstrated that in the sheep, aerosolized PAF produced a pulmonary dysfunction that could be suppressed by a LTD 4 antagonist (FPL55712) but that was insensitive to an antihistamine (chlorpheniramine). However, these data differ in that a cyclooxygenase inhibitor (piroxicam) blocked a significant fraction of the obstructive response to aerosolized PAF in the guinea pig, whereas the sheep's pulmonary functional response was unaffected by indomethacin. This difference in the sensitivity to cyclooxygenase inhibitors may be species related, as Lefort and coworkers (32) have also demonstrated that the pulmonary dysfunction exhibited by guinea pigs after aerosolized PAF challenge can be inhibited by aspirin. We also observed that the effect of a LTD 4 antagonist (ICI-204,219), a cyclooxygenase inhibitor (piroxicam), and an H 1 antagonist (pyrilamine) on the pulmonary obstructive response to aerosolized PAF was unaffected by passive sensitization with IgG r- Pretolani and coworkers (33) have reported that the lungs of actively sensitized guinea pigs respond to PAP challenge by releasing large quantities of histamine. However, our data are not inconsistent, in that in a previous study, Pretolani and coworkers (34) found that lungs from passivelysensitized guinea pigs, like lungs from nonimmunized guinea pigs, do not release histamine in response to PAF challenge. In conclusion, we have shown that both PAF antagonists and LTD 4 antagonists block, whereas cyclooxygenase inhibitors enhance, the IgGcmediated pulmonary obstructive response to aerosolized antigen challenge of pyrilamine- and propranolol-pretreated guinea pigs. The observation that the selective LTD4 antagonist ICI-204,219 inhibits a significant fraction of the pulmonary obstructive response to an aerosolized PAF challenge is consistent with the hypothesis that part of the LTD 4 which mediates the aerosolized antigen-driven, IgG I-dependent obstructive response is generated in response to PAF production.
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Acknowledgment The writers thank Gerry Antognoli and Robin Breslow for their technical assistance. References 1. Regal JF. IgG vs. IgE: mediators of antigeninduced guinea pig lung parenchymal contraction. Immunopharmacology 1985; 10:137-46. 2. Udem BJ, Buckner CK, Harley P, Graziano FM. Smooth muscle contraction and release of histamine and slow-reacting substance of anaphylaxis in pulmonary tissues isolated from guinea pigs passively sensitized with IgO I or IgE antibodies. Am Rev Respir Dis 1985; 131:260-6. 3. Anderson P, Brattsand R. Protective effects of the glucocorticoid, budesonide, on lung anaphylaxis in actively sensitized guinea pigs: Inhibition of IgBbut not of IgO-mediated anaphylaxis. Br J Pharmacol 1982; 76:139-47. 4. Cheng JB, Pillar JS, Conklyn MJ, Breslow R, Shirley JT, Showell HJ. Involvement of peptidoleukotrienes in antigen-dependent thromboxane ('IX) synthesis in IgG I-sensitized guinea pig lungs. Adv Prostaglandins Leukotriene Res 1989; 19:187-90. 5. Regal JF. Immunoglobulin G- and immunoglobulin E-mediated airway smooth muscle contraction in the guinea pig. J Pharmacol Exp Ther 1983; 228:116-20. 6. Martin LN. Separation of guinea pig IgO subclasses by affinity chromatography on protein A-sepharose. J Immunol Methods 1982; 52:205. 7. Silbaugh SA, Stengel PW, Dillard RD, Bemis KG. Pulmonary gas trapping in the guinea pig and its application for pharmacological testing. J Pharmacol Methods 1987; 18:295-303. 8. Stengel PW, Pechous PA, Silbaugh SA. Mechanism of A23187-induced airway obstruction in the guinea pig. Prostaglandins 1987; 33:567-77. 9. Stengel PW, Silbaugh SA. Mechanisms of gas loss from normal and hyperinflated excised guinea pig lungs. Respir Physiol 1986; 63:129-38. 10. Casals-Stenzel J. Effects of WEB-2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis. Immunopharmacology 1987; 13:117-24. 11. Hay DWP, Muccitelli RM, Tucker SS, et al. Pharmacologic profile of SK&F 104353: A novel, potent and selectivepeptidoleukotriene receptor an-
tagonist in guinea pig and human airways. J Pharmacol Exp Ther 1987; 243:474-81. 12. Hwang S, Lam M, Alberts AW, Bugianesi R, Chabala JC, Ponpipom MM. Biochemical and pharmacological characterization of L-659,989: An extremely potent, selective and competitive receptor antagonist of platelet-activating factor. J Pharmacol Exp Ther 1988; 246:534-41. 13. Krell RD, Buckner CK, Keith RA, et al. ICI204,219: a potent, selective peptide leukotriene receptor antagonist. J Allergy Clin Immunol1988; 81:276. 14. Zar JH. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall, Inc., 1974. 15. Casals-Stenzel J, Muaceuic G, Weber K. Pharmacological actions of WEB-2086, a new specific antagonist of platelet activating factor. J Pharmacol Exp Ther 1987; 241:974-81. 16. Cirino M, Lagente V, Lefort J, Vargaftig BB. A study with BN-52021 demonstrates the involvement of PAF-acether in Igfi-dependent anaphylactic bronchoconstriction. Prostaglandins 1986;32:121-6. 17. Lagente V, Cirino M, Desquand S, Lefort J, Vargaftig BB. Involvement of PAF-acether in anaphylactic bronchoconstriction induced in guinea pigs by aerosolized antigen. Int Arch Allergy Appl Immunol 1988; 85:14-9. 18. Lagente V, Touvay C, Randon J, et al. Interference of the PAF-acether antagonist BN-52021 with passive anaphylaxis in the guinea pig. Prostaglandins 1987; 33:265-74. 19. Michoud MC, Fraser RS, Pare PO, Hogg JC. Effect of indomethacin and atropine in experimental asthma in conscious guinea pigs. J Appl Physiol 1976; 40:889-94. 20. Adcock JJ, Oarland LO, Moncada S, Salomon JA. The mechanism of enhancement of fatty acid hydroperoxides of anaphylactic mediator release. Prostaglandins 1978; 16:179-87. 21. Burka JF, Paterson NAM. Evidence for lipoxygenase pathway involvement in allergic tracheal concentration. Prostaglandins 1980; 19:499-515. 22. Hitchcock M. Effect of inhibitors of prostaglandin synthesis and prostaglandins E 1 and FlU on the immunologic release of mediators of inflammation from actively sensitized guinea-pig lung. J Pharmacol Exp Ther 1978; 207:630-40. 23. Marin MO. Pharmacology of airway secretion. Pharmacol Rev 1986; 38:273-89.
