Lymphokine-induced Airway Hyperresponsiveness in the Rat1- 3
PAOLO M. RENZI, SANTO SAPIENZA, TAO OU, NAI SAN WANG, and JAMES G. MARTIN Introduction
Asthma is a chronic disease characterized by reversible airway obstruction and airway hyperresponsiveness to a variety of physical and chemical stimuli (1, 2). Recent investigations of the mechanisms of hyperresponsiveness after acute airway insults such as allergen provocation and viral infections suggest an important pathogenetic role for inflammation (3, 4). Despite its probable importance in causing sustained increases in airway responsiveness, chronic inflammation such as is seen in the airways of asthmatic patients has received relatively little attention. One of the key cells in chronic inflammation is the lymphocyte, and therefore it is reasonable to hypothesize that it may be involved, directly or indirectly, in airway hyperresponsiveness. A number of lines of evidence, albeit circumstantial, suggest that the lymphocyte may be important in altered airway responsiveness. First, the lymphocyte is present and increased in the inflammatory infiltrate in the airways of patients both with severe fatal asthma (5) and with less severe chronic asthma (3, 6, 7). Second, the lymphocyte plays a role in the regulation of atopy (8) and in the defense against viral infections (4), two conditions known to be associated with airway hyperresponsiveness (1-4). Third, lymphocytes in vitro produce lymphokines capable of activating or inducing the proliferation of eosinophils (9-11), basophils (11-13), neutrophils (14, 15), and macrophages (16), all potential participants in asthma. To explore the potential role of the lymphocyte in airway responsiveness we examined the effects of interleukin-2 (IL-2), a major growth factor for T lymphocytes, on the response of the rat to inhaled aerosolized methacholine (MCh). We report the effects of subcutaneous administration of IL-2 for 4.5 days on airway responsiveness to MCh, lung histology, and lung lavage. Methods Preparation of Animals and Measurement of Pulmonary Mechanics Eighteen pathogen-free male Lewisrats (mean
SUMMARY We evaluated the potential role of the lymphocyte in chronic airway inflammation and responsiveness by repeated administration to rats of interleukln-2 (IL-2), the principallymphoklne responsible for lymphocyte proliferation. Lewis rats (mean weight, 184 ± 2 g) received either 120,000 10) or vehicle (n 7) subcutaneously twice a day for 4.5 days. Animals were units of IL-2 (n anesthetized with urethane and intubated for measurements of pulmonary resistance (RL) and airway responsiveness to aerosol methacholine (MCh). Lung lavage was performed, the animals were exsanguinated, and the lungs were fixed in 10% formalin. Histologic edema and the extent of infiltration of the bronchi, pulmonary veins, and arteries by cells was scored blindly. IL-2 increased airway responsiveness to MCh; the concentrations of MCh causing a doubling of RL were 0.14versus 1.39 mg/ml (geometric mean) for the IL-2 and vehicle group, respectively (p 0.001). IL-2 significantly increased total cellular return and the percentage of lymphocytes, neutrophlls, and eosinophils in lavage. IL-2 caused edema and a mixed cellular infiltration of the bronchovascular tree. Lymphocytes predominated around the airways and veins. A correlation (r 0.50) was present between airway responsiveness and airway inflammation but not with edema or vascular infiltration. Release of IL-2 by lymphocytes in the airways may be an important mediator of airway hyperresponslveness.
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AM REV RESPIR DIS 1991; 143:375-379
weight, 184 ± 2 g) were randomized to receive either recombinant human IL-2 (generously provided by the Glaxo Institute of Molecular Biology, Switzerland) or vehicle (8 nglml of Escherichiacoli endotoxin in 50 mM acetic acid). Eight rats received 120,000units of IL-2, and eight rats received the vehicle subcutaneously twice daily in 150/0 porcine gelatin for 4.5 days. Two rats received IL-2 without gelatin, and although histologic changes and airway responsiveness weresimilar to those obtained with IL-2 in gelatin, these animals were included only in the analysis of histological changes and airway responsiveness. One vehicle-treated animal was excluded from analysis because of pneumonia. Pilot experiments were performed to determine the optimal amount of IL-2 that could be administered to rats for 4.5 days. One male Lewisrat received480,000units of IL-2 in 150/0 porcine gelatin subcutaneously twice a day and died on the third day. Two male Lewis rats received240,000units of IL-2 in 15% gelatin subcutaneously twice a day and died at 4.5 days prior to the measurement of pulmonary mechanics. All three rats lost weight and showed severe mononuclear cell infiltration of the lungs on histologic examination. A dose of 120,000 units of IL-2 administered subcutaneously in 150/0 gelatin twice a day was chosen because this dose did not affect the behavior of the rats, nor did it impede their normal weight gain. Within 30 min of the last dose of IL-2 or vehicle, anesthesia was induced with urethane (0.9 to 1.1 g/kg, intraperitoneally). Blind endotracheal intubation was then performed
using a 6-cm length of PE-240 polyethylene catheter. A heating pad was used to maintain body temperature constant. Pulmonary resistance was measured during spontaneous tidal breathing with the animals in the left lateral decubitus position. Flow was measured by placing the tip of the tracheal tube inside a small Plexiglas's box (265 ml in volume) to the end of which a Fleisch no. 0 pneumotachograph coupled to a differential transducer (MP-45, ± 2 em H 2 0 ; Validyne Corp., Northridge, CA) was attached. Volume was obtained by electrical integration of the flow signal using a respiratory inteftrator (HP8815A; Hewlett-Packard, Wal.tham, MA). Pulmonary resistance (RL) was obtained by the electrical subtraction .technique of Mead and Whittenberger (17). Endotracheal tube resistance was 0.11 em H 20/ml/s at the flow of 25 mIls. Tube resistance was subtracted from all values of RL.
