Eosinophils are not required to induce airway hyperresponsiveness after nematode infection

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1 2640 A.J.Coyleetal. Eur.J.Immunol : Eosinophils are not required to induce airway hyperresponsiveness after nematode infection Anthony J. Coyle 1, Gabriele Köhler 2, Shogo Tsuyuki 3, Frank Brombacher 4 and Manfred Kopf 5 1 Department of Biology, Inflammation division, Millenium Pharmaceuticals, Cambridge, Boston, USA 2 Department of Pathology, University of Freiburg, Freiburg, Germany 3 R&D Department Kissei Pharmaceutical Co. Ltd., Matsumoto, Japan 4 Max-Planck Institute for Immunobiology, Freiburg, Germany 5 Basel Institute for Immunology, Basel, Switzerland Eosinophilic inflammation of the airways is believed to play a central role in the pathogenesis of bronchial asthma. Inoculation of mice with the nematode Nippostrongylus brasiliensis induces pulmonary inflammation, characterized by a marked infiltration of eosinophils, subsequent to the migration of parasites through the lungs. Infection is associated with polarized Th2 responses in different strains of mice tested. Thus, this model may be useful to determine the relationship between established pulmonary eosinophilic inflammation, Th2 immune responses and airway changes in a nonallergic background. In the present study, we have used IL-5-deficient mice to evaluate the role of IL-5 in eosinophilic lung inflammation and airway hyperresponsiveness (AHR). In wild-type C57BL/6 mice, infection with N. brasiliensis resulted in eosinophil accumulation, associated with extensive lung damage characterized by hemorrhage and alveolar wall destruction, and a strong AHR following methacholine treatment. In IL-5-deficient mice, eosinophil infiltration and the associated lung damage was abrogated. Nonetheless, AHR was unimpaired. Our results suggest that eosinophil accumulation plays a central role in lung damage but is not responsible for the induction of airway constriction following N. brasiliensis infection. Key words: IL-5 / Eosinophil / Lung inflammation / Asthma Received 16/4/98 Accepted 18/5/98 1 Introduction There is now a substantial body of evidence to suggest that airway inflammation, characterized by an infiltration of Th2 cells, eosinophils and mast cells, is a central event in the pathogenesis of asthma [1, 2]. Th2 cells produce a panel of cytokines including IL-4, IL-5, IL-10 and IL-13, which together regulate an allergic type of response. IL-4 is responsible for the production of specific IgE antibodies, which by cross-linking Fc 4 RI on mast cells and basophils induce the release of mediators such as histamine and leukotrienes. IL-5 effects the maturation, survival and migration of eosinophils [3 7]. In situ hybridization for IL-5 in biopsies from asthmatic patients showed a correlation between IL-5 detection, eosinophil number, and the clinical severity of the disease [8]. Activated Abbreviations: AHR: Airway hyperresponsiveness Bronchoalveolar lavage [I 18260] BAL: eosinophils and mast cells elaborate inflammatory mediators including eicosanoids, histamine, platelet activating factor (PAF), and various proteases [9, 10]. It has been postulated that the secretion of an array of highly charged cationic proteins such as major basic protein (MBP) by eosinophils plays an important role in the induction of asthma [11 13]. Although the cellular and inflammatory changes accompanying asthma are fairly well documented, the mechanism by which activated eosinophils and mast cells contribute to the physiological changes that we recognize as asthma, namely lung inflammatory damage, reversible airflow limitation, and airway hyperresponsiveness (AHR), still remains to be fully elucidated. In particular, it remains to be demonstrated that eosinophil accumulation is directly responsible for the associated tissue damage and the asthmatic response. The most commonly used mouse model for the study of extrinsic asthma involves systemic OVA sensitization and repeated aerosol provocation followed by a bron /98/ $ /0 WILEY-VCH Verlag GmbH, D Weinheim, 1998

