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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 257-264, 2002
© 2002 American Thoracic Society

Immunomodulatory Effects of Antigen-Pulsed Macrophages in a Murine Model of Allergic Asthma

Edith M. Janssen, Marca H. M. Wauben, Frans P. Nijkamp, Willem van Eden and Antoon J. M. van Oosterhout

Department of Pharmacology and Pathophysiology, Faculty of Pharmacy, Utrecht University; and Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Address correspondence to: Dr. A. J. M. van Oosterhout, Ph.D., Department of Pharmacology & Pathophysiology, Faculty of Pharmacy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands. E-mail: A.J.M.VanOosterhout{at}Pharm.uu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophages (M{phi}) play an unique role in the activation and regulation of T cells through their ability to modulate specific costimulatory and cytokine signals. Here we investigated the immunomodulatory effects of allergen presentation by M{phi} in a murine model of allergic asthma. Purified peritoneal M{phi} were pulsed with ovalbumin (OVA) (OVA-M{phi}), or the immunodominant epitope OVA323–339 (OVA323–339-M{phi}), and characterized for cell surface markers, cytokine production, and antigen-presenting capacity toward OVA323–339-specific DO11.10 T cells. Antigen-pulsed M{phi} were injected (intravenously) in OVA-sensitized Balb/c mice that were repeatedly challenged with OVA or saline aerosol. Administration of OVA-M{phi} inhibited airway eosinophilia and hyperresponsiveness to methacholine concomitant with a reduced interleukin (IL)-4 and IL-5 production by T cells upon OVA stimulation in vitro. Interestingly, OVA-induced IL-10 levels remained unchanged, whereas interferon-{gamma} could not be detected. In contrast to OVA-M{phi}, OVA323–339-M{phi} administration had no effects on these asthma manifestations. Additional in vitro studies demonstrated that OVA-M{phi}, but not OVA323–339-M{phi}, produced high levels of IL-10 upon interaction with the DO11.10 T cells. This IL-10 production by the OVA-M{phi} was dependent on MHC–TCR and CD86–CD28, but not CD80–CD28 or CD40–CD154 interactions. Our data suggest that IL-10 production by allergen presenting M{phi} plays a crucial role in successful immunotherapy.

Abbreviations: airway hyperresponsiveness, AHR • bronchoalveolar lavage fluid, BALF • enzyme-linked immunosorbent assay, ELISA • fluorescence-activated cell sorter, FACS • heat-inactivated rat serum, hRS • interferon-{gamma}, IFN-{gamma} • immunoglobulin, Ig • interleukin, IL • Iscove's Modified Dulbecco's Medium, IMDM • lymph node, LN • lipopolysaccharide, LPS • macrophage, M{phi} • ovalbumin, OVA • phosphate-buffered saline, PBS • enhanced pause, Penh • T-cell receptor, TCR • transforming growth factor, TGF • T helper 2 cells, Th2 cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic asthma is characterized by elevated levels of allergen-specific immunoglobulin (Ig)E, reversible airway obstruction, chronic airway inflammation, and airway hyperresponsiveness to various bronchoconstrictive stimuli (1). Allergen-specific CD4+ T helper 2 (Th2) cells that produce predominantly interleukin (IL)-4, IL-5, and IL-13, have been found to control the cellular and molecular events underlying the initiation and progression of the symptoms of chronic asthma (2–4). Ample studies demonstrated that IL-4 promotes the development of Th2 cells and plays an important role in B cell activation and immunoglobulin class switching to IgE (5, 6). In addition, IL-5 has been described to affect the induction, maturation, activation, and survival of eosinophils (7, 8). More recently IL-13 has been shown to activate eosinophils and B cells, promote IgE production, and promote the induction of airway hyperresponsiveness (AHR) and mucus hypersecretion in experimentally induced asthma (4, 9–11). Consequently, modulation or inhibition of the allergen-specific Th2 response by either inducing Th2 cell anergy or skewing the Th2 response into a Th1 or regulatory T cell response has become an interesting strategy for intervention in the development or progression of allergic disorder.

