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. 2001 Oct;126(1):9–15. doi: 10.1046/j.1365-2249.2001.01643.x

Alveolar macrophages of allergic resistant and susceptible strains of rats show distinct cytokine profiles

J Sirois 1, E Y Bissonnette 1
PMCID: PMC1906159  PMID: 11678894

Abstract

Brown Norway rats are widely used as a model of asthma, whereas Sprague Dawley rats do not develop allergic reactions under the same conditions. Given the importance of alveolar macrophages (AM) in down-regulating cellular immune responses in the lung, we postulated that the different susceptibilities in the development of airway allergic reactions in these rat strains may be related to functional differences in their AM. We investigated the production of important mediators in asthma, namely tumour necrosis factor (TNF), interleukin-10 (IL-10), IL-12, IL-13, nitric oxide (NO) and macrophage inflammatory protein-1α (MIP-1α), by AM of unsensitized Sprague Dawley and Brown Norway rats. AM were purified by adherence and stimulated with OX8 (anti-CD8 antibody) or LPS. OX8 stimulation significantly increased the release of TNF, IL-10 and NO in both strains of rats, whereas MIP-1α and IL-12 release were increased in Brown Norway rats only. Interestingly, stimulated AM from Sprague Dawley rats released significantly more TNF and less IL-10, IL-12, IL-13, MIP-1α and NO compared with AM from Brown Norway rats. These differences were also observed at the mRNA level, except for TNF. Thus, AM from Brown Norway and Sprague Dawley rats are functionally different. Furthermore, LPS- and OX8-stimulated AM from Brown Norway rats produce more Th2 type cytokines (IL-10 and IL-13) than AM from Sprague Dawley rats, suggesting that these cells may play an important role in creating a cytokine milieu that may favour the development of allergic reactions.

Keywords: alveolar macrophages, Th1/, Th2cytokines, nitric oxide, asthma, rats

Introduction

Recent advances in the understanding of inflammatory reactions have demonstrated that distinct patterns of cytokine production and T helper (Th) lymphocytes are associated with different kinds of inflammatory responses. On this basis, cytokines and Th cells have been divided into Th1 and Th2 categories [1]. Th1 cytokines (interferon-γ [IFN-γ], interleukin-2 [IL-2], IL-12 and lymphotoxin) are mainly implicated in cell-mediated inflammatory reactions whereas Th2 cytokines (IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13) favour antibody production (particularly IgE), enhance both eosinophil proliferation and function, and are closely associated with allergic responses [1]. Development of successful immune responses requires a balance between Th1 and Th2 cells whereas an imbalance is associated with pathology. Thus, parameters that control Th1/Th2 development may play a crucial role in susceptibility and resistance to disease conditions.

Alveolar macrophages (AM) are found throughout the respiratory tract from the alveolus to the larynx, and represent the most abundant cells not only in the alveoli and distal airspaces, but also in the conducting airways [2]. They are the first line of defence against inhaled allergens, infectious agents and other immunological insults. AM play a key role in the maintenance of homeostasis in the lungs by suppressing T-cell activation and the antigen presentation activities of dendritic cells [3,4].

The pathophysiology of asthma involves different cell types and numerous cytokines, chemokines and mediators [5]. There is increasing evidence suggesting that AM participate in the production and maintenance of airway inflammation in asthma and allergic diseases [6]. Following antigen challenge, the increase in AM numbers in bronchoalveolar lavage exceeds that of other cell types, including cells such as eosinophils which are characteristic of asthma [7]. Moreover, animals with immunological memory for an allergen, depleted of AM, show highly elevated IgE responses and a large influx of T cells in the airways, as well as an increase in IgE-secreting B cells in the draining lymph nodes, following challenge with the aerosolized allergen [8–10]. However, in wild-type animals, i.e. possessing AM, there is no increase in either IgE production or T-cell influx. Furthermore, AM can produce both Th1‐ (IL-12) and Th2‐ (IL-10 and IL-13) type cytokines [6,11], suggesting that AM may create the cytokine milieu that will determine the type of immune response developed.

Several animal models that mimic human asthma have been developed to investigate this disease. There is a well established model of allergic asthma in Brown Norway rats that reflects many features of human allergic asthma, including both early and late phase reactions [12], elevated antigen-specific IgE [13], airway inflammation [14] and increased bronchial responsiveness to several stimuli [15]. In contrast, only a low percentage of Sprague Dawley rats develop an early airway response (< 20%) or detectable serum specific IgE (< 10%) after challenge with ovalbumin [16]. These two strains of rats represent a valuable tool to investigate the role of AM in the development of allergic asthma. Thus, we hypothesized that the susceptibility of Brown Norway rats to develop allergic asthma may be related to functional differences in their AM as compared with those of Sprague Dawley rats. Stimulated AM from unsensitized Sprague Dawley rats released significantly more TNF but less IL-10, IL-12, IL-13, nitric oxide (NO) and macrophage inflammatory protein-1α (MIP-1α) than AM from unsensitized Brown Norway rats. These differences may play an important role in the development of allergic asthma.