24. Vermue NA, Hertog AD. The prostaglandin response in guinea-pig taenia caeci and tracheal smooth muscle of different ages. Eur J Pharmacol 1988; 147:273-7. 25. Lefer AM, Roth OM, Lefer OJ, Smith JB. Potentiation of leukotriene formation in pulmonary and vascular tissue. Naunyn Schmiedebergs Arch Pharmacol 1984; 326:186-9. 26. Voelkel NF, Worthen S, Reeves JT, Henson PM, Murphy RC. Nonimmunological production of leukotrienes induced by platelet-activating factor. Science 1982; 218:286-8. 27. Abraham WM, Stevenson JS, Garrido R. A possible role for PAF in allergen-induced late responses: Modification by a selective antagonist. J Appl Physiol 1989; 66:2351-7. 28. Bonnet J, Thibaudeau 0, Bessin P. Dependency of the PAF-acether induced bronchospasm on the lipoxygenase pathway in the guinea-pig. Prostaglandins 1983; 2(:457-66. 29. Jancar S, Theriault P, Provencal B, Cloutier S, Sirois P. Mechanism of action of plateletactivating factor on guinea-pig lung parenchymal strips. Can J Physiol Pharmacol1988; 66: 1187- 91. 30. Lewis AJ, Dervinis A, Cheng J. The effects of antiallergic and bronchodilator drugs on plateletactivating factor (PAF-acether) induced bronchospasm and platelet aggregation. Agents Actions 1984; 15:636-42. 31. Moodley J, Stuttle A. Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leukocytes. Prostaglandins 1987; 33:253-64. 32. Lefort J, Rotilio 0, Vargaftig BB. The platelet independent release of thromboxane A 1 by PAFaceter from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br J Pharmacol 1984; 82:565-75. 33. Pretolani M, Lefort J, Vargaftig BB. Limited interference of specific PAF antagonists with hyperresponsiveness to PAF itself of lungs from actively sensitized guinea-pigs. Br J Pharmacol 1989; 97:433-42. 34. Pretolani M, Lefort J, Vargaftig BB. Active sensitization induces lung hyperresponsiveness in the guinea pigs: Pharmacological modulation and triggering role of the booster injection. Am Rev Respir Dis 1988; 138:1572-78.
