Therapeutic efficacy of an anti IL-5 monoclonal antibody delivered into the respiratory tract in a murine model of

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1 Therapeutic efficacy of an anti IL-5 monoclonal antibody delivered into the respiratory tract in a murine model of asthma Felix R. Shardonofsky, MD, Joe Venzor III, MD, Roberto Barrios, MD, Khai-Pang Leong, MD, and David P. Huston, MD Houston, Texas Background: IL-5 is central to the pathogenesis of airway eosinophilic inflammation and hyperresponsiveness associated with both atopic and nonatopic asthma. The therapeutic potential of IL-5 antagonists in asthma is supported by the inhibition of airway eosinophilia and hyperresponsiveness in animal models receiving neutralizing anti-il-5 mabs intravenously or intraperitoneally. Objective: The purpose of this study was to test the hypothesis that mabs against IL-5 delivered by way of the respiratory tract are as effective as those delivered intraperitoneally in diminishing the pulmonary eosinophilic inflammation and airway hyperresponsiveness in a murine model of ovalbumininduced asthma. Methods: Ovalbumin-sensitized Balb/c mice were given an anti IL-5 mab delivered intranasally or an isotype-matched control mab delivered intranasally before respiratory challenge with ovalbumin. Outcome variables included respiratory system resistance responses to methacholine, bronchoalveolar lavage fluid cellularity, and lung histopathology. Results: Anti IL-5 mabs administered intranasally to ovalbumin-sensitized and challenged mice significantly decreased eosinophil counts in bronchoalveolar lavage fluid and lung tissue and significantly reduced airway hyperresponsiveness relative to ovalbumin-sensitized and challenged mice that received either no mab treatment or an isotype-matched control mab. Similar results were obtained when an anti IL-5 mab was given intraperitoneally. Conclusion: This is the first study to demonstrate that delivery of anti IL-5 mabs into the respiratory tract is efficacious in attenuating the asthma phenotype in a murine model. These results provide impetus for the development of inhaled IL-5 antagonists for the treatment of human asthma. (J Allergy Clin Immunol 1999;104: ) Key words: Asthma, IL-5, airway hyperresponsiveness, inhaled therapy, eosinophils From the Departments of Pediatrics, Medicine, Pathology, and Microbiology and Immunology, Baylor College of Medicine, Houston. Supported by the American Lung Association (F.R.S.), the American Academy of Allergy and Immunology Under-Represented Minority Investigator in Asthma and Allergy Award (J.V.), the National Institutes of Health (AI36936, D.P.H.; HL03784, F.R.S.), the Singapore Ministry of Health (K.L.), the Cullen Foundation (D.P.H.), and the Trammell Foundation (D.P.H.). Received for publication Sep 10, 1998; revised Feb 22, 1999; accepted for publication Feb 24, Reprint requests: Felix R. Shardonofsky, MD, Section of Pediatric Pulmonology, Baylor College of Medicine, 1102 Bates St, Suite 410, Houston, TX Copyright 1999 by Mosby, Inc /99 $ /1/98183 Abbreviations used BALF: Bronchoalveolar lavage fluid MR: Maximal bronchoconstrictor response PD 100 : Provocation dose 100% Rrs: Respiratory system resistance Investigations into the mechanisms of asthmatic inflammation have focused on IL-5 because eosinophils are considered to be important effector cells in the pathobiology of asthma, 1 and IL-5 plays a central role in virtually all aspects of eosinophil biology. 2-4 The significance of IL-5 to asthma is derived from studies that show an increased expression of IL-5 mrna and protein product in cells recovered from bronchial biopsy specimens and bronchoalveolar lavage fluid (BALF) from both atopic and nonatopic asthmatic subjects. 5-7 Expression of these products is also enhanced after bronchopulmonary provocation of sensitized subjects with specific antigens. 