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FIGURE 2. Serial anastomotic bronchialblood How (QBr) in control and experimental dogs. Arrow indicates times of delivery of PGF.. aerosol. Toppanel indicates peak airwaypressure. In the 3 animals with bilateral cervical vagotomy, there was a tendency for a slight increase in bronchial blood flow, although this did not reach statistical significance. Since it is not very clear whether this effect on QBr was due to the bronchoconstriction or to a direct effect of methacholine on the bronchial vasculature, we used prostaglandin F 2> which is known to be a potent bronchoconstrictor. Although a potent pulmonary vasoconstrictor, it does not appreciably change systemic and thus presumably bronchial artery tone. One hundred nanograms of PG F...dissolved in 10 ml saline solution was nebulized into the LLL airway (Fig 2). In 5 additional dogs (labeled "controls" in Fig 2), 10 ml of saline solution was nebulized to the LLL airway using the same compressed air-driven nebulizer. Bronchial blood flow measured by the overflow method and LLL peak airway pressure was constant fur the initial 3O-minute control period. When PGF... was aerosolized into the LLL, peak airway pressure always increased significantly and remained elevated for the duration of the experiment. Anastomotic bronchial blood flow increased promptly and this increase is statistically significant at p < 0.05 level. At 45 minutes, bronchial blood flow was still significantly elevated compared to control. In the control dogs, where 10 ml of saline solution was nebulized into the LLL airway, bronchial blood flow did not change appreciably over the duration of75-90 minutes. Thus, we conclude that two bronchoconstrictor aerosols, methacholine and PG F..., both increase bronchial blood flow. However, since PGF..., unlike methacholine, has no effect on systemic arteries, we believe that it is bronchoconstriction rather than a direct action of PG F... on the artery that increases anastomotic bronchial blood flow. The response that the bronchial blood flow to bronchial constriction is in 1848

1 McLaughlin RF. Bronchial artery distribution in variousmammals and in humans. Am Rev Respir Dis 1983;128:557-8 2 Martinez L, de LetonaJ, Castro de la MataR. Aviado OM. Local and reflex effects of bronchial arterial injection of drugs. J Pharmacal Exp Ther 1961; 133:295-303 3 Arrnendia P, Martinez L. de Letona J, Aviado OM. Responses of the bronchial veins in a heart-lung-bronchial preparation. Circ Res 1962; 10:3-10 4 Jindal SK. Lakshminarayan S. KirkW, Butler J. Acuteincrease in anastomotic bronchialblood How after pulmonaryartery obstruction. J Appl Physioll984; 57:424-28 5 Baile EM. Albert RK. Kirk w Lakshminarayan S, Wiggs BJR. Pare PD. Positive end-expiratory pressure decreases bronchial blood How in the dog. J Appl Physiol 1984; 56(5):1289-93

The Rabbit Model of the Late Asthmatic Response* Gary L. Larsen. M.D. t

Asthma is a common chronic disease characterized by reversible obstruction of the airways. In patients with asthma who develop symptoms after allergen exposure, different temporal patterns of obstruction are recognized, including an immediate asthmatic response (IAR) which occurs 10-30 minutes after allergen exposure and represents an IgE-mediated reaction to allergen, and a late asthmatic response (LAR) occurring 2-12 hours after allergen exposure. Although the LAR was first described over a century ago, it was Herxheimer' in 1952 who first pointed out that the LAR was seen in patients with more severe asthma. Subsequently, several other studies came to the same conclusion based on the fullowing observations. First, LARs resemble chronic, severe bronchial asthma where steroids are required to decrease or abolish symptoms and abnormalities in pulmonary physiology. 2.3 Second, Warner' observed a significant correlation between frequent attacks of asthma and the presence of LARs in children challenged with house dust mite. Third, Cockcroft and associates' reported that allergen-induced LARs are associated with an increase in bronchial responsiveness to histamine and methacholine, while subjects with only lARs after inhalation challenge do not exhibit this heightened responsiveness. More recently, these observations have been extended by Cartier and eo-workers," who demonstrated the increased responsiveness after LARs was not due