JOHN W. WATSON, MARYROSE CONKLYN, and HENRY J. SHOWELL
Introduction
Guinea pigs have long been used as a model of pulmonary immediate hypersensitivity reactions. These reactions are triggered by the interaction of antigen with cytophilic immunoglobulins attached to resident pulmonary cells, such as mast cells, and alveolar macrophages as well as circulating cells, such as basophils. The anaphylactic release of mediators subsequent to cross-linking of surface-bound antibody produces bronchoconstriction, pulmonary vascular leak, and mucus secretion which, in turn, results in airway obstruction, dyspnea, and death. Both IgG, and IgE immunoglobulins sensitize pulmonary tissue in this species. However, in vitro studies using sensitized pulmonary tissue indicate that the mediators released and sensitivity to pharmacologic agents may depend on the species of sensitizing antibody (1,2). In vivo studies using partially purified serum preparations to selectively sensitize guinea pigs with either IgG or IgE antibodies also indicate that there may be marked antibody-dependent differences in the sensitivity of the acute antigen-driven pulmonary hypersensitivity responses to various pharmacologic agents (3). We have isolated and purified an antiovalbumin (anti-OA) IgG l antibody from the serum of guinea pigs actively sensitized to ovalbumin. Specific in vitro antigen challenge of lung tissue derived from animals passively sensitized with this anti-Oe-IgG, has previously been shown to result in the release of LTB4 , LTD4 , and histamine (4). In this study, we have characterized the in vivo pharmacologic modulation of an acute pulmonary hypersensitivity response mediated by this antibody to determine the involvement, if any, of platelet-activating factor (PAF), LTD4 , and the cyclooxygenase products of arachidonic acid (AA)
SUMMARY We determined the pulmonary obstructive response to aerosolized antigen challenge, and Its sensitivity to antagonists of specific lipid mediators, In IgG 1 passively sensitized (lgG 1-PS) guinea pigs. Antlovalbumln (OA)-lgG1 was Isolated by affinity chromatography from serum derived from actively Immunized Hartley guinea pigs. Propranolol and pyrilamine pretreated, IgG1-PS guinea pigs were challenged with aerosolized antigen and pulmonary obstruction was quantified by measurements of excised lung gas volume (ELGV). ELGVIncreased between 150 and 1,035% In a doseproportional fashion with Increasing antigen exposure (0.001 to 0.1% nebulizer concentration). The leukotrlene antagonists ICI-204,219 and SKF-104,353 exhibited dose-proportional Inhibitions In antigen-Induced elevations In ELGV, Inhibiting up to 65 and 87% at the maximal concentrations examined. Similarly, the platelet-activating factor (PAF) antagonists WEB-2086 and L-659,989 Inhibited antigen-Induced elevations In ELGV, Inhibiting up to 94 and 59% at the maximal concentrations examined. In contrast, the cyclooxygenase (CO) Inhibitor plroxlcam significantly enhanced (p < 0.05)the OA-Induced elevations In ELGV. Aerosolized PAFchallenge produced dose-proportional elevations In ELGVthat were significantly Inhibited by the LTD. antagonist ICI-204,219(38 and 43% Inhibition) and the CO Inhibitor plroxlcam (62 and 48% Inhibition) In sensitized and nonsensltlzed animals, respectively. We hypothesize that IgG1-dependent airway obstruction Is mediated In part by LTD. produced In response to PAF generation. AM REV RESPIR DIS 1990; 142:1093-1098
metabolism. To this end, guinea pigs were passivelysensitized with purified homologous anti-OA-IgG h pretreated with pyrilamine and propranolol, and challenged with aerosolized OA antigen. The involvement of PAF, the leukotrienes, and the cyclooxygenase products of arachidonic acid metabolism in the resulting pulmonary obstructive response was inferred by treating these animals with specific antagonists and enzyme inhibitors before antigen challenge. Since our data indicated that both PAF antagonists and LTD4 antagonists could block 85 to 95070 of the pyrilamine-and propranololmodified IgGrmediated obstructive response, we also evaluated the pharmacology of aerosolized PAF-induced airway obstruction to determine if PAF produces airway obstruction by stimulating LTD4 synthesis. Methods Isolation of IgOl IgG l antibodies were raised in male Hartley guinea pigs, immunized according to the method of Regal (5). Blood was collected by cardiac puncture and the serum stored at
-20 0 C. Antiovalbumin (OA)-IgG l was isolated by affinity chromatography using protein A-Sepharose CL-4B (Pharmacia Chemicals, Uppsala, Sweden) and OA-Sepharose 4B columns as follows. Antiserum (250to 300 ml) was applied to a protein A-Sepharose CL4B column (1.5 x 17ern) which flowed directly onto the OA-Sepharose 4B column (1.5 x 15 em), Both columns had been equilibrated with phosphate-buffered saline (PBS), pH 7.4. After washing with PBS, the columns were separated and independently eluted with 0.1 M glycine, pH = 3.0. The eluted protein fractions from the OA-Sepharose 4B column that consisted primarily of anti-OA IgG l were combined and dialyzed against citrate-phosphate buffer (CPB), pH = 7.4 (6). This sample was then reapplied to the protein ASepharose CL-4B column that had been (Received in original form June 19, 1989 and in revised form February 28, 1990) 1 From the Department of Immunology and Infectious Diseases,Central ResearchDivision, Pfizer Inc., Groton, Connecticut. 2 Correspondence and requests for reprints should be addressed to John W. Watson, Ph.D., Department of Immunology and Infectious Diseases, Central Research Division, Pfizer Inc., Groton, CT 06340.
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WATSON, CONKLYN, AND SHOWELL
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equilibrated in CPB, pH 7.4. The protein was then isolated by pH gradient using CPB. The anti-OA IgG. eluted between pH 6 and pH 5. The fractions containing anti-OA IgG. were combined and dialyzed against PBS. The purity of the preparation was determined by electrophoresis on an 8 to 25070 acrylamide gel using the Pharmacia Phast System. Specific activity was established by the ability to sensitize guinea pig tracheal rings with an ED so of 3.7 ± 0.5 (x ± SE) ug/ml.