Recently the involvement of macrophages (M{phi}) in the induction of the phenotype and effector function of naive and previously committed T cells has become a topic of research. Although M{phi} can induce both Th1 and Th2 phenotypes in naive T cells (12, 13), various studies demonstrated that M{phi} favor the induction of Th1 cell responses (14, 15) and can actively suppress the induction of Th2 cells through their IL-12 production (16). On the other hand M{phi} have also been reported to exert immunosuppressive effects toward Th1-mediated immune responses by secreting anti-inflammatory mediators such as IL-10, prostaglandin E2, and transforming growth factor (TGF)-ß, resulting in a T cell population with a regulatory phenotype (17, 18). Depending on the type of M{phi}, their activation state and the interaction and communication with the T cells, the M{phi} will provide either an immunostimulatory or an immunosuppressive environment in which the T cells become activated.

Here we examined the immunoregulatory effects of antigen presentation by M{phi} in a well-characterized murine model of allergic asthma (19). In this model, ovalbumin (OVA)-sensitized and OVA-challenged BALB/c mice display AHR, airway eosinophilia, and high levels of OVA-specific IgE in serum. In addition, OVA-specific Th2 cells that produce IL-4, IL-5, and IL-13 upon stimulation with OVA are present in lung-draining lymph nodes (LN) and in infiltrated lung tissue (20). We demonstrated that the immunodominant epitope of OVA, OVA323–339, accounts for 50% of the OVA-specific B cells and 60–70% of the OVA-specific T cell response (21). In this article we studied the immunoregulatory capacity of allergen-pulsed M{phi}, by administration of OVA or OVA323–339-pulsed peritoneal M{phi} in previously sensitized mice and by monitoring the development of airway manifestations and OVA-specific Th2 responses upon repeated OVA challenge.

Our data show that processing and presentation of OVA, but not OVA323–339 by M{phi}, inhibits airway eosinophilia and hyperresponsiveness concomitant with a reduced IL-4 and IL-5 production by OVA-specific T cells. In addition, the in vitro data demonstrate that M{phi}–T cell interaction upon OVA processing and presentation resulted in reciprocal activation of the T cell and the M{phi} and the induction of IL-10 production by the M{phi}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Specific pathogen–free male BALB/c mice (6–8 wk) and OVA323–339 T-cell receptor (TCR) transgenic DO11.10 mice on a BALB/c background (22) were bred at the Central Animal Laboratory (Utrecht, The Netherlands), and housed in macrolon cages and provided with OVA-free food and water ad libitum.

Isolation and Pulsing of Antigen-Presenting Cells
M{phi} were obtained from naive Balb/c mice by extensive washing of the peritoneal cavity with cold phosphate-buffered saline (PBS). The peritoneal cells were cultured for 2 h in glass petri dishes in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco; Paisley UK) containing 2% heat-inactivated rat serum (hRS; Central Animal Laboratory), 100 E.U. penicillin, and 100 µg/ml streptomycin. Nonadherent cells were removed, and adherent cells were collected using a rubber cell scraper. The collected cells contained > 90% M{phi} as determined by fluorescence-activated cell sorter (FACS) analysis using CD11b and MHC class II as markers. M{phi} were pulsed with medium, OVA (2 mg/ml; Grade V; Sigma Chemical Co., St. Louis, MO), or the immunodominant epitope OVA323–339 (OVA323–339; 10 µg/ml; Isogen Bioscience, Maarn, The Netherlands) for 3 h at 37°C and 5% CO2. It cannot be excluded that the OVA used for the experiments did contain lipopolysaccharide (LPS). However, to exclude that LPS/endotoxins in the OVA preparation affected the outcome of the experiments, parallel in vivo experiments were performed using anti-endotoxin–treated OVA and double high-performance liquid chromatography–purified OVA. After pulsing, the cells were thoroughly washed to remove all residual soluble OVA and OVA323–339.

Characterization of M{phi} by Surface Molecules and Cytokine Production
Analysis of surface molecules was performed by flow cytometric analysis. Pulsed M{phi} were incubated with FACS buffer (PBS, 1% BSA, 0.1% sodium azide) containing 5% hRS and 1:500 diluted supernatant of 24G2 hybridoma cells ({alpha}Fc{gamma}RII/III). Subsequently, cells were stained with fluorescein isothiocyanate–coupled antibody for MHC class II, CD80, CD86, CD11b, CD11c, or the relevant isotype control (PharMingen, San Diego, CA). Dead cells were excluded using propidium iodide. Cells were analyzed on a FACS scan using Cell Quest (Becton-Dickinson, San Jose, CA).