8,9 Data from animal models further support an important role of IL-5 in the asthmatic response Rationale to target IL-5 in the treatment of asthma is provided by the capacity of neutralizing anti IL-5 mabs administered intravenously or intraperitoneally to inhibit antigen- or virus-induced bronchial hyperreactivity and airway eosinophilic inflammation in several species The purpose of the present study was to test the hypothesis that neutralization of IL-5 in a murine model of asthma can be achieved by directly delivering anti IL- 5 mab into the respiratory tract. This hypothesis was based on evidence that protein molecules can be absorbed intact across the respiratory epithelium. 18,19 METHODS Animals Male Balb/c mice 6 to 10 weeks of age from Jackson Laboratory (Bar Harbor, Me) were housed under conventional conditions for the studies. The experiments described in this report were approved by the Animal Research Ethics Board of Baylor College of Medicine. Monoclonal antibodies The rat IgG1-κ anti-murine IL-5 mab, TRFK-5, was purified from hybridoma ascites fluid by affinity chromatography, as previously described The isotype-matched control mab for TRFK- 5 was GL-113, a rat IgG1-κ anti-escherichia coli β-galactosidase that was prepared in a similar manner. Affinity-purified TRFK-5 215

2 216 Shardonofsky et al J ALLERGY CLIN IMMUNOL JULY 1999 A B FIG 1. Number of leukocytes (A) and differential of leukocytes (B) in BALF obtained from sham-sensitized and challenged mice (n = 9), ovalbumin-sensitized and challenged mice that either were not treated with mab (n = 10) or were treated with anti IL-5 mab (TRFK-5) intranasally (n = 11) or intraperitoneally (n = 10), and from ovalbumin-sensitized and challenged mice treated with an isotypematched control mab (GL-113; n = 10). Data are expressed as the means ± SEM of number of cells mm -3 (A) and percentage of leukocyte subpopulations (B). ANOVA followed by Tukey s test: *Different from sham-sensitized and challenged mice (P <.05); different from ovalbumin-sensitized and challenged mice either not treated with mab or treated with GL-113 mab (P <.05). and GL-113 were diluted in PBS to the desired final concentrations immediately before use. ELISA Serum concentrations of TRFK-5 were determined by a sandwich ELISA, as previously described Induction of airway inflammation and hyperresponsiveness Ovalbumin-induced eosinophilic pulmonary inflammation and airway hyperresponsiveness were used as a murine paradigm of asthma. 23 Mice were primed with an intraperitoneal injection of 50 µl of PBS containing 4 µg of ovalbumin (Sigma Chemical Co, St Louis, Mo) adsorbed to 4 mg of aluminum hydroxide (Sigma Chemical Co) on protocol days 0 and 14. On days 14, 25, 26, 27, and 28, mice were challenged intranasally with 40 µl of PBS containing 4 µg of ovalbumin. 23 Sham-sensitized and challenged mice received intraperitoneal injections of 4 mg of aluminum hydroxide in 50 µl of PBS on days 0 and 14, and nasal drops of PBS on days 14 and 25 to 28. Before intraperitoneal and intranasal administrations, animals were lightly anesthetized with inhaled Metafane (Pittman-Moore, Mundelein, Ill). Nasal drops were delivered into the nose of lightly anesthetized mice. Antibody treatment On day 24, lightly anesthetized mice were given 40 µl of either PBS containing 100 µg of TRFK-5 intranasally, PBS containing 100 µg of GL-113 intranasally, or PBS containing 100 µg of TRFK- 5 intraperitoneally. Assessment of airway responsiveness to methacholine On day 29, mice were anesthetized by an intraperitoneal injection of ketamine, xylazine, and acepromazine; tracheotomized; placed in the supine position inside a 0.37-L Plexiglass pressure-sensitive body plethysmograph; paralyzed with pancuronium bromide; and mechanically ventilated (Harvard Apparatus 687). The ventilatory settings consisted of supplemental oxygen, tidal volume of 13 ± 1 ml kg 1 (mean ± SD), and respiratory rate of 120 minutes 1. Respiratory system resistance (Rrs) and dynamic elastance as a function of time were derived from the body plethysmograph and tracheal pressure signals by applying a recursive least-square algorithm off-line to the equation of motion of the single-compartment linear model of the respiratory system. 