(Received in original form January 10, 1990 and in revised form June 27, 1990) 1 From the Meakins-Christie Laboratories and the Department of Pathology, Royal Victoria Hospital, McGill University, and the Pulmonary Service,St. Luc Hospital, Universityof Montreal, Montreal, Quebec, Canada. 2 Supported by Grant No. MA-I0637 from the Medical Research Council of Canada. 3 Correspondence 'and requests for reprints should be addressed to Dr. P. M. Renzi, MeakinsChristie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2.
375
RENZI, SAPIENZA, DU, WANG, AND MARTIN
376
Tidal volume, breathing frequency, and minute ventilation were also calculated.
Methacholine Challenge Aerosols were generated using a Hudson nebulizer containing 3 ml of solution, which was driven by a compressed air source at an airflow of 7.5 L/min. The nebulizer output was 0.18 ml/min. The nebulizer was connected to one side port of the Plexiglas box, and during aerosolization, airflow was diverted through a second side port. To assess airway responsiveness to MCh, rats inhaled aerosols of normal saline and progressively doubling concentrations of MCh in normal saline for 30 s. MCh concentrations ranged from 0.063 to 16 mg/rnl, The peak value of RLwas measured before and after inhalation of saline and after each of the concentrations ofMCh. An interval of approximately 3 min elapsed between the administration of each concentration of MCh. The concentration of MCh required to double RL(EC2ooRL)was obtained by linear interpolation between the two concentrations bounding the point at which RL reached 200070 of control.
Lung Lavage At the end of the experiment, lung lavage was performed with 25 ml of saline at room temperature by instillation and gentle aspiration of 5-ml aliquots through the endotracheal tube. Total cell count was determined using a hemacytometer. Differential cell counts were assessed on cytologicpreparations. Slideswere prepared using a Cytospin (Model Cytospin II; Shandon, Pittsburgh, PAl and stained with Wright-Giemsa. Two hundred cells were examined by light microscopy.
Total White Blood Cell Determination and Assessment of Lytic Activity After lung lavage, the animals were killed by exsanguination, blood was retrieved in heparinized tubes, and the total white blood cell count was determined in a Coulter counter (Coulter Electronics, Hialeah, FL). Lytic activity was measured by a modified 4-h chromium-51 release assay (18). Tumor target cells were from the YAC-l mouse lymphoma cell line and the K562 human chronic myelogenous leukemia cell line obtained from American Type Culture Collection (Rockville, MD). These cells were mycoplasma-free and maintained as suspension cultures in tissue culture medium (TCM) with 10070 heat inactivated fetal calf serum (HIFCS). The tumor cells were radiolabeled by incubation with 50 J,lCi of (5tCr]sodium chromate (New England Nuclear, Boston, MA) for 2 h at 37° C in humidified air plus 5070 CO 2 washed twice in phosphate-buffered saline, once in RPMI 1640, and resuspended in TCM plus 20070 HIFCS at a concentration of lOS cells/ml. One hundred microliters of this cell suspension containing lQ4 target cells were placed in each microwell of a round-bottom microtiter plate (Flow Laboratories, McLean, VA). Peripheral blood effector cells were obtained by centrifugation (450 g for 40 min
Statistical Analysis
at 22° C) of the buffy coat of rat blood over a Ficoll-Hypaque density gradient (Organon Teknika, Durham, NC), washed twice in phosphate-buffered saline (pH 7.3), and resuspended in TCM. Blood effector cells were added in quadruplicate to the target cells at four different effector-to-target-cell ratios, 80:1, 40:1, 20:1, and 10:1. Target cells incubated in TCM without effector cells were used as controls for spontaneous stCr release during the assay. Spontaneous release averaged 8 ± 1.2070 for all experiments. After a 4-h incubation at 37° C in humidified air plus 5070 CO 2 , the plates were centrifuged at 450 g for 5 min, 100 ul of the supernatant were carefully removed from each well, and the radioactivity was determined using a gamma counter. Lytic activity of blood effector cells was determined as percent specific release calculated as [(ER - CR/T - CR) x 100], where ER = StCrreleased by experimental cells, CR = StCr released spontaneously, and T = total StCr. Standard errors were less than 1070.
Group results are expressed as mean ± SE except for values of EC 2ooRL, which are reported as geometric means. The significance of differences was evaluated using unpaired t tests or the Mann-Whitney U test for pathology scores. Correlation between histology scores and airway responsiveness was examined using Spearman's rank test. Significance was considered to be established with a p value < 0.05.
Results
Effect of IL-2 on Airway Responsiveness to Methacholine After general anesthesia and endotracheal intubation there was no significant difference between the baseline RL, respiratory frequency, tidal volume, and minute ventilation of the IL-2 and vehicle-treated animals (table 1). IL-2 increased airway responsiveness (figure 1). The concentration of MCh necessary to cause a doubling of lung resistance (EC 2ooRL) was much less in the IL-2 treated than in the vehicle-treated animals (0.137 mg/ml versus 1.39 mg/ml; p = 0.001).
Pathologic Assessment of the Lungs After exsanguination of the rats, the lungs were fixed in 10070 formalin at a pressure of 25 em H 2 0 for 48 h. Slides from paraffinembedded sections of the lungs were stained with hematoxylin-eosin. Using a scoring system, the extent of the pathologic changes in the bronchi, veins, arteries, and alveoli were scored by two observers blinded to the group status of the specimens. Edema, cellular infiltration, and epithelial detachment were assessed. A score of zero was given if none of the specific structures (airways or vessels) examined on the slide had the changes looked for; 1+ ~ 25070; 2+ ~ 50070 but> 25070; 3 + > 50070 of the specific structures had the changes. There was good interindividual reproducibility of the scoring (r = 0.957).
Effects of IL-2 on Lung Histology The administration of IL-2 caused peribronchial and perivascular edema with a mixed cellular infiltration (figures 1 and 2). No overlap was found between the cellular infiltration scores of the IL-2and vehicle-treated animals. Although some overlap was noted for bronchovascular edema, the difference between the IL-2- and vehicle-treated groups was still significantly different (p < 0.02). IL-2
TABLE 1 EFFECT OF INTERLEUKIN-2 ON LUNG MECHANICS· Baseline Lung Resistance (em H20/mils)
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control animals was included in our anal ysis (r = 0.65, p < 0.05). No correlation was found between bronchial edema and airway responsiveness (r = 0.02) or between venous edema, cellular infiltration, and airway responsiveness. We also compared airway responsiveness of IL-2-treated animals with a pathology score ~ 1 to that of IL-2treated animals with a pathology score > 1. A significant difference in airway responsiveness was found only for cellular infiltration of the bronchi (p = 0.024).