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·From the Department of Pediatrics, National Jewish Hospital! National AsthmaCenter. and Department of Pediatrics, School of Medicine.UniversityofColoradoHealth SciencesCenter. Denver. t Associate Professor of Pediatrics. Supported by grants HL-27063 and HL-31376 from the National Heart, Lung and BloodInstitute. Reprint requests: Dr. Larsen. Department ofPediatrics, 3800 East

Colfax, Denver 80206

27lh Amu8J Aspen Lung ConfenInce

to a reduction in baseline airways caliber alone. Work from the same laboratory by Boulet and associates' found natural exposure to ragweed in ragweed-sensitive patients with asthma led to seasonal increases in methacholine responsiveness that were limited to a group with dual asthmatic responses. Thus, a clearer understanding of the pathogenesis of LARs might give insight into mechanisms of disease present in patients with severe or frequent asthma. The pathogenesis of LARs has been unclear with both IgEmediated as well as non- IgE mediated mechanisms proposed to explain these reactions.f" Early observations on pathogenesis by Pepys and eo-workers" in patients with allergic bronchopulmonary aspergillosis who had late reactions in both skin and lungs led to the conclusion these late responses were Arthus reactions (type 3) based on deposition of IgG, IgM, and C3 in biopsies of the cutaneous reactions. Subsequent observations regarding the late cutaneous response (LCR) have suggested many of these reactions are not type 3 events. For example, Dolovich and Little'' showed that LCRs occurred after injection of Bacdlu« subtilis enzymes, and that the late reaction did not appear to require the participation of antibody isotypes other than IgE. Subsequently, Dolovich and eo-workers" demonstrated that LCRs also occurred after injection of anti-IgE, demonstrating that complement activation was not essential for the response to occur. The hypothesis that LCRs could be mediated by IgE related mechanisms was further strengthened by Solley and eo-workers" in passive transfer experiments, where heating of the passively transferred serum for 4 hours at 56°C to denature IgE led to ablation of the capacity to transfer both the immediate and late cutaneous responses. These investigators also found that removal of IgE by passage of serum over an anti-IgE immunosorbent also abolished the ability to passively transfer the reaction, while IgE recovered from the immunosorbent restored the response when passively transferred. From the work of Oertel and Kaliner," an inflammatory factor from rat peritoneal mast cell granules has been identified that upon injection into rat skin produces the same lesions produced by either injection of anti-IgE or compound 48/80. Thus, in terms of late reactions to antigen in the skin, experimental evidence strongly suggests that IgE and the mast cell are important in the production of this response. The advancement in our understanding of LARs during the past decade has not been as marked as our progress regarding LCRs. However; several observations have suggested that the responses in the lung, as in the skin, are not all Arthus reactions. For example, pollen antigens such as ragweed, which may not cause production oflarge quantities of IgG antibody, can provoke LARs. 8 In addition, cromolyn sodium inhalation prior to antigen inhalation will prevent both lARs and LARs, thus raising questions about the role of mast cells in the pathogenesis of LARs. 2 Neutrophil chemotactic activity, an activity that is thought to be mast-cell associated," has been recently demonstrated by Nagy and eo-workers" to appear in 2 peaks in sera of patients undergoing allergen bronchial provocation. The peaks in the activity coincided with early and late allergen-induced asthmatic reactions, suggesting that mast cells may participate in both responses. The effects of steroids on LARs parallels that seen with LCRs in that the immediate reaction is not altered, while the late response is abolished, suggesting that the skin