To determine the cytokine production profile of the pulsed M{phi}, cells were cultured in 96-well plates (1 x 105 cells/well) in the absence or presence of 2 µg/ml LPS (Escherichia coli, 0111:B4; Sigma) and after 12–72 h cytokines in the supernatants were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) or bioassay.

Isolation and Culture of OVA323–339-Specific T Cells
OVA323–339-specific DO11.10 T cells were obtained from TCR transgenic mice by negative selection, as described previously (23). Cells obtained after depletion were shown to be > 90% OVA323–339-specific T cells, as demonstrated by FACS analysis using the clonotype-specific Ab KJ1.26 (24). Viability of the cells was > 95%. T cells were cultured in IMDM supplemented with 10% FCS, 2 nM L-glutamine, 100 E.U. penicillin, 100 µg/ml streptomycin, and 50 µM ß-mercaptoethanol.

Determination of M{phi}–T Cell Interaction
To investigate the antigen-presenting capacity, OVA-, OVA323–339-, and medium-pulsed M{phi} were cultured with 1 x 105 DO11.10 T cells in a range of 101–105 APC/well. After 72 h cytokine production and proliferation were determined. In parallel cultures DO11.10 T cells were cultured with M{phi} that were treated with Mitomycin C after pulsing. After fixing, M{phi} were still functional antigen-presenting cells, but failed to produce cytokines upon stimulation with LPS (data not shown). To determine the requirement for costimulation during antigen presentation and T cell activation, pulsed M{phi} were cultured with DO11.10 T cells in the presence of 20 µg/ml mCTLA4-IgG, blocking antibodies to CD80 (16–10A1), CD86 (GL1), and MHC class II (MKD6) on the M{phi}, CD154 (MR1) on the T cells, or the appropriate isotype controls. At various time points cytokine levels in the supernatant were determined by sandwich ELISA or bioassay.

Cytokine Analysis
IL-6 production was determined by the IL-6–dependent B9 clone as described by Helle and coworkers (25). Levels of IL-4, IL-5, IL-10, interferon (IFN)-{gamma} (PharMingen), and IL-12 p40 and p70 (Genzyme, Cambridge, MA) were determined by sandwich ELISA as described by the manufacturer. The detection limits of the ELISAs were 16 pg/ml for IL-4 and IL-5, and 100 pg/ml for IL-10, IL-12 (p40 and p70), and IFN-{gamma}.

Induction of Airway Symptoms and Treatment Protocol
Previously, we described the development of an OVA-based murine model with features reminiscent of allergic asthma (19). In this model, BALB/c mice are actively sensitized by 7 intraperitoneal injections of 10 µg OVA in 0.5 ml pyrogen-free saline without adjuvant on alternate days. Treatment was performed 14 d after the last sensitization by intravenous administration of 2 x 105 M{phi} (medium-, OVA-, or OVA323–339-pulsed) in 50 µl saline. As an additional control group, mice were intravenously injected with 50 µl saline. Seven days later, mice were exposed to OVA (2 mg/ml) or saline aerosol challenges for 5 min on 8 consecutive days. Aerosols were performed in a plexiglass exposure chamber coupled to a Jet nebulizer (Pari IS-2 Jet nebulizer, particle size 2–3 µ; PARI Respiratory Equipment, Richmond, VA) driven by compressed air at a flow rate of 6 liters/min.

Analysis of In Vivo Antigen-Induced Airway Manifestations
Airway responses to inhaled methacholine were measured in conscious, unrestrained mice using barometric whole-body plethysmography (Buxco, Sharon, CT) as described previously (26). As parameter of airway hyperresponsiveness, increases in enhanced pause (Penh) were determined. In short, mice were placed in a whole-body chamber and basal readings were obtained and averaged for 3 min. Aerosolized saline and increasing concentrations of methacholine (range 1.5–50 mg/ml) were nebulized for 3 min, and readings were taken and averaged for 3 min after each nebulization. Airway responsiveness was expressed as the Penh per dose methacholine.

After finishing the methacholine dose–response curve mice were bled and bronchoalveolar lavages were performed. Lavage cells were differentiated into mononuclear cells, neutrophils, and eosinophils by standard morphology and Diff-Quik staining (Merz and Dade, Düdingen, Switzerland).