2 Eur. J. Immunol : Asthma in the absence of eosinophils 2641 choconstrictor agent such as methacholine. However, by using this immunization regimen, the development of exaggerated airway responsiveness is dependent on the genetic background and only observed in Th2 responder strains (e.g. BALB/c, A/J) [2, 14, 15]. Infection with the nematode Nippostrongylus brasiliensis is a well-established model to investigate Th2 responses. Infective third stage (L3) larvae are injected through the skin and migrate to the lungs (days 1 2), where a strong eosinophilic inflammatory response is induced. Larvae are coughed up and swallowed (days 2 4), and mature into egg-laying adults in the jejunum (days 5 8). Adult worms are expelled by day 9 11 after inoculation. Independent of the genetic background of mice, infection is accompanied by blood and lung eosinophilia, intestinal mastocytosis, and high IgE levels, the three cardinal features of a polarized Th2 response [16, 17]. In the present study, we have used N. brasiliensis to establish an animal model of pulmonary inflammation and AHR on a nonallergic background. Further, we have used this model to elucidate the role of IL-5 and eosinophils in lung tissue damage and airway changes in IL-5- deficient C57BL/6 mice [18]. 2 Results 2.1 Impaired eosinophil production in the bone marrow and a lack of eosinophil accumulation in the blood and in the BAL fluid of infected IL-5-deficient mice We followed the time course of eosinophilia in mice infected with N. brasiliensis. Prior to infection, there were no significant differences in the number of eosinophils in theblood(17.1± eosinophils/ml and 16.8 ± eosinophils/ml, n j 12 14, p G 0.05) (Fig. 1A) or bone marrow (3.2 ± eosinophils/ml and 3.6 ± eosinophils/ml, n j 3, p G 0.05) (Fig. 1B) between wild-type and IL-5-deficient mice, respectively. Following infection, the number of eosinophils both in the blood and bone marrow increased sixfold in wildtype mice. In contrast, IL-5-deficient mice failed to develop either a an increased number of eosinophils in the bone marrow (Fig. 1B) or peripheral blood eosinophilia (Fig. 1A). Infection of wild-type mice increased the number of eosinophils in the bronchoalveolar lavage (BAL)atday13(312± /ml, n j 4) compared to noninfected mice (0.1 ± /ml, n j 5, p X 0.01). In contrast, eosinophil infiltration was abrogated in the lungs of IL-5-deficient mice (noninfected 0.1 ± /ml, n j 3; infected 1.0 ± /ml, n j 3, p G 0.05). There was also a small, but significant Figure 1. Eosinophil numbers in peripheral blood (A), bone marrow (B) and BAL (C) from wild-type mice (closed columns) or IL-5-deficient mice (open columns) at days indicated following infection with N. brasiliensis. Results are expressed as eosinophils/ 10 3 ml and represent data from n j 3 5 mice. Statistical significance was determined by a Student s t-test and shown as * between noninfected and c between wild-type mice and IL-5-deficient mice with a p value of X 0.05 considered significant. increase in the number of neutrophils and lymphocytes in the BAL after infection in wild-type mice, which was also observed in the BAL fluid of IL-5-deficient mice (Fig. 2). Likewise, the macrophage number was increased twofold in the BAL fluid of wild-type mice after infection (noninfected ± /ml, n j 4, infected ± /ml, p X 0.05). However, this increase in macrophage number was not observed in the BAL fluid of infected IL-5-deficient mice (noninfected ± /ml, infected ± /ml, n j 4, p G 0.05). From these results, we conclude that the mechanism by which IL-5-deficient mice have an

3 2642 A.J.Coyleetal. Eur.J.Immunol : lungs as illustrated under high power magnification in Fig. 3 (middle panel). We next examined whether these eosinophils exhibited normal morphological characteristics. As shown in Fig. 3 (lower panel), lung eosinophils from IL-5-deficient mice exhibited normal morphology with large specific granules with electron-dense core and band-shaped nucleus shown as two separate lobes and were similar to those identified in the lungs of wild-type mice (Fig. 3, lower panel). 2.4 IL-5-deficient mice develop AHR after infection Figure 2. Cellular composition of the BAL fluid before (closed bars) or day 13 post infection with N. brasiliensis (hatched bars) in wild-type (WT) and IL-5-deficient mice. Data shown are for n j 3 5 mice per group and expressed as mean ± SEM of cells/ 10 3 ml. Significance (p X 0.05) wasdeterminedbyastudent st-test and indicated as * betweenday0andday13or c between wild-type and IL-5- deficient mice. attenuated lung eosinophil infiltration is related to a failure to generate eosinophils in the bone marrow and that IL-5 is not involved in the accumulation of eosinophils in the lungs per se. To understand more fully the relationship between eosinophil-mediated histopathological changes in the lung and AHR, we recorded the breathing pattern in unrestrained mice in response to aerosolized methacholine. N. brasiliensis-infected compared to uninfected wild-type mice showed a strong increase in AHR, which correlated to increasing concentrations of methacholine inhaled (Fig. 4). Infected IL-5-deficient mice showed an airflow obstruction, which was comparable to that in wild-type mice. We conclude that infection with N. brasiliensis is sufficient to induce AHR in C57BL/6 mice without a requirement for intranasal antigen challenge. AHR is induced in the absence of eosinophils and obvious histopathological changes but in the presence of a strong local IL-4 response (not shown). 