Characterization of Lung Hyperresponsiveness, Inflammation, and Alveolar Macrophage Mediator Production in Allergy Resistant and Susceptible Rats

Characterization of Lung Hyperresponsiveness, Inflammation, and Alveolar Macrophage Mediator Production in Allergy Resistant and Susceptible Rats Eric Careau, Jocelyne Sirois, and Elyse Y. Bissonnette Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l Université Laval, Québec, Canada To better understand asthma pathogenesis, we characterized airway responsiveness, lung inflammation, and mediator production of alveolar macrophages (AM) after allergen sensitization and challenge in two strains of rats showing different susceptibilities in developing airway allergic reactions. Airway responsiveness to acethylcholine was measured 24 h after ovalbumin (OVA) challenge, whereas bronchoalveolar lavages were performed 5 min, 8 h, and 24 h after challenge. Brown Norway rats showed airway hyperresponsiveness after challenge, whereas lung resistance remained unchanged in Sprague- Dawley rats. Interestingly, Sprague-Dawley rats developed a neutrophilic inflammation, whereas both neutrophils and eosinophils were increased in Brown Norway rats. AM mediator production varied with time with a lower tumor necrosis factor (TNF) and interleukin (IL)-10 release at 8 h after challenge. OVA challenge stimulated spontaneous TNF and IL-10 release by AM isolated 24 h after challenge in both strains of rats, although AM from Brown Norway rats released significantly more IL-10 and TNF. Furthermore, nitric oxide production was increased only in OVA-challenged (24 h) Brown Norway rats. Our results suggest that AM may participate to the expansion of Th2 inflammation in Brown Norway rats and that differences in AM mediator production may explain, in part, distinct allergic susceptibilities in these two strains of rats. Asthma is a chronic illness characterized by inflammation, reversible airway obstruction, and increased airway responsiveness to various stimuli (1). There is growing evidence suggesting an important role for T helper (Th)2 cytokine-producing cells in asthma pathogenesis (2). A shift in Th1/Th2 balance has been demonstrated in asthmatic subjects (3) and in ovalbumin (OVA)-sensitized and -challenged Brown Norway rats (4). Although T cells are well known to produce these cytokines and being involved in asthma pathogenesis (5), other cell types may be important in Th2 cytokine production. Furthermore, the cytokine milieu in the lung may play a crucial role in determining the type of primary immune response, Th1 or Th2, to inhaled antigen. Alveolar macrophages (AM) are the most abundant cells in the alveoli, distal airspaces, and conducting airways. They are the first line of defense against infectious agents and other immunologic insults, and one of their functions is to downregulate the immune response in the (Received in original form October 2, 2001 and in revised form January 25, 2002) Address correspondence to: Dr. Elyse Bissonnette, Hôpital Laval 2725, chemin Sainte-Foy, Sainte-Foy, PQ, G1V 4G5 Canada. E-mail: elyse. bissonnette@med.ulaval.ca Abbreviations: acetylcholine, ACh; alveolar macrophages, AM; bronchoalveolar lavage, BAL; interferon-, IFN- ; immunoglobulin, Ig; interleukin, IL; macrophage inflammatory protein, MIP; nitric oxide, NO; ovalbumin, OVA; phosphate-buffered saline, PBS; pulmonary resistance, RL; T helper, Th; tumor necrosis factor, TNF. Am. J. Respir. Cell Mol. Biol. Vol. 26, pp. 579 586, 2002 Internet address: www.atsjournals.org lung (6). Depletion of AM in rats with immunologic memory results in a profound state of hyperresponsiveness to inhaled antigens characterized by an increased IgE response and an influx of activated T and B cells into the lung, showing the importance of AM in preventing airway allergic reaction development (7, 8). Interestingly, AM can produce both Th1 (tumor necrosis factor [TNF] and interleukin [IL]-12) and Th2 cytokines (IL-6, IL-10, and IL-13) in addition to a broad range of inflammatory mediators, including nitric oxide (NO), which has been shown to inhibit Th1 response (9 11). Although there is some evidence suggesting that TNF plays an important role in the development of Th1 response (12), TNF has also been shown to increase airway responsiveness in experimental animals and in human subjects, which raises questions as to its role in asthma pathogenesis (13, 14). In asthma, AM are activated (15) and show a decreased inhibitory effect of T cell proliferation (16). Furthermore, they release more proinflammatory cytokines and they enhance IL-5 production by Th cells (17, 18). Thus, AM may participate to create the cytokine milieu, predisposing the development of Th2 cells and promoting allergic inflammation. There is a well-established model of allergen-induced airway hyperresponsiveness in Brown Norway rats (19) that reflects many features of human allergic asthma, including both early and late (70% of animals) phase reactions, increase in antigen-specific IgE following