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Published ahead of print on February 12, 2004, doi:10.1165/rcmb.2003-0229OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 22-27, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0229OC

Original Article

Adoptive Transfer of Alveolar Macrophages Abrogates Bronchial Hyperresponsiveness

Eric Careau and Elyse Y. Bissonnette

Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l'Université Laval, Québec, Canada

Address correspondence to: Eric Careau, Centre de Recherche en Pneumologie, Hôpital Laval, 2725, Chemin Sainte-Foy, Sainte-Foy, QC, Canada G1V 4G5. E-mail: eric.careau{at}crhl.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increasing evidence suggests that alveolar macrophages (AM) are involved in asthma pathogenesis. To better understand the role that these cells play, we investigated the capacity of AM from allergy-resistant rat, Sprague Dawley (SD), to modulate airway hyperresponsiveness of allergy-susceptible rat, Brown Norway (BN). AM of ovalbumin (OVA)-sensitized BN rats were eliminated by intratracheal instillation of liposomes containing clodronate. AM from OVA-sensitized SD rats were transferred into AM-depleted BN rats 24 h before allergen challenge. Airway responsiveness to methacholine was measured the following day. Instillation of liposomes containing clodronate in BN rats eliminated 85% AM after 3 d compared with saline liposomes. Methacholine concentration needed to increase lung resistance by 200% (EC200RL) was significantly lower in OVA-challenged BN rats (27.9 ± 2.8 mg/ml) compared with SD rats (63.9 ± 8.6 mg/ml). However, when AM from SD rats were transferred into AM-depleted BN rats, airway responsiveness (64.0 ± 11.3 mg/ml) was reduced to the level of naïve rats (54.4 ± 3.7 mg/ml) in a dose-dependent manner. Interestingly, transfer of AM from BN rats into SD rats did not modulate airway responsiveness. To our knowledge, this is the first direct evidence showing that AM may protect against the development of airway hyperresponsiveness.

Abbreviations: alveolar macrophages, AM • bronchoalveolar lavage, BAL • Brown Norway, BN • dichloromethylene-diphosphate, Cl2MDP • methacholine concentration needed to increase lung resistance by 200%, EC200RL • immunoglobulin, Ig • interleukin, IL • ovalbumin, OVA • pulmonary resistance, RL • Sprague Dawley, SD • T helper, Th • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Patients with asthma exposed to allergen develop an early-phase reaction, mainly mediated by mast cells (1); a late-phase reaction 6–8 h later; and an eosinophilic inflammation 24 h after allergen challenge (2). There is a close relationship between airway inflammation and airway hyperresponsiveness in asthma (3, 4), indicating that measurement of airway hyperresponsiveness may reveal valuable information on the inflammatory status of the patient. Furthermore, airway hyperresponsiveness to a wide variety of nonspecific stimuli is a characteristic feature of asthma (5) and is closely related to the severity and frequency of asthma symptoms (6).

Alveolar macrophages (AM) play a key role in the maintenance of immunologic homeostasis in the respiratory tract and represent the most abundant cells in the conducting airways (7). Although they are well known to suppress T cell activation and antigen presentation activities of dendritic cells (8, 9), their role in asthma pathogenesis is still debated (10). However, the technology has become available to selectively deplete macrophage populations in vivo with liposomes containing clodronate (dichloromethylene-diphosphate or Cl2MDP) (11), revealing the importance of these cells during allergen challenge. These liposomes are phagocytosed by macrophages that are selectively killed by apoptosis without damaging the surrounding tissues (12–15). Animals with immunologic memory for an allergen, depleted of their AM using this technique, show highly elevated immunoglobulin (Ig)E response and a large influx of T cells in the airways, as well as an increase in IgE-secreting B cells in draining lymph nodes following challenge with aerosolized allergen (14–16). In contrast, in sham-treated animals, i.e., possessing AM, there is no cell influx or IgE-producing cells. These data suggest that AM may play a role in the prevention of airway inflammation in asthma and allergic diseases.

Ovalbumin (OVA)-sensitized Brown Norway (BN) rat is a well-characterized animal model of asthma that reflects many features of human allergic asthma, including both early- and late-phase reactions, an increase in bronchial responsiveness to methacholine, and an increase of OVA-specific IgE (17–19). Interestingly, Sprague Dawley (SD) rats do not develop allergic reactions or an increase in IgE production under the same conditions (20, 21). This difference between these two strains of rats is a valuable tool for studying the role of AM in the development of allergic asthma.