and lung may have more in common in terms of mechanism than just the temporal development of the reactions.' 7 The rabbit model of the LAR was developed to address the pathogenesis of this disorder. Studies from separate laboratories had shown that rabbits produce a homocytotropic antibody analogous to human IgE.l8.19 From the work of Pinkard and co-workers, 20 we knew if rabbits were immunized within the first 24 hours of life, many would produce only IgE antibody to the antigen, while rabbits immunized beginning at 7 days of age would produce several antibody isotypes to the antigen. In our initial study," rabbits were immunized with Alternaria tenuis, a common antigen found to induce LARs in our population of chronic, severe asthmatic subjects (Shampain, unpublished observations). Subsequently, ragweed antigen has been used as the sensitizing antigen with similar results." The immunization schedule is outlined in Table I. Antibody status of immunized rabbits was assessed at 3 months of age. Homocytotropic antibody (IgE) was measured by homologous passive cutaneous anaphylaxis (PCA) after a latent period of 3 days, while rabbit antigen-specific IgG was defined by heterologous PCA in guinea pigs as well as by precipitation assay." Once the antibody status was defined, antigen bronchial provocation challenges were performed in intubated rabbits who were awake and spontaneously breathing. Assessment oflung function was obtained by measuring airflow at the mouth with a pneumotachograph, and transpulmonary pressure employing an esophageal balloon. The pressure and flow signals were sent to an analog computer (Hewlett-Packard Respiratory Analyzer Model 8816A) which integrated flow to tidal volume and computed dynamic compliance (Cdyn) by the technique of Von Neergaard and WilT' and total pulmonary resistance (RL) by the method of electrical subtraction of Mead and Whittenberger. "" As described in detail in the study by Shampain et al," biphasic alterations in lung function (RL, Cdyn) were noted with an early phase increase in RL and decrease in Cdyn, beginning within 15 minutes of challenge and lasting through approximately 30 minutes. A later phase of increased RL and decreased Cdyn began at approximately 120 minutes and persisted through the remainder of the 6 hour study. Control rabbits, ie, nonimmunized rabbits challenged with saline or A tenuis or rabbits immunized with bovine serum albumin but challenged with saline or Alternaria, did not exhibit significant alterations in lung function. Rabbits with multiple antibody isotypes also had early and Table I-Immunization Schedule cfNew Zealand White Rabbm for Preferential Production if Homocytotropic (lgE) Antibody Age, days

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-Antigen extract = Altemar1a tenuiB or ragweed diluted 1:20weight to volume; aluminum hydroxide = 10 mglml suspended in normal saline. tIP = intraperitoneally; SC = subcutaneously. CHEST I 87 I 5 I MAY. 1985 I SUpplement

185S

late phase changes in RL and Cdyn that were of less magnitude than those seen in rabbits with IgE only. The above observations showed that rabbits with only IgE antibody to an antigen can have not only an immediate but also a late asthmatic response to antigen challenge. This would argue against this response being an Arthus phenomenon. Indeed, the observation that the presence of antigen-specific IgG led to decreased pulmonary responses suggested the IgG might be protecting against this response. To investigate this in more detail, additional experiments were conducted." Rabbits who were litter mates with homologous PCA titers of 1:8 were directly compared to rabbits with higher titers of IgE but also detectable IgG. Despite having lower IgE titers, animals with only IgE had more marked responses than rabbits with IgE and IgG. Passive transfusion of "IgE" rabbits with serum containing IgG against Alternaria blunted the response of the animals to challenge, again demonstrating that IgG inhibited and did not enhance the reaction in this model. To determine if the LAR in this model could occur in the absence of cellular immune mechanisms associated with immunization, nonimmunized rabbits were transfused with plasma containing IgE antibodies to A tenuis followed by bronchial challenge to the antigen." Compared with matched control rabbits transfused with nonimmune plasma, rabbits receiving antigen-specific plasmas showed biphasic early and late phase changes in RL and Cdyn similar to actively immunized rabbits. Animals sensitized by passive transfer of A tenuis immune sera have also been employed to study the role of antigenspecific IgE and IgG in producing the late response......7 Again, one goal was to study these humoral factors in a system where cellular immune mechanisms associated with immunization were absent. Groups of recipient rabbits were categorized according to the immune status of the transfused serum: controls were transfused with normal serum while the remainder received Alternaria immune serum that contained either antigen-specific IgE or both antigen-specific IgE and IgG. Another group was designated "G;' and received heat-treated serum (to minimize IgE) that still 220 200 180 160 Q) 140 c 120 100 Q) rn 80 as 60