Effect of IL-2 on the Cellular Composition of Lung Lavage The volume of lavage fluid retrieved averaged 830;0 of that instilled in IL-2-treated animals and 85% in vehicle-treated animals. IL-2 increased the total cellular return in lung lavage as well as the percentage of lymphocytes, neutrophils, eosinophils, and basophils (table 2). Eosinophils and basophils were found only in the lavage of IL-2-treated animals. Effect of IL-2 on Total White Blood Cells and Effector Cell Lytic Activity The administration of IL-2 caused a leukocytosis, with 9.6 ± 0.4 versus 4.2 ± 0.8 x 106 white blood cells/ml for IL-2treated versus vehicle-treated animals. The subcutaneous administration of IL-2 also increased the lytic activity of peripheral blood mononuclear cells against the YAC-I and K562 target cells (figure 4). Discussion
Fig. 2. Lung histology of IL-2 treated animals. An example of edema and cellula r infiltrat ion of the bronchi (A) and veins (8) of an IL-2-treated rat is shown . A predom inantly lymphocytic infiltration is present around the airway walls and the veins.
also appeared to increase epithelial detachment, but the difference was not significant compared with that in the control group. Little change was noted around the pulmonary arteries or in lung parenchyma. In pilot experiments, when higher doses of IL-2 were administered, the cellular infiltrate and edema weremore diffuse. The predominant cell around the airways and pulmonary veins was the lymphocyte, but macrophages, eosinophils,
neutrophils, and basophils were also present. When edema or the extent of cellular infiltration of the airways or pulmonary veins were compared with airway responsiveness to MCh, a weak correlation was found only between cellular infiltration of the bronchi and airway responsiveness (r = 0.50) (figure 3). This correlation became significant only when the scoring of cellular infiltration of the airways of
Wehave shown that the systemic administration of 120,000U of IL-2 to rats twice a day for 4.5 days caused a peribronchial and perivenous cellular infiltration with edema. Lung lavage contained an increased cellular return and an increase in the number of lymphocytes, neutrophils, eosinophils, and basophils retrieved. These changes were associated with an increase in airway responsiveness to the cholinergic bronchoconstrictor, methacholine. The histologic changes in the lungs that we observed are consistent with the results of others. In mice (19), the systemic administration of IL-2 causes a predominantly lymphocytic infiltration of the lungs, liver, spleen, and kidneys. In rats (20), IL-2 has been reported to cause a marked tissue infiltration with lympho cytes and eosinophils. In our preliminary experiments, higher doses of IL-2 caused diffuse infiltration of the bronchovascu-
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lar tree and the lung parenchyma. For the experiments reported, we chose a dose of IL-2 that affected predominantly the bronchovascular tree. This dose did not cause significant changes in lung resistance or minute ventilation, but it did increase airway responsiveness. When relationships between the histologic changes and airway responsiveness were examined, a correlation was found only with cellular infiltration of the bronchi. In previousexperiments,wehaveshown that an acute , high dose, intravenous administration of IL-2 also causes an increase in airway responsivenesswithin I h before any cellular infiltration occurs (21). The only significant pathologic change observed in the acutely treated animals was bronchial epithelial detachment. Histologic changes differed after chronic administration of IL-2; at the doses reported there was no significant bronchial epithelial detachment, where-
as edema and cellular infiltration of the bronchovascular tree were prominent (figures 1 and 2). The mechanism of the observed increase in airway responsiveness is not clear. Under the influence of IL-2, lymphocytes may have secreted lymphokines that affect smooth muscle directly, but evidence for the existence of such bronchoactive cytokines is lacking. An alternate explanation is that in vivo release of lymphokines from lymphocytes stimulated by IL-2 caused activation or proliferation of eosinophils, neutrophils, macrophages, and basophils (9-16, 2225), and that these cells, in turn, released one or more of several possible lipid- or granule-associated bronchoactive mediators (26-30). It is possible that structural changes in the airways rather than synthesis of bronchoactive substances may have contributed to the altered bronchial respon-
siveness. Thickening of the airway wall secondary to inflammatory cell infiltration and edema could amplify the effects of airway smooth muscle shortening on pulmonary resistance (31). Such a mechanism has been postulated to be important in asthma, a condition that is associated with thickening of the airway walls (32). Recent evidence suggests an important role for mechanical interdependence between the airways and parenchyma in limiting bronchoconstrictive responses (33). It is possible that peribronchial edema may haveuncoupled the alveolar attachments from the airways, reducing the tethering effect of the parenchyma and permitting greater airway narrowing. Our results suggest that the effects of IL-2 were mediated by its action on specific receptors and not related to the nonspecific effect of the administration of a foreign protein. IL-2 had similar immunologic effects on the peripheral blood of rats as reported in humans, causing a leukocytosis and increasing natural killer and lymphokine-activated killer activity against YAC-l and K-562 target cells by approximately 7-fold. Weadministered IL-2 in combination with porcine gelatin. This method of administration served two purposes, one to maintain a constant serum level of IL-2 (34) and the other to control for the reaction of the animals to foreign protein. Both IL-2 and control animals received a substantial excess of protein in the form of gelatin (270 mg of gelatin versus 0.36 mg of IL-2).
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Interleukin-2 Diluent Significance, p value
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2.75 ± 0.29 1.64 ± 0.23 0.014
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, Lung lavage was performed by injection and immediate retrieval of five s-rnt aliquots of saline after the methacholine challenge . Total cells were counted on a hemacytometer , and differential was assessed by a blinded observer on a Wright Giemsa stain of cytocentrifuged slides by count ing 200 cells under oil immersion microscopy. t 40% of the lymphocytes were large granular lymphocytes.