active immunization (20), airway inflammation (21), and increased bronchial responsiveness to several stimuli following allergen challenge (22). In contrast, a low percentage of Sprague- Dawley rats develops an early airway response ( 20%) or detectable serum-specific IgE under the same conditions (23). Furthermore, we have demonstrated for the first time that stimulated AM from naive Sprague-Dawley rats produce significantly more TNF and less IL-10, IL-13, macrophage inflammatory protein (MIP)-1, and NO compared with AM from allergy susceptible Brown Norway rats, suggesting an important role for these cells in the pathogenesis of asthma (10). Using this model, we have investigated AM cytokine production in allergy resistant (Sprague-Dawley) and susceptible (Brown Norway) rats after allergen challenge. Sensitized animals were challenged with aerosolized OVA (5%) for 5 min and AM were isolated 5 min, 8 h, or 24 h later, corresponding to early phase, late phase, and eosinophilic inflammation, respectively (4, 19). Brown Norway rats showed airway hyperresponsiveness to acetylcholine (ACh) after OVA challenge compared with Sprague-Dawley rats, although the latter developed a neutrophilic inflammation. Airway inflammation, measured 24 h after challenge, was higher in Brown Norway rats and their AM released more TNF, IL-10, and NO compared with Sprague-Dawley rats,

580 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 26 2002 suggesting that AM may participate to the distinct allergy susceptibilities observed in these two strains of rats. Materials and Methods Animals Brown Norway rats (BN/Ssn) aged 72 82 d were obtained from Harlan-Sprague Dawley (Indianapolis, IN) whereas age matched Sprague-Dawley rats were obtained from Charles River (St. Constant, PQ, Canada). All animals were maintained in filter top cages in virus/antigen free conditions in Laval Hospital animal facility on a 12 h light/dark cycle. Animals were given food and water ad libitum. This experimental protocol was approved by University Laval Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. In vivo OVA Sensitization and Challenge Animals were sensitized by intraperitoneal injection of 1 ml OVA grade V (1 mg/ml; Sigma Chemical Co, St. Louis, MO) and Al(OH 3 ) (100 mg/ml; BDH Laboratory Supply, UK) in sterile 0.9% saline solution. Control animals received saline only. Three weeks later, animals were challenged with 5% aerosolized OVA. OVA was aerosolized for 5 min using a Hudson micromist 880 nebulizer (Hudson RCI, Temecula, CA) and an airflow of 6 liters/min air. Control animals were challenged with 0.9% saline solution. Measurement of Airway Responsiveness Measurement of airway hyperresponsiveness was performed with animals that were anesthetized intraperitoneally with an initial dose of 1.5 g/kg urethane (Sigma). Additional urethane was administered as required to maintain adequate anesthesia. Animals were laid on a heating pad and rectal temperature was continuously monitored and maintained between 36 and 38 C. A 6-cm length of PE240 tube (Becton Dickinson Diagnostics, Sparks, MD) was used as endotracheal tube. The end of the intratracheal tube was connected to a Quatra-T CombiChamber (Scireq; Montreal, PQ, Canada), which uses mouth flow and esophageal pressure measurements to accurately calculate pulmonary resistance (R L ). Changes in oesophageal pressure were measured using a saline-filled catheter PE160 (Becton Dickinson) connected to a differential pressure transducer, and mouth flow from intratracheal tube was measured by a pneumotachograph. R L was simultaneously calculated using the software package included with the Quatra-T apparatus. Animals were exposed to aerosolized doubling concentrations of ACh for 30 s and R L measurements were taken every min for 5 min. Peak value of R L was measured after each concentration and the challenge was stopped at 128 mg/ml ACh. The concentration of ACh inducing 200% increase of R L over the initial baseline (EC 200 R L ) was calculated by interpolation of concentration response curve from individual animal. AM Isolation AM were isolated as previously described (10). Cells from bronchoalveolar lavage (BAL) were centrifuged and resuspended in RPMI 1640 medium (Gibco BRL, Burlington, ON, Canada) supplemented with 5% fetal bovine serum (Sigma), penicillin (100 U/ml), streptomycin (100 U/ml) and 10 mm HEPES buffer (Gibco). Purity of AM was determined according to Diff-Quik coloration and nonspecific esterase staining. Minimum of 200 cells/ slide were counted to determine cell type. Viability always exceeded 95% according to Trypan blue exclusion. AM were further purified by adherence. Briefly, AM (10 5 /well) were incubated at 37 C for 2 h and wells were vigorously washed with RPMI 1640 medium, giving a purity of 98%. Cytokines AM were treated with anti-cd8 (OX8; Serotec, Kidlington Oxford, UK), known to stimulate AM cytokine production (10, 24), or isotype control at 5 g/ml for 4 h and 20 h for TNF and IL-10 release, respectively. Cell-free