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contained high titers of antigen-specific IgG (1:2048). Assessment of lung function after the challenge with A tenuis showed the largest responses were in the group that received sera with antigen-specific IgE alone. The group with both antigen-specific IgE and IgG showed blunting of the LAR in a dose-dependent fashion with increasing titers of IgG antibody. The animals receiving "G;' like controls, showed no IAR or LAR.1& Subsequent immunofluorescent studies in selected animals from each group failed to show any granular deposition of immunoglobulin or C3 in either the airways or blood vessels of the lung.F'Ihus, just as in animals sensitized by immunization, passively sensitized rabbits also demonstrated a decrease in the physiologic abnormalities when antigen-specific IgG was present. Other observations that have been made with the animal model relate to the effects of various classes of drugs on the IAR and LAR. In a study cited previously," the adrenergic drug isoproterenol was given to 9 rabbits for 5 minutes at the end of the 6-hour study. A dose of 0.5 J.l.g!kglmin did not reverse alterations in RLand Cdyn (Fig 1). Other adrenergic agents which have been employed to reverse the airways obstruction have included inhaled 1%isoetharine HCl as well as epinephrine given IV (Behrens and Marsh, unpublished observations). Again, neither drug caused significant reversal of airways obstruction when the LAR had developed. Similar observations have been made in clinical studies.v' Twoother classes of drugs have been studied to determine their effect on the LAR. Pretreatment of animals before antigen challenge with disodium cromoglycate has been noted to prevent both the IAR and LAR, while pretreatment with placebo prevented neither. U A similar effect in humans has been noted...lIS On the other hand, IV steroids given before antigen challenge of sensitized rabbits did not prevent an IAR, but ablated the LAR. Intravenous saline (placebo) did not prevent either. uThe same effect has again been noted in patients with LARs."lIS Thus, in terms of the response in the animals compared to the response in humans, the effects are similar. What remains unanswered is how these medications produce these changes in response. The future ability to assess mediator release as well as monitor the pathologic events in

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FIGURE 1. The response of 9 immunized rabbits to challenge with Alternaria ten"ia is shown as a percent change from baseline in pulmonary resistance and dynamic compliance. Six hours after challenge, IV infusion of isoproterenol did not reverse these lung function abnormalities.