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Fig. 4. Effect of IL-2 on cytotoxic activity of peripheral blood mononuclear cells. Lytic activity from the effector cells of the blood of two rats receiving IL·2 was tested against YAC·1 (open squares) and K562 (closed squares) target cells. Lytic activity from the effector cells of the blood of two rats receiving the vehicle was also tested at the same time against YAC-1 (open circles) and K562 (closed circles) target cells.
379
INTERLEUKIN-2 AND AIRWAY RESPONSIVENESS
When a T lymphocyte is activated by antigen, it upregulates its receptors for IL-2 and increases its secretion of this lymphokine (35). T lymphocytes are activated in patients with severeasthma and have increased IL-2 receptors (36). Lymphocytes from asthmatic but not from normal children develop an increase in proliferation to IL-2 when exposed to a specific antigen (37). Insofar as IL-2 has been shown to increase the production of some lymphokines in vitro (38), it is reasonable to postulate that IL-2 may also increase the secretion of other lymphokines that affect the inflammatory cells found in asthma (9-16). Indeed, chronic administration of IL-2 to humans for the treatment of cancer causes leukocytosis, eosinophilia, and activation of the eosinophils (39). Interestingly, bronchospasm has also been reported as an occasional side effect (40). It is well known that antigen provocation (1) and viral infections (4) can also increase airway responsiveness. The mechanism by which asthma is triggered under these circumstances is unknown, but in both instances T lymphocyte activation and IL-2 secretion is probable. Indeed, it has recently been shown that high levelsof IL-2 can be produced during systemic viral infections in vivo (41). The extrapolation of our results to these clinical conditions is complicated by the lack of data on the amount of IL-2 produced locally by activated lymphocytes. However, we are tempted to speculate that lymphocyte activation and secretion of IL-2, as it occurs during the immune response to antigen or viruses in the genetically predisposed person, may playa role in the development of abnormal airway responsiveness. Acknowledgment The writers would like to thank Mrs. Maria Makroyanni and Ms. Barbara Kidd for their assistance in preparation of the manuscript. References 1. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987; 136:740-51. 2. Nadel JA, Sheppard D. Mechanisms of bronchial hyperreactivity in asthma. In: Weiss EB, Segal MS, Stein M, eds. Bronchial asthma. Mechanisms and therapeutics. Boston: Little, Brown, 1985; 30-6. 3. Rocklin RE, Findlay SR. Immunologic mechanisms and recent advances in asthma. In: Weiss EB, Segal MS, Stein M, eds. Bronchial asthma. Mechanisms and therapeutics. Boston: Little, Brown, 1985; 41-51. 4. Busse WW. Clinical implications of basic research. The contribution of viral respiratory infec-
tions to the pathogenesis of airway hyperreactivity. Chest 1988; 93:1076-82. 5. Reed CEo Symposium. Basic mechanisms of asthma. Role of inflammation. Chest 1988; 94: 175-90. 6. Salvato G. Some histological changes in chronic bronchitis and asthma. Thorax 1968; 23:168-72. 7. Sobonya RE. Quantitative structural alterations in long-standing allergic asthma. Am Rev Respir Dis 1984; 130:289-92. 8. Buckley RH, Becker WG. Abnormalities in the regulation of human IgE synthesis. Immunol Rev 41;288-314. 9. Silberstein DS, Owen WF, Gasson JC, et al. Enhancement of human eosinophil CYtotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor. J Immunol 1986; 137:3290-4. 10. Warren DJ, Moore MAS. Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells. J Immunol1988; 140:94-9. 11. Tanno Y, Bienenstock J, Richardson M, Lee TDG, Befus AD, Denburg JA. Reciprocal regulation of human basophil and eosinophil differentiation by separate T-cell-derived factors. Exp Hematol 1987; 15:24-33. 12. Hirai K, Morita Y, Misaki Y, et al. Modulation of human basophil histamine release by hemopoietic growth factors. J Immunol 1988; 141:3958-64. 13. Bienenstock J. An update on mast cell heterogeneity. J Allergy Clin Immunol 1987; 81:763-9. 14. DiPersio JF, Billing P, Williams R, Gasson JC. Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. J Immunol 1988; 140:4315-22. 15. Dahinden CA, Zingg J, Maly FE, de Weck AL. Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMtp as second signals. J Exp Med 1988; 167:1281-95. 16. Fischer HG, Frosch S, Reske K, Reske-Kunz AB. Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis ofMHC class II molecules and to augmented antigen presentation function. J Immunol 1988; 14:3882-8. 17. Mead J, Whittenberger J. Physical properties of human lung measured during spontaneous respiration. J Appl Physiol 1953; 5:770-96. 18. Ginns LC, Ryu JH, Rogol PR, Sprince N, Oliver LC, Larsson CJ. Natural killer cell activity in cigarette smokers and asbestos workers. Am Rev Respir Dis 1985; 131:831-4. 19. Ettinghausen SE, Lipford EH III, Mule JJ, Rosenberg SA. Systemic administration of recombinant interleukin 2 stimulates in vivo lymphoid cell proliferation in tissues. J Immunol 1985; 135: 1488-97. 20. Anderson TD, Hayes TJ. Toxicity of human recombinant interleukin-2 in rats. Pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement. Lab Invest 1989; 60:331-46. 21. Renzi PM, Sapienza S, Du T, Wang NS, Martin JG. Acute effects of interleukin-2 on airway responsiveness (abstract). FASEB 1989; 3(Part II: AI049). 22. Valone FH, Epstein LB. Biphasic plateletactivating factor synthesis by human monocytes stimulated with IL-I-B, tumor necrosis factor or IFN-a 1. J Immunol 1988; 141:3945-50.