Index of Airway Obstruction The volume of air trapped behind closed airways in excised guinea pig lungs was utilized as an index of a physiologic response to antigen and/or mediator challenge. In this species, excisedlung gas volume (ELGV) has been previously shown to increase in a dose-proportional manner with increasing aerosol concentrations of LTD.., methacholine, and histamine (7), and it has been shown to be highly correlated with measurements of pulmonary resistance and dynamic compliance (8). The rapidity with which ELGV increases following challenge, and its reversibility with isoproterenol infusion, are consistent with the hypothesis that smooth muscle constriction plays a role in mediator-driven elevations in ELGV (8). However, it is likely that ELGV could be influenced by any force that would contribute to the obstruction of airways at low lung volumes such as mucus secretion, peribronchial edema, a reduction in lung elasticity, or an alteration in airway surface forces. What is important in the context of this study is that ELGV is sensitive to LTD.., PAF, and histamine challenge and therefore can be used to indicate the presence of a physiologic concentration of these substances following antigen or mediator challenge. ELGV was determined by measuring the stable volume of air trapped within the excised lung at a transpulmonary pressure of zero em HzO. Immediately after death, the pleural space was opened and the lungs allowed to collapse to a stable volume. The lungs were surgically removed, the nonpulmonary tissues dissected away, then the trachea was shortened and tied just above the carina. The loss of trapped air from lungs treated in this manner is approximately 50J0/h and independent of the amount of gas within the lung (9). The volume of air within the lung was determined by measuring the negative force exerted by the lung when submerged in saline as described by Stengel and Silbaugh (9). Because the density of lung tissue is approximately equal to the density of saline, this force (in grams) is equal to the volume of air (in milliliters) trapped within the lung (Archimedes' principle). All volume measurements were completed within 5 min of excision. Volumes were normalized to animal weight to give excised lung gas volume in units of milliliters per kilogram. Aerosol Generation Aerosols were generated by a Pulmosonic model 25 nebulizer (Devilbiss Co., Somerset, PA) using 3 ml of test solution. This type of
nebulizer produces a 7 to 8 urn particle with a geometric standard deviation (SD) of 2 to 3. However, particle size may be altered before respiration as aerosols are recirculated in a closed loop system that consists of the nebulizer, a l-L expansion chamber, a circulating fan, and a 9-L exposure chamber. To determine the respirability of particles generated in this system, a group of five guinea pigs were exposed to aerosolized Evans blue dye for 10 min (nebulizer concentration 30 mg/ ml). Following death, their upper airways (nose to trachea) and lower airways (trachea and lungs) werehomogenized and dye extracted with formamide/acetonitrile. Spectrophotometric analysis of these supernatants indicated that, after substracting for background interference, the upper and lower airways contained an average of 2.5 and 32.0 f.1g Evans blue dye, respectively. Clearly, the particles generated by this system are preferentially distributed to the lung rather than to the upper airways.
Sensitizing Dose of IgG l Groups of guinea pigs were sensitized subcutaneously with varying doses of anti-OAIgG. (0.01, 0.03, 0.1, 0.3 mg/kg) 72 h before antigen challenge. Thirty minutes before challenge, guinea pigs weremedicated with pyrilamine maleate salt (5 mg/kg subcutaneously) and propranolol (5 mg/kg subcutaneously). During challenge, groups of three animals were allowed to breathe a 0.1810 ovalbumin aerosol (nebulizer concentration) for 5 min. Eight minutes after aerosol challenge, animals were killed with 130 mg sodium pentobarbital given intraperitoneally. ELGV was determined after death (""2 min after sodium pentobarbital). Antigen Dose-Response Curve Guinea pigs were sensitized with 0.375 mg/kg anti-OA-IgG. 72 h before antigen challenge. Thirty minutes before challenge, they were pretreated with propranolol (5 mg/kg subcutaneously) and pyrilamine maleate salt (5 mg/kg subcutaneously). Five animals were placed in the exposure chamber simultaneously and allowed to breathe antigen aerosol for 5 min. The aerosol antigen concentration was varied with the exposure group by stepwise increases in the nebulizer ovalbumin concentration. Eight minutes after challenge, animals were given 130mg Na pentobarbital intraperitoneally and ELGV determined following death. Pharmacology of Antigen-induced A irway Obstruction We chose a combination of anti-OA-IgG. (0.375 mg/kg) and aerosol antigen challenge (0.02 to 0.022%) that resulted in an average ELGV near 80870 of the maximal response (producing apnea and death). Guinea pigs were pretreated with pharmacologic agents at prechallenge time points that are consistent with published usage (10-13). Guinea pigs were also pretreated with propranolol (5 mg/kg subcutaneously) and pyrilamine male-
ate salt (5 mg/kg subcutaneously) 30 min before challenge. Control animals, orally dosed with vehicle, and test drug-pretreated animals (oral or aerosol dosing) werechallenged simultaneously in groups of four to six animals. During challenge, animals were placed in the aerosol exposure chamber and allowed to breathe ovalbumin aerosol for 5 min. Eight minutes after exposure, a time previously determined as the period of maximal bronchoconstriction, animals were killed with 130 mg sodium pentobarbital intraperitoneally and ELGV determined.