2.2 IL-5-deficient mice have a markedly reduced lung damage after infection Histological examination of lung tissue from infected wild-type mice supported the data obtained by BAL and revealed intense multifocal eosinophilic lesions which were mainly perivascular, but also marked in the bronchial submucosa and lung interstitium. In contrast, eosinophilic inflammation was abrogated in the lungs obtained from IL-5-deficient mice (Fig. 3, upper panel). In addition to the eosinophilic inflammation, the lung response to infection with N. brasiliensis was characterized by severe hemorrhage, pulmonary edema and destruction of the alveolar septal wall. In contrast, there was a strikingly reduced degree of histopathologic changes in the lungs obtained from IL-5-deficient mice (Fig. 3, upper panel). 2.3 Electron microscopy of lung eosinophils reveals normal morphology It is important to note that while eosinophil infiltration was greatly reduced in the lungs of IL-5-deficient mice, we were still able to identify some eosinophils in the 3 Discussion In the present study, we have used a well-characterized model, namely infection with the nematode N. brasiliensis, to investigate the role of IL-5 in lung eosinophilic inflammation using IL-5-deficient mice. In agreement with previous studies, the results of this study indicate that IL-5 plays a critical role in the development of blood and tissue eosinophilia following infection [4, 18]. This defect appears to be at the level of the bone marrow as following infection, IL-5-deficient mice also exhibited a greatly reduced capacity to generate eosinophils. Nonetheless, IL-5 is not essential for eosinopoiesis under control conditions, since basal numbers of eosinophils in the peripheral blood and bone marrow in IL-5-deficient mice were comparable to that observed in wild-type mice prior to infection. It is important to note that occasionally some eosinophils were detected in the lungs in IL-5- deficient mice after infection. Electron microscopy on lung tissue from these mice indicated that these eosinophils exhibited normal morphology with intact characteristic electron-dense core. In view of the observation that the basal number of eosinophils in these mice is normal, we speculate that these eosinophils found in the lungs of

4 Eur. J. Immunol : Asthma in the absence of eosinophils 2643 Figure 3. Photomicrograph of lungs taken on day 13 from N. brasiliensis-infected wild-type (a,c,e) or IL-5-deficient (b,d,f) mice. Upper panel, magnification 6.25; middle panel, magnification 250; lower panel, electron microscopy of eosinophils from the lungs, magnification Photographs are representative of lungs from three to four animals. IL-5-deficient mice are derived from the basal circulating pool of eosinophils in the blood which are recruited into the lungs following infection and that the signals for eosinophil tissue recruitment are intact in IL-5-deficient mice. Indeed, eotaxin, an eosinophil-specific chemokine, was expressed comparably in lungs of IL-5- deficient and wild-type mice during the course of infection (not shown). The reduced cellular infiltrate observed in the BAL fluid of IL-5-deficient mice was due principally to a reduced eosinophil accumulation with no significant reduction in either neutrophil or lymphocyte number in the lung, indicating that these cells migrate into the airways independent of IL-5. However, while infection resulted in an increase in the number of macrophages in the BAL fluid of wild-type mice, this was not observed in IL-5-deficient mice. As macrophages have been demonstrated to play an important role in immune surveillance by engulfing unwanted cells in an attempt to resolve inflammatory responses [19, 20], we speculate that the reduced macrophage number in IL-5-deficient mice may be a consequence of abrogated eosinophil accumulation. However, further studies are required to more precisely elucidate the mechanisms by which macrophages infiltrate into the lung after infection. The data obtained by BAL are further supported by histological examination of lung tissue. Infection of wildtype mice induced an intense inflammation of the lungs