Analysis of OVA-Specific Immunoglobulins
OVA-specific IgE and IgG2a in the serum were measured as described previously (21). The detection levels of the ELISAs were 0.05 U/ml for IgG2a, and 0.5 U/ml for IgE.

Analysis of T Cell Responses in Lungs and Lung-Draining LN Cells
Lungs were lavaged with sterile PBS, and perfused via the right ventricle with 5 ml saline containing 100 U/ml heparin to remove blood and intravascular leukocytes (20). Lungs were minced and digested with 3 ml RPMI containing 2.4 mg/ml collagenase and 1 mg/ml DNase (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37%. The digest was filtered through a 70-µm cell strainer with 10 ml IMDM containing 20% FCS. Lung-draining LN cells were homogenized over a 70-µm cell strainer to obtain a single cell suspension. Lung-draining LN cells (2 x 105 cells/well) and lung cells (8 x 105 cells/well) were cultured in IMDM supplemented with 10% FCS, 2 nM L-glutamine, 100 E.U. penicillin, 100 µg/ml streptomycin, and 50 µM ß-mercaptoethanol in 96-well plates, in the presence of OVA (10 µg/ml) or medium. As a positive control, cells were cultured with immobilized CD3 antibody ({alpha}CD3, clone 17A2, 50 µg/ml). After 120 h of culture, supernatant was collected and cytokine levels were determined.

Data Analysis
Unless stated otherwise, data are expressed as mean ± standard error of the mean and evaluated using an analysis of variance followed by a Dunnett test. A probability value P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic Characterization of M{phi}
Purified peritoneal lavage M{phi} displayed high levels of MHC class II and the M{phi} marker CD11b, whereas expression of the DC marker CD11c was absent (Figure 1) . In addition, M{phi} expressed high levels of the costimulatory molecule CD86, whereas expression of CD80 was hardly detectable (Figure 1). For further characterization, M{phi} were cultured with medium or LPS and cytokine production was analyzed. Freshly isolated M{phi} produced low levels of IL-6 and IL-10 (14.0 ± 0.3 U/ml and 165 ± 29 pg/ml, respectively), whereas IL-12 (p40 and p70) was undetectable. LPS stimulation of the M{phi} resulted in a 22-fold increase in IL-6 and a 40-fold increase in IL-10 production (324.6 ± 88.3 U/ml and 6429 ± 381 ng/ml, respectively), whereas IL-12p40 was hardly and IL-12p70 was not detectable. Pulsing M{phi} with OVA or OVA323–339 did not affect the expression of the surface markers or cytokine production compared with non-pulsed M{phi} (data not shown).



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  		Figure 1. Flow cytometric analysis of macrophages (M{phi}). M{phi} were prepared as described in MATERIALS AND METHODS. Solid lines represent the staining with antibodies to MHC class II, CD11b, CD11c, CD80, and CD86, and dotted lines represent the corresponding isotype control. The results of a representative experiment are shown. Results were reproduced in four independent experiments.

 
Analysis of M{phi}–T Cell Interactions
To study the antigen-presenting capacity, OVA-, OVA323–339-, or nonpulsed M{phi} were cultured with freshly isolated OVA323–339-specific DO11.10 T cells. Nonpulsed (control) M{phi} did not induce proliferation or cytokine production in DO11.10 T cells, whereas OVA-M{phi} and OVA323–339-M{phi} induced a M{phi} number–dependent proliferation and cytokine production (Figure 2) . Interestingly, OVA323–339-M{phi} but not OVA-M{phi} induced production of IL-4 by the DO11.10 T cells (Figure 2). When fixed OVA323–339-M{phi} were used, IFN-{gamma} and IL-10 levels in the supernatant were not affected, indicating that these cytokines were produced by the DO11.10 T cells (Figure 3A) . Also culturing DO11.10 T cells with fixed OVA-M{phi} did not affect the IFN-{gamma} production, indicating that the IFN-{gamma} was produced by the T cells (Figure 3A). In contrast, fixing of the OVA-M{phi} markedly reduced levels of IL-10 production upon DO11.10 T cell interaction, suggesting that the IL-10 was largely produced by the OVA-M{phi} (Figure 3A).