24 Values for Rrs were corrected for equipment resistance. Data were obtained under baseline conditions and during tail vein intravenous bolus injections of methacholine chloride (Sigma Chemical Co), with dosage doubled from 6 µg kg 1 up to 1600 µg kg 1. Methacholine dose-response curves were constructed with Rrs as an index of airway caliber. The provocation dose 100% (PD 100 ) and the maximal constrictor response (MR) were taken as indices of airway responsiveness. The PD 100, which is the methacholine dose that caused the Rrs to increase by 100% relative to the postsaline solution value, was obtained by linear interpolation. A plateau or MR was considered to be present when, for 3 consecutive increasing doses of methacholine, Rrs either increased by less than 15% or decreased after attaining a maximum value. Bronchoalveolar lavage and cytology After completion of the methacholine challenge, mice were killed, and BAL was performed by 5 repeated washings with 200 µl of PBS through the tracheal cannula. Approximately 150 µl of fluid was recovered after each lavage. Total cell numbers were counted with the use of a hemocytometer. The BALF was then centrifuged at 1180g for 5 minutes, and the cell pellet were resuspended in 200 µl of PBS. Cytoprep smears of BAL cells were stained with Wright-Giemsa (EM Diagnostic Systems, Gibbtown, NJ). Differential cell counts were determined from at least 3 separate analysis of 100 leukocytes. Lung histopathology features The lungs were excised, fixed with 10% formalin, embedded in paraffin, sectioned at 5 µm thickness, and stained with hematoxylin and eosin. Eosinophils, neutrophils, macrophages, and lymphocytes were identified by morphologic criteria 25 and enumerated by highpower microscopy ( 200) in bronchial and bronchiolar walls, perivascular tissues, and alveoli. Data were expressed as the average number of cells per highpower field from 5 different fields for each lung structure. Investigators involved with pathology and pulmonary physiology studies were masked to experimental interventions. Statistical analysis Comparisons among experimental conditions were performed using one-way ANOVA and covariance. When a significant difference was found, the Tukey s test was performed to determine significant differences between different experimental conditions. Probability (P) values of less than.05 were accepted as statistically signif-

3 J ALLERGY CLIN IMMUNOL VOLUME 104, NUMBER 1 Shardonofsky et al 217 FIG 2. Lung histopathology. A, Lung section from a sham-sensitized and challenged mouse shows bronchioli, small pulmonary vessels, and alveoli with no evidence of inflammation. B, Lung section from an ovalbumin-sensitized mouse shows prominent inflammatory cell infiltrates composed of eosinophils, macrophages, and lymphocytes surrounding bronchioli and vessels. Patchy alveolar inflammatory cell infiltrates are seen. Inflammatory cells and debris are observed within the airway lumen. C, Lung section from an ovalbumin-sensitized mouse treated intranasally with an anti IL-5 mab (TRFK-5). There is a marked decrease of the intensity of inflammatory infiltrate in comparison with that seen in the ovalbumin-sensitized animal that received no mab treatment (B). D, Lung section from an ovalbumin-sensitized and challenged mouse treated intranasally with an isotype-matched control mab (GL-113). Peribronchiolar and perivascular inflammatory changes are similar to those observed in the ovalbumin-sensitized and challenged mouse that received no mab treatment (B). Lungs were fixed after BAL was performed. (Hematoxylin and eosin stain; original magnification: 35.) icant. Analysis was performed with the SPSS statistical package (SPSS Inc). Values are means ± SEM unless otherwise specified. RESULTS Cellular changes in BALF The values for total leukocyte number and differential leukocyte count in BALF are shown in Fig 1. The ovalbumin sensitization and airway challenges of mice resulted in significant increases (P <.05) in both the total cell number and percentage of eosinophils and a decrease in the percentage of macrophages (P <.05) in BALF relative to those obtained in sham-sensitized/challenged counterparts. No statistically significant differences were observed for the percentage of neutrophils and lymphocytes among any of the experimental groups. When ovalbumin-primed mice were treated with TRFK- 5 that was delivered either intranasally or intraperitoneally before