5 2644 A.J.Coyleetal. Eur.J.Immunol : Figure 4. AHR on day 13 after infection with N. brasiliensis. Airway reactivity was measured immediately after exposure to incremental doses of aerosolized methacholine in infected and control uninfected mice by whole body plethysmography (Buxco ). Values for the enhanced pause (PenH) were calculated and used as index of airway responsiveness (see Sect. 4.6). with focal lesions rich in eosinophils surrounding blood vessels, airways and in the interstitial parenchyma. Moreover, in agreement with previous observations [21], there was also evidence of widespread lung tissue damage characterized by pulmonary edema, hemorrhage and destruction of the alveolar septal walls. In contrast, eosinophil infiltration was not only abrogated in the lungs of IL-5-deficient mice, but there was a marked reduction in the associated lung destruction. These observations provide direct evidence of the causal link between IL-5 eosinophil infiltration and tissue damage observed in the lungs after infection with N. brasiliensis. Eosinophils have the capacity to secrete an array of preformed highly toxic cationic proteins such as MBP, which has been shown to induce airway constriction [11], and eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO), the latter of which has the property to catalyze the formation of hydrogen peroxide and toxic hypohalide radicals, in particular HOBr ions [22]. However, while it has been demonstrated that these eosinophil-derived mediators can induce damage to epithelial cells in vitro [13], their precise involvement in eosinophil-mediated lung damage following infection remains to be determined. The association between eosinophilic inflammation and bronchial asthma has long been recognized and a body of evidence suggests that accumulation of eosinophils in the lungs of individuals with bronchial asthma is a central event in the pathogenesis of this disease. In view of the observed relationship between eosinophil infiltration and lung damage after infection, we next determined whether eosinophilic inflammation of the lungs was associated with AHR. Infected C57BL/6 mice showed a strong increase in AHR, which was comparable in intensity to AHR measured in BALB/c mice after repeated OVA aerosol challenge (data not shown). Interestingly, AHR was unimpaired in IL-5-deficient mice lacking pulmonary eosinophilic inflammation and associated tissue damage. This demonstrates that eosinophils were not responsible for AHR induced by parasite infection. However, we cannot exclude the possibility that the presence of some apparently intact eosinophils in lungs of IL-5- deficient mice was sufficient to induce full AHR. The importance of eosinophils for the induction of AHR has been a matter of great debate. Studies utilizing anti-il-5 mab have reported abrogated eosinophil influx in the absence of attenuated AHR [14, 23]. In contrast, OVA challenge of IL-5-deficient mice has been reported to inhibit eosinophil influx and AHR [24]. The reason for these conflicting results may reside in the different genetic mouse strains used for the experiments [2]. It has previously been shown that continuous administration of soluble protein antigen resulted in the selective development of Th2 cells in BALB/c but not in C57BL/6 mice [25, 26]. BALB/c Th2 commitment is due to intrinsic extinction of IL-12 signaling [27 29]. In accordance with this, OVA sensitization and repeated aerosol challenge induces consistently greater AHR in BALB/c compared to C57BL/6 mice ([14], our unpublished results). Inter-strain differences in the ability to develop Th2 cells are not seen after N. brasiliensis infection, at least not when comparing C57BL/6, 129Sv, and BALB/c mice (M. Barner, M. Kopf, unpublished). In this context, this model may be useful to test airway changes in different knockout mice, which are commonly generated on an asthmahyporesponsive background (e.g. 129Sv, C57BL/6). We are currently investigating which cytokines regulate airway responses after N. brasiliensis infection. Conversely, the possibility exists that migrating larvae directly damage the epithelium leading to AHR as has been proposed for exposure to chemical irritants such as ozone [30] or toluene diisocyanate [31]. However, it should be noted that increased AHR was maintained for more than 1 week after the larvae disappeared from the lungs. Furthermore, exposure of mice to Schistosoma mansoni, another parasitic helminth, which migrates intravascularly to lodge in the lung, causes lung eosinophilia but does not increase AHR (A. Finlay, A. Wilson, unpublished results). Notably, S. mansoni induces pulmonary eosinophil influx in the presence of a Th1-biased response (high IFN- +, low IL-4 and IgE levels). In conclusion, we have demonstrated that eosinophil infiltration into the lungs is dependent on IL-5. The primary role of this cytokine is to generate eosinophils in