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  		Figure 2. Proliferation and cytokine production in cultures of freshly isolated DO11.10 T cells after stimulation with different M{phi} cell numbers. DO11.10 T cells (1 x 105 per well) were cultured with (1 x 101 to 1 x 105) of control- (closed diamonds), OVA- (closed circles), or OVA323–339-pulsed M{phi} (open circles). Proliferation was determined after 72 h of culture by pulsing the cells for another 16 h with 3[H]thymidine. Proliferation is expressed as counts per minute (CPM ± SEM of triplicate wells). Cytokines in the supernatant were determined in parallel cultures by sandwich ELISA. The results of a representative experiment are shown. Results were reproduced in three independent experiments.

 


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  		Figure 3. Cytokine production after stimulation with antigen-pulsed M{phi}. (A) Cytokine levels in the supernatants after stimulation with untreated (closed symbols) or fixed (open symbols) OVA323–339-pulsed and OVA-pulsed M{phi}. DO11.10 T cells (1 x 105 cells/well) were cultured with a dose range of untreated or fixed M{phi}. After 96 h of culture, IFN-{gamma} and IL-10 production were determined by ELISA (mean ± SEM of triplicate wells). (B) Effect of blocking of different costimulatory pathways on IL-10 production by OVA-M{phi} upon interaction with DO11.10 T cells. DO11.10 T cells (1 x 105 cells/well) were cultured with 1 x 105 OVA-M{phi} in the presence of CTLA-4-Ig or blocking antibodies to MHC class II, CD80, CD86, and CD40L. After 96 h of culture, IL-10 production was determined by ELISA (mean ± SEM of triplicate wells). Closed diamonds, control M{phi}; closed circles, OVA-M{phi}; open circles, fixed OVA-M{phi}; closed triangles, OVA323–339-M{phi}; open triangles, fixed OVA323–339-M{phi}.

 
Culturing the OVA-M{phi} and DO11.10 T cells in the presence of blocking antibodies to MHC class II or CD86 almost completely inhibited IL-10 production by the M{phi}, whereas blocking of costimulation via CD80 or CD40, by using an antibody against CD154 did not affect the IL-10 production by the M{phi} (Figure 3B).

In Vivo Airway Hyperresponsiveness and Eosinophilia
To study the immune regulatory capacity of antigen-pulsed M{phi} in vivo, we used a well-established murine model of asthma. OVA-sensitized Balb/c mice were treated intravenously with M{phi} (OVA-, OVA323–339-, or nonpulsed) and repeatedly challenged with either OVA or saline. Twenty-four hours after the last challenge airway responsiveness to methacholine (MCh), eosinophil infiltration in the bronchoalveolar lavage, and OVA-specific Th2 cytokine production by lung cells and lung-draining LN cells were determined.

In control and control-M{phi} treated mice, repeated OVA challenge induced a significant increase in AHR to methacholine compared with the corresponding saline-challenged mice at doses ranging from 12.5–50 mg/ml methacholine (Figure 4) . Interestingly, treatment with OVA-M{phi} inhibited this AHR completely, resulting in similar Penh values in OVA- and saline-challenged animals on all tested doses of methacholine. In contrast, treatment with OVA323–339-M{phi} did not inhibit AHR compared with the control groups (Figure 4).



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  		Figure 4. Airway hyperresponsiveness to aerosolized methacholine in M{phi}-treated mice. Mice were sensitized with OVA and later challenged with saline (white bars) or OVA (black bars) eight times. Before challenge, mice were treated intravenously with 2 x 105 control-, OVA-, or OVA323–339-pulsed M{phi}. Values are expressed as mean ± SEM (n = 8 mice per group). *P < 0.05 compared with corresponding saline-challenged mice; #P < 0.05 compared with control-M{phi} treated OVA-challenged mice.

 
Sensitization followed by saline challenge did not result in eosinophil infiltration in the bronchoalveolar lavage fluid (BALF), whereas OVA challenge induced high numbers of eosinophils and mononuclear cells in the BALF of all treatment groups (Table 1) . Treatment of mice with control-M{phi} or OVA323–339-M{phi} did not alter the number of inflammatory cells in the BALF, whereas treatment with OVA-M{phi} resulted in a 75% reduction (P < 0.05) of eosinophils in the BALF compared with control-treated mice (Table 1).


