Introduction

Abstract Introduction References

Alveolar macrophages are found throughout the respiratory tract from the alveolus to the larynx, and represent the most abundant cells in the airway lumen. They are believed to be the major defenders of the lung against infectious agents and other immunologic insults (1). Their functions are mediated by the release of several mediators, including nitric oxide (2), -glucuronidase, superoxide anion, platelet-activating factor (3), and leukotriene B₄ (4), as well as chemotactic factors for neutrophils and eosinophils (5) that play important roles in inflammation. Furthermore, alveolar macrophages have been identified as potent producers of several cytokines, including macrophage inflammatory protein-1 (MIP-1) (6) and tumor necrosis factor- (TNF-), which possess a broad range of proinflammatory properties (7).

In addition to its inflammatory effects, TNF- causes bronchial hyperresponsiveness in experimental animals (8) and in humans (9). It promotes the expression of endothelial and intercellular adhesion molecules that increase endothelial permeability and facilitate recruitment of leukocytes to the site of inflammation, such as in asthma (10). Furthermore, TNF- can stimulate these cells and the production of many inflammatory mediators (11), amplifying the inflammatory response.

MIP-1, a member of the C-C chemokine family, is important in pulmonary inflammation and lung diseases (12, 13). It is produced by a variety of cell types, including alveolar macrophages (6), and induces the recruitment of monocytes, neutrophils, lymphocytes, and eosinophils (14) to stimulate the production of TNF-, interleukin (IL)-1, and IL-6 (15); to stimulate surface immunoglobulin (Ig)E⁺ B cells; and to enhance IgE production (16). Furthermore, MIP-1 plays an important role in the inflammatory response to viral infection (12) and activates basophils and mast cells (17), suggesting also a role for this cytokine in allergic reactions.

Interferons (IFNs) were identified and defined by their ability to prevent viral replication, and play a crucial role in host defense against virus infections (18). IFN- and IFN- are produced by leukocytes and fibroblasts, respectively, whereas IFN- is produced by natural killer cells and lymphocytes (Th1) after mitogenic or antigenic stimulation (19). In addition to its potent antiviral activity, IFN- can modulate immune functions such as regulation of cell growth and differentiation; regulation of biosynthesis and expression of the major histocompatibility complex; and enhancement of macrophage phagocytosis, cytotoxicity, and antimicrobial activity (20). Furthermore, IFN- increases the numbers of IgE low-affinity receptors (Fc RII) on monocytes (21). Activation of these receptors by IgE-immune complexes stimulates the release of inflammatory mediators such as leukotrienes (22), platelet-activating factor (23), IL-1, and TNF- (24). The release of the latter is also enhanced by IgE-dependent stimulation of alveolar macrophages (25).

Interestingly, there is an increase in the expression of Fc RII on alveolar macrophages of asthmatic patients (26). Thus, given the production of IFN- during viral infections, we hypothesized that IFN- potentiates the release of inflammatory mediators by alveolar macrophages stimulated with IgE/anti-IgE, which will contribute to allergic inflammation in the lung. IFN- stimulated the release of TNF- by alveolar macrophages in a time- and dose-dependent manner, and increased the release of MIP-1. Stimulation of alveolar macrophages through Fc RII resulted in the release of TNF- without MIP-1 release. However, IFN- pretreatment potentiated the release of both cytokines when alveolar macrophages were stimulated with IgE/anti-IgE.

	Materials and Methods

Animals

Male Sprague-Dawley rats were obtained from Health Sciences Laboratory Animal Services (University of Alberta, Edmonton, AB, Canada) and maintained in an isolation room with filter-topped cages to minimize unwanted infections. Rats of 200 to 250 g were infected 5 to 6 wk before use with 3,000 third-stage larvae of Nippostrongylus brasiliensis by a single subcutaneous injection. This infection sensitized mast cells from different anatomic sites to the worm antigen (27) and increased the number of Fc RII on alveolar macrophages in rats (28). This experimental protocol was approved by the University of Alberta Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.

Reagents

Purified (> 90%) rat myeloma IgE protein (clone IR162, code PRP07) and monoclonal antibody to the heavy chain of IgE were purchased from Serotec, Ltd. (Toronto, ON, Canada). Rat recombinant IFN- was obtained from Immunocorp (Montréal, PQ, Canada). Recombinant murine MIP-1 and goat antimurine MIP-1 polyclonal antibody were purchased from R&D Systems (Minneapolis, MN). Mouse antirat recombinant IFN- (18 mg/ml) was a gift from Dr. M. Belosevic of the University of Alberta (Edmonton, AB, Canada). Polymyxin B was purchased from Sigma (St. Louis, MO). RPMI-1640 medium, phosphate-buffered saline (PBS), and IFN- contained less than 0.05 endotoxin units when tested by E-Toxate kit (Sigma).