Given the importance of AM in the immune response, we investigated the modulation of airway responsiveness of allergic rats by AM from nonallergic rats. Differences in airway responsiveness between OVA-sensitized and -challenged SD and BN rats demonstrated that BN rats are more reactive to methacholine than SD rats. However, when the AM population of sensitized BN rats was depleted and reconstituted with AM from sensitized SD rats, airway hyperresponsiveness in these BN rats was reduced to the level of naïve rats. These data demonstrate the importance of AM in preventing the development of airway hyperresponsiveness in sensitized animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Sensitization
Male BN rats (BN/Ssn), 80 d old, were obtained from Harlan Sprague Dawley (Indianapolis, IN), and age-matched SD rats were obtained from Charles River (St. Constant, QC, Canada). All animals were maintained in filter-top cages in virus/antigen-free conditions in Laval Hospital animal facility. Animals were sensitized and challenged with OVA grade V (Sigma Chemical Co., St. Louis, MO) as previously described (21). This sensitization protocol has been shown to cause airway hyperresponsiveness and an increase in eosinophil and lymphocyte numbers in bronchoalveolar lavage (BAL) after OVA challenge three weeks later (22). Control rats were sensitized or challenged with saline. The Laval University Animal Care Committee approved this experimental protocol in accordance with the guidelines of the Canadian Council on Animal Care.

Liposome Preparation
Suspensions of multilamellar liposomes encapsulating Cl2MDP (gift from Roche Diagnostics, Mannheim, Germany) were prepared according to Van Rooijen method (11). Briefly, 86 mg L-{alpha}-phosphatidylcholine (egg) and 8 mg cholesterol (Avanti Polar-Lipid, Alabaster, AL) were dissolved in 10 ml chloroform in a round-bottom flask connected to a rotary evaporator. After evaporation, 10 ml phosphate-buffered saline (PBS) containing Cl2MDP (250 mg/ml) or PBS (for control liposomes) was added and the suspension was sonicated 2 h later. Liposomes were maintained in suspension for 2 h at room temperature (for liposome swelling) and nonencapsulated Cl2MDP was removed by centrifugation (100,000 x g) for 30 min. Liposomes were washed three times with PBS and finally suspended in 4 ml PBS and stored at 4°C under nitrogen atmosphere.

Liposome Administration
The liposome solution was given using an intratracheal intubation method. Briefly, animals were anaesthetized and a 5-cm long #14 venous catheter with its blunted needle (Becton Dickinson Diagnostics, Sparks, MD) was inserted through the glottis using a laryngoscope (Welch Allyn, Buffalo Grove, IL). When the catheter was inserted, the needle was quickly removed to let the animal breathe. To instill the liposomes into the right or left lung, a 9-cm long polyethylene PE50 tube (Becton Dickinson Diagnostics) with a right or left curve, respectively, was inserted through the catheter. This method ensured that both the right and left lungs received the same quantity of Cl2MDP liposomes, a fact that we verified with colorant delivery (unpublished data). Liposome suspension was given in each lung using a 1-ml syringe containing 100 µl liposomes connected to the PE50 tube.

Cells
AM were isolated as previously described (23). Trypan blue exclusion and crystal violet coloration were used to evaluate viability and cell count, respectively. Cytospins were performed and stained with Diff-Quik (Gibco BRL, Burlington, ON, Canada) for cell differentiation. The number of living AM present in BAL was calculated using the formula: Total cell count (crystal violet) x % viability (trypan blue) x % AM (cytospins).

AM Transfer
To verify the presence of transferred AM in the lung of recipient rats, AM from SD rats were labeled with 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes Inc., Eugene, OR) before being transferred into BN rats. BAL was performed in the recipient rats 48 h later and fluorescent AM were evaluated using an EPICS ELITE ESP cytometer (Beckman-Coulter, Miami, FL). Acquisition of fluorescence data was gated by forward angle light scatter and side scatter and the data rate was set at less than 500 events per second. Samples were allowed to run approximately one minute before the acquisition of a minimum of 5,000 events.