23. Tsai J J, Cromwell 0, Maestrelli P, O'Hehir RE, Moq bel R, Kay AB. Leukotriene release enhancing factor. Purification, specific allergen induction and further biologic properties. J Immunol 1989; 142:1661-8. 24. Schell S, Novak K, Tone G, Fitch F. Interleukin 2 stimulates GM-CSF production in murine helper T cell clones through an antigen independent pathway (abstract). FASEB 1989; 3:AI269. 25. Wirthmueller U, De Weck AL, Dahinden CA. Platelet-activating factor production in human neutrophils by sequential stimulation with granulocytemacrophage colony-stimulating factor and the chemotactic factors C5A or formylmethionylleucylphenylalanine. J Immunol1989; 142:3213-8. 26. Nogrady SG, Bevan C. H2 receptor blockade and bronchial hyperreactivity to histamine in asthma. Thorax 1981; 36:268-71. 27. Cuss FM, Dixon CMS, Barnes PJ. Effects of inhaled platelet activating factor on pulmonary function and bronchial responsivenessin man. Lancet 1986; 2:189-92. 28. Murray JJ, Tonnel AB, Brash AR, et al. Release of prostaglandin D 2 into human airways during acute antigen challenge. N Engl J Med 1986; 315:800-4. 29. Holroyde MC, Altounyan REC, Cole M, Dixon M, Elliott EV. Bronchoconstriction produced in man by leukotrienes C and D. Lancet 1981; 2:17-8. 30. Moncada S, Flower RJ, Vane JR. Prostaglandins, prostacyclin, thromboxane A 2 , and leukotrienes. In: Gilman AG, Goodman LS, eds. The pharmacological basis of therapeutics. New York: MacMillan, 1985; 660-73. 31. Moreno RH, Hogg JC, Pare PD. Mechanisms of airway narrowing. Am Rev Respir Dis 1986; 133:1171-80. 32. James AL, Pare PD, Hogg JC. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989; 139:242-6. 33. Ding DJ, Martin JG, Macklem PT. The effects of lung volume on maximal methacholine induced bronchoconstriction in normal humans. J Appl Physiol 1987; 62:1324-30. 34. Chang AE, Hyatt CL, RosenbergSA. Systemic administration of recombinant human interleukin-2 in mice. J BioI Response Mod 1984; 3:561-72. 35. Cantrell DA, Smith KA. Transient expression of interleukin 2 receptors. Consequences of T cell growth. J Exp Med 1983; 158:1895-911. 36. Corrigan CJ, Hartnell A, Kay AB. T lymphocyte activation in acute severeasthma. Lancet 1988; 1:1129-32. 37. Yoshizawa I, Noma T, Kawano Y, Yata J. Allergen-specific induction of interleukin-2 (IL-2) responsiveness in lymphocytes from children with asthma. 1. Antigen specifics and initial events of induction. J AllergyClin Immunol1989; 84:246-55. 38. Dunn DE, Jin J, Lancki DW, Fitch FW. An alternative pathway of induction of lymphokine production by T lymphocyte clones. J Immunol 1989; 142:3847-56. 39. Silberstein DS, Schoof DD, Rodrick ML, et al. Activation of eosinophils in cancer patients treated with IL-2 and IL-2-generated lymphokineactivated killer cells. J Immunol1989; 142:2162-7. 40. Rosenberg SA, Lotze MT, Muel LM, et al. Observations on the systemic administration of autologous lymphokine activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 1985; 313:1485-92. 41. Kasaian M, Biron CA. The role of interleukin-2 in T cellactivation and proliferation in vivo. FASEB 1989; 3:A478.
PAOLO M. RENZI, SANTO SAPIENZA, TAO OU, NAI SAN WANG, and JAMES G. MARTIN Introduction
Asthma is a chronic disease characterized by reversible airway obstruction and airway hyperresponsiveness to a variety of physical and chemical stimuli (1, 2). Recent investigations of the mechanisms of hyperresponsiveness after acute airway insults such as allergen provocation and viral infections suggest an important pathogenetic role for inflammation (3, 4). Despite its probable importance in causing sustained increases in airway responsiveness, chronic inflammation such as is seen in the airways of asthmatic patients has received relatively little attention. One of the key cells in chronic inflammation is the lymphocyte, and therefore it is reasonable to hypothesize that it may be involved, directly or indirectly, in airway hyperresponsiveness. A number of lines of evidence, albeit circumstantial, suggest that the lymphocyte may be important in altered airway responsiveness. First, the lymphocyte is present and increased in the inflammatory infiltrate in the airways of patients both with severe fatal asthma (5) and with less severe chronic asthma (3, 6, 7). Second, the lymphocyte plays a role in the regulation of atopy (8) and in the defense against viral infections (4), two conditions known to be associated with airway hyperresponsiveness (1-4). Third, lymphocytes in vitro produce lymphokines capable of activating or inducing the proliferation of eosinophils (9-11), basophils (11-13), neutrophils (14, 15), and macrophages (16), all potential participants in asthma. To explore the potential role of the lymphocyte in airway responsiveness we examined the effects of interleukin-2 (IL-2), a major growth factor for T lymphocytes, on the response of the rat to inhaled aerosolized methacholine (MCh). We report the effects of subcutaneous administration of IL-2 for 4.5 days on airway responsiveness to MCh, lung histology, and lung lavage. Methods Preparation of Animals and Measurement of Pulmonary Mechanics Eighteen pathogen-free male Lewisrats (mean
SUMMARY We evaluated the potential role of the lymphocyte in chronic airway inflammation and responsiveness by repeated administration to rats of interleukln-2 (IL-2), the principallymphoklne responsible for lymphocyte proliferation. Lewis rats (mean weight, 184 ± 2 g) received either 120,000 10) or vehicle (n 7) subcutaneously twice a day for 4.5 days. Animals were units of IL-2 (n anesthetized with urethane and intubated for measurements of pulmonary resistance (RL) and airway responsiveness to aerosol methacholine (MCh). Lung lavage was performed, the animals were exsanguinated, and the lungs were fixed in 10% formalin. Histologic edema and the extent of infiltration of the bronchi, pulmonary veins, and arteries by cells was scored blindly. IL-2 increased airway responsiveness to MCh; the concentrations of MCh causing a doubling of RL were 0.14versus 1.39 mg/ml (geometric mean) for the IL-2 and vehicle group, respectively (p 0.001). IL-2 significantly increased total cellular return and the percentage of lymphocytes, neutrophlls, and eosinophils in lavage. IL-2 caused edema and a mixed cellular infiltration of the bronchovascular tree. Lymphocytes predominated around the airways and veins. A correlation (r 0.50) was present between airway responsiveness and airway inflammation but not with edema or vascular infiltration. Release of IL-2 by lymphocytes in the airways may be an important mediator of airway hyperresponslveness.