Alveolar Macrophage Isolation and Stimulation

Alveolar macrophages were isolated as previously described (29). Briefly, the abdominal aorta of an anesthetized Sprague-Dawley rat was severed and the animal was exsanguinated. The trachea was cannulated and the lung was lavaged with a total volume of 60 ml cold PBS in 5- to 8-ml aliquots. About 90% of the lavage fluid was recovered and the purity of alveolar macrophages was 95 ± 3% according to May-Grünwald-Giemsa and nonspecific esterase staining. Viability always exceeded 97% according to Trypan blue exclusion. For the release of mediators, alveolar macrophages were treated with IFN- for different periods of time before treatment with IgE for 2 h. At the end of this treatment, cells were washed and finally stimulated with anti-IgE for 18 h (determined from preliminary time-course experiments). Cell-free supernatants were frozen at 70°C until assayed for mediator content.

TNF- and MIP-1 Enzyme-Linked Immunosorbent Assay

Supernatants were tested for TNF- content by enzyme-linked immunosorbent assay (ELISA; Biosource International, Montréal, PQ, Canada) using polyclonal antibodies which can detect 4 pg/ml of total rat TNF-. MIP-1 was quantitated using a modification of a double-ligand method as previously described (30). Briefly, a flat-bottom 96-well microtiter plate (Nunc Maxisorp; NUNC, Naperville, IL) was coated with 100 µl/well of goat anti-MIP-1 antibody (1 µg/ml) in PBS for 20 h at 4°C. The plate was then washed three times with PBS and nonspecific binding was blocked with 5% bovine serum albumin in PBS containing 0.02% sodium azide (blocking buffer) for 20 h at 24°C. The plate was washed three times with PBS and duplicates of cell-free supernatants were added to the plate for 2 h at 25°C. At the end of the incubation, the plate was washed three times, 100 µl/well of biotinylated goat anti-MIP-1 antibody at 1 µg/ml in blocking buffer was added, and the plate was incubated for 1 h at 25°C. After washes, 100 µl/ well of streptavidin-peroxidase conjugate (Sigma) at 1 µg/ ml was added for 30 min and the plate was washed to remove unbound conjugate before adding the substrate tetramethyl benzidine (Sigma). The reaction was stopped 30 min later by adding 100 µl H₂SO₄ 1 M, and the plate was read at 450 nm with a correction wavelength of 570 nm in a Vmax kinetic microplate reader (Molecular Devices Co., Menlo Park, CA). A standard curve was done for each plate using different concentrations of recombinant murine MIP-1 (from 7.1 to 2,000 pg/ml). This ELISA method consistently detected MIP-1 concentrations > 7 pg/ml.

Reverse Transcription-Polymerase Chain Reaction

Alveolar macrophages from normal and infected rats were isolated and treated or not with IgE/anti-IgE as specified in the text, and total RNA was extracted using TRIzol reagent (Life Technologies, Burlington, ON, Canada), yielding 3.5 ± 0.3 µg RNA/10⁶ alveolar macrophages, with an optical density_260/280 ratio of 1.8. For complementary DNA synthesis, 1 µg of total messenger RNA (mRNA) was reverse transcribed by SuperScript RNase (GIBCO BRL), using a Genamp 2400 Programmable Thermal Controller (Perkin Elmer, Mississauga, ON, Canada) according to the manufacturer's protocols. Polymerase chain reaction (PCR) was a modification of the GIBCO BRL Taq DNA polymerase protocol, with changes in the concentration of deoxynucleotide triphosphate (1.23 µM) and 10× PCR buffer (67 mM Tris [pH 8.8], 1.5 mM MgCl₂, 16.6 mM [NH₄]₂SO₄, and 10 mM -mercaptoethanol) in a total volume of 20 µl. The primers used were: (1) rat -actin 5' primer: 5'-GTG GGG CGC CCC AGG CAC CA-3', and 3' primer: 5'-GTC CTT AAT GTC ACG CAC GAT TTC-3'; (2) rat TNF- 5' primer: 5'-TTC TGT CTA CTG AAC TTC GGG GTG ATG GGT CC-3', and 3' primer: 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3'; and (3) rat Fc RI chain 5' primer: ATC GTC ACA TTG GGT CAT TGT G and 3' primer: 5'-TGT TCA AAT AGC CTG TGC AGT G-3'. The PCR products for -actin, TNF-, and high-affinity receptor for IgE (Fc RI) were 526, 295, and 323 base pairs, respectively. Rat peritoneal mast cells were used as control for the expression of Fc RI. The temperatures and times were: 95°C for 45 s, 62°C for 45 s, and 72°C for 2 min for -actin; 94°C for 45 s, 62°C for 45 s, and 72°C for 2 min for TNF-; 94°C for 45 s, 53°C for 45 s, and 72°C for 2 min for Fc RI. After preliminary tests of PCR cycle numbers, 30 cycles were performed. Products were run on a 2% agarose gel and stained with ethidium bromide.