the airways should help evaluate basic mechanisms of action of these drugs in this animal model. The histopathology of the immediate and late asthmatic and cutaneous responses in this rabbit model are areas of ongoing study by Behrens and co-workers. ".30 As summarized by Behrens et al30 in a previous presentation, while there are good descriptions of the histology of immediate and late cutaneous responses (ICR and LCR, respectively) in humans, there is no description of an evolving IAR and LAR in either human or animal studies. In the rabbit, we have reported that both the ICR and IAR are characterized by interstitial edema and vessel dilatation, while the LCR and LAR showed a mixed cellular lnflltration." In the lungs, the cellular infiltrate is located about airways as well as perivascularly. These cutaneous findings in the rabbit are similar to those described in human cutaneous responses, a.13 and thus raise the possibility that the pathology in the airways in the rabbit may be similar to that found in humans with asthma. The last area of study employing the rabbit model of LARs that will be reviewed deals with airway reactivity. As noted above, studies from Hargreaves laboratory'" have associated increases in nonspecific airways reactivity with the LAR and not the 1AR.Marsh and others" in our laboratory have looked at airways reactivity to histamine 3 days before and 3 days after ragweed challenge in two groups of rabbits: a control group transfused with nonimmune serum, and an immunized group who received serum containing ragweed-specific IgE. While the control group had no LAR after antigen challenge, and did not alter airway reactivity, the sensitized animals all developed LARs and had increases in airways reactivity that were marked in most instances. In addition, pulmonary inflammation as assessed by analysis ofcells obtained by bronchoalveolar lavage was significantly greater in the sensitized animals. The temporal association of the alterations in reactivity with the alterations in cells in lavage suggested the inflammatory process may contribute to the increases in reactivity by mechanisms remaining to be explained. Other features of the LAR in this rabbit model will be studied in the future. A more detailed assessment of pulmonary physiology including measure of pressure-volume curves will be employed to address functions of small vs large airways during the IAR and LAR. Correlation of structural (pathology) with functional (Physiology) changes in the same animal may give important infOrmationregarding the mechanisms involved in the development ofairways obstruction. In addition, the availability of an animal model of LARs should allow fur a systematic evaluation of mediators that may contribute to the late asthmatic response. If; as suggested by the above observations, the LAR is an inflammatory event, we can expect the process to be complex and influenced by many separate mediators or systems of mediators. 31,33 ACKNOWLEDGMENT: I wish to thank Georgia Wheeler for her assistance in pre~g this manuscript and B. Lyn Behrens and William R. Marsh for their review of the manuscript.

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REFERENCES 1 Herxheimer H. The late bronchial reaction in induced asthma. Int Arch Allergy Appl Immunol 1952; 3:323-8 2 Booij-Noord H, Orie NGM, deVries K. Immediate and late bronchial obstructive reactions to inhaIation of house dust and

22

protective effects of disodium cromoglycate and prednisolone. J Allergy Clin Immunol 1971; 48:344-54 Hargreave FE, Dolovich J, Robertson DG, Kerigan AT. The late asthmatic responses. Can Med Assoc J 1974; 110:415-21 Warner Jo. Significance of late reactions after bronchial challenge with house dust mite. Arch Dis Child 1976; 51:905-11 Cockcroft D\v, Ruffin RE, Dolovich J, Hargreave FE. Allergeninduced increase in non-allergic bronchial reactivity. Clin Allergy 1977; 7:503-13 Cartier A, Thomson NC, Frith PA, Roberts R, Hargreave FE. Allergen-induced increase in bronchial responsiveness to histamine: relationship to the late asthmatic response and change in airway caliber. J Allergy Clin Immunol 1982; 70:170-7 Boulet LP, Cartier A, Thomson NC, Roberts RS, Dolovich J, Hargreave FE. Asthma and increases in nonallergic bronchial responsiveness from seasonal pollen exposure. J Allergy Clin Immunol 1983; 71:399-406 Robertson DG, Keugan Al: Hargreave FE, Chalmers R, Dolovich J. Late asthmatic responses induced by ragweed allergen. J Allergy Clin Imunol 1974; 54:244-54 Pepys J. Immunopathology of allergic lung disease. Clin Allergy 1973; 3:1-22 Pepys J, Turner-Warwick M, Dawson PL, Hinson KF\V, Arthus (type III) skin test reactions in man: clinical and immunopathological features. In: Rose B, et ai, eds. Allergology: Pr0ceedings of the sixth congress of the international association of allergology. Amsterdam: Excerpta Medica Foundation, 1968; 221-35 Dolovich J, Little DC. Correlates of skin test reactions to Bacillus subtilis enzyme preparations. J Allergy Clin Immunol 1972; 49: 43-53 Dolovich J, Hargreave FE, Chalmers R, Shier KJ, Gauldie J, Bienenstock J. Late cutaneous allergic responses in isolated 19Edependent reactions. J Allergy Clin Immunol 1973; 52:38-46 Solley GO, Gleich GJ, Jordan RE, Schroeter AL. The late phase of the immediate wheal and Bare skin reaction: its dependence upon 19E antibodies. J Clin Invest 1976; 58:408-20 Oertel HL, Kaliner M. The biologic activity of mast cell granules: III. Purification of inflammatory factors of anaphylaxis (IF-A) responsible for causing late-phase responses. J Immunol 1981; 127:1398-402 Schenkel E, Atkins PC, Yost R, Zweiman B. Antigen-induced neutrophil chemotactic activity from sensitized lung. J Allergy Clin Immunol 1982; 70:321-5 Nagy L, Lee TH, Kay AB. Neutrophil chemotactic activity in antigen-induced late asthmatic reactions. N Engl J Med 1982; 306:497-501 Gleich GJ. The late phase of the immunoglobulin E-mediated reaction: a link between anaphylaxis and common allergic disease? J Allergy Clin Immunol 1982; 70:160-9 ZvaiHer NJ, Robinson JO. Rabbit homocytotropic antibody. A unique rabbit immunoglobulin analogous to human 19E. J Exp Med 1969; 130:907-29 Ishizaka K, Ishizaka'I; Hornbrook MM. A unique rabbit immunoglobulin having homocytotropic antibody activity. Immunochemistry 1970; 7:515-28 Pinckard RN, Halonen M, Meng AL. Preferential expression of anti-bovine serum albumin 19E homocytotropic antibody synthesis and anaphylactic sensitivity in the neonatal rabbit. J Allergy Clin Immunol 1972; 49:301-10 Shampain Mp, Behrens BL, Larsen GL, Henson PM. An animal model of late pulmonary responses to Alternaria challenge. Am Rev Respir Dis 1982; 126:493-8 Larsen GL, Shampain Mp, Marsh WR, Behrens BL. An animal model of the late asthmatic response to antigen challenge. In: Kay AB, Austen KF, Lichtenstein LM, eds. Asthma: Immunology, Immunopharmacology and 'Ueatment. London: Academic CHEST I 87 I 5 I MAY. 1985 I Supplement