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AM REV RESPIR DIS 1991; 143:375-379
weight, 184 ± 2 g) were randomized to receive either recombinant human IL-2 (generously provided by the Glaxo Institute of Molecular Biology, Switzerland) or vehicle (8 nglml of Escherichiacoli endotoxin in 50 mM acetic acid). Eight rats received 120,000units of IL-2, and eight rats received the vehicle subcutaneously twice daily in 150/0 porcine gelatin for 4.5 days. Two rats received IL-2 without gelatin, and although histologic changes and airway responsiveness weresimilar to those obtained with IL-2 in gelatin, these animals were included only in the analysis of histological changes and airway responsiveness. One vehicle-treated animal was excluded from analysis because of pneumonia. Pilot experiments were performed to determine the optimal amount of IL-2 that could be administered to rats for 4.5 days. One male Lewisrat received480,000units of IL-2 in 150/0 porcine gelatin subcutaneously twice a day and died on the third day. Two male Lewis rats received240,000units of IL-2 in 15% gelatin subcutaneously twice a day and died at 4.5 days prior to the measurement of pulmonary mechanics. All three rats lost weight and showed severe mononuclear cell infiltration of the lungs on histologic examination. A dose of 120,000 units of IL-2 administered subcutaneously in 150/0 gelatin twice a day was chosen because this dose did not affect the behavior of the rats, nor did it impede their normal weight gain. Within 30 min of the last dose of IL-2 or vehicle, anesthesia was induced with urethane (0.9 to 1.1 g/kg, intraperitoneally). Blind endotracheal intubation was then performed
using a 6-cm length of PE-240 polyethylene catheter. A heating pad was used to maintain body temperature constant. Pulmonary resistance was measured during spontaneous tidal breathing with the animals in the left lateral decubitus position. Flow was measured by placing the tip of the tracheal tube inside a small Plexiglas's box (265 ml in volume) to the end of which a Fleisch no. 0 pneumotachograph coupled to a differential transducer (MP-45, ± 2 em H 2 0 ; Validyne Corp., Northridge, CA) was attached. Volume was obtained by electrical integration of the flow signal using a respiratory inteftrator (HP8815A; Hewlett-Packard, Wal.tham, MA). Pulmonary resistance (RL) was obtained by the electrical subtraction .technique of Mead and Whittenberger (17). Endotracheal tube resistance was 0.11 em H 20/ml/s at the flow of 25 mIls. Tube resistance was subtracted from all values of RL.
(Received in original form January 10, 1990 and in revised form June 27, 1990) 1 From the Meakins-Christie Laboratories and the Department of Pathology, Royal Victoria Hospital, McGill University, and the Pulmonary Service,St. Luc Hospital, Universityof Montreal, Montreal, Quebec, Canada. 2 Supported by Grant No. MA-I0637 from the Medical Research Council of Canada. 3 Correspondence 'and requests for reprints should be addressed to Dr. P. M. Renzi, MeakinsChristie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2.
375
RENZI, SAPIENZA, DU, WANG, AND MARTIN
376
Tidal volume, breathing frequency, and minute ventilation were also calculated.
Methacholine Challenge Aerosols were generated using a Hudson nebulizer containing 3 ml of solution, which was driven by a compressed air source at an airflow of 7.5 L/min. The nebulizer output was 0.18 ml/min. The nebulizer was connected to one side port of the Plexiglas box, and during aerosolization, airflow was diverted through a second side port. To assess airway responsiveness to MCh, rats inhaled aerosols of normal saline and progressively doubling concentrations of MCh in normal saline for 30 s. MCh concentrations ranged from 0.063 to 16 mg/rnl, The peak value of RLwas measured before and after inhalation of saline and after each of the concentrations ofMCh. An interval of approximately 3 min elapsed between the administration of each concentration of MCh. The concentration of MCh required to double RL(EC2ooRL)was obtained by linear interpolation between the two concentrations bounding the point at which RL reached 200070 of control.
Lung Lavage At the end of the experiment, lung lavage was performed with 25 ml of saline at room temperature by instillation and gentle aspiration of 5-ml aliquots through the endotracheal tube. Total cell count was determined using a hemacytometer. Differential cell counts were assessed on cytologicpreparations. Slideswere prepared using a Cytospin (Model Cytospin II; Shandon, Pittsburgh, PAl and stained with Wright-Giemsa. Two hundred cells were examined by light microscopy.