supernatants were tested for TNF and IL-10 content using immunoassay kit for rat TNF and IL-10 (Biosource International, Camarillo, CA) with a sensitivity of 4 pg/ml and 5 pg/ml, respectively. BAL fluids were concentrated 80 times using centrifugal filter devices Centricon PLUS-20 with ultracel PL-10 membranes (Millipore, Nepean, ON, Canada) before measuring TNF and IL-10 levels. Measurement of Nitric Oxide Production After AM treatment (48 h) with OX8 or isotype control at 5 g/ml, cell-free supernatants were assayed for NO 2 using Griess reaction as previously shown (25). NO 2 concentration, propor- tional to OD 540, was determined using a Molecular Device (Menlo Park, CA) V max Kinetic Microplate Reader with reference to a standard curve (NaNO 2 ). Statistical Analysis The hyperresponsiveness data were analyzed using a one-way analysis of variance, whereas data from BAL were analyzed using a two-way analysis of variance. Scheffe s multiple technique was used for a posteriori comparisons. The normality assumption was verified with the Shapiro-Wilk test and the Bartlett s statistic was used to verify the homogeneity of variances for each tested effect. Differences were considered significant when P was 0.05. The data were analyzed using the statistical package program SAS v8.2 (SAS Institute Inc., Cary, NC). Results Airway Hyperresponsiveness Sensitized Brown Norway and Sprague-Dawley rats were exposed to aerosolized OVA or saline for 5 min and airway reactivity to ACh was measured 24 h later. ACh concentration needed to provoke 200% increase of airway resistance (EC 200 R L ) in saline challenged Sprague-Dawley and Brown Norway rats was 60 11 mg/ml and 56 14 mg/ml respectively (Figure 1). OVA challenge did not modulate responsiveness to ACh of Sprague-Dawley rats (64 14 mg/ml). However, airway hyperresponsiveness was significantly higher in OVA challenged Brown Norway rats with an EC 200 R L of 27 10 mg/ml compared with 56 14 mg/ml in saline challenged rats. These data demonstrate the development of airway hyperresponsiveness after allergen challenge in OVA-sensitized Brown Norway rats, but not in Sprague-Dawley rats. Airway Inflammation BALs were performed at different times after OVA and saline challenge. Although Brown Norway rats had significantly more cells in BALs than Sprague-Dawley rats, total cell number of each strain of rat did not significantly change after saline challenge (Figure 2). OVA challenge significantly increased number of cells recovered at 24 h in both strains of rats. However, cell recovery was significantly higher in Brown Norway rats (10.5 1.7 10 6 cells) compared with Sprague-Dawley rats (7.0 0.8 10 6 cells). Interestingly, OVA challenge caused a significant decrease of cell number in BAL of Brown Norway rats 5 min

Careau, Sirois, and Bissonnette: Alveolar Macrophages in Asthma Pathogenesis 581 Figure 1. Concentration of acethylcholine (ACh) required to increase pulmonary resistance (R L ) to 200% of control value (EC 200 R L ). Animals were exposed to ovalbumin (OVA) 21 d after sensitization and ACh airway responsiveness was measured 24 h later. Mean SEM of five animals in each group. Results with different letters are significantly different. and 8 h after allergen challenge, whereas cell number remained unchanged in Sprague-Dawley rats. Saline challenge did not significantly modify cell profile in BAL, although a significant increase in lymphocytes was observed 24 h after saline challenge in both strains of rats (Table 1). An increase in eosinophil was observed at 8 h and 24 h after OVA challenge in Brown Norway rats, and at 24 h only in Sprague-Dawley rats, although the latter had 5.9-fold less eosinophils than Brown Norway rats. Interestingly, neutrophils were significantly increased in both strains of rats at 8 h and 24 h after allergen challenge. However, this increase in neutrophils was significantly higher in Sprague-Dawley rats compared with Brown Norway rats. Number of AM decreased at 5 min and 8 h after OVA challenge in Brown Norway rats and at 8 h in Sprague- Dawley rats, whereas a significant increase was observed at 24 h in both strains of rats (Table 1). Furthermore, there was a 2-fold increase in total number of lymphocytes in bronchoalveolar lavages 24 h after OVA challenge both strains of rats. Figure 2. Total number of cells in bronchoalveolar lavage (BAL) performed at different times after OVA challenge. Saline challenge did not significantly modify cell number but there was significantly ( P 0.01) more cells in BAL of Brown Norway rats compared with the one of Sprague-Dawley rats. There was a significant ( P 0.01) increase of cell number in BAL 24 h after ovalbumin challenge in both strains of rats compared with salinechallenged animals. Significant difference (*P 0.05) between cell number over time within the same strain of rats is indicated by *. Mean SEM of eight experiments. Open bars, 5 min after challenge; striped bars, 8 h after challenge; cross-hatched bars, 24 h after challenge. AM Mediator