6 Eur. J. Immunol : Asthma in the absence of eosinophils 2645 the bone marrow in response to infection and not for the accumulation of eosinophils in the lungs per se. Moreover, we demonstrate that eosinophils cause the pathological changes in lung tissue but are not responsible for airway responses following N. brasiliensis infection. This infection animal model expressing AHR can provide useful information on the importance of specific mechanisms in the development of asthma. 4 Materials and methods Lungs were prepared for histological examination on day 13 after infection. Mice were anesthetized with urethane, the trachea cannulated, the chest opened and the lungs perfused via the trachea with 4 % buffered formaldehyde in PBS (ph 7.0, formal saline) at exactly 15 cm H 2 O maintained by a pressure seal for 60 min. The fixed lungs were trimmed off the heart and large blood vessels and removed en bloc into formal saline and processed into paraffin. Sections (15? m) were cut and stained with hematoxylin and eosin and examined at low power to study gross histopathological changes. To investigate regions of further interest, slides were stained with Biebrich Scarlet [32] and examined at higher power ( 250). Slides were photographed using a Zeiss Axiophoto photomicroscope. 4.1 Mice IL-5-deficient mice (C57BL/6) used in this study were generated using conventional gene targeting techniques and have been previously described in detail [18]. C57BL/6 control mice were provided by IFFA-Credo (Saint Germain-surl Abresle, France). Mice were maintained pathogen free at the Basel Institute for Immunology. For experiments week-old mice of either sex were used and kept in microisolator cages. 4.2 Infection with N. brasiliensis N. brasiliensis wasmaintainedbypassagethroughtifrai rats. Mice were inoculated with 750 L3 infective larvae by s.c. injection. Blood was taken from the tail vein either before infection or at day 4, 7, 10 and 13 post infection. Total cell counts and blood smears were performed, stained with Diff-Quik R (Baxter Dade AG., Dudingen, Switzerland) and a total of 200 cells counted differentially using standard morphological criteria. 4.3 Quantification of eosinophil accumulation Bone marrow eosinophil number was assessed by washing the bone marrow from each femur with 1 ml of heparinized PBS, either before or on day 7 and day 13 after infection. Total counts were made and cytospin preparations prepared (Shandon Scientific Ltd., Cheshire, GB), stained and the percentage of eosin-positive cells determined. To assess the accumulation of eosinophils in the lung after infection with N. brasiliensis, mice were anesthetized with urethane and the trachea cannulated. BAL was performed by injecting 0.3 ml PBS into the lungs via the trachea. The fluid was then immediately withdrawn and this procedure repeated a total of four times and the number of eosinophils quantified as described above. 4.4 Lung histology 4.5 Electron microscopy analysis Lung tissue was fixed for 12 h at 4 C in 3 % cacodylatebuffered glutaraldehyde at ph 7.35 and then transferred to a 5 % saccharose solution. Postfixation was performed with 1 % osmium tetroxide and 50 mm potassium ferricyanide. The specimens were then washed with distilled water, dehydrated in graded alcohols and embedded in araldit resin. Ultrathin sections of 50 nm were cut, placed on copper grids, stained with 5 % uranyl acetate and 0.2 % lead citrate and examined using a Philips CM10 (Philips, Eindhoven, The Netherlands) electron microscope operating at 80 kv. 4.6 Measurement of airway responsiveness Airway responsiveness was assessed in infected and uninfected mice by inducing airflow obstruction with a methacholine aerosol using a noninvasive method [33, 34]. This procedure estimates total pulmonary airflow in the upper and lower respiratory tracts. Minute volume, tidal volume, breathing frequency, and enhanced pause (PenH) were determined from conscious mice placed in a whole body plethysmograph (Buxco, EMKA Technologies, Paris, France). The chamber pressure was used as a measure of the difference between thoracic expansion (or contraction) and airvolume removed from (or added to) the chamber during inspiration (or expiration). Mice were placed in each chamber and pulmonary airflow obstruction was assessed by measuring PenH using the following formula as recommended by the manufacturer: PenH j ([T E /0.3T R ] 1) (2PEF/3PIF), where PenH j enhanced pause (dimensionless), T E j expiratory time (s), T R j relaxation time (s), PEF j peak expiratory flow (ml/x), PIF j peak inspiratory flow (ml/ x). The breathing pattern was collected for 2 min and an average of each variable was derived from 30 s. The peak PenH value was recorded. Measurements of methacholine responsiveness were obtained by exposing mice for 30 min to saline followed by incremental doses of aerosolized methacholine (Aldrich Chemie, Steinheim, Germany) and monitoring the breathing pattern for 10 min after challenge.

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