Materials and methods

Animals

Brown Norway rats (BN/Ssn) aged 72–82 days were obtained from Harlan-Sprague Dawley (Indianapolis, IN, USA) whereas age-matched Sprague Dawley rats were obtained from Charles River (St Constant, QC, Canada). All animals were maintained in filter-top cages in virus–antigen-free conditions in the Laval Hospital animal facility on a 12 h light/dark cycle. Animals were given food and water ad libidum.

In vivo ovalbumin challenge

Some rats were sensitized to ovalbumin by intraperitoneal injection of 1 ml ovalbumin Fraction V (1 mg/ml) (Sigma, St Louis, MO, USA) and Al(OH3) (100 mg/ml) (BDH Laboratory Supply, Poole, UK) in sterile 0·9% saline solution. This sensitization protocol has been shown to cause airway hyperresponsiveness and an increase in eosinophil and lymphocyte numbers in bronchoalveolar lavage after aerosol exposure to ovalbumin [17]. Three weeks after ovalbumin sensitization, animals were challenged with 5% aerosolized ovalbumin whilst in a transparent plastic box to permit observation of the reaction to the allergen challenge. Ovalbumin was aerosolized for 5 min with a Hudson micromist 880 nebulizer (Hudson RCI, Temecula, CA, USA), using compressed air at a pressure giving an output of 0·3–0·4 ml/min. This experimental protocol was approved by University Laval Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.

AM isolation

AM from unsensitized Brown Norway and Sprague Dawley rats were isolated as previously described [18]. Briefly, animals were anaesthetized and exsanguinated by cutting the abdominal aorta. The trachea was cannulated and the lung was lavaged with a total volume of 50 ml cold, endotoxin-free saline. About 90% of the lavage fluid was recovered. Cells, with a viability of 97% according to Trypan blue exclusion, were stained with May-Grünwald Giemsa.

Cytokines

AM from naive rats were incubated in RPMI 1640 medium (supplemented with 1 mm glutamine, 5% FBS and antibiotic) for 2 h at 37°C in a humidified atmosphere of 5% CO2 in air to allow adherence. After this incubation, the cells were washed gently with warm medium and resuspended in fresh medium. AM (99% pure) were then treated at 37°C with either OX8 (anti-CD8 antibody) or isotype control (IgG1) at 5 µg/ml or LPS (1 ng/ml) for 6 h for TNF release, or 20 h for IL-10, IL-12, IL-13 and MIP-1α release. The time used to study the release of cytokines by AM was determined in preliminary experiments. Cell-free supernatant fluids were tested for cytokine content using immunoassay kits for rat TNF, IL-10, IL-12 (recognizing both p70 and p40) and IL-13 (Biosource International, Camarillo, CA, USA) with a sensitivity of 4 pg/ml, 5 pg/ml, 5 pg/ml and 1·5 pg/ml, respectively. MIP-1α content was measured using a double-ligand method as previously described [18]. This ELISA consistently detected MIP-1α concentrations > 7 pg/ml.

Measurement of nitric oxide (NO) production

AM were treated with OX8 or an isotype control at 5 µg/ml or LPS at 1 ng/ml for 24 h in phenol red-free RPMI, and cell-free supernatant fluids were assayed for NO2− using the Griess reaction as previously described [19]. Plates were read at 540 nm using a Molecular Devices (Menlo Park, CA, USA) V max Kinetic Microplate Reader, and NO2− concentrations were determined by reference to a standard curve (NaNO2).

RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)

Given that mRNA is expressed before the release of the protein, AM were treated either with or without OX8 (5 µg/ml) for 4 h and total RNA was extracted using TRIzol reagent (Life Technologies, Burlington, ON, Canada). For cDNA synthesis, 2 µg total RNA were reverse transcribed by Moloney murine leukaemia virus reverse transcriptase (Gibco-BRL, Burlington, ON, Canada) using a Peltier Thermal Cycler 200 (MJ Research Inc, Watertown, MA, USA). The PCR was performed using the Qiagen Taq DNA polymerase protocol. The primers used were: (a) rat β-actin sense primer: 5′-GTG GGG CGC CCC AGG CAC CA-3′ and antisense primer 5′-GTC CTT AAT GTC ACG CAC GAT TTC-3′ (526 bp); (b) murine TNF sense primer: 5′-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3′ and antisense primer 5′-TTC ATG GCC TTG TAG ACA CC-3′ (354 bp); (c) rat IL-10 sense primer: 5′-CAC TGC TAT GTT GCC TGC TC-3′ and antisense primer 5′-TGA GTG TCA CGT AGG CTT CTA-3′ (286 bp); (d) rat IL-12 p40 sense primer: 5′-CCG ATG CCC CTG GAG AAA C-3′ and antisense primer 5′-CCT TCT TGT GGA GCA GCA G-3′ (207 bp); (e) rat iNOS sense primer: 5′-ACA ACA GGA ACC TAC CAG CTC A-3′ and antisense primer 5′GAT GTT GTA GCG CTG TGT GTC A-3′ (651 bp); (f) rat MIP-1α sense: 5′-ATG AAG GTC TCC ACC ACT-3′ and antisense primer 5′-TCA GGC ATT CAG TTC CAG-3′ (279 bp). Primers for rat IL-13 (368 bp) and IL-12 p35 (308 bp) were purchased from Biosource. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Relative mRNA expression was quantified by densitometric scanning analysis using NIH Image 1·61 and normalized against β-actin which gave semi-quantitative results.

Statistical analysis

Analysis of variance, combined with Fisher's protected least significant difference test, was used to compare the different treatments on AM from either Brown Norway or Sprague Dawley rats (Figs 1,2,3,4). The Student's test for paired data was used to compare cell types in bronchoalveolar lavage between the two strains of rats. Differences were considered significant when P was < 0·05.

Fig. 1.

Fig. 1

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Production of TNF, MIP-1α and NO by alveolar macrophages (AM). AM were stimulated with OX8 (5 µg/ml) or an isotype control (IgG), and cell-free supernatant fluids were tested for TNF, MIP-1α and NO content. OX8 significantly (‡P < 0·002) increased TNF and NO release from AM of both strains of rats, whereas it significantly (‡P < 0·002) increased MIP-1α release from AM of Brown Norway rats only when compared with the isotype control. TNF release was significantly (*P < 0·01) higher, whereas MIP-1α and NO release was significantly lower, in Sprague Dawley rats compared with Brown Norway rats. A significant difference between both strains of rats is indicated by *P < 0·01. Mean ± s.e.m. of 5–10 experiments. (□), Brown Norway; (Inline graphic), Sprague Dawley.

Fig. 2.

Fig. 2

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Differences in mRNA levels for TNF, MIP-1α and NOS2 in alveolar macrophages (AM) from Brown Norway and Sprague Dawley rats. Cells were treated with or without OX8 for 4 h and RT-PCR was performed for TNF, MIP-1α, NOS2 and β-actin (a). Lane 1, sham-treated AM and lane 2, OX8-treated AM. Relative mRNA expression of TNF, MIP-1α and NOS2 was quantified by densitometric analysis normalized against β-actin. Significant differences (*P < 0·05) between AM from Brown Norway and Sprague Dawley rats and significant increases (‡P < 0·05) in mRNA levels compared with the sham-treated control are shown. Results of a representative experiment are presented in (a) whereas mean ± s.e.m. of 7–10 experiments is presented in (b). (□), Brown Norway; (Inline graphic), Sprague Dawley.

Fig. 3.

Fig. 3

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Production of Th1 and Th2 cytokines by alveolar macrophages (AM). Cells were stimulated with OX8 (5 µg/ml) or with an isotype control (IgG) for 20 h, and IL-12, IL-10 and IL-13 were measured in cell-free supernatant fluids. OX8 significantly increased (‡P < 0·01) IL-12 release from AM of Brown Norway rats only, whereas it significantly increased (‡P < 0·01) IL-10 release from AM of both rat strains compared with the isotype control. IL-10 and IL-12 release from AM in the presence of the isotype control and from OX8-stimulated AM were significantly (*P > 0·05) higher in Brown Norway compared with Sprague Dawley rats. IL-13 was not modulated by OX8 but its release was significantly (*P > 0·05) higher in AM from Brown Norway compared with Sprague Dawley rats. Mean ± s.e.m. of 5–6 experiments. (□), Brown Norway; (Inline graphic), Sprague Dawley.

Fig. 4.