ovalbumin airway challenges, the total number of cells and the percentage of eosinophils recovered from BALF were significantly decreased (P <.05), and the percentage of macrophages were increased (P <.05) relative to those obtained in ovalbumin-sensitized and challenged animals that received no mab treatment (Fig 1). The BALF cellularity in ovalbumin-sensitized and challenged mice treated with GL-113 intranasally was similar to that found in ovalbumin-sensitized and challenged mice that received no mab treatment. Lung histopathologicy Lung sections from representative mice that received either sham or ovalbumin sensitization and challenge are shown in Figs 2 and 3, A and B, respectively. The ovalbumin-sensitized and challenged mice had a dense subepithelial, peribronchiolar, and perivascular inflammatory cellular infiltrate composed of eosinophils, macrophages, and lymphocytes. Patchy cellular infiltrates were also noted in the lung parenchyma. Mucus and debris were observed in the airway lumina. By contrast, sham-sensitized and challenged mice lacked such inflammation. The number of leukocytes in lung tissues obtained in the experimental groups are displayed in Fig 4. Mice sensitized and challenged with ovalbumin had significantly more eosinophils and lymphocytes per high-power field in the airway s wall (P <.05), perivascular tissues (P <.05), and alveoli (P <.05) and an increase in the number of macrophages per high-power field in bronchioli (P <.05) and perivascular tissues (P <.05) compared with those obtained in sham-sensitized and challenged mice. The intranasal treatment of ovalbumin-primed mice

4 218 Shardonofsky et al J ALLERGY CLIN IMMUNOL JULY 1999 FIG 3. Lung histopathology. A, Lung section from a sham-sensitized and challenged mouse shows no inflammatory changes. B, Lung section from an ovalbumin-sensitized mouse. Inflammatory cells, including eosinophils, macrophages, and lymphocytes, infiltrate the interstitium and adventitia of a bronchiole. The latter contains debris within its lumen. C, Lung section from an ovalbumin-sensitized mouse treated intranasally with an anti IL-5 mab (TRFK-5). The magnitude of the peribronchiolar inflammatory cellular infiltrate is substantially decreased relative to that observed in the ovalbumin-sensitized and challenged mouse that received no mab treatment(b). D, Lung section from an ovalbumin-sensitized and challenged mouse treated intranasally with an isotype-matched control mab (GL-113). A bronchiole whose lumen is filled with mucus is encased by a dense inflammatory infiltrate similar to that seen in ovalbumin-sensitized and challenged mice that received no mab treatment (B). Lungs were fixed after bronchoalveolar lavage was performed. (Hematoxylin and eosin stain; original magnification, 70.) with TRFK-5 was associated with a significant reduction in the magnitude of the lung tissue inflammatory cell infiltration elicited by ovalbumin airway challenges (Figs 2; 3, E; and 4). In these animals, the numbers of eosinophils and lymphocytes in airways and perivascular tissues were significantly decreased (P <.05) relative to those obtained in ovalbumin-sensitized and challenged mice that received no mab treatment. The effects associated with TRFK-5 delivered intranasally were similar to those obtained with intraperitoneal injections of TRFK-5, except that the number of eosinophils per high-power field in bronchi and perivascular tissues was lower (P <.05) with intranasal, compared with intraperitoneal, TRFK-5 treatment (Fig 4). Whereas the changes of lung tissue eosinophil numbers induced by the experimental interventions paralleled the changes of eosinophil counts in BALF, the variations in the number of lymphocytes and macrophages in lung tissues were not reflected in the corresponding cell counts in BALF. GL-113 delivered intranasally to antigen-sensitized animals did not alter the inflammatory response induced by respiratory antigen challenges (Figs 2 and 3, E). GL-113 treated animals showed a greater number of macrophages per high-power field in lung tissues than ovalbumin-primed and challenged animals that received no mab treatment (Fig 4). Airway responsiveness to methacholine Compared