BAL were done in SD rats 20 d after sensitization and 2 x 106 cells were suspended in 200 µl PBS. These cells were transferred by intratracheal intubation into BN rats that had received Cl2MDP-liposomes 3 d earlier using the same technique. Animals were allowed to recuperate for 24 h before being challenged with 5% aerosolized OVA. Airway responsiveness to methacholine was measured 24 h later. The same protocol was used for the transfer of BN AM into SD rats. Figure 1 schematizes the protocol used for the transfer of AM from SD rats into BN rats.



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  		Figure 1. AM transfer protocol. AM from Sprague Dawley rats were transferred into Brown Norway rats 20 d after OVA sensitization. OVA challenge (for 5 min) was performed and methacholine airway responsiveness was measured 24 h later.

 
Measurement of Airway Responsiveness
Measurement of airway responsiveness was performed as previously described (22). Briefly, animals were anaesthetized intraperitoneally with urethane (1.5 g/kg; Sigma Chemical) and laid on a heating pad. A 6-cm length of PE240 (Becton Dickinson Diagnostics) was used as the endotracheal tube. The end of the intratracheal tube was connected to a Quatra-T CombiChamber (Scireq, Montreal, QC, Canada). The Quadra-T uses measurements of mouth flow and esophageal pressure to accurately calculate pulmonary resistance (RL). Changes in esophageal pressure were measured using a saline-filled catheter PE160 tube (Becton Dickinson Diagnostics) connected to a differential pressure transducer. Mouth flow, from the intratracheal tube, was measured by a pneumotachograph. RL was simultaneously calculated using the software package included with the Quatra-T.

Animals were exposed to doubling concentrations of methacholine for 30 s with airflow of 6 liters/min air. Then, RL measurements were taken every min for 5 min. The peak value of RL was measured after each concentration and the challenge was stopped at 128 mg/ml. The concentration of methacholine required to cause 200% increase in resistance, EC200RL, was calculated by interpolation of concentration–response curve from individual animal.

OVA-Specific IgE and IgG2a Levels in Sera
Blood (4–5 ml) was drawn from abdominal aorta in the absence of anticoagulant. Serum samples were collected and OVA-specific IgE and IgG2a levels were measured by ELISA as previously described (24). Plates were read at 540 nm using a Molecular Device (Menlo Park, CA) Vmax Kinetic Microplate Reader.

Statistical Analysis
To compare AM depletion in rats for days 0–21, a one-way analysis of variance was used. Comparisons between days and hyperresponsiveness data were performed using Tukey's studentized range test. Comparison between groups was performed using contrast to perform custom hypothesis tests. A one-way analysis of variance was used to determine the amount of AM needed to modulate airway responsiveness, and comparisons between groups were performed with the Dunnett's technique. The normality assumption was verified with the Shapiro-Wilk test and Bartlett's statistic was used to verify the homogeneity of variances for each tested effect. The data were considered significant when P values were <= 0.05. The data were analyzed using the statistical package program SAS v8.2 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OVA-Specific IgE and IgG2a Levels
To characterize the animal model, OVA-specific IgE and IgG2a levels were determined. Saline-sensitized BN and SD rats had very low levels of IgE (data not shown), and OVA sensitization did not significantly increase OVA-specific IgE levels in SD rats. However, OVA-specific IgE levels were significantly higher (P < 0.03) in OVA-sensitized BN rats compared with SD rats after saline and OVA challenge (Figure 2). OVA challenge and the transfer of AM of SD rats into AM-depleted BN did not significantly modify IgE levels. OVA-specific IgG2a was the same in both strains of rats used (data not shown). These results suggest that BN rats developed an allergic response when sensitized to OVA, but SD rats did not. Moreover, OVA challenge did not modulate OVA-specific IgE concentration in either group.



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  		Figure 2. OVA-specific IgE concentrations after sensitization and challenge. Sera were collected 24 h after OVA or saline challenge. Serum amounts of IgE were significantly (*P < 0.03) higher in BN rats compared with SD rats. OVA challenge and the transfer of alveolar macrophages from SD into BN rats did not modulate IgE concentration. Mean ± SEM of five rats.