Fig. 4

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Differences in mRNA levels for IL-10, IL-12 and IL-13 in alveolar macrophages (AM) of Brown Norway and Sprague Dawley rats. Cells were treated with or without OX8 for 4 h, and RT-PCR was performed for IL-10, IL-12, IL-13 and β-actin. Lane 1, sham-treated AM and lane 2, OX8-treated AM. Relative mRNA expression of IL-10, IL-12 and IL-13 was quantified by densitometric analysis normalized against β-actin. Significant differences (*P < 0·05) between AM from Brown Norway and Sprague Dawley rats, and significant increases (‡P < 0·02) in mRNA levels compared with the control, are shown. Results of a representative experiment are presented in (a) whereas mean ± s.e.m. of 6–8 experiments is presented in (b).(□), Brown Norway; (Inline graphic), Sprague Dawley.

Results

Aerosol ovalbumin exposure

Sensitized Brown Norway and Sprague Dawley rats were exposed to aerosolized ovalbumin for 5 min. Brown Norway rats demonstrated obvious respiratory distress characterized by defensive posture, opening of the mouth, exaggerated laboured breathing motions and wheezing. These reactions started within 2–3 min, lasted for a few minutes before recovery of the animals and were observed in 97% of Brown Norway rats (n = 30). In contrast, aerosolized ovalbumin did not cause any detectable reaction in immunized Sprague Dawley rats (n = 30). Animals from both strains of rats exposed to saline, or sensitized with saline and exposed to ovalbumin, did not demonstrate any respiratory problems.

Mediator release by AM

Bronchoalveolar lavages were performed on unsensitized rats. There was no significant difference in cell numbers or percentages of AM, lymphocytes and neutrophils between the two strains of rats (Table 1). However, there were significantly more eosinophils in the bronchoalveolar lavage of Brown Norway rats (3·0 ± 1·7%) compared with Sprague Dawley rats (0·1 ± 0·1%).

Table 1.

Characterization of cells in bronchoalveolar lavage (BAL) of naive Brown Norway and Sprague Dawley rats

Rat strain AM Lymphocyte Neutrophil Eosinophil
Brown Norway 94·7 ± 0·2% 1·7 ± 0·6% 0·6 ± 0·2% 3·0 ± 1·7%*
Sprague Dawley 94·6 ± 1·6% 4·2 ± 1·7% 1·1 ± 0·7% 0·1 ± 0·1%
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*

Cytospins of bronchoalveolar lavage cells were stained with May-Grünwald Geisma and the percentage of each cell type was calculated. There were significantly (P < 0·01) more eosinophils in bronchoalveolar lavage from Brown Norway rats than Sprague Dawley rats. Mean (%) ± s.e.m. of five experiments.

Stimulation of AM through their membrane CD8 has been shown to increase the release of TNF, NO and IL-1β [19–21]. Thus, AM were stimulated with OX8 (anti-CD8 antibody) or an isotype control for 6, 20 and 24 h to assess the production of TNF, MIP-1α and NO, respectively. The isotype control did not significantly modulate the release of TNF whereas OX8 significantly stimulated (P < 0·002) its release from AM of both strains of rats (Fig. 1). Furthermore, OX8-stimulated AM from Sprague Dawley rats released significantly more TNF (2·2 ± 0·5 ng/106 AM) than those from Brown Norway rats (0·9 ± 0·1 ng/106 AM). In contrast, isotype control and OX8-stimulated AM of Brown Norway rats released significantly more MIP-1α than those from Sprague Dawley rats (Fig. 1). Although OX8 did not significantly stimulate AM MIP-1α release in Sprague Dawley rats, it significantly increased it in Brown Norway rats. Similar results were observed using LPS, a common stimulus for macrophages. Significantly more TNF (418 ± 93 pg/106 AM; P < 0·05) and less MIP-1α (7448 ± 440 pg/106 AM; P < 0·03) was released from LPS-stimulated AM from Sprague Dawley rats compared with those from Brown Norway rats (224 ± 51 and 8298 ± 246 pg/106 AM, respectively).

The same stimuli were used to investigate the production of NO by AM. OX8 treatment significantly increased NO release compared with AM treated with an isotype control (Fig. 1). However, OX8-stimulated AM from Brown Norway rats released significantly more NO (32·7 ± 3·1 µm/106 AM) than those from Sprague Dawley rats (17·2 ± 3·6 µm/106 AM). Similarly, LPS-stimulated AM from Brown Norway rats released significantly (P < 0·02) more NO (33 ± 4 µm/106 AM) than those from Sprague Dawley rats (22 ± 3 µm/106 AM).