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  		TABLE 1 Absolute numbers of mononuclear cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid

 
Serum Levels of OVA-Specific Immunoglobulins
Sensitization of mice with OVA followed by saline challenge resulted in clearly detectable levels of OVA-specific IgE and IgG2a in all treatment groups. Compared with saline challenge, OVA challenge significantly enhanced OVA-specific IgE but not IgG2a production (Table 2) . Treatment with control-M{phi} or OVA323–339-M{phi} did not affect OVA-specific immunoglobulin levels. However, in OVA-M{phi}–treated saline-challenged mice an increase of OVA-specific IgE level was observed (Table 2). Importantly, OVA-specific IgG2a was not affected by any of the treatments.


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  		TABLE 2 OVA-specific immunoglobulins in serum

 
OVA-Specific Cytokine Production by Lung-Draining LN Cells
Lung-draining LN and lungs were collected 24 h after the last OVA or saline challenge and T cell responses upon OVA stimulation were determined. Lung-draining LN cell cultures of saline-challenged mice did not produce any detectable levels of cytokines after in vitro stimulation with OVA (data not shown). In contrast, OVA-challenged mice produced high levels of Th2-associated cytokines (e.g., IL-4, IL-5, IL-10) upon OVA stimulation. Compared with control treatment, treatment with control-M{phi} or OVA323–339-M{phi} did not alter cytokine production upon stimulation with OVA (Figure 5A) . Remarkably, treatment with OVA-M{phi} resulted in significantly decreased production of IL-4 and IL-5 upon OVA stimulation, whereas production of IL-10 was unaffected (Figure 5A).



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  		Figure 5. OVA-specific cytokine production in vitro by lung-draining LN cells (A) and lung cells (B) after M{phi} therapy. Lung-draining LN cells and lung cells obtained from OVA-challenged mice were cultured with OVA (10 µg/ml) for 120 h. Cytokines in the supernatants were determined by ELISA. No cytokines were detected in cell cultures obtained from saline-challenged mice (data not shown). IFN-{gamma} production could be detected after stimulation with immobilized {alpha}CD3, but not after stimulation with OVA (data not shown). Data are expressed as mean ± SEM (n = 8 per group). *P < 0.05 compared with OVA-challenged mice.

 
Comparable results were found in lung cultures. Lung cultures of saline-challenged mice did not produce detectable levels of cytokines after in vitro stimulation with OVA, whereas lung cells from OVA-challenged mice produced high levels of IL-5 and IL-10 (Figure 5B). Treatment with control-M{phi} or OVA323–339-M{phi} did not affect OVA-specific IL-5 or IL-10 production, whereas OVA-M{phi} treatment resulted in significantly reduced levels of OVA-specific IL-5 but not IL-10 (Figure 5B). Interestingly, IFN-{gamma} could not be detected in any of the LN cultures or lung cultures after stimulation with OVA, whereas high levels of IFN-{gamma} were observed in all cultures after stimulation with {alpha}CD3 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
M{phi} play an unique role in the activation and regulation of T cells through their ability to modulate specific costimulatory and cytokine signals. Here we showed that antigen presentation by OVA-pulsed M{phi} can reduce antigen-specific Th2 responses and inhibit antigen-induced airway manifestations as AHR and airway eosinophilia in a murine model of allergic asthma.

The M{phi} used in this study share many features with alternatively activated M{phi} that have been described to actively participate in anti-inflammatory processes and tolerance induction (reviewed in Ref. 17). The peritoneal M{phi} expressed various surface markers characteristic for M{phi} in general (27), and had the capacity to phagocytose and present exogenous antigen to naive CD4+ T cells. Like alternatively activated M{phi}, our M{phi} expressed very high levels of MHC class II and CD86 compared with M{phi} derived from various lymphoid compartments (data not shown). In addition, the M{phi} failed to produce TNF-{alpha}, NO (data not shown), and the Th1-skewing cytokine IL-12 upon LPS stimulation. Instead of these proinflammatory mediators, they produced IL-6 and very high levels of the immunoregulatory cytokine IL-10 upon LPS stimulation.