 
Time–Course Analysis of AM Depletion
To determine the best time to perform transfer of AM, time–course of AM depletion was investigated. Cl2MDP- or PBS-liposomes were instilled in BN rats, BAL was performed at different times after the instillation procedure, and AM were counted. Instillation of PBS-liposomes did not modulate the number of AM in BAL (Figure 3, hatched bar). However, instillation of Cl2MDP-liposomes in both right and left lungs caused a significant (P < 0.0001) reduction (85.1 ± 2.3%) of AM population 3 d after the instillation compared with rats that received liposomes containing PBS. No significant difference was observed in the number of eosinophils, neutrophils, and lymphocytes present in BAL (data not shown). Similar results were obtained with SD rats (data not shown). AM depletion lasted for 7–9 d (65%). The number of AM increased slowly after 5 d and reached their normal concentration between 14 and 17 d, at a rate of ~ 0.35 x 106 AM/d.



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  		Figure 3. Time–course analysis of AM disappearance and reappearance after instillation of clodronate. Bronchoalveolar lavages were performed at different times after liposome instillation. After 3 d, 85.1% AM were eliminated. Hatched bar represents number of AM ± SEM in bronchoalveolar lavages of animals that received PBS-liposomes. Significant reduction (P < 0.0001) in AM number was observed from Days 1–14 after clodronate instillation. Mean ± SEM of three different rats/time.

 
Bronchoalveolar Lavage Composition before Transfer
To limit the manipulation and activation of cells before being instilled into the rats, AM were not purified. Table 1 shows the number of AM and different cell types present in BAL of OVA-sensitized animals before transfer into AM-depleted animals. Although there were some other cell types, AM represented the most abundant cells in BAL of OVA-sensitized SD (> 98%) as observed in BAL of saline-sensitized SD rats (21). In some experiments, no lymphocytes were detected in cell counts of SD rats before transfer. Given the number of AM in SD BAL (Table 1), 2.0 x 106 AM were used for adoptive transfer to respect the physiologic amount of AM. The proportion of transferred AM compared with remaining endogenous AM after depletion was ~ 4:1. This was confirmed by transferring CMFDA-labeled AM. BAL of recipient rats contained 79.9 ± 4.4% (n = 3) CMFDA-labeled AM 48 h after transfer.


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  		TABLE 1. Cell population in bronchoalveolar lavage before transfer

 
Aerosolized OVA Challenge
Sensitized BN and SD rats were observed when they were exposed to aerosolize OVA for 5 min. As we previously described (21), 2–3 min after starting OVA challenge BN rats demonstrated obvious respiratory distress, characterized by defensive posture, opening of the mouth, exaggerated labored breathing motions, and wheezing. These reactions lasted for several minutes. In contrast, SD rats and saline-challenged rats did not show these reactions. Furthermore, AM-depleted BN rats that had received AM from SD rats or from saline-sensitized rats 24 h earlier did not demonstrate any respiratory distress, suggesting that the transfer of AM eliminated the early-phase allergic reaction.

RL of BN and SD Rats
OVA-sensitized SD and BN rats that were challenged with saline or OVA were exposed to doubling concentrations of aerosolized methacholine for 30 s and lung resistance was measured every min for 5 min. The highest RL obtained for each methacholine concentration was plotted to compare airway responsiveness in the two rat strains (Figure 4). OVA challenge did not modify RL of SD rats. However, RL was significantly higher in OVA-challenged BN rats compared with saline-challenged animals and OVA-challenged SD rats. Thus, OVA challenge increased airway responsiveness in BN rats, but not in SD rats.



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  		Figure 4. Airway responsiveness of BN and SD rats. Animals were exposed to doubling concentrations of methacholine during 30 s, and RL was measured every minute for 5 min. The highest RL for each concentration was plotted. Results from a representative experiment are presented. OVA-challenged BN rats (squares) showed higher RL to methacholine than saline-challenged BN rats (diamonds), saline-challenged SD rats (circles), and OVA-challenged SD rats (triangles).