To explore the mechanism by which OX8 stimulated TNF and MIP-1α production further, we investigated the effect of this stimulation at the mRNA level using RT-PCR. OX8 stimulation increased TNF mRNA levels in AM of both strains of rats, whereas the MIP-1α mRNA level was significantly increased only in AM from Brown Norway rats (Fig. 2). In accordance with MIP-1α release, the MIP-1α mRNA level was significantly higher in sham- and OX8-stimulated AM from Brown Norway rats compared with those from Sprague Dawley rats. However, in contrast to TNF release, no significant difference was observed in OX8-stimulated AM from Sprague Dawley and Brown Norway rats at the level of TNF mRNA.

In macrophages, NO synthesis is mainly catalysed by the inducible form of NO synthase, NOS2 [22]. Thus, we investigated the expression of NOS2 mRNA levels in the same samples as mentioned above (Fig. 2). Although unstimulated AM from Brown Norway rats expressed slightly more NOS2 mRNA than those from Sprague Dawley rats, the difference was not significant (Fig. 2). OX8 stimulation caused an increase in NOS2 mRNA expression in both strains of rats, but there was three times more NOS2 mRNA expressed in AM from Brown Norway rats compared with Sprague Dawley rats (Fig. 2). These data on TNF, MIP-1α and NO production demonstrate differences in AM mediator production from strains of rats with distinct susceptibilities to develop allergic reactions.

Th1 and Th2 cytokine production by AM

AM were treated with OX8 or an isotype control for 20 h and cell-free supernatant fluids were tested for IL-12, IL-10 and IL-13. Unstimulated AM from Brown Norway rats released significantly (P < 0·05) more IL-12, a Th1 cytokine, than AM from Sprague Dawley rats (Fig. 3). OX8 treatment stimulated (P < 0·01) IL-12 release from AM of Brown Norway rats, but not from those of Sprague Dawley rats.

AM from Brown Norway rats released significantly more IL-10 than those from Sprague Dawley rats (Fig. 3). OX8 significantly stimulated (P < 0·01) the release of IL-10 in both strains of rats, causing a significantly higher increase in Brown Norway rats. However, IL-13, another Th2 cytokine, was not modulated by OX8 stimulation (Fig. 3). Nonetheless, isotype control and OX8-stimulated AM from Brown Norway rats released significantly more IL-13 than AM from Sprague Dawley rats. Furthermore, LPS did not increase IL-10 and IL-13 release by AM of Sprague Dawley rats (2·9 ± 2 pg/106 AM and 2·1 ± 0·8 pg/106 AM, respectively) but significantly stimulated IL-13 release by AM of Brown Norway rats (control, 6·8 ± 2·6 pg/106 AM and LPS-stimulated AM, 8·6 ± 3·8 pg/106 AM). These data raise the possibility of the presence of different cytokine profiles for AM in these two strains of rats.

To determine whether the cytokine release observed in culture supernatant fluids reflected an increase in their steady state levels of mRNA, RT-PCR analysis was performed on RNA isolated from cells treated with or without OX8 for 4 h (Fig. 4a). Results observed for mRNA were similar to those obtained at the protein level. However, although OX8 significantly (P < 0·01) increased IL-12 p40 mRNA levels (Fig. 4b), the increase in IL-12 p35 mRNA levels (0·389 ± 0·104 for the control and 1·057 ± 0·322 for OX8 stimulation, n = 6) was not significant.

Discussion

There is increasing evidence to suggest that Th2-type cytokines play a pivotal role in the induction and persistence of airway inflammatory processes in allergic asthma [5]. However, there is limited information on the parameters involved in orientating this immune response. AM are an important source of cytokines. Among such cytokines, IL-12 may play an important role in the control of IgE production and in leading the immune response towards a Th1 pattern [23]. AM also produce Th2-type cytokines, such as IL-10 and IL-13, which may guide the immune response towards a Th2 type [1]. Furthermore, AM are potent producers of TNF [11] and NO [19], both of which possess a broad range of proinflammatory properties, but there is some evidence suggesting that TNF plays an important role in the development of Th1 responses [24] whereas NO has the opposite effect [25]. Thus, given the role of these AM mediators in the development of an immune response, it is tempting to speculate that there may be different cytokine profiles for AM which may play an important role in the susceptibility of developing allergic asthma.