Production after OVA Sensitization To investigate the immunomodulatory effect of the sensitization protocol on AM mediator production, both strains of rats were sensitized with saline or OVA. Animals were killed 21 d after the sensitization without challenge and AM were purified from BAL. Cells were stimulated with OX8 for 4 h and TNF was measured in cell-free supernatants (Figure 3A). OX8 significantly increased TNF release in both strains of rats. However, stimulated AM from Sprague-Dawley rats released significantly more TNF than AM from Brown Norway rats. Interestingly, OVA sensitization reduced spontaneous and OX8-stimulated TNF release in both Brown Norway and Sprague-Dawley rats. Production of IL-10 was measured in cell-free supernatants of AM stimulated with and without OX8 for 20 h. OX8 significantly increased IL-10 release, but this augmentation was significantly more substantial in Brown Norway rats compared with Sprague-Dawley rats (Figure 3B). OVA sensitization significantly reduced OX8-stimulated IL-10 release by AM in both strains of rats without affecting the spontaneous release. NO production was not modulated by OVA sensitization (Figure 3C). OX8-stimulated AM from Brown Norway rats released significantly more NO than AM from Sprague-Dawley rats. TNF Production by AM Isolated after OVA and Saline Challenge Rats were sensitized with OVA and challenged 21 d later for 5 min with aerosolized saline or OVA. Animals were killed at different times after the challenge (5 min, 8 h, and 24 h) and BAL was performed. AM were purified and stimulated with and without OX8 and levels of TNF in cellfree supernatants were measured. In general, AM from both strains of rats released significantly more TNF after saline challenge compared with unchallenged OVA-sensitized animals (Figures 3A and 4). OX8 significantly increased TNF release by AM from rats challenged with saline and OVA. AM from Sprague-Dawley rats, isolated 5 min and 8 h after saline challenge, released significantly more TNF when stimulated with OX8 than AM from Brown Norway rats (Figures 4A and 4B). Spontaneous release of TNF was not modulated by OVA challenge when cells were isolated 5 min and 8 h after. However, there was a significant increase in spontaneous TNF release when AM were isolated 24 h after OVA challenge (Figure 4C). Interestingly, levels of this cytokine were significantly lower 8 h after challenge compared with 5 min and 24 h. Furthermore, OX8- stimulated AM from Sprague-Dawley rats exposed to saline released significantly more TNF than AM from Brown Norway rats at all times studied (Figure 4). In contrast, OX8-stimulated AM of OVA-challenged Sprague-Dawley rats released a similar amount of TNF and significantly

582 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 26 2002 TABLE 1 Millions of cells in bronchoalveolar lavage of sensitized rats after ovalbumin or saline challenge Time Strain Challenge AM Ly Eo PMN 5 min BN Saline 3.68 0.07* 0.14 0.03 0.14 0.04* 0.08 0.05 OVA 1.44 0.04* 0.10 0.02 0.11 0.04* 0.03 0.02 SD Saline 2.53 0.02 0.08 0.01 0.02 0.01 0.02 0.01 OVA 2.41 0.03 0.08 0.03 0.02 0.01 0.02 0.01 8 h BN Saline 3.95 0.10* 0.08 0.03 0.11 0.03* 0.05 0.03 OVA 2.39 0.08* 0.09 0.04 0.31 0.08* 0.37 0.06* SD Saline 2.75 0.02 0.05 0.01 0.00 0.00 0.03 0.01 OVA 1.82 0.14 0.04 0.02 0.05 0.02 0.67 0.12 24 h BN Saline 3.72 0.08* 0.28 0.07 0.31 0.10* 0.06 0.02 OVA 5.60 0.34* 0.56 0.14 2.13 0.35* 2.26 0.19* SD Saline 2.58 0.04 0.20 0.05 0.00 0.00 0.04 0.02 OVA 3.26 0.40 0.50 0.10 0.36 0.06 2.85 0.30 Definition of abbreviations: Alveolar macrophages, AM; Brown Norway, BN; eosinophils, Eo; lymphocytes, Ly; neutrophils, PMN; Sprague Dawley, SD. Bronchoalveolar lavage was performed at different times after saline or ovalbumin (OVA) challenge and cells were stained for differential cell counts. * Significant difference (P 0.05) between BN and SD rats. OVA challenge significantly (P 0.05) decreased number of AM at 5 min and 8 h but increased it at 24 h. Number of Eo and PMN significantly increased at 24 h in both strains of rats. AM, Ly, Eo, and PMN number significantly changed over time (P 0.05). Mean SEM of eight animals. less TNF than AM from Brown Norway rats at 5 min and 24 h, respectively (Figure 4). Moreover, AM of OVAchallenged rats released significantly more TNF when stimulated with OX8- compared with saline-challenged animals in both strains of rats after 8 h and 24 h (Figure 4B and 4C). BALs performed 24 h after challenge were concentrated 80 times and TNF levels were measured. There were low levels of TNF in saline-challenged animals (9.7 5.7 pg/ml and 2.5 2.5 pg/ml in Brown Norway and Sprague-Dawley rats, respectively, n 4). However, OVA challenge significantly increased TNF levels in BAL of Brown Norway rats (68.3 13.0 pg/ml), whereas similar TNF levels were observed in OVA-challenged Sprague-Dawley rats (6.8 5.1 pg/ml). Production of IL-10 by AM Isolated after OVA and Saline Challenge AM were isolated after OVA challenge as mentioned above and stimulated with and without