Statistical Analysis

Analysis of variance, combined with Fisher's protected least significant difference test or Student's tests for paired data, were used to compare treatments. Differences were considered significant when P < 0.05.

	Results

Release of TNF- from Alveolar Macrophages Stimulated through Fc RII

Previous studies have shown that the percentage of alveolar macrophages bearing Fc RII in normal rats is about 26%, whereas it increases to about 60% in N. brasiliensis- infected rats (28). Thus, to investigate the stimulation of alveolar macrophages through Fc RII, N. brasiliensis- infected rats were used. Alveolar macrophages from infected (5 wk after infection) and age-matched uninfected rats were isolated, incubated with IgE (1 µg/ml) for 2 h, gently washed, and stimulated with anti-IgE (0.5 µg/ml) for 18 h (optimal time as determined by preliminary experiments). IgE and anti-IgE alone did not significantly modulate the release of TNF- by alveolar macrophages (Figure 1). However, there was a significant increase in TNF- release from alveolar macrophages stimulated with IgE/anti-IgE. These results were observed only with alveolar macrophages from infected rats, suggesting that the number of alveolar macrophages bearing Fc RII may be too low in uninfected rats (as previously shown) for the stimulation to occur. Thus, infected rats were used to investigate the influence of IFN- on the release of mediators by alveolar macrophages.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Stimulation of TNF- release through Fc RII. Alveolar macrophages (AM) from normal and N. brasiliensis-infected rats were treated with and without IgE (1 µg/ml) for 2 h and stimulated with anti-IgE (0.5 µg/ml) for 18 h. Stimulation with IgE and anti-IgE significantly increased (P < 0.01) the release of TNF- by alveolar macrophages from infected rats. Mean ± SEM of seven to 10 experiments done in duplicate.

To explore further the mechanisms by which IgE/anti-IgE stimulates the release of TNF-, we investigated the effect of this stimulation at the mRNA level using reverse transcription (RT)-PCR analysis. Alveolar macrophages from infected rats were treated or not with IgE for 2 h, gently washed, and stimulated with anti-IgE for 2 h. Total RNA was isolated, reverse-transcribed, and analyzed by PCR. Unstimulated alveolar macrophages showed small amounts of TNF- PCR product compared with the strong PCR signal in cells stimulated with IgE/anti-IgE (Figure 2). The -actin RT-PCR product was unchanged by this stimulation.

View larger version (30K): [in this window] [in a new window]   Figure 2.   RT-PCR analysis of TNF- and -actin in unstimulated and IgE/anti-IgE-stimulated alveolar macrophages from infected rats. Lanes 1 and 7, DNA ladder; lane 2, negative control, lanes 3 and 5, unstimulated alveolar macrophages; lanes 4 and 6, IgE (1 µg/ml, 2 h)/anti-IgE (0.5 µg/ml, 2 h)-stimulated alveolar macrophages.

The presence of Fc RI has been shown on monocytes of atopic individuals (31). Thus, the presence of Fc RI on alveolar macrophages of normal and infected rats was investigated using RT-PCR. Rat peritoneal mast cells were used as a positive control. Even after 45 cycles, no Fc RI PCR product was identified in alveolar macrophages from either normal or infected rats, whereas it was present as early as 15 cycles in mast cells (data not shown).

Potentiation of TNF- Release by IFN- Treatment

To investigate the effect of IFN- on the release of TNF- by alveolar macrophages during allergic reactions, alveolar macrophages from N. brasiliensis-infected rats were treated with different concentrations of IFN- for 20 h, treated or not with IgE (1 µg/ml), washed, and stimulated with anti-IgE (0.5 µg/ml) for 18 h. IFN- treatment alone significantly (P < 0.01) increased the release of TNF- in a dose-dependent manner (14 ± 8, 109 ± 31, 242 ± 58, and 245 ± 55 pg/10⁶ cells in the presence of 0, 400, 600, and 800 U/ml IFN-, respectively; data not shown). This stimulation was not due to endotoxin contamination because all reagents tested negative for the presence of endotoxin and the addition of polymixin B (10 µg/ml) did not modify the results (data not shown).