with sham-sensitized and challenged mice, the average response to methacholine of ovalbumin-sensitized and challenged mice was significantly enhanced at doses of methacholine greater than or equal to 200 µg kg 1 (Fig 5). When ovalbumin-primed mice received TRFK-5 either intranasally or intraperitoneally before airway challenges with ovalbumin, Rrs responses to methacholine were significantly lower than those obtained in ovalbumin-sensitized and challenged mice that were not treated with mab and similar to those observed in sham-sensitized and challenged mice. TRFK-5 treated mice showed average values for PD 100 and MR: that were significantly greater and lower, respectively, than those obtained in ovalbuminsensitized and challenged animals that were not treated with an mab (log 10 PD 100 : 1.35 ± 0.17 µg kg 1 vs 0.96 ± 0.13 µg kg 1 [P <.05]; log 10 MR: 2.91% ± 0.08% vs 3.26% ± 0.08% [P <.05]). The indices of airway responsiveness for the former were not different from those in sham-sensitized mice. The administration of the GL-113 control mab caused no significant changes in the degree of airway responsiveness of the murine model (Fig 5).

5 J ALLERGY CLIN IMMUNOL VOLUME 104, NUMBER 1 Shardonofsky et al 219 A B C FIG 5. Effects of anti IL-5 mab on airway responsiveness to methacholine. Data are expressed as means ± SEM of methacholine dose/airway response relationships using Rrs as an index of airway caliber from sham-sensitized and challenged mice (n = 9), ovalbumin-sensitized and challenged mice that either were not treated with mab (n = 10) or were treated with anti IL-5 mab (TRFK-5) intranasally (n = 11) or intraperitoneally (n = 10), and from ovalbumin-sensitized and challenged mice treated with an isotype-matched control mab (GL-113; n = 10). S indicates administration of saline solution. ANOVA followed by Tukey s test: * Different from sham-sensitized and challenged mice (P <.05) and different from nonsensitized mice (P <.05); different from ovalbumin-sensitized and challenged mice not treated with mab or treated with GL-113 mab (P <.05). ip, Intraperitoneally. 40 µg ml 1, whereas mice given TRFK-5 intranasally had serum concentrations of 1226 ± 165 ng ml 1. Serum obtained from the same mice before ovalbuminsensitization and TRFK-5 treatment had no detectable TRFK-5. FIG 4. Number of leukocytes in lung tissue. Data are expressed as means ± SEM of number of eosinophils (A), lymphocytes (B), and macrophages (C) per high-power field (original magnification, 450) in bronchi (Br), bronchioli (Bl), alveoli (Alv), and perivascular tissues (PeriV) of the lungs from sham-sensitized and challenged mice (n = 9) from ovalbumin-sensitized and challenged mice that either were not treated with mab (n = 10) or were treated with anti IL-5 mab (TRFK-5) intranasally (n = 11) or intraperitoneally (ip; n = 10), and from ovalbumin-sensitized mice treated with an isotype-matched control mab (GL-113; n = 10). ANOVA followed by Tukey s test: * Different from sham-sensitized and challenged mice (P <.05) and different from nonsensitized mice (P <.05); different from ovalbumin-sensitized and challenged mice not treated with mab or treated with GL-113 mab (P <.05). TRFK-5 serum concentrations Serum concentrations of TRFK-5 were determined 5 days after mice were given 100 µg of TRFK-5 intraperitoneally or intranasally. Mice given TRFK-5 intraperitoneally had TRFK-5 serum concentrations of more than DISCUSSION These studies in a murine model of asthma demonstrate that delivery of anti IL-5 mab into the respiratory tract can significantly attenuate airway eosinophilic inflammation and hyperresponsiveness. There are only a few published studies on the therapeutic effects of neutralizing mab delivered into the airways in animal models of pulmonary inflammation. Our results are consistent with those of Abraham et al 26 who have shown that an aerosolized neutralizing mab to α 4 -integrins is effective in blocking the late-phase allergic response and airway inflammation in a sheep model of asthma. Similarly, Henderson et al 27 have demonstrated that the intranasal delivery of an anti-cd49d mab abrogates the asthma phenotype in a murine model. In our studies, the decline in the lung tissue and BALF eosinophilia and the decrease in the severity of lung histopathologic changes associated with anti IL-5 mab treatment likely reflect neutralization of the IL-5 promoting effects on eosinophil biology. However, we also