OX8 to release IL-10 (Figure 5). OX8 significantly increased AM IL-10 release except in saline-challenged Sprague-Dawley rats and OVA-challenged Brown Norway rats (24 h). OX8- stimulated IL-10 production was significantly higher in Brown Norway rats compared with Sprague-Dawley rats when AM were isolated 5 min and 8 h after saline challenge and 5 min and 24 h after OVA challenge. However, OVA challenge did not modulate AM IL-10 release in Sprague-Dawley rats 5 min after challenge (Figure 5A), but significantly increased both spontaneous and OX8- stimulated IL-10 release from AM of Brown Norway rats. Furthermore, spontaneous IL-10 release 24 h after OVA challenge was significantly increased in both strains of rats compared with 8 h, and this spontaneous release was higher in Brown Norway rats (Figure 5C). Although OVA challenge did not modulate spontaneous release of IL-10 when AM were isolated 8 h after challenge, IL-10 levels were significantly increased 24 h after OVA challenge. IL-10 levels were significantly lower 8 h after challenge compared with 5 min and 24 h, as shown for TNF release. Levels of these cytokines were similar in both strains of rats 8 h after OVA challenge (Figure 5B). IL-10 levels were measured in concentrated BALs performed 24 h after challenge. Similar levels of IL-10 were found in BALs of saline-challenged Brown Norway and Sprague-Dawley rats (15.8 2.9 pg/ml and 18.9 4.7 pg/ml respectively, n 4). Although IL-10 levels were not significantly modulated by OVA challenge in Sprague-Dawley rats (28.5 9.3 pg/ml), these levels were significantly increased in Brown Norway rats (38.3 8.8 pg/ml). NO Production by AM after Challenge AM were isolated at different times after OVA challenge as mentioned above and stimulated with and without OX8 to release NO (Figure 6). OVA challenge significantly increased NO release of OX8-stimulated AM from Brown Norway rats at all time but only after 5 min in Sprague- Dawley rat. OX8-stimulated AM from Brown Norway rats released significantly more NO than AM from Sprague- Dawley rats when isolated after OVA challenge at all times studied. In contrast to IL-10 and TNF release, AM NO production was significantly reduced in all groups of animals killed 24 h after OVA challenge compared with 8 h (Figure 6B and 6C). Furthermore, OVA challenge increased the spontaneous release of NO in both strains of rats at 8 h after the challenge and in Brown Norway rats at 24 h. There was no significant difference in OX8-stimulated AM NO release between saline-challenged Brown Norway and Sprague-Dawley rats at all times studied. Interestingly, our results suggest that AM take more time to produce IL-10 and TNF than NO. Discussion Brown Norway rats have been widely used as a model of asthma and airway hyperresponsiveness using various sensitization protocol (19 22). Although this model has been previously characterized regarding inflammation and airway responsiveness, there are few publications demon-

Careau, Sirois, and Bissonnette: Alveolar Macrophages in Asthma Pathogenesis 583 Figure 3. Mediator production by alveolar macrophages (AM) from saline- and OVA-sensitized rats. BAL was performed 21 d after sensitization, and AM were purified and stimulated with OX8 (5 g/ml) for 4, 20, and 48 h for the release of TNF (A), IL-10 (B), and NO (C), respectively. OX8 significantly (*P 0.05) stimulated the release of these mediators compared with sham-treated cells. Significant differences (P 0.05) between Brown Norway (hatched bars) and Sprague-Dawley (open bars) rats within the same treatment (saline or OVA) are indicated by, whereas significant differences (P 0.02) between saline and OVA sensitization within the same strain of rats are indicated by. Mean SEM of eight to ten experiments. Figure 4. TNF production by AM from OVA-sensitized rats challenged with saline or OVA for 5 min. BAL was performed 5 min (A), 8 h (B), or 24 h (C) after challenge, and AM were purified and stimulated with OX8 for the release of TNF compared with sham-treated cells. OX8 significantly (*P 0.05) stimulated the release of this mediator. Significant differences (P 0.05) between Brown Norway (hatched bars) and Sprague-Dawley (open bars) rats within the same treatment (saline or OVA) are indicated by, whereas significant differences (P 0.01) between saline and OVA challenge within the same strain of rats are indicated by. Mean SEM of seven experiments. strating the inflammatory response after allergen challenge in allergic-resistant and -susceptible rats in parallel (26). We have previously demonstrated that AM isolated from naive Brown Norway rats produce more NO and Th2-type cytokines (IL-10 and IL-13) and less Th1-type cytokines (TNF) than AM from Sprague-Dawley rats (10). Furthermore, OVA challenge provokes an early allergic response characterized by defensive posture, opening of the mouth, exaggerated labored breathing motions, and wheezing in OVA-sensitized Brown Norway rats, but not in Sprague- Dawley rats, showing their different susceptibilities to develop allergy. In the present study, we have characterized airway hyperresponsiveness, lung inflammation, and cyto-