Total White Blood Cell Determination and Assessment of Lytic Activity After lung lavage, the animals were killed by exsanguination, blood was retrieved in heparinized tubes, and the total white blood cell count was determined in a Coulter counter (Coulter Electronics, Hialeah, FL). Lytic activity was measured by a modified 4-h chromium-51 release assay (18). Tumor target cells were from the YAC-l mouse lymphoma cell line and the K562 human chronic myelogenous leukemia cell line obtained from American Type Culture Collection (Rockville, MD). These cells were mycoplasma-free and maintained as suspension cultures in tissue culture medium (TCM) with 10070 heat inactivated fetal calf serum (HIFCS). The tumor cells were radiolabeled by incubation with 50 J,lCi of (5tCr]sodium chromate (New England Nuclear, Boston, MA) for 2 h at 37° C in humidified air plus 5070 CO 2 washed twice in phosphate-buffered saline, once in RPMI 1640, and resuspended in TCM plus 20070 HIFCS at a concentration of lOS cells/ml. One hundred microliters of this cell suspension containing lQ4 target cells were placed in each microwell of a round-bottom microtiter plate (Flow Laboratories, McLean, VA). Peripheral blood effector cells were obtained by centrifugation (450 g for 40 min
Statistical Analysis
at 22° C) of the buffy coat of rat blood over a Ficoll-Hypaque density gradient (Organon Teknika, Durham, NC), washed twice in phosphate-buffered saline (pH 7.3), and resuspended in TCM. Blood effector cells were added in quadruplicate to the target cells at four different effector-to-target-cell ratios, 80:1, 40:1, 20:1, and 10:1. Target cells incubated in TCM without effector cells were used as controls for spontaneous stCr release during the assay. Spontaneous release averaged 8 ± 1.2070 for all experiments. After a 4-h incubation at 37° C in humidified air plus 5070 CO 2 , the plates were centrifuged at 450 g for 5 min, 100 ul of the supernatant were carefully removed from each well, and the radioactivity was determined using a gamma counter. Lytic activity of blood effector cells was determined as percent specific release calculated as [(ER - CR/T - CR) x 100], where ER = StCrreleased by experimental cells, CR = StCr released spontaneously, and T = total StCr. Standard errors were less than 1070.
Group results are expressed as mean ± SE except for values of EC 2ooRL, which are reported as geometric means. The significance of differences was evaluated using unpaired t tests or the Mann-Whitney U test for pathology scores. Correlation between histology scores and airway responsiveness was examined using Spearman's rank test. Significance was considered to be established with a p value < 0.05.
Results
Effect of IL-2 on Airway Responsiveness to Methacholine After general anesthesia and endotracheal intubation there was no significant difference between the baseline RL, respiratory frequency, tidal volume, and minute ventilation of the IL-2 and vehicle-treated animals (table 1). IL-2 increased airway responsiveness (figure 1). The concentration of MCh necessary to cause a doubling of lung resistance (EC 2ooRL) was much less in the IL-2 treated than in the vehicle-treated animals (0.137 mg/ml versus 1.39 mg/ml; p = 0.001).
Pathologic Assessment of the Lungs After exsanguination of the rats, the lungs were fixed in 10070 formalin at a pressure of 25 em H 2 0 for 48 h. Slides from paraffinembedded sections of the lungs were stained with hematoxylin-eosin. Using a scoring system, the extent of the pathologic changes in the bronchi, veins, arteries, and alveoli were scored by two observers blinded to the group status of the specimens. Edema, cellular infiltration, and epithelial detachment were assessed. A score of zero was given if none of the specific structures (airways or vessels) examined on the slide had the changes looked for; 1+ ~ 25070; 2+ ~ 50070 but> 25070; 3 + > 50070 of the specific structures had the changes. There was good interindividual reproducibility of the scoring (r = 0.957).
Effects of IL-2 on Lung Histology The administration of IL-2 caused peribronchial and perivascular edema with a mixed cellular infiltration (figures 1 and 2). No overlap was found between the cellular infiltration scores of the IL-2and vehicle-treated animals. Although some overlap was noted for bronchovascular edema, the difference between the IL-2- and vehicle-treated groups was still significantly different (p < 0.02). IL-2
TABLE 1 EFFECT OF INTERLEUKIN-2 ON LUNG MECHANICS· Baseline Lung Resistance (em H20/mils)
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control animals was included in our anal ysis (r = 0.65, p < 0.05). No correlation was found between bronchial edema and airway responsiveness (r = 0.02) or between venous edema, cellular infiltration, and airway responsiveness. We also compared airway responsiveness of IL-2-treated animals with a pathology score ~ 1 to that of IL-2treated animals with a pathology score > 1. A significant difference in airway responsiveness was found only for cellular infiltration of the bronchi (p = 0.024).
Effect of IL-2 on the Cellular Composition of Lung Lavage The volume of lavage fluid retrieved averaged 830;0 of that instilled in IL-2-treated animals and 85% in vehicle-treated animals. IL-2 increased the total cellular return in lung lavage as well as the percentage of lymphocytes, neutrophils, eosinophils, and basophils (table 2). Eosinophils and basophils were found only in the lavage of IL-2-treated animals. Effect of IL-2 on Total White Blood Cells and Effector Cell Lytic Activity The administration of IL-2 caused a leukocytosis, with 9.6 ± 0.4 versus 4.2 ± 0.8 x 106 white blood cells/ml for IL-2treated versus vehicle-treated animals. The subcutaneous administration of IL-2 also increased the lytic activity of peripheral blood mononuclear cells against the YAC-I and K562 target cells (figure 4). Discussion
Fig. 2. Lung histology of IL-2 treated animals. An example of edema and cellula r infiltrat ion of the bronchi (A) and veins (8) of an IL-2-treated rat is shown . A predom inantly lymphocytic infiltration is present around the airway walls and the veins.