Aerosolized PAF Dose-Response Curve PAF was dissolved in normal saline containing 0.25 0J0 bovine serum albumin and stored on ice until it was added to the nebulizer. Normal, unsensitized guinea pigs were premedicated with propranolol (5 mg/kg subcutaneously for 30 min), placed in the aerosol chamber in groups of five, and allowed to breathe PAF aerosol for 5 min. The aerosol PAF concentration varied with the exposure group by stepwise increases in the nebulizer PAF concentration. Following challenge, guinea pigs were killed and the ELGV was determined. Pharmacology of Aerosolized PAF; LTD4 , or Methacholine-induced A irway Obstruction Animals were dosed with pharmacologic agents at pretreatment times consistent with previously published usage (10-13). Propranolol (5 mg/kg subcutaneously) pretreatment was given 30 min before challenge except in animals challenged with methacholine. During challenge, animals were placed in the exposure chamber and allowed to breathe PAF aerosol (300 ug/ml saline nebulizer concentration), LTD..aerosol (0.06 ug/ml), or methacholine Cl aerosol (0.02 mg/ml) for 5 min. Control animals, orally dosed with vehicle, and test drug-pretreated animals were challenged simultaneously in groups of four to six. Immediately after challenge, guinea pigs were killed and the ELGV was determined. Source of Reagents Ovalbumin (grade V), propranolol (DLpropranolol hydrochloride), and pyrilamine (maleate salt) were purchased from Sigma Chemical Company (S1. Louis, MO). WEB2086wasobtained from Boehringer Ingelheim (Ridgefield, CT). SKF-I04,353 was obtained from Smith, Kline and French Laboratories. L-659,989 was obtained from Merck, Sharp and Dohme Inc. (Rahway, NJ). Piroxicam and ICI-204,219 were synthesized by the medicinal chemistry division at Pfizer Inc. (Groton, CT). Statistical Significance Statistical significance was determined using Dunnett's t test for multiple comparisons with controls (14). Student's t test was utilized in cases where only two groups existed. Mean values were considered significantly different for p < 0.05.
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Results
The excised lung gas volume from normal control guinea pigs was 1.95 ± 0.47 ml/kg (x ± SD, n = 6), and this value was unchanged by sensitization with 0.375 mg/kg anti-OA-IgG t (1.86 ± 0.3; x ± SD, n = 5). Aerosol antigen chalenge of guinea pigs sensitized 72 h before challenge resulted in an elevation in the BLGV. Antigen-induced elevations in BLGV were associated with respiratory distress as the values for ELGV exceeded 18ml/kg. Deaths resulting from ventilatory insufficiency were associated with ELGV values above 20 ml/kg, The optimal dose of anti-Oa-Igfl, for sensitization was determined by varying the dose of IgG t , given72 h before challenge, with 0.1% ovalbumin aerosol. The threshold for sensitization with anti-OAIgG t was between 0.03 and 0.1 mg/kg, and the response plateaued above 0.1 mg/kg, Increasing levels of aerosolized antigen exposure produced a dose-proportional increase in ELGV (figure 1). Threshold pulmonary response as measured by ELGV occurred at nebulizer concentrations of 0.001 to 0.0030/0 ovalbumin. Maximal and lethal pulmonary responses to aerosolized antigen occurred at nebulizer concentrations of 0.03 to 0.1% ovalbumin. At the aerosolized antigen concentration used for the pharmacology experiments, 3 of 10animals died before being killed. The ELGV determined from these animals wereincluded in the control group's average ELGV. The elevations in ELGV produced by aerosol antigen challenge of anti-OAIgG t sensitized, propranolol, and pyrilamine-pretreated guinea pigs could be suppressed by bothleukotriene modulators and PAF antagonist. The leukotriene antagonists ICI-204,219 (ED so = 3.5 mg/kg given orally 2 h before challenge)
(figure 2A) and SKF-I04,353 (ED so = 0.37 mg/ml nebulizer concentration 5 min aerosol) (figure 2B), and the PAF antagonists WEB-2086 (ED so = 1mg/kg
given orally 45 min before challenge) (figure 3A) and L-659,989 (ED so = 7 mg/kg given orally 2 h before challenge) (figure 3B) each produced dose-proportional inhibitions of aerosolized antigen-induced elevations in ELGV. The cyclooxygenase inhibitor piroxicam had a tendency to exacerbate the antigen-induced elevations in ELGV,although this enhancement was not statistically significant at the usual antigen stimulus because this produced a near-maximal airway obstruction. Lowering the antigen concentration to 0.0120/0 uncovered a potent cyclooxygenase inhibitor enhancement (p < 0.05) of the antigen-induced airway obstruction where the ELGV after antigen challenge was 13.4 ± 1.6 (x ± SE, n = 8) ml/kg in control guinea pigs and 19.6 ± 1.4 (x ± SE, n = 8) ml/kg in guinea pigs pretreated with piroxicam (20 mg/kg given orally 2 h before challenge).
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before challenge) did not alter this response (EWV = 14.0 ± 0.8, n = 5). To determine if any of the antagonists or enzyme inhibitors exhibited nonspecific pulmonary effects at the dosages and exposure times utilized in our other protocols, we evaluated the effect of these compounds for which we had sufficient supplies on an aerosolized methacholine CI-induced elevation in ELGV (figure 5). A reduction in methacholine-induced elevations in ELGV could result from suppression of minute ventilation, nonspecific bronchodilatory activity, or muscarinic antagonism. Piroxicam, pyrilamine, WEB2086, and ICI-204,219 did not have a statistically significant effect on methacholine-induced bronchoconstriction.