584 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 26 2002 Figure 5. IL-10 production by AM from OVA-sensitized rats challenged with saline or OVA for 5 min. BAL was performed 5 min (A), 8 h (B), or 24 h (C) after challenge, and AM were purified and stimulated with OX8 for the release of IL-10. OX8 significantly (*P 0.05) stimulated IL-10 release compared with sham-treated cells. Significant differences (P 0.05) between Brown Norway (hatched bars) and Sprague Dawley (open bars) rats within the same treatment (saline or OVA) are indicated by, whereas significant differences (P 0.01) between saline and OVA challenge within the same strain of rats are indicated by. Mean SEM of seven experiments. Figure 6. NO production by AM from OVA-sensitized rats challenged with saline or OVA for 5 min. BAL was performed 5 min (A), 8 h (B), or 24 h (C) after challenge, and AM were purified and stimulated with OX8 for the release of NO. OX8 significantly (*P 0.05) stimulated NO release compared with shamtreated cells. Significant differences (P 0.05) between Brown Norway (hatched bars) and Sprague-Dawley (open bars) rats within the same treatment (saline or OVA) are indicated by, whereas significant differences (P 0.05) between saline and OVA challenge within the same strain of rats are indicated by. Mean SEM of seven experiments. kine production in these two strains of rats after allergen challenge. OVA-sensitized and -challenged Brown Norway rats showed airway hyperresponsiveness to ACh as demonstrated by other studies (19, 27), but airway responsiveness of Sprague-Dawley rats was not modified by OVA challenge, showing the difference in these two strains of rats with respect to the development of airway hyperresponsiveness. To further understand the resistance of Sprague- Dawley rats to develop allergic asthma, we characterized the inflammatory response after allergen challenge in both strains of rats. To our knowledge, this is the first study demonstrating the modulation of AM functions by intraperitoneal allergen sensitization and the time-course of

Careau, Sirois, and Bissonnette: Alveolar Macrophages in Asthma Pathogenesis 585 airway inflammation and cytokine production after allergen challenge in two strains of rats showing different susceptibilities to develop allergic asthma. AM were isolated from saline- and OVA-sensitized rats before allergen challenge to determine the effect of the immunization protocol on mediator production by AM. Interestingly, the allergen sensitization caused a reduction in AM TNF and IL-10 production, whereas NO release was not affected. Local OVA sensitization has been shown to stimulate the production of both IgG and IgE (28). Thus, it is possible that local immunization (intraperitoneally) caused a systemic immune response that may affect AM cytokine production. Although lavage sampling of the airway does not always reflect the milieu of the interstitium, it provides samples that are in close approximation to it (29). Thus, BALs were performed during the early phase (5 min), late phase (8 h), and inflammatory response phase (24 h) after allergen challenge. Interestingly, there were less cells in BAL of Brown Norway rats 5 min and 8 h after OVA challenge compared with saline challenge. It is possible that the bronchoconstriction observed during the early and late phase in Brown Norway rats, but not in Sprague-Dawley rats, reduced cell recovery in BAL of these rats. However, both strains of rats showed an inflammatory response 24 h after allergen challenge, although the inflammation was significantly higher in Brown Norway rats compared with Sprague- Dawley rats. The characterization of the inflammatory responses 24 h after OVA challenge showed interesting differences between the two strains of rats. Brown Norway rats showed a neutrophilic and eosinophilic inflammation (27), whereas Sprague-Dawley rats had an important neutrophilic but a limited eosinophilic inflammation. Furthermore, there was a similar increase in number of lymphocytes in both strains of rats but there was a significant increase of AM in Brown Norway rats only. Thus, the difference in the susceptibilities of these two strains of rats to develop allergic asthma may depend on the intensity of the inflammatory response in the lung as well as the cell types involved in this inflammation. Production of mediators by AM may play an important role in asthma pathogenesis (7). AM TNF production varies over time with a minimum production 8 h after challenge, but there was a significant increase in AM TNF release in both strains of rats 24 h after OVA challenge. In general, Sprague-Dawley rats produce more TNF than Brown Norway rats except at 24 h after OVA challenge, suggesting a protective role early in time. The role of TNF as a protective or potentiating mediator in asthma is controversial (12 14). Our data suggest that the timing of TNF production