Importantly, the M{phi} could also be activated by interaction with antigen-specific T cells. Upon interaction with the DO11.10 T cells OVA-M{phi}, but not OVA323–339-M{phi}, produced high levels of the immunoregulatory cytokine IL-10. The production of IL-10 by OVA-M{phi} upon M{phi}–T cell interaction may greatly affect the activation state and the phenotype of the T cells. By blocking of surface and costimulatory molecules on the OVA-M{phi} or the T cells we showed that M{phi}–T cell interaction via MHC-TCR and CD28-CD86, but not CD28-CD80 or CD154-CD40, were crucial for this IL-10 production by the OVA-M{phi}. These observations on bidirectional communication between T cells and M{phi} are in line with the findings of Chabot and coworkers, who recently demonstrated that M{phi}-like microglia cells produce high levels of IL-10 upon interaction with T cells (28). However, they showed that this IL-10 production required microglia–T cell interaction through both CD28-CD80/CD86 and CD154-CD40 (28). We are currently investigating the role of other costimulatory pathways, including other members of the B7 family, PD-L1/2:PD-1 and B7 h/B7RP-1:ICOS (29–32), that have been shown to regulate T cell responses on bidirectional communication between T cells and APC.

Explanations for this different activation of the T cells and M{phi} might be found in the intrinsic characteristic of the peptide and protein. Whereas OVA323–339 can bind directly to MHC class II, OVA needs to be taken up and processed by the M{phi} to be presented in the MHC. Uptake and processing may engage different receptors and intracellular pathways that lead to a different activation state of the M{phi}. As a result, the expression of soluble or membrane-bound costimulatory molecules that are involved in T cell activation and regulation could differ between the differently pulsed M{phi} populations. Although we cannot exclude that other cell-surface molecules were upregulated upon pulsing with OVA or OVA323–339, our data showed that the expression of MHC class I and II and the important costimulatory molecules, CD80, CD86, and CD40 was comparable in all pulsed and unpulsed M{phi} populations. In addition, spontaneous or LPS-induced cytokine production by the M{phi} was not altered after OVA or OVA323–339 pulsing. These observations suggest that another factor than M{phi} activation contributed to the altered M{phi}–T cell interaction.

Another explanation could be a different alignment of the immunodominant OVA323–339 epitope in the MHC after processing of OVA and exogenously binding of the synthetic peptide. Scott and coworkers showed the existence of at least three different OVA323–339 alignments after crystallization of MHC class II I-Ad with OVA323–339 (33). Antigen processing might favor a specific alignment of the peptide, resulting in specific flanking residues and an altered TCR affinity and TCR interaction. Alternatively, the ligand density on the M{phi} could play a role. OVA323–339 is a peptide with a relative strong MHC-binding affinity, and pulsing the M{phi} with soluble peptide will most likely result in a higher peptide-MHC density than found after processing of the entire OVA. Different degrees of TCR occupancy have been shown to lead to different intracellular signaling and a different phenotypic response by the T cells (34). A role for the ligand density hypothesis is supported by our in vitro data that OVA323–339-M{phi} are more potent in inducing proliferation and cytokine production than a comparable number of OVA-pulsed M{phi}. Because M{phi}–T cell communication appears to be a reciprocal event, differences in ligand density may not only affect the T cell response, but also the M{phi} response. Together these data suggest that antigen presentation of OVA or OVA323–339 can result in a different reciprocal activation of the T cells and M{phi} and thereby in a different phenotype and effector function in both cell types.

The differences in M{phi} and T cell effector function may explain why treatment with OVA-M{phi} but not with OVA323–339-M{phi} resulted in a complete inhibition of the AHR and a strong reduction of the eosinophilia and Th2 cytokine production by the OVA-specific T cells.

Subcutaneous administration of entire allergens, or their dominant epitopes during conventional immunotherapy in humans and mice, indicate that presentation of allergenic components modulate the airway symptoms and T cell responses (21, 35). Most clinical studies report a reduction in cytokine production by allergen-specific Th2 cells after successful immunotherapy (36, 37). Especially cytokines such as IL-4 and IL-5 are inhibited, whereas IL-10 production often remains unaffected, or has increased. In our study we also see a clear reduction of IL-4 and IL-5 production by LN cultures upon stimulation with OVA, whereas IL-10 production is unaffected. The two most popular hypotheses on the reduction of the allergen-specific Th2 response encompass (i) the induction of allergen-specific Th1 responses that by cytokine production counterbalance the Th2 response, and (ii) the induction of a regulatory T cell population that will act on the Th2 cells through immunomodulatory cytokines like IL-10 or TGF-ß. Whether these Th1 or regulatory T cells arise from modulation of the existing Th2 response or the recruitment of naive CD4+ T cells from the endogenous repertoire is still under investigation.