IFN- treatment (800 U/ml, 20 h) significantly potentiated the release of TNF- by alveolar macrophages stimulated with IgE/anti-IgE (962 ± 218 pg/10⁶ cells compared with 164 ± 45 pg/10⁶ cells without IFN-). The release of TNF- stimulated with IFN- alone was subtracted from all samples in Figure 3. Interestingly, IFN- treatment with anti-IgE without added IgE significantly stimulated the release of TNF-, suggesting that some host-derived IgE stays on the surface of alveolar macrophages even after a long incubation period and that IFN- primes alveolar macrophages to release TNF- after the stimulation with IgE/anti-IgE. The potentiation of TNF- release by IFN- reached a plateau at 800 U/ml because treatment of alveolar macrophages with 1,000 U/ml IFN- for 20 h showed a similar amount of TNF- release as did 800 U/ml (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 3.   Potentiation of TNF- release by IFN- treatment. Alveolar macrophages (AM) from N. brasiliensis-infected rats were treated for 20 h with different concentrations of IFN-, treated or not with IgE (1 µg/ml) for 2 h, washed, and stimulated with anti-IgE (0.5 µg/ml) for 18 h. Spontaneous release of TNF- was subtracted. IFN- significantly increased (*P < 0.05) the release of TNF- from alveolar macrophages when stimulated by anti-IgE or IgE/anti-IgE. Mean ± SEM of three to four experiments done in duplicate.

Shorter treatment (4 h) with 800 U/ml IFN- also significantly increased the release of TNF- by IgE/anti-IgE- stimulated and unstimulated alveolar macrophages (Figure 4). However, IFN- treatment did not significantly increase the release of TNF- from alveolar macrophages of uninfected rats. IgE-dependent TNF- release stimulated by IFN- was lower after 4 h (121 ± 6 pg/10⁶ cells) than after 20 h (962 ± 245 pg/10⁶ cells), suggesting a time-dependent effect of IFN- on the release of TNF- by alveolar macrophages stimulated through Fc RII. This stimulation was specific to IFN- because the addition of antirat IFN- abrogated the increase of TNF- release (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 4.   Potentiation of TNF- release by short treatment with IFN-. Alveolar macrophages (AM) from N. brasiliensis-infected rats were treated for 4 h with 800 U/ml IFN-, treated or not with IgE (1 µg/ml) for 2 h, washed, and stimulated with anti-IgE (aIgE, 0.5 µg/ml) for 18 h. Stimulation with IgE/anti-IgE and treatment with IFN- significantly increased (P < 0.01 and **P < 0.002, respectively) the release of TNF-. Mean ± SEM of eight experiments done in duplicate.

Stimulation of MIP-1 Release from Alveolar Macrophages by IFN- Treatment

To verify whether alveolar macrophages could be stimulated by IgE/anti-IgE to release MIP-1, cells were treated with IgE for 2 h, washed, and stimulated with anti-IgE (18 h). The spontaneous release of MIP-1 was 13 ± 2 pg/ 10⁶ alveolar macrophages, and this release was not increased by IgE/anti-IgE stimulation (15 ± 5 pg/10⁶ cells, Figure 5). However, as a positive control, alveolar macrophages could be stimulated with 2 ng/ml lipopolysaccharide to release MIP-1 (840 ± 95 pg/10⁶ cells). IFN- treatment alone (4 h) significantly stimulated the release of MIP-1 by alveolar macrophages of infected rats (35 ± 6 pg/10⁶ cells in the presence of IFN-, compared with 15 ± 3 pg/10⁶ cells), but not from alveolar macrophages of normal rats (30 ± 9 pg/10⁶ cells in the presence of IFN-, compared with 13 ± 2 pg/10⁶ cells; n = 7). Interestingly, the release of MIP-1 in the presence of IFN- was significantly increased (98 ± 40 pg/10⁶ cells) by IgE/anti-IgE stimulation (Figure 5). Given the similarity between 4 and 18 h of IFN- treatment in the results of TNF- release, 18 h treatment of IFN- was not tested for MIP-1 release.