Our results (summarized in Table 2) demonstrate that AM from Brown Norway and Sprague Dawley rats are functionally different. Brown Norway rats are widely used as a model of asthma and hyperresponsiveness [12–16]. However, to our knowledge, their AM have never been compared with those of resistant rats. AM from susceptible and resistant rats showed significant distinctions in their cytokine profile. AM from resistant rats produced significantly more TNF than susceptible rats when stimulated with anti-CD8 antibody (OX8). We have previously demonstrated that Sprague Dawley rats have significantly more AM CD8+ than Brown Norway rats (83 ± 5% and 53 ± 5%, respectively) [26]. The difference in TNF release (43%) by OX8-stimulated AM from Sprague Dawley rats compared with AM from Brown Norway rats may be accounted for by the greater number of CD8+ AM (35%) observed in the former rats. However, similar results were obtained with OX8- and LPS-stimulated AM, suggesting that AM from Sprague Dawley rats release more TNF than AM from Brown Norway rats, independent of the stimuli. Although TNF is mostly considered as an inflammatory cytokine, it also plays an important role in the development of a Th1 response in animal models [24]. Thus, increased TNF production by AM from Sprague Dawley rats may lead the immune response towards a Th1 phenotype, making this strain of rats more resistant to the development of allergic asthma.

Table 2.

Summary of alveolar macrophage cytokine production in Brown Norway and Sprague Dawley rats

Brown Norway Sprague Dawley
Mediators Protein mRNA level Protein mRNA level
TNF – = + =
MIP-1α + + – –
NO + + – –
IL-10 + + – –
IL-13 + + – –
IL-12 + –
IL-12p35 = =
IL-12p40 + –
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Alveolar macrophages (AM) from Brown Norway rats produce more (+) MIP-1α, NO, IL-12p40, IL-10 and IL-13, an equal amount (=) of IL-12p35 but less (–) TNF compared with AM from Sprague Dawley rats.

MIP-1α has been shown to recruit and stimulate eosinophils [27]. Elevated production of MIP-1α by unstimulated AM of Brown Norway rats may explain, in part, the increased number of eosinophils in the BAL of these rats compared with Sprague Dawley rats. Furthermore, stimulated AM from Brown Norway rats release even more MIP-1α which may participate in the recruitment of eosinophils observed after allergen challenge in these rats [14].

An increase in exhaled NO has been demonstrated in asthmatic patients [28] and after challenge in sensitized animals [29]. This increased NO production is associated with elevated NOS2 (inducible isoform of NO synthase) expression in bronchial tissue [30]. In animal models, a high concentration of NO has been shown to inhibit both the proliferation of Th1 cells and the production of IL-2 and IFN-γ [30]. Thus, increased NO production by AM in Brown Norway rats may contribute to the inhibition of the Th1 response, allowing the development of a Th2 response.

IL-12 is a 70 kDa heterodimeric cytokine composed of 35 kDa and 40 kDa subunits linked by disulphide bonds [31]. Although each subunit is independently controlled, the expression of p35 determines the level of active IL-12 protein [32]. By contrast, p40 subunits can exist as dimers which can bind IL-12 receptors and act as IL-12 antagonists in vivo and in vitro [33,34]. Our data show that AM from Brown Norway rats release more IL-12 (measured by ELISA) and express more IL-12 p40 mRNA than AM from Sprague Dawley rats, whereas there was no significant difference in IL-12 p35 mRNA levels because of the high variation. Given that the ELISA for IL-12 recognizes both IL-12 p70 and the p40 subunit either free or as a dimer, our data suggest that AM from Brown Norway rats may produce more IL-12 p40, which may act as an IL-12 antagonist and contribute to the inhibition of the Th1 response. However, further investigations are needed to confirm this observation.

IL-10, usually considered an immunosuppressive and anti-inflammatory cytokine [1], may potentiate the Th2-type response by inhibiting Th1 cytokine production such as IFN-γ and IL-12 [35,36]. Interestingly, AM from Brown Norway rats produced more IL-10 and IL-13, another Th2 cytokine produced in increased amounts in allergic diseases [37], than AM from Sprague Dawley rats, suggesting a role for AM IL-10 and IL-13 production in the initiation of allergic asthma development in Brown Norway rats.

The development of immune responses involves a complex interplay between immune cells, mediators and genetics. The microenvironment plays a crucial role in the development of Th1 and Th2 responses. Our data suggest that mediators released by AM may play an important role in creating the cytokine milieu in the lung that will guide the immune response towards Th1 or Th2. Our results lead to the possibility of the existence of AM with distinct cytokine profiles, namely AM-1 and AM-2, as suggested by Mill et al. [38]. However, further investigations are needed to clarify the role of AM-1 and AM-2 in the development of allergic reactions.

Acknowledgments

This work was supported by the Medical Research Council of Canada. One author (E.Y.B.) is a Medical Research Council of Canada and Canadian Lung Association Scholar.