We and others previously demonstrated that treatment with the Th1-inducing cytokines IL-12/and IL-18, or the Th1-derived cytokine IFN-{gamma}, before and during challenge significantly reduced AHR and eosinophilia in murine asthma models (38–41). These findings clearly indicate that Th1 cells and Th1-associated cytokines can play an important role in the downregulation of Th2 cells. However, because IFN-{gamma} and IL-12 are pleotropic cytokines, it also has been suggested that their effects on airway manifestations are not solely mediated by the promotion of Th1 responses. This hypothesis is supported by studies in which adoptive transfer of OVA-specific Th1 cells, administered either before or after sensitization, failed to counterbalance allergen-specific Th2 cells, and even enhanced airway inflammation (42, 43). Although the induction of a Th1-associated response could play a role in the inhibition of the airway manifestations and reduction of the Th2 response, our data do not support the induction of a Th1-associated response by OVA-M{phi}. Lung-draining LN cells did not produce detectable levels of IFN-{gamma} upon OVA stimulation, and serum levels of OVA-specific IgG2a, an IFN-{gamma}–associated immunoglobulin, were unaltered by OVA-M{phi} treatment. In addition, IL-12 could not be detected in any stage of the in vitro and in vivo experiments, whereas the M{phi} produced clearly detectable levels of IL-10, a cytokine known for its suppressive effect on Th1 development.

Alternatively, macrophage treatment could induce a regulatory T cell population that is able to suppress the OVA-specific Th2 cells. Different types of regulatory CD4+ T cells have been described, based on their cytokine profile and suppressive effects on antigen-specific immune responses. Th3 regulatory T cells have been reported to arise during the induction of oral tolerance or after stimulation in the presence of TGF-ß, and IL-10 in vitro and suppress T cell responses by their TGF-ß production (44). Tr1 CD4+ regulatory cells as described by Groux and coworkers (45) are generated by stimulation in the presence of IL-10 and inhibit the proliferative response of T cells both in vivo and vitro by their IL-10 production. More importantly, transfer of these IL-10–producing Tr1 cells coincident with OVA immunization inhibited OVA-specific Th2 cell priming both in terms of proliferative responses and cytokine production (46).

Because OVA-M{phi}, but not OVA323–339-M{phi}, produced high levels of IL-10 upon T cell interaction and activation in vitro, it is possible that OVA-M{phi}–T cell interaction favored the induction of T cells with a regulatory phenotype. This is in line with the finding of Akbari and colleagues (47), who demonstrated recently that pulmonary DCs from IL-10+/+, but not IL-10-/-, mice exposed to respiratory antigen induced antigen-specific IL-10–producing regulatory T cells in recipient mice, indicating a critical role for IL-10 production by the APC for the induction of tolerance. Moreover, Akdis and coworkers (48) described that the reduction of allergen-specific Th2 responses in successful conventional allergen immunotherapy in humans was initiated and maintained by IL-10 produced by allergen-specific T cells and monocytes. In addition, there is ample evidence for a regulatory role of IL-10 in allergen-induced airway manifestations because in various animal models administration of IL-10 inhibited AHR, cell recruitment into the airways, and Th2 cytokine production (49–52).

In our study there was no indication for an increased Th1 response to the OVA-M{phi} therapy that could counterbalance the Th2 response. Because OVA-M{phi} therapy resulted in inhibition of IL-4 and IL-5 production by OVA-specific T cells, whereas IL-10 production remained unchanged, it is most likely that the effect of OVA-M{phi} therapy is mediated by IL-10 produced by the OVA-pulsed macrophages, resulting in an IL-10–producing/regulatory T cell population that positively contributes to the amelioration of the disease.


   Acknowledgments
 
G. Hofman and K. Hoebe are acknowledged for technical assistance, and Prof. Dr. L. Adorini for the gift of the DO11.10 transgenic mice. This study was supported by NWO (grant GB-MW 901-06). The research of M. H. M. Wauben has been made possible by a fellowship of the Royal Netherlands Academy of Arts & Sciences.

Received in original form November 26, 2001

Received in final form April 17, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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