also appeared to increase epithelial detachment, but the difference was not significant compared with that in the control group. Little change was noted around the pulmonary arteries or in lung parenchyma. In pilot experiments, when higher doses of IL-2 were administered, the cellular infiltrate and edema weremore diffuse. The predominant cell around the airways and pulmonary veins was the lymphocyte, but macrophages, eosinophils,
neutrophils, and basophils were also present. When edema or the extent of cellular infiltration of the airways or pulmonary veins were compared with airway responsiveness to MCh, a weak correlation was found only between cellular infiltration of the bronchi and airway responsiveness (r = 0.50) (figure 3). This correlation became significant only when the scoring of cellular infiltration of the airways of
Wehave shown that the systemic administration of 120,000U of IL-2 to rats twice a day for 4.5 days caused a peribronchial and perivenous cellular infiltration with edema. Lung lavage contained an increased cellular return and an increase in the number of lymphocytes, neutrophils, eosinophils, and basophils retrieved. These changes were associated with an increase in airway responsiveness to the cholinergic bronchoconstrictor, methacholine. The histologic changes in the lungs that we observed are consistent with the results of others. In mice (19), the systemic administration of IL-2 causes a predominantly lymphocytic infiltration of the lungs, liver, spleen, and kidneys. In rats (20), IL-2 has been reported to cause a marked tissue infiltration with lympho cytes and eosinophils. In our preliminary experiments, higher doses of IL-2 caused diffuse infiltration of the bronchovascu-
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lar tree and the lung parenchyma. For the experiments reported, we chose a dose of IL-2 that affected predominantly the bronchovascular tree. This dose did not cause significant changes in lung resistance or minute ventilation, but it did increase airway responsiveness. When relationships between the histologic changes and airway responsiveness were examined, a correlation was found only with cellular infiltration of the bronchi. In previousexperiments,wehaveshown that an acute , high dose, intravenous administration of IL-2 also causes an increase in airway responsivenesswithin I h before any cellular infiltration occurs (21). The only significant pathologic change observed in the acutely treated animals was bronchial epithelial detachment. Histologic changes differed after chronic administration of IL-2; at the doses reported there was no significant bronchial epithelial detachment, where-
as edema and cellular infiltration of the bronchovascular tree were prominent (figures 1 and 2). The mechanism of the observed increase in airway responsiveness is not clear. Under the influence of IL-2, lymphocytes may have secreted lymphokines that affect smooth muscle directly, but evidence for the existence of such bronchoactive cytokines is lacking. An alternate explanation is that in vivo release of lymphokines from lymphocytes stimulated by IL-2 caused activation or proliferation of eosinophils, neutrophils, macrophages, and basophils (9-16, 2225), and that these cells, in turn, released one or more of several possible lipid- or granule-associated bronchoactive mediators (26-30). It is possible that structural changes in the airways rather than synthesis of bronchoactive substances may have contributed to the altered bronchial respon-
siveness. Thickening of the airway wall secondary to inflammatory cell infiltration and edema could amplify the effects of airway smooth muscle shortening on pulmonary resistance (31). Such a mechanism has been postulated to be important in asthma, a condition that is associated with thickening of the airway walls (32). Recent evidence suggests an important role for mechanical interdependence between the airways and parenchyma in limiting bronchoconstrictive responses (33). It is possible that peribronchial edema may haveuncoupled the alveolar attachments from the airways, reducing the tethering effect of the parenchyma and permitting greater airway narrowing. Our results suggest that the effects of IL-2 were mediated by its action on specific receptors and not related to the nonspecific effect of the administration of a foreign protein. IL-2 had similar immunologic effects on the peripheral blood of rats as reported in humans, causing a leukocytosis and increasing natural killer and lymphokine-activated killer activity against YAC-l and K-562 target cells by approximately 7-fold. Weadministered IL-2 in combination with porcine gelatin. This method of administration served two purposes, one to maintain a constant serum level of IL-2 (34) and the other to control for the reaction of the animals to foreign protein. Both IL-2 and control animals received a substantial excess of protein in the form of gelatin (270 mg of gelatin versus 0.36 mg of IL-2).
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2.75 ± 0.29 1.64 ± 0.23 0.014
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, Lung lavage was performed by injection and immediate retrieval of five s-rnt aliquots of saline after the methacholine challenge . Total cells were counted on a hemacytometer , and differential was assessed by a blinded observer on a Wright Giemsa stain of cytocentrifuged slides by count ing 200 cells under oil immersion microscopy. t 40% of the lymphocytes were large granular lymphocytes.
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Fig. 4. Effect of IL-2 on cytotoxic activity of peripheral blood mononuclear cells. Lytic activity from the effector cells of the blood of two rats receiving IL·2 was tested against YAC·1 (open squares) and K562 (closed squares) target cells. Lytic activity from the effector cells of the blood of two rats receiving the vehicle was also tested at the same time against YAC-1 (open circles) and K562 (closed circles) target cells.
379
INTERLEUKIN-2 AND AIRWAY RESPONSIVENESS
When a T lymphocyte is activated by antigen, it upregulates its receptors for IL-2 and increases its secretion of this lymphokine (35). T lymphocytes are activated in patients with severeasthma and have increased IL-2 receptors (36). Lymphocytes from asthmatic but not from normal children develop an increase in proliferation to IL-2 when exposed to a specific antigen (37). Insofar as IL-2 has been shown to increase the production of some lymphokines in vitro (38), it is reasonable to postulate that IL-2 may also increase the secretion of other lymphokines that affect the inflammatory cells found in asthma (9-16). Indeed, chronic administration of IL-2 to humans for the treatment of cancer causes leukocytosis, eosinophilia, and activation of the eosinophils (39). Interestingly, bronchospasm has also been reported as an occasional side effect (40). It is well known that antigen provocation (1) and viral infections (4) can also increase airway responsiveness. The mechanism by which asthma is triggered under these circumstances is unknown, but in both instances T lymphocyte activation and IL-2 secretion is probable. Indeed, it has recently been shown that high levelsof IL-2 can be produced during systemic viral infections in vivo (41). The extrapolation of our results to these clinical conditions is complicated by the lack of data on the amount of IL-2 produced locally by activated lymphocytes. However, we are tempted to speculate that lymphocyte activation and secretion of IL-2, as it occurs during the immune response to antigen or viruses in the genetically predisposed person, may playa role in the development of abnormal airway responsiveness. Acknowledgment The writers would like to thank Mrs. Maria Makroyanni and Ms. Barbara Kidd for their assistance in preparation of the manuscript. References 1. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987; 136:740-51. 2. Nadel JA, Sheppard D. Mechanisms of bronchial hyperreactivity in asthma. In: Weiss EB, Segal MS, Stein M, eds. Bronchial asthma. Mechanisms and therapeutics. Boston: Little, Brown, 1985; 30-6. 3. Rocklin RE, Findlay SR. Immunologic mechanisms and recent advances in asthma. In: Weiss EB, Segal MS, Stein M, eds. Bronchial asthma. Mechanisms and therapeutics. Boston: Little, Brown, 1985; 41-51. 4. Busse WW. Clinical implications of basic research. The contribution of viral respiratory infec-
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