1178

Press, 1984; 245-62 23 Halonen M, Fisher HK, Blair C, Batter C, Pinckard RN. IgEinduced respiratory and circulatory changes during systemic anaphylaxis in the rabbit. Am Rev Respir Dis 1976; 114:961-70 24 Von Neergaard K, Win K. Uber eine methode zur messung der lungenel astizitat am lebenden menschen, inbesondere beim ernphysim. Z Klin Med 1927; 105:35-50 25 Mead J, Whittenberger JL. Physical properties of human lungs measured during spontaneous respiration. J Appl Physioll953; 5:779-96 26 Behrens BL, Marsh WR, Henson PM, Larsen GL. Passive transfer of the late pulmonary response in an animal modelrelationship of immunologic status to pulmonary physiologic changes [Abstract). Am Rev Respir Dis 1983; 127(2):65 27 Behrens BL, Clark RAF, Marsh WR, Larsen GL. Modulation of the late asthmatic response by antigen specific IgG in an animal model. Am Rev Respir Dis (in press) 28 Pepys J, Hutchcroft BJ. Bronchial provocation tests in the etiologic diagnosis and analysis of asthma. Am Rev Respir Dis 1975; 112:829-59 29 Behrens BL, Clark RAF, Feldsien DC, Presley DM, Glezen LS, Graves JP, et al. Histopathology of the immediate and late asthmatic response in an animal model [Abstract). Am Rev Respir Dis 1984; 129:4 30 Behrens BL, Clark RAF, Feldsien DC, Presley DM, Glezen LS, Graves JP, et al. Comparison of the histopathology of the immediate and late asthmatic and cutaneous responses in an animal model. Chest 1985; 87:153S-55S 31 Marsh WR, Irvin CG, Behrens BL, Larsen GL. Pulmonary inHammation and changes in airways reactivity after late asthmatic responses in an animal model [Abstract). Am Rev Respir Dis 1984; 129:4 32 Larsen GL, Parrish DA, Henson PM. Lung defense: the paradox of inHammation. Chest 1983; 83(suppl):1-5 33 Larsen GL, Henson PM. Mediators of inHammation. Ann Rev Immunol 1983; 1:335-59

Immunologic Challenge and Epithelial Ion Transport in Canine Trachea* Stephen c. Lazarus, M.D.; George D. Leikauf, Ph.D.; Laurence J McCabe, AB.; Keith Paige; K. Fan Chung, M.D.; }ayA Nadel, M.D.; andWamm M. Gold, M.D.