may be important in the inflammatory process to determine which type of inflammatory response, Th1 or Th2, will take place. However, TNF seems to be involved during the allergic inflammatory response (24 h) as measured in BAL fluids. The complexity of TNF effects on lung functions has already been suggested by Martin and coworkers (30). IL-10 production was reduced 8 h after challenge, but no difference was observed in OVA-challenged animals at that time. However, at 5 min and 24 h after challenge, stimulated AM from Brown Norway rats produced more IL-10 than AM from Sprague-Dawley rats. OVA challenge (24 h) stimulated AM spontaneous release of IL-10 release in both strains of rats, although the increase was significantly stronger in Brown Norway rats compared with Sprague-Dawley rats. Higher IL-10 production observed in Brown Norway rats may potentiate the Th2-type response by donwregulating interferon- (IFN- ) and IL-12 production (26, 31 32). Increased exhaled NO has been demonstrated after challenge in sensitized animals (33). Furthermore, exhaled NO is increased in patients with asthma, and there is a strong correlation between NO levels and airway hyperresponsiveness (34). NO production by stimulated AM decreased with time after challenge in our model. However, OX8-stimulated AM from Brown Norway rats released significantly more NO than AM from Sprague-Dawley rats at all times investigated. In vivo OVA challenge stimulated the spontaneous release of NO from AM of Brown Norway rats at 8 h and 24 h, but not of Sprague-Dawley rats, compared with saline-challenged animals, suggesting that AM NO production may play an important role in the development of allergic asthma. This increase in AM NO production may contribute to the inhibition of Th1 response, as previously suggested (35). The production of Th2 cytokines after antigen challenge of sensitized rats has been previously shown (4, 26). An increase of IL-4 and IL-5 mrna levels in the lung has been observed in Brown Norway rats after OVA challenge, whereas increase in IL-2 and IFN- mrna levels has been observed in Sprague-Dawley rats (26). However, protein levels have not been investigated. Furthermore, to our knowledge, mediator production by AM at different times after allergen challenge has never been demonstrated. Our results suggest that AM may participate to the expansion of Th2 inflammation in Brown Norway rats. Differences in mediator production by AM of Brown Norway and Sprague-Dawley rats may play an important role in the development of airway inflammation. Furthermore, AM of Sprague-Dawley rats may protect the animal against the development of allergic asthma, as previously suggested (7, 8). However, further investigations are needed to better understand the importance of AM in the development of airway responsiveness. Acknowledgments: The authors thank Geneviève Ménard for her technical support and Dr. Jamila Chakir for critical reading of the manuscript. This work was supported by the Medical Research Council of Canada and the Réseau en Santé Respiratoire du FRSQ-axe asthme. E.Y.B. is a senior FRSQ Scholar. References 1. Goldstein, R. A., W. E. Paul, D. D. Metcalfe, W. W. Busse, and E. R. Reece. 1994. NIH conference: asthma. Ann. Intern. Med. 121:698 708. 2. Le Gros, G., K. Erb, N. Harris, J. Holloway, K. McCoy, and F. Ronchese. 1998. Immunoregulatory networks in asthma. Clin. Exp. Allergy 28:92 96. 3. Paul, W. 1994. Determination of the lymphokine-producing phenotype of CD4 T cells: a potential control point in allergic and infectious diseases. Ann. Intern. Med. 121:698 708. 4. Romagnani, S. 1994. Regulation of the development of type 2 T-helper cells in allergy. Curr. Opin. Immunol. 6:838 846. 5. Corrigan, C. J., and A. B. Kay. 1992. T cells and eosinophils in the pathogenesis of asthma. Immunol. Today 13:501 507. 6. Schauble, T. L., W. H. Boom, C. K. Finegan, and E. A. Rich. 1993. Characterization of suppressor function of human alveolar macrophages for T lymphocyte responses to phyto-hemagglutinin: cellular selectivity, reversibility, and early events in T cell activation. Am. J. Respir. Cell Mol. Biol. 8: 89 97.

586 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 26 2002 7. Strickland, D. H., T. Thepen, U. R. Kees, G. Kraal, and P. G. Holt. 1993. Regulation of T-cell function in lung tissue by pulmonary alveolar macrophages. Immunology 80:266 272. 8. Thepen, T., C. McMenamin, B. Girn, G. Kraal, and P. G. Holt. 1992. Regulation of IgE production in presensitised animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen. Clin. Exp. Allergy 22:1107 1114. 9. Mills, C. D., K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill. 2000. M-1/ M-2 macrophages and the Th1/Th2 paradigm. J. Immunol. 164:6166 6173. 10. Sirois, J., and E. Y. Bissonnette. 2001. Alveolar macrophages of allergic resistant and susceptible strains of rats show distinct cytokine profiles. Clin. Exp. Immunol. 126:9 15. 11. Taylor-Robinson, A. W., F. Y. Liew, A. Severn, D. Xu, S. J. McSorley, P. Garside, J. Padron, and R. S. Phillips. 