 
EC200RL was calculated for each animal. Saline- and OVA-challenged SD rats had an EC200RL of 59.7 ± 6.2 mg/ml and 63.9 ± 8.6 mg/ml, respectively (Figure 5), whereas saline- and OVA-challenged BN rats showed an EC200RL of 54.4 ± 3.7 mg/ml and 27.9 ± 2.8 mg/ml (Figure 6). There is a significant difference (P < 0.001) between OVA-challenged BN and SD rats. These results indicated that BN rats developed airway hyperresponsiveness (lower concentration of methacholine needed to obtain EC200RL) in response to inflammation that occurred during the 24 h after OVA challenge.



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  		Figure 5. Airway responsiveness (EC200RL) of SD rats with and without AM. OVA challenge was performed 3 d after Cl2MDP-liposome instillation on OVA-sensitized SD rats. The animals were exposed to methacholine 24 h later to measure airway responsiveness. OVA-sensitized SD rats without AM demonstrated significant (*P < 0.003) airway hyperresponsiveness after OVA challenge compared with saline-challenged rats or rats with AM. EC200RL represents the methacholine concentration needed to increase airway resistance by 200%. Mean ± SEM of four rats.

 


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  		Figure 6. Airway responsiveness (EC200RL) of BN rats with and without AM. OVA challenge was performed 3 d after Cl2MDP-liposome instillation on OVA-sensitized BN rats. The animals were exposed to methacholine 24 h later to measure airway responsiveness. OVA-sensitized BN rats with or without AM demonstrated significant (*P < 0.001) airway hyperresponsiveness after OVA challenge compared with saline-challenged rats. EC200RL represents the methacholine concentration needed to increase airway resistance by 200%. Mean ± SEM of five rats.

 
To investigate the role of AM in airway responsiveness, OVA-sensitized SD and BN rats were depleted of their AM, challenged with OVA, and exposed to methacholine. AM depletion of OVA-challenged SD rats caused a significant increase (P < 0.003) in airway responsiveness (Figure 5). Interestingly, EC200RL of OVA-challenged SD rats depleted of their AM (36.8 ± 1.3 mg/ml) was higher (P < 0.006) than EC200RL of AM-depleted BN rats (24.3 ± 6.9 mg/ml), suggesting that the residual AM (~ 15%) in SD rats may influence airway hyperresponsiveness development. Saline challenge did not change airway responsiveness of OVA-sensitized rats with or without AM (Figures 5 and 6). Furthermore, AM depletion and OVA challenge of saline-sensitized rats did not alter airway responsiveness to methacholine.

To further investigate the role AM play in airway hyperresponsiveness development, transfers of AM from allergy-resistant and allergy-susceptible rats were done (Figure 7). AM from OVA-sensitized BN rats were transferred into AM-depleted BN rats, and EC200RL (24.1 ± 5.6 mg/ml) was comparable to OVA-challenged BN rats (27.9 ± 2.8 mg/ml). No change in airway responsiveness was observed when AM from OVA-sensitized SD rats were transferred into AM-depleted SD rats. However, when AM from OVA-sensitized SD rats were transferred into AM-depleted OVA-sensitized BN rats 24 h before OVA challenge, BN rats showed normal levels of airway responsiveness to methacholine with an EC200RL of 64.0 ± 11.3 mg/ml (Figure 7). These results demonstrated that AM from SD rats may protect BN rats against the development of airway hyperresponsiveness. Furthermore, transfer of AM from BN rats into AM-depleted SD rats did not modulate airway responsiveness of these animals (Figure 7), suggesting that AM cannot transfer susceptibility to allergy.



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  		Figure 7. Airway responsiveness (EC200RL) after transfer of AM. Bronchoalveolar lavage cells from OVA-sensitized SD or BN rats were transferred into OVA-sensitized rats 3 d after Cl2MDP-liposome instillation. The animals were exposed to OVA 24 h later and airway responsiveness to methacholine was measured the following day. Transfer of AM from OVA-sensitized BN rats did not reduce BN rat hyperresponsiveness whereas transfer of AM from SD rats (*P < 0.02) reduced BN rat hyperresponsiveness. However, transfer of AM from OVA-sensitized BN rats into SD rats did not modulate airway responsiveness of the latter. EC200RL represents the methacholine concentration needed to increase airway resistance by 200%. Mean ± SEM of four rats.