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Inhalation of a PAF aerosol produced PAF (300 ug/ml nebulizer concentration) elevations in ELGV in propranolol-pre- did not elevate the ELGV above control treated guinea pigs, which were propor- . levels (control 2.3 ± 0.3 ml/kg, n = 5, tional to the nebulizer concentration of lyso-PAF 2.8 ± 0.3 ml/kg, n = 5). The PAP. In nonsensitized guinea pigs, the elevations in ELGV produced by PAF threshold for elevation of ELGV oc- challenge could be partially suppressed curred at 30 ug/ml, The nebulizer con- by a cyclooxygenase inhibitor or a leucentration of PAF producing a maximal kotriene antagonist (figure 4). The speresponse was 1,000 ug/ml, After chal- cific PAF antagonist WEB-2086 blocked lenge with aerosolized PAF (300 ug/ml 97.1 ± 3.5070 (x ± SE, n = 4) and 77.5 nebulizer concentration), the ELGV was ± 5.5070 (x ± SE, n = 4) of the PAFelevated to 15.8 ± 0.8 ml/kg (x ± SE, induced elevation in ELGV when nonn = 15)in nonsensitized animals and to sensitized animals were pretreated at 3 15.7 ± 0.9 ml/kg (x ± SE, n = 10) in and 0.3 mg/kg given orally 45 min besensitized animals. Challenge with lyso- fore challenge, respectively. The leukotriene antagonist ICI-204,219 (10mg/kg, given orally 2 h before challenge) inhibited 43.2 ± 5.0010 (x ± SE, n = 10, p < 100 0.01) and 38.3 ± 7.9% (x ± SE, n = 10, 90 p < 0.01) of the PAF-induced increases w 80 in EWV in nonsensitized and sensitized Z f 70 animals, respectively. The cyclooxygenase ~ 60 inhibitor piroxicam (20 mg/kg given orali 50 ly 2 h before challenge) inhibited 47.8 ± ~ 40 u 3.4070 (x ± SE, n = 5, p < 0.01) and 62.1 'S 30 ± 5.8% (x ± SE, n = 10, p < 0.01) of 20 the PAF-induced elevations in ELGV in 10 nonsensitized and sensitized animals, respectively. The HI antagonist pyrilaAEROSOLIZED PAF CHALLENGE mine did not produce a statistically sigFig. 4. Effect of several antagonists/inhibitors on aeronificant change in the PAF-induced elesolized PAF (300 I-&glml, 5 min)-induced elevations in vation in ELGV in either nonsensitized ELGV in sensitized (open bars) and nonsensitized (stipor sensitized animals. pled bars) guinea pigs. Control group n = 10sensitized, A 5-min challenge with aerosolized n = 15 nonsensitized. ICI-204,219 10 mglkg given orally 2 h before challenge, n = 10sensitized, n = 10 nonLTD4 (nebulizer concentration 0.06 J.Lg/ sensitized. Piroxicam 20 mglkg given orally 2 h, n = ml) produced an elevation of the EWV 10sensitized, n = 5 nonsensitized. Pyrilamine 5 mglkg to 14.0 ± 0.4 ml/kg (x ± SE, n = 5). subcutaneously 30 min, n = 10 sensitized, n = 4 nonPretreatment with the PAF antagonist sensitized. ** p < 0.01,NS = not statistically significant. Data expressed as x ± SE. WEB 2086 (10mg/kg given orally 45 min til
Discussion
L-659.989 mg/kg (p.o. 2 hrs)
Aerosolized OA challenge of guinea pigs passively sensitized with homologous antiovalbumin IgG., and pretreated with antihistamines and a ~-adrenergic blocker, produced dose-dependent increases in EWV. The PAF antagonists WEB-2086 (10, IS) and L-659,989 (12), as well as the LTD4 antagonists ICI-204,219 (13) and SKF-I04,353 (11), inhibited this response in a dose-proportional fashion (figures 2 and 3). These data are therefore consistent with the hypothesis that both PAF and LTD4 are released in IgG 1-mediated pulmonary hypersensitivity reactions. These studies were conducted under the protection of an HI antagonist because preliminary studies indicated that the IgG 1-mediated pulmonary hypersensitivity response is dominated by histamine, at least during and immediately af-
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19G1"MEDIATED ACUTE PULMONARY OBSTRUCTION
ter antigen challenge. Similarly, animals were pretreated with propranolol because it significantly increased the sensitivity and maximal obstructive response to both antigen and mediator challenge. Therefore, although the data presented in figures 2 and 3 indicate that specific antagonists of LTD 4 and PAF block virtually all of the pulmonary obstructive response to antigen challenge, in reality this model has been configured to enhance the lipid mediator component of this response. Our observations that the histamineindependent airway obstructive response to aerosolized antigen challenge of IgG 1sensitized guinea pigs could be suppressed by LTD 4 antagonists are consistent with previous in vitro findings. Udem and coworkers (2) and Regal (1)observed leukotriene-associated bioactivity and leukotriene antagonist-suppressible bioactivity, following antigen challenge of pulmonary tissue derived from animals passively sensitized with purified IgG i More recently, Cheng and coworkers (4) have documented elevated levels of histamine, LTD4 , and LTB4 after antigen challenge of chopped lung from IgG 1-sensitized guinea pigs. To date, the role played by PAF in IgG Imediated pulmonary tissue responses has not been investigated. However, the presence of a PAF antagonist suppressible bronchoconstriction has been documented in both actively (10, 16) and passively (10, 17, 18) sensitized guinea pigs. Keep in mind that both the actively sensitized and the crude-serum sensitized animals used in previous studies possessed both IgE and IgG 1 homocytotropic antibodies and therefore did not demonstrate a PAF-antagonist suppressible pulmonary response to sensitization by a specific antibody as described here. The role, if any, played by PAF in IgE-mediated pulmonary hypersensitivity responses has yet to be established. In this study, pretreatment of IgG 1sensitized guinea pigs with the cyclooxygenase inhibitor piroxicam significantly enhanced the aerosolized antigeninduced airway obstruction. These data are consistent with previous studies using actively sensitized guinea pigs (19) and guinea pigs passively sensitized with either crude IgG 1 - or IgE-rich serum (3). Investigations into the mechanism of this enhancement have indicated that indomethacin increases histamine and leukotriene release while suppressing the release of PGEz-like material after antigen challenge of pulmonary tissue derived