1994. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. Eur. J. Immunol. 24:980 984. 12. Becher, B., M. Blain, P. S. Giacomini, and J. P. Antel. 1999. Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12. J. Immunol. 162:684 688. 13. Kips, J. C., J. Tavernier, and R. A. Pauwels. 1992. Tumor necrosis factor (TNF) causes bronchial hyperresponsiveness in rats. Am. Rev. Respir. Dis. 145:332 336. 14. Thomas, P. S., D. H. Yates, and P. J. Barnes. 1995. Tumor necrosis factor- increases airway responsiveness and sputum neutrophilia in normal human subjects. Am. J. Respir. Crit. Care Med. 152:76 80. 15. Oosterhoff, Y., H. C. Hoogsteden, B. Rutgers, H. F. Kauffman, and D. S. Postma. 1995. Lymphocyte and macrophage activation in bronchoalveolar lavage fluid in nocturnal asthma. Am. J. Respir. Crit. Care Med. 151:75 81. 16. Aubas, P., B. Cosso, P. Godard, F. B. Michel, and J. Clot. 1984. Decreased suppressor cell activity of alveolar macrophages in bronchial asthma. Am. Rev. Respir. Dis. 130:875 878. 17. John, M., S. Lim, J. Seybold, P. Jose, A. Robichaud, B. O Connor, P. J. Barnes, and K. F. Chung. 1998. Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1, granulocyte-macrophage colony-stimulating factor, and interferon- release from alveolar macrophages in asthma. Am. J. Respir. Crit. Care Med. 157:256 262. 18. Tang, C., J. M. Rolland, X. Li, C. Ward, R. Bish, and E. H. Walters. 1998. Alveolar macrophages from atopic asthmatics, but not atopic nonasthmatics, enhance interleukin-5 production by CD4 T cells. Am. J. Respir. Crit. Care Med. 157:1120 1126. 19. Elwood, W., J. O. Lötvall, P. J. Barnes, and K. F. Chung. 1991. Characterization of allergen-induced bronchial hyperresponsiveness and airway inflammation in actively sensitized Brown Norway rats. J. Allergy Clin. Immunol. 88:951 960. 20. Waserman, S., R. Olivenstein, P. M. Renzi, L. J. Xu, and J. G. Martin. 1992. The relationship between late asthmatic responses and antigen-specific immunoglobulin. J. Allergy Clin. Immunol. 90:661 669. 21. Renzi, P. M., R. Olivenstein, and J. G. Martin. 1993. Inflammatory cell populations in the airways and parenchyma after antigen challenge in the rat. Am. Rev. Respir. Dis. 147:967 974. 22. Bellofiore, S., and J. G. Martin. 1988. Antigen challenge of sensitised rats increases airway responsiveness to methacholine. J. Appl. Physiol. 65:1642 1646. 23. Olivenstein, R., P. M. Renzi, J. P. Yang, P. Rossi, S. Laberge, S. Waserman, and J. G. Martin. 1993. Depletion of OX-8 lymphocytes from the blood and airways using monoclonal antibodies enhances the late airway response in rats. J. Clin. Invest. 92:1477 1482. 24. Lin, T. J., N. Hirji, G. R. Stenton, M. Gilchrist, B. J. Grill, A. D. Schreiber, and A. D. Befus. 2000. Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production. J. Immunol. 164:1783 1792. 25. Bissonnette, E. Y., C. M. Hogaboam, J. L. Wallace, and A. D. Befus. 1991. Potentiation of TNF- mediated cytotoxicity of mast cells by their production of nitric oxide. J. Immunol. 147:3060 3065. 26. Renzi, P. M., A. S. al Assaad, J. Yang, Z. Yasruel, and Q. Hamid. 1996. Cytokine expression in the presence or absence of late airway responses after antigen challenge of sensitized rats. Am. J. Respir. Cell Mol. Biol. 15: 367 373. 27. Haczku, A., J. Sun, K. F. Chung, P. J. Barnes, A. B. Kay, and R. Moqbel. 1995. Airway hyperresponsiveness, elevation of serum-specific IgE and activation of T cells following allergen exposure in sensitized Brown-Norway rats. Immunology 85:598 603. 28. Waserman, S., R. Olivenstein, P. Renzi, L. J. Xu, and J. G. Martin. 1992. The relationship between late asthmatic responses and antigen-specific immunoglobulin. J. Allergy Clin. Immunol. 90:661 669. 29. Reynolds, H. Y. 2000. Use of bronchoalveolar lavage in humans past necessity and future imperative. Lung 178:271 293. 30. Martin, C., A. Wohlsen, and S. Uhlig. 2001. Changes in airway resistance by simultaneous exposure to TNF-alpha and IL-1beta in perfused rat lungs. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L595 601. 31. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M. Howard, K. W. Moore, and A. O Garra. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444 3451. 32. D Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, and G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon -production by suppressing natural killer cell stimulatory factor/ IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041 1048. 33. Persson, M. G., and L. E. Gustafsson. 1993. Allergen-induced airway obstruction in guinea-pigs is associated with change in nitric oxide levels in exhaled air. Acta Physiol. Scand. 149:461 466. 34. Xiong, Y., G. Karupiah, S. P. Hogan, P. S. Foster, and A. J. Ramsay. 1999. Inhibition of allergic airway inflammation in mice lacking nitric oxide synthase 2. J. Immunol. 162:445 452. 35. Yan, Z. Q., G. K. Hansson, B. E. Skoogh, and J. O. Lötvall. 1995. Induction of nitric oxide synthase in a model of allergic occupational asthma. Allergy 50:760 764.