References

  • 1.Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today. 1996;17:138–46. doi: 10.1016/0167-5699(96)80606-2. 10.1016/0167-5699(96)80606-2. [DOI] [PubMed] [Google Scholar]
  • 2.Eschenbacher WL, Gravelyn TR. A technique for isolated airway segment lavage. Chest. 1987;92:105–9. doi: 10.1378/chest.92.1.105. [DOI] [PubMed] [Google Scholar]
  • 3.Schauble TL, Boom WH, Finegan CK, Rich EA. Characterization of suppressor function of human alveolar macrophages for T lymphocyte responses to phytohemagglutinin: cellular selectivity, reversibility, and early events in T cell activation. Am J Respir Cell Mol Biol. 1993;8:89–97. doi: 10.1165/ajrcmb/8.1.89. [DOI] [PubMed] [Google Scholar]
  • 4.Holt PG, Oliver J, Bilyk N, et al. Downregulation of the antigen presenting cell function (s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J Exp Med. 1988;167:262–74. doi: 10.1084/jem.177.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Barnes P. Cytokines as mediators of chronic asthma. Am J Respir Crit Care Med. 1994;150:S42–S49. doi: 10.1164/ajrccm/150.5_Pt_2.S42. [DOI] [PubMed] [Google Scholar]
  • 6.Daftary SS, Calhoun WJ. Macrophages and macrophage diversity in asthma. In: Holgate ST, Busse WW, editors. Inflammatory mechanisms in asthma. New York: Marcel Dekker Inc.; 1998. pp. 361–86. [Google Scholar]
  • 7.Liu MC, Hubbard WC, Proud D, et al. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airway in allergic asthmatics: cellular, mediator, and permeability changes. Am Rev Respir Dis. 1991;144:51–8. doi: 10.1164/ajrccm/144.1.51. [DOI] [PubMed] [Google Scholar]
  • 8.Thepen T, Van Rooijen N, Kraal G. Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice. J Exp Med. 1989;170:499–509. doi: 10.1084/jem.170.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Thepen T, Hoeben K, Breve J, Kraal G. Alveolar macrophages down-regulate local pulmonary immune responses against intratracheally administered T-cell-dependent, but not T-cell-independent antigens. Immunology. 1992;76:60–4. [PMC free article] [PubMed] [Google Scholar]
  • 10.Thepen T, McMenamin C, Girn B, Kraal G, Holt PG. Regulation of IgE production in pre-sensitized animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen. Clin Exp Allergy. 1992;22:1107–14. doi: 10.1111/j.1365-2222.1992.tb00137.x. [DOI] [PubMed] [Google Scholar]
  • 11.Kelley J. Cytokines of the lung. Am Rev Respir Dis. 1990;141:765–88. doi: 10.1164/ajrccm/141.3.765. [DOI] [PubMed] [Google Scholar]
  • 12.Eidelman DH, Bellofiore S, Martin JG. Late airway responses to antigen challenge in sensitized inbred rats. Am Rev Respir Dis. 1988;137:1033–7. doi: 10.1164/ajrccm/137.5.1033. [DOI] [PubMed] [Google Scholar]
  • 13.Waserman S, Olivenstein R, Renzi PM, Xu LJ, Martin JG. The relationship between late asthmatic responses and antigen-specific immunoglobulin. J Allergy Clin Immunol. 1992;90:661–9. doi: 10.1016/0091-6749(92)90140-w. [DOI] [PubMed] [Google Scholar]
  • 14.Renzi PM, Olivenstein R, Martin JG. Inflammatory cell populations in the airways and parenchyma after antigen challenge in the rat. Am Rev Respir Dis. 1993;147:967–74. doi: 10.1164/ajrccm/147.4.967. [DOI] [PubMed] [Google Scholar]
  • 15.Bellofiore S, Martin JG. Antigen challenge of sensitized rats increases airway responsiveness to methacholine. J Appl Physiol. 1988;65:1642–6. doi: 10.1152/jappl.1988.65.4.1642. [DOI] [PubMed] [Google Scholar]
  • 16.Olivenstein R, Renzi PM, Yang JP, et al. Depletion of OX-8 lymphocytes from the blood and airways using monoclonal antibodies enhances the late airway response in rats. J Clin Invest. 1993;92:1477–82. doi: 10.1172/JCI116725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Elwood W, Lötvall JO, Barnes PJ, Chung KF. Characterization of allergen-induced bronchial hyperresponsiveness and airway inflammation in actively sensitized Brown-Norway rats. J Allergy Clin Immuno. 1991;88:951–60. doi: 10.1016/0091-6749(91)90253-k. [DOI] [PubMed] [Google Scholar]