C

PiOUS tracheobronchial secretions are an important concomitant of asthma and other inflammatory pulmonary diseases. These secretions are a complex mixture of ions, water, and high molecular weight glycoproteins. Antigen challenge! and a number of mast cell-derived mediators'" have been shown to increase glycoprotein secretion from sensitized human airways in vitro. Antigen also increases glycoprotein secretion and produces large changes in ion flux in the tracheas of allergic sheep.' ·From the Cardiovascular Research Institute and Department of Medicine, University of CalifOrnia, San Francisco. Supported in part by National Heart, Lung, and Blood Institute Program Project Grant No. HL-24136 and HL-29877, and grants from the Council for Tobacco Research- V . S.A., Inc., the California Research and Medical Education Fund of the American Lung Association of CalifOrnia, and the Research Evaluation and Allocation Committee, University of CalifOrnia, San Francisco, School of Medicine. Reprint requests: Dr: Lazarus, Cardiovascular Research Institute, I315-MC?fJitt Hospital, UCSF, San Francisco 94143

1888

Specific antigen binding to IgE on the surface of mast cells can stimulate these cells to secrete a number of preformed and newly formed mediators. Recent studies suggest that many nonimmunologic stimuli can also cause mast cell secretion. We believe that the mast cell is of major importance in the regulation of respiratory tract secretions. These studies were designed to examine the interaction between mast cell mediators and ion and water transport across canine tracheal epithelium. We describe two preparations from which mast cell mediators were generated. One involved the use of chopped lung parenchyma and has been well described in the literature by many investigators. This method is useful both to examine the feasibility of mast cell-target cell interactions and to provide a comparison fur studies of isolated mast cells. We also describe an isolated mast cell preparation which we have developed from the canine mastocytoma. We have successfully propagated these cells in nude mice and can easily obtain large numbers of nearly pure mast cells which are morphologically and functionally identical. METHODS

Samples of canine lung were dissected free of pleura and bronchi and minced into fragments (3 x 3 x 3 mm), These fragments were sensitized passively by incubating for 90 minutes at 37"C with serum containing a high titer of dog anti-ragweed IgE and then challenged with ragweed antigen. Aliquots of mediators released by this challenge were assayed for histamine or transferred to Ussing chambers. Pieces of the membranous portion of dog trachea, freed of muscle, were mounted in Ussing chambers (1.33 em' surface area). Short-circuit current was monitored continuously, and potential difference was measured every 10 minutes." To examine the effect of cyclooxygenase blockade in the epithelium, indomethacin (IO-6M) was added to the medium, bathing both the submucosal and luminal surfaces, and the short-circuit current was allowed to stabilize before aliquots of mediators were added. Mastocytoma cells from cutaneous lesions of dogs were characterized as described previously," Suspensions of physically dispersed cells were transferred to subcutaneous sites in nude mice. The nodules which developed were excised and disaggregated, and cells were used for morphologic and functional studies, or were passaged to a subsequent generation of mice. RESULTS

Neither ragweed antigen alone nor supernatant from lung fragments incubated without ragweed (spontaneous release) produced a significant change in short-circuit current. The mediators released by ragweed challenge of lung fragments produced an increase in short-circuit current, the magnitude of which was dependent upon the concentration of antigen used. Ragweed antigen, 50 fJ.g x ml", produced a maximum response, and the mediators released increased short-circuit current from 39.7±3.0 to 58.9±4.5 fJ.A mps x cm " (mean ± SE; n = 19; p