 
To determine the number of AM from SD rat required to modulate airway responsiveness of BN rat, transfer of different amounts of AM was performed (Figure 8). Depleted OVA-sensitized BN rats were reconstituted with 0.5, 1.0, or 2.0 x 106 AM from OVA-sensitized SD rats and challenged with OVA the following day. BN rats that received 0.5 and 1.0 x 106 AM showed a diminution of airway responsiveness to methacholine (41.1 ± 4.9 and 47.1 ± 5.8 mg/ml, respectively) that was not significantly different than OVA-sensitized and -challenged BN rats (30.7 ± 2.2 mg/ml) and saline-challenged BN rats (51.9 ± 2.5 mg/ml). These rats showed an intermediate level of airway responsiveness between normal and hyperresponsive rats. Transfer of 2.0 x 106 AM from SD into BN rats was required to significantly normalize airway responsiveness in BN rats with an EC200RL of 57.0 ± 6.2 mg/ml (Figure 8). Thus, AM modulate airway responsiveness in a dose–dependent manner.



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  		Figure 8. Airway responsiveness (EC200RL) after transfer of different amounts of AM. Bronchoalveolar lavage cells (0.5, 1.0, or 2.0 x106) from OVA-sensitized SD rats were transferred into OVA-sensitized BN rats 3 d after Cl2MDP-liposome instillation. The animals were exposed to OVA or saline 24 h later and airway responsiveness to methacholine was measured the following day. Transfer of 0.5 or 1.0 x 106 AM from OVA-sensitized BN rats did not significantly reduce BN rat hyperresponsiveness whereas transfer of 2.0 x 106 AM from SD rats (*P < 0.003) abrogated BN rat hyperresponsiveness. EC200RL represents methacholine concentration needed to increase airway resistance by 200%. Mean ± SEM of five rats. Columns with different letters are significantly different.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Different cell types play important roles in inflammatory diseases such as asthma. Given the controversial role of AM population, we have developed an animal model in which AM can be depleted and reconstituted to investigate the role of these cells in asthma. For this study, BN rats, which, in contrast to SD rats, reflect many features of human allergic asthma, were used. Our data on OVA-specific IgE levels confirmed the different allergy susceptibilities of these two strains of rats. To our knowledge, the reconstitution of AM population of allergic animals with AM from nonallergic animals to study airway hyperresponsiveness development has never been investigated.

The characterization of AM depletion following clodronate instillation was done to determine the best time to transfer AM from allergy-resistant rats into allergy-susceptible rats. Our data showed that the number of neutrophils, eosinophils, and lymphocytes was not modified by clodronate instillation and that 3 d were necessary to eliminate 85.1% AM. The time required to deplete AM may be due to apoptosis, which requires a longer process than necrosis (25). Indeed, apoptosis has been shown to be the main mechanism of cell death of phagocytic cells after ingestion of liposome-encapsulated clodronate (12, 13, 26). However, liposome clearance was important in our model to prevent the effects of free clodronate or liposomes on reconstituted AM population. A previous study (27) showed that no radiolabeled free clodronate was detected 3 h after injection of liposomes and that macrophage cell debris was present only during the first two days after liposome administration, suggesting that in vivo experiments could be performed 48 h after liposome treatment. Thus, given these observations and the time–course of AM depletion with Cl2MDP-liposomes (Figure 3), the transfer of AM of SD rats was performed 3 d after liposome instillation in BN rats.

We have previously demonstrated that OVA-sensitized BN rats developed airway hyperresponsiveness to inhaled methacholine after OVA challenge in contrast to SD rats (22). In the present study, we demonstrated that AM-depleted SD rats showed an increased airway responsiveness, supporting the result of Thepen and colleagues (15), who demonstrated an increase in IgE response and large influx of T cells in the airways of AM-depleted SD rats. The significant difference in airway responsiveness between AM-depleted BN and SD rats suggests that the presence of 15% AM in the SD rats is sufficient to influence airway responsiveness. These results strengthen the important role of AM in the development of airway hyperresponsiveness.