  • 18.Déry RE, Bissonnette EY. IFN-γ potentiates the release of TNF-α and MIP-1α by alveolar macrophages during allergic reactions. Am J Respir Cell Mol Biol. 1998;20:407–12. doi: 10.1165/ajrcmb.20.3.3252. [DOI] [PubMed] [Google Scholar]
  • 19.Hirji N, Lin TJ, Befus AD. A novel CD8 molecule expressed by alveolar and peritoneal macrophages stimulates nitric oxide production. J Immunol. 1996;158:1833–40. [PubMed] [Google Scholar]
  • 20.Hirji N, Lin TJ, Bissonnette EY, Belosevic M, Befus AD. Mechanisms of macrophage stimulation through CD8: macrophage CD8α and β induce nitric oxide production and associated killing of the parasite Leishmania major. J Immunol. 1998;160:6004–11. [PubMed] [Google Scholar]
  • 21.Lin TJ, Hirji N, Stenton GR, et al. Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production. J Immunol. 2000;164:1783–92. doi: 10.4049/jimmunol.164.4.1783. [DOI] [PubMed] [Google Scholar]
  • 22.Pendino KJ, Laskin JD, Shuler RL, Punjabi CJ, Laskin DL. Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase. J Immunol. 1993;151:7196–205. [PubMed] [Google Scholar]
  • 23.Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today. 1993;14:335–8. doi: 10.1016/0167-5699(93)90230-I. [DOI] [PubMed] [Google Scholar]
  • 24.Becher B, Blain M, Giacomini PS, Antel JP. Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12. J Immunol. 1999;162:684–8. [PubMed] [Google Scholar]
  • 25.Taylor-Robinson AW, Liew FY, Severn A, et al. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. Eur J Immunol. 1994;24:980–4. doi: 10.1002/eji.1830240430. [DOI] [PubMed] [Google Scholar]
  • 26.Bissonnette EY, Déry RE, Sirois J. Heterogeneity of alveolar macrophages may explain susceptibility to develop allergic asthma. Clin Invest Med. 1999;22:S4. [Google Scholar]
  • 27.Rot A, Krieger M, Brunner T, Bischoff SC, Schall TJ, Dahinden CA. RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes. J Exp Med. 1992;176:1489–95. doi: 10.1084/jem.176.6.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Dupont LJ, Rochette F, Demedts MG, Verleden GM. Exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma. Am J Respir Crit Care Med. 1998;157:894–8. doi: 10.1164/ajrccm.157.3.9709064. [DOI] [PubMed] [Google Scholar]
  • 29.Persson MG, Gustafsson LE. Allergen-induced airway obstruction in guinea-pigs is associated with change in nitric oxide levels in exhaled air. Acta Physiol Scand. 1993;149:461–6. doi: 10.1111/j.1748-1716.1993.tb09643.x. [DOI] [PubMed] [Google Scholar]
  • 30.Yan ZQ, Hansson GK, Skoogh BE, Lötvall JO. Induction of nitric oxide synthase in a model of allergic occupational asthma. Allergy. 1995;50:760–4. doi: 10.1111/j.1398-9995.1995.tb01221.x. [DOI] [PubMed] [Google Scholar]
  • 31.Kobayashi M, Fitz L, Ryan M, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med. 1989;170:827–45. doi: 10.1084/jem.170.3.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Hayes MP, Wang J, Norcross MA. Regulation of interleukin-12 expression in human monocytes: selective priming by interferon-gamma of lipopolysaccharide-inducible p35 and p40 genes. Blood. 1995;86:646–50. [PubMed] [Google Scholar]
  • 33.Gately MK, Carvajal DM, Connaughton SE, et al. Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo. Ann NY Acad Sci. 1996;795:1–12. doi: 10.1111/j.1749-6632.1996.tb52650.x. [DOI] [PubMed] [Google Scholar]
  • 34.Ling P, Gately MK, Gubler U, et al. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J Immunol. 1995;154:116–27. [PubMed] [Google Scholar]
  • 35.Fiorentino DF, Zlotnik A, Vieira P, et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol. 1991;146:3444–51. [PubMed] [Google Scholar]
  • 36.D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon γ-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993;178:1041–8. doi: 10.1084/jem.178.3.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Till S, Durham S, Dickason R, et al. IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-γ expression. Immunology. 1997;91:53–7. doi: 10.1046/j.1365-2567.1997.00218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164:6166–73. doi: 10.4049/jimmunol.164.12.6166. [DOI] [PubMed] [Google Scholar]

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