6 220 Shardonofsky et al J ALLERGY CLIN IMMUNOL JULY 1999 observed a decrease in the number of lymphocytes in bronchi and perivascular tissues. This may have resulted from a decrease in the number and biologic function of eosinophils, because eosinophils may also act as antigenprocessing cells and activate CD4 + T cells after cytokine priming at the site of inflammation. 28 The dose of anti IL-5 mab used either intranasally or intraperitoneally in our mice was identical to that injected intraperitoneally or intravenously in previously published studies and was at least as efficacious. Most intriguing was the tendency for even greater efficacy of intranasal anti IL-5 mab on bronchial and perivascular eosinophilic inflammation (Fig 4). These differential effects on inflammation, depending on the route of administration of an IL-5 antagonist, are in contrast to the similar effects of either route of administration on airway responsiveness (Fig 5). The true amount of mab delivered to the respiratory epithelia in our mice remains to be studied, because variable proportions of anti IL-5 mab given intranasally likely remained in the nasal cavity, were cleared from the nose by the mucociliary escalator, were inhaled and partly expectorated, or swallowed. Absorption of the mab through the respiratory epithelium was confirmed by the serum concentrations of the anti IL-5 mab, which were lower than those achieved in our mice after intraperitoneal delivery but above the reported minimum serum concentration of anti IL-5 mab needed to inhibit IL-5 induced peripheral blood eosinophilia in mice. 29 The relationship between airway eosinophilic inflammation and bronchial hyperresponsiveness may be partly related to dysfunction of inhibitory M2-muscarinic autoreceptors resulting from positively charged products of eosinophil activation in the airways 30 and activation of airways neurokinin-2 receptors. 31 The linkage between airway dysfunction and IL-5 mediated pulmonary eosinophilia is further supported by an elegant study by Foster et al, 11 showing that IL-5 knockout mice were unable to mount a pulmonary eosinophilic response or develop airway hyperresponsiveness after antigen sensitization and challenge. The asthma phenotype was restored in those mice when an IL-5 producing vaccinia virus was delivered to the lungs. However, airway hyperresponsiveness has been observed in models of asthma in the absence of eosinophil accumulation in the airways, 13,32,33 suggesting that eosinophil-independent mechanisms may also contribute to airway hyperresponsiveness. Such eosinophilindependent (and IL-5 independent) mechanisms contributing to airway hyperresponsiveness include leukotrienes 32 and IL Recently, a role for CD4 + T cells independent of IL-4 and IL-5, perhaps by means of the production of IL-13, 35,36 has also been shown to be important for the development of airway hyperreactivity. In addition, the genetic background may affect the contribution of various pathways to the development of airway hyperreactivity. 34 Thus although a pivotal role of the eosinophil in asthmatic inflammation is well established, 1,5,8,16,30,31,33,38 the causal role of eosinophils in airway hyperresponsiveness remains controversial. In conclusion, this is the first study to demonstrate that respiratory delivery of anti IL-5 mab can significantly attenuate pulmonary eosinophilic inflammation and airway hyperresponsiveness. Although IL-5 likely acts in concert with other proinflammatory molecules, including chemokines and other cytokines, in the pathophysiologic features of asthma, our data demonstrate that neutralization of IL-5 related effects has therapeutic relevance and, importantly, that therapeutic efficacy can be achieved by delivering IL-5 antagonists via the airways. We thank Dr Joe Rodarte for advice and critical review, Jana Clark for editing the original manuscript, and J. 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