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from actively sensitized guinea pigs (20, 21). Although leukotrienes clearly mediate airway obstruction in this model, the effect of PGEz has not been established. However, PGE z has been shown to reduce mediator release after antigen challenge of pulmonary tissue from actively sensitized guinea pigs (22), reduce mucus secretion in a number of species (23), and relax carbachol-contracted guinea pig tracheal preparations (24), all of which could reduce the pulmonary obstructive response to antigen challenge. Although our study does not address mechanism, our observations are consistent with a cyclooxygenase inhibitormediated enhancement of leukotriene formation/ release and/or inhibition of PGE z formation/release. Our observation that both PAF antagonists and LTD 4 antagonists each inhibit up to 85 to 95010 of the pyrilamine-modified IgG 1-mediated airway obstructive response, and literature reports documenting PAF-mediated leukotriene synthesis (25, 26) and Ll'Da-antagonist-suppressible PAF activity (27-31), led us to hypothesize that leukotrienes, synthesized subsequent to PAF generation, were responsible for the IgG 1-mediated airway obstruction. To clarify the role that PAF plays in this response, we challenged control and drug pretreated guinea pigs with aerosolized PAP. We found that aerosolized PAF - but not lyso-PAF -induced a dose-proportional airway obstruction. The obstructive response to PAF was partially blocked by the LTD 4 antagonist ICI-204,219 and the cylooxygenase inhibitor piroxicam, whereas the histamine antagonist pyrilamine did not have a significant effect (figure 4). Conversely, the airway obstructive response to aerosolized LTD 4 was unaffected by pretreatment with the PAF antagonist WEB-2086. Therefore, these data are consistent with the hypothesis that airway obstruction produced by aerosolized antigen challenge of IgG 1-sensitized guinea pigs is mediated in part by Ll'Ds-syntheslzed secondary to PAF synthesis in that a significant fraction of the obstructive response to aerosolized PAF can be inhibited by a LTD4 antagonist. However, these data are inconsistent with this hypothesis in that a significant fraction of the PAF-mediated airway obstruction could . be blocked by the cylooxygenase inhibitor piroxicam, which enhanced rather than reduced antigen-induced airway obstruction. One possible explanation for these data would invoke PAF, functioning as an intracellular messenger, to pro-
mote 5-lipoxygenase product formation within the cells that have been activated by antigen. However, when PAF is exogenously applied to respiratory epithelium, other cells, whose response to PAF includes both cyclooxygenase and 5-lipoxygenase product formation, may be involved in the pulmonary obstructive response. Our observations, in part, are consistent with a report by Abraham and coworkers (27), which demonstrated that in the sheep, aerosolized PAF produced a pulmonary dysfunction that could be suppressed by a LTD 4 antagonist (FPL55712) but that was insensitive to an antihistamine (chlorpheniramine). However, these data differ in that a cyclooxygenase inhibitor (piroxicam) blocked a significant fraction of the obstructive response to aerosolized PAF in the guinea pig, whereas the sheep's pulmonary functional response was unaffected by indomethacin. This difference in the sensitivity to cyclooxygenase inhibitors may be species related, as Lefort and coworkers (32) have also demonstrated that the pulmonary dysfunction exhibited by guinea pigs after aerosolized PAF challenge can be inhibited by aspirin. We also observed that the effect of a LTD 4 antagonist (ICI-204,219), a cyclooxygenase inhibitor (piroxicam), and an H 1 antagonist (pyrilamine) on the pulmonary obstructive response to aerosolized PAF was unaffected by passive sensitization with IgG r- Pretolani and coworkers (33) have reported that the lungs of actively sensitized guinea pigs respond to PAP challenge by releasing large quantities of histamine. However, our data are not inconsistent, in that in a previous study, Pretolani and coworkers (34) found that lungs from passivelysensitized guinea pigs, like lungs from nonimmunized guinea pigs, do not release histamine in response to PAF challenge. In conclusion, we have shown that both PAF antagonists and LTD 4 antagonists block, whereas cyclooxygenase inhibitors enhance, the IgGcmediated pulmonary obstructive response to aerosolized antigen challenge of pyrilamine- and propranolol-pretreated guinea pigs. The observation that the selective LTD4 antagonist ICI-204,219 inhibits a significant fraction of the pulmonary obstructive response to an aerosolized PAF challenge is consistent with the hypothesis that part of the LTD 4 which mediates the aerosolized antigen-driven, IgG I-dependent obstructive response is generated in response to PAF production.