In previous studies, we have demonstrated that AM from BN and SD rats produce different cytokine profiles (21) and that airway inflammation after allergen challenge in these two strains of rats is different (22). Thus, to further investigate the role of AM in asthma, AM from allergy-resistant SD rats were transferred into allergy-susceptible AM-depleted BN rats before allergen challenge. Interestingly, BN rats that received AM of SD rats showed normal levels of airway responsiveness, which was dependent on the number of SD AM transferred. However, the transfer of AM from BN rats into BN rats did not modulate airway hyperresponsiveness, suggesting that animal manipulation was not responsible for the protection observed with AM from SD rats. A recent study demonstrated that lung–cell transfer reduced allergic airway inflammation in a murine model (28). However, cells transferred in that study were composed of 88% pulmonary macrophages containing a mixture of AM, interstitial, and intravascular macrophages. Our study used a population of cells containing 98% AM, suggesting that AM are the cells responsible for this protection.

Numerous studies have shown that T lymphocytes and IgE are important in airway hyperresponsiveness (29–31). Naïve animals that received antigen-primed T lymphocytes responded to inhaled specific antigen with an increase in airway responsiveness (29). Furthermore, adoptive transfer of sensitized CD4+ T cells induced T helper-type cytokine profile in response to OVA challenge in BN rats (30), suggesting that T lymphocytes may transfer allergy susceptibility. Interestingly, transfer of AM from BN rats into SD rats did not modulate airway responsiveness in the latter, suggesting that AM and the small number of lymphocytes in BAL of BN rats do not transfer allergy susceptibility in this model. Although there is increasing evidence suggesting an important role of T lymphocytes in transferring allergy susceptibility, there is limited information on the transfer of allergy resistance. Our data suggest that AM may play that role.

We have previously demonstrated that AM from BN and SD rats are functionally different. AM from naïve BN rats produced more macrophage inflammatory protein-1{alpha}, nitric oxide, interleukin (IL)-10, IL-13, and IL-12p40 but less tumor necrosis factor (TNF) than AM from naïve SD rat (21). OVA sensitization reduced levels of TNF and IL-10 released by AM in both strains of rats, but it did not change the differences between BN and SD rats. However, AM from OVA-sensitized BN rats released significantly more TNF, IL-10, and nitric oxide than AM from SD rats when isolated 24 h after OVA challenge (22). A recent study has also suggested that lung macrophages are important to reduce airway inflammation by enhancing interferon-{gamma} production (28). Given that cytokines have been shown to contribute to airway events that determine asthma symptoms and airway responsiveness (32), differences in AM cytokine production between BN and SD rats may explain, in part, the allergy protection provided by AM from SD rats. Higher IL-10 AM production observed in BN rats may potentiate Th2-type response by downregulating interferon-{gamma} and IL-12 production (33). Furthermore, it has been previously demonstrated (34) that AM from patients with asthma have phagocytosis dysfunction, suggesting that altered AM functions may be involved in asthma pathogenesis. These results, coupled with our data, demonstrate the vital role of AM in the pathogenesis of asthma and support the idea that AM function may prevent SD rats from developing airway hyperresponsiveness. Mechanisms involved in this protecting effect, cytokine production and phagocytosis activity, are currently under investigation.

Although the measurement of early-phase reaction was not part of the investigation, we have observed that AM transfer from SD rats into AM-depleted BN rats eliminated respiratory distress associated with the early-phase reaction, which is mainly mediated by mast cells. These results suggest that AM from allergy-resistant rats may downregulate mast cell functions or may release less histamine releasing factor (35, 36), reducing the inflammatory responses and airway hyperresponsiveness. Further investigations are needed to better understand the modulation of mast cell functions by AM in asthma.

In summary, the transfer of AM from allergy-resistant rats into allergy-susceptible rats abrogates the airway hyperresponsiveness of the latter. These results suggest that functions of AM from BN rats may be altered during the allergen sensitization process. These changes in AM function could lead to the difference of cytokine production observed between AM from BN and SD rats after sensitization and challenge (22). They may explain, in part, the susceptibility of BN rats to develop airway hyperresponsiveness.


   Acknowledgments
 
The authors thank André Blouin, Geneviève Ménard, and Philippe Pouliot for their technical support, Dr. Guy Tremblay and Dr. Michel Laviolette for their critical reading of the manuscript, and Serge Simard for the statistical analysis of the results. This work was supported by the Réseau en Santé Respiratoire du Fonds de la Recherche en Santé du Québec axe asthme and by the Canadian Institutes of Health Research. E.Y.B. is a senior FRSQ scholar.

Received in original form June 17, 2003

Received in final form January 12, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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