View larger version (31K): [in this window] [in a new window]   Figure 5.   Stimulation of MIP-1 release by IFN- treatment. Alveolar macrophages (AM) from N. brasiliensis-infected rats were treated for 4 h with or without 800 U/ml IFN-, treated or not with IgE (1 µg/ml) for 2 h, washed, and stimulated with anti-IgE (aIgE, 0.5 µg/ml) for 18 h. IFN- significantly increased (*P < 0.05) and potentiated the release of MIP-1. Mean ± SEM of four to six experiments done in duplicate.

	Discussion

A panoply of mediators and cell types are involved in the pathogenesis of asthma, which seems to be mediated by a Th2-type response leading to the release of IL-4 and IL-5 (32). Interestingly, viral infections stimulate Th1-type cytokines such as IFNs and IL-2 (33, 34), and IFN- is known to downregulate Th2-type cytokines (35). Thus, according to the Th1/Th2 model, the production of IFN- should be beneficial for asthmatic patients. This hypothesis was tested in atopic dermatitis and hyper-IgE syndrome patients, and some therapeutic effects have been reported (36, 37). However, few studies have been done in asthmatic patients. Some preliminary data suggest that IFN- treatment in childhood asthma has no significant therapeutic effects (38). In contrast, nebulized IFN- has been shown to normalize airways responsiveness in a mouse model of allergen sensitization, but only when given 3 d before allergen sensitization (39), suggesting that IFN- modifies the sensitization process to an allergen. Thus, although IFN- downregulates Th2-type cytokines, it does not normalize airways responsiveness when given after allergen sensitization. Furthermore, treatment with antibodies to IFN- has recently been shown to abolish the development of airway hyperresponsiveness in ovalbumin-challenged mice (40), questioning the role of IFN- and Th2 hypothesis in the pathogenesis of asthma (41). In addition, several studies have shown an increase in serum levels of IFN- during severe asthma (42) and in bronchoalveolar lavage fluids even in mild asthma (43). The majority of T cells from the airway lumen of patients with asthma produced IL-2 and IFN-, and only a small proportion produced IL-4 (44). Thus, the role of IFN- in the pathogenesis of asthma seems to be paradoxical to its role in the Th1/Th2 model.

Alveolar macrophages play an important role in the initiation and perpetuation of lung inflammation as seen in allergic asthma (45). Fc RII may play an important role in this process. IgE/anti-IgE stimulation increased the level of TNF- mRNA and the release of the protein by alveolar macrophages (Figures 1 and 2). The increase in TNF- mRNA could be explained by an increase in transcription or in the stability of the message. However, it has been suggested that TNF- mRNA stability is not an important factor in the accumulation of the message within macrophages (46), suggesting that the upregulation of TNF- is modulated at the transcriptional level.

Interestingly, the number of Fc RII-bearing alveolar macrophages is increased in asthmatic patients (69%) compared with nonasthmatic subjects (3%), suggesting a role for this receptor in allergic reactions (21). Furthermore, the expression of Fc RII on alveolar macrophages has been shown to be increased from 12 to 74% in a rat model of allergic asthma (47). Although Fc RI has been shown on human monocytes, no message for Fc RI was detectable in rat alveolar macrophages using RT-PCR. Given this evidence, we conclude that rat alveolar macrophages can be stimulated through Fc RII. However, we cannot completely rule out the possibility that IgE binds, with low affinity, to Fc receptors.

Because of the importance of TNF- in the inflammatory response (7) and bronchial hyperresponsiveness (8, 9), its modulation should be tightly controlled. Our results show that IFN- treatment potentiates in a concentration- and time-dependent manner the production of TNF- by alveolar macrophages (Figures 3 and 4) and that this effect was abrogated by anti-IFN- treatment. Thus, TNF- may be involved in the perpetuation of inflammation by recruiting and stimulating inflammatory cells. Although an increased amount of MIP-1 has been observed in bronchoalveolar lavage fluid of asthmatic subjects (48), in our experiments stimulation of alveolar macrophages with IgE/anti-IgE did not increase release of MIP-1 (Figure 5). Interestingly, if alveolar macrophages were pretreated with IFN- there was a potentiation of MIP-1 release when the cells were stimulated through their Fc RII. This suggests that alveolar macrophages may not be an important source of MIP-1 during allergic reactions unless IFN-, perhaps from a viral infection, is present.

Although IFN- is a member of the Th1 spectrum of cytokines, its role in asthma is still controversial (41). Thus, we propose that IFN-, produced during viral infection, primes alveolar macrophages to release more inflammatory mediators during allergic reactions. Excessive production of IFN- may be responsible for macrophage-mediated exaggerated airway inflammation and hyperresponsiveness in susceptible individuals with virus- or allergen-induced asthma.