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Acknowledgment The writers thank Gerry Antognoli and Robin Breslow for their technical assistance. References 1. Regal JF. IgG vs. IgE: mediators of antigeninduced guinea pig lung parenchymal contraction. Immunopharmacology 1985; 10:137-46. 2. Udem BJ, Buckner CK, Harley P, Graziano FM. Smooth muscle contraction and release of histamine and slow-reacting substance of anaphylaxis in pulmonary tissues isolated from guinea pigs passively sensitized with IgO I or IgE antibodies. Am Rev Respir Dis 1985; 131:260-6. 3. Anderson P, Brattsand R. Protective effects of the glucocorticoid, budesonide, on lung anaphylaxis in actively sensitized guinea pigs: Inhibition of IgBbut not of IgO-mediated anaphylaxis. Br J Pharmacol 1982; 76:139-47. 4. Cheng JB, Pillar JS, Conklyn MJ, Breslow R, Shirley JT, Showell HJ. Involvement of peptidoleukotrienes in antigen-dependent thromboxane ('IX) synthesis in IgG I-sensitized guinea pig lungs. Adv Prostaglandins Leukotriene Res 1989; 19:187-90. 5. Regal JF. Immunoglobulin G- and immunoglobulin E-mediated airway smooth muscle contraction in the guinea pig. J Pharmacol Exp Ther 1983; 228:116-20. 6. Martin LN. Separation of guinea pig IgO subclasses by affinity chromatography on protein A-sepharose. J Immunol Methods 1982; 52:205. 7. Silbaugh SA, Stengel PW, Dillard RD, Bemis KG. Pulmonary gas trapping in the guinea pig and its application for pharmacological testing. J Pharmacol Methods 1987; 18:295-303. 8. Stengel PW, Pechous PA, Silbaugh SA. Mechanism of A23187-induced airway obstruction in the guinea pig. Prostaglandins 1987; 33:567-77. 9. Stengel PW, Silbaugh SA. Mechanisms of gas loss from normal and hyperinflated excised guinea pig lungs. Respir Physiol 1986; 63:129-38. 10. Casals-Stenzel J. Effects of WEB-2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis. Immunopharmacology 1987; 13:117-24. 11. Hay DWP, Muccitelli RM, Tucker SS, et al. Pharmacologic profile of SK&F 104353: A novel, potent and selectivepeptidoleukotriene receptor an-
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24. Vermue NA, Hertog AD. The prostaglandin response in guinea-pig taenia caeci and tracheal smooth muscle of different ages. Eur J Pharmacol 1988; 147:273-7. 25. Lefer AM, Roth OM, Lefer OJ, Smith JB. Potentiation of leukotriene formation in pulmonary and vascular tissue. Naunyn Schmiedebergs Arch Pharmacol 1984; 326:186-9. 26. Voelkel NF, Worthen S, Reeves JT, Henson PM, Murphy RC. Nonimmunological production of leukotrienes induced by platelet-activating factor. Science 1982; 218:286-8. 27. Abraham WM, Stevenson JS, Garrido R. A possible role for PAF in allergen-induced late responses: Modification by a selective antagonist. J Appl Physiol 1989; 66:2351-7. 28. Bonnet J, Thibaudeau 0, Bessin P. Dependency of the PAF-acether induced bronchospasm on the lipoxygenase pathway in the guinea-pig. Prostaglandins 1983; 2(:457-66. 29. Jancar S, Theriault P, Provencal B, Cloutier S, Sirois P. Mechanism of action of plateletactivating factor on guinea-pig lung parenchymal strips. Can J Physiol Pharmacol1988; 66: 1187- 91. 30. Lewis AJ, Dervinis A, Cheng J. The effects of antiallergic and bronchodilator drugs on plateletactivating factor (PAF-acether) induced bronchospasm and platelet aggregation. Agents Actions 1984; 15:636-42. 31. Moodley J, Stuttle A. Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leukocytes. Prostaglandins 1987; 33:253-64. 32. Lefort J, Rotilio 0, Vargaftig BB. The platelet independent release of thromboxane A 1 by PAFaceter from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br J Pharmacol 1984; 82:565-75. 33. Pretolani M, Lefort J, Vargaftig BB. Limited interference of specific PAF antagonists with hyperresponsiveness to PAF itself of lungs from actively sensitized guinea-pigs. Br J Pharmacol 1989; 97:433-42. 34. Pretolani M, Lefort J, Vargaftig BB. Active sensitization induces lung hyperresponsiveness in the guinea pigs: Pharmacological modulation and triggering role of the booster injection. Am Rev Respir Dis 1988; 138:1572-78.
