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The Journal of Immunology, 2002, 168: 890-899.
Copyright © 2002 by The American Association of Immunologists

Allergic Inflammatory Response to Short Ragweed Allergenic Extract in HLA-DQ Transgenic Mice Lacking CD4 Gene1

Svetlana P. Chapoval*, Koji Iijima{dagger}, Eric V. Marietta*, Michele K. Smart*, Andrei I. Chapoval*, Amy G. Andrews{ddagger} and Chella S. David2,*

* Department of Immunology, {dagger} Allergic Diseases Research Laboratory, and {ddagger} Section of Veterinary Medicine, Mayo Clinic, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the role of HLA-DQ molecules and/or CD4+ T cells in the pathogenesis of allergic asthma, we generated HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous class II (A{beta}null) and CD4 genes and challenged them intranasally with short ragweed allergenic extract (SRW). We found that DQ6/CD4null mice developed a strong eosinophilic infiltration into the bronchoalveolar lavage and lung tissue, while DQ8/CD4null mice were normal. However, neither cytokines nor eosinophil peroxidase in the bronchoalveolar lavage of DQ6/CD4null mice was found. In addition, the airway reactivity to methacholine was elevated moderately in DQ6/CD4null mice compared with the high response in DQ/CD4+ counterparts and was only partially augmented by CD4+ T cell transfer. The DQ6/CD4null mice showed Th1/Th2-type cytokines and SRW-specific Abs in the immune sera in contrast to a direct Th2 response observed in DQ6/CD4+ mice. The proliferative response of spleen mononuclear cells and peribronchial lymph node cells demonstrated that the response to SRW in DQ6/CD4null mice was mediated by HLA-DQ-restricted CD4-CD8-NK1.1- T cells. FACS analysis of PBMC and spleen mononuclear cells demonstrated an expansion of double-negative (DN) CD4-CD8-TCR{alpha}{beta}+ T cells in SRW-treated DQ6/CD4null mice. These cells produced IL-4, IL-5, IL-13, and IFN-{gamma} when stimulated with immobilized anti-CD3. IL-5 ELISPOT assay revealed that DN T cells were the cellular origin of IL-5 in allergen-challenged DQ6/CD4null mice. Our study shows a role for HLA-DQ-restricted CD4+ and DN T cells in the allergic response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic asthma is a chronic airway inflammatory disease characterized by reversible airway obstruction, hyperreactivity, and eosinophil/lymphocyte influx. There is increasing evidence that the asthmatic process is driven and maintained by chronically activated T cells with a Th2 phenotype (1, 2, 3, 4) that promote the activation and recruitment of eosinophils (1, 2, 3, 4, 5, 6) and regulate the Ig class switch to the development of Ag-specific IgE Ab responses (7).

Studies with murine models of Ag-induced asthma have shown that pulmonary eosinophilia and airway hyperreactivity (AHR)3 can be significantly inhibited or attenuated by treatment with mAb to CD4 (8); Thy-1 (9); Th2-type cytokines such as IL-4 (10), IL-5 (11, 12), and IL-13 (13, 14); or their receptors (15, 16, 17). An allergic response can be induced with an adoptive transfer of Ag-primed CD4+ Th2-type cells (18, 19). However, in vivo depletion of CD4+ T cells decreased, but did not abolish, Ag-induced recruitment of eosinophils to the site of challenge (20). Moreover, depletion of CD3+ cells results in prevention of AHR, whereas depletion of CD4+ cells results in its partial inhibition (21). Therefore, a definitive role for CD4+ T cells in the pathogenesis of allergic disease is not established. Recent investigations demonstrated a critical role for CD8+ T cells (22) and NK cells (23) in the regulation of pulmonary eosinophilia and AHR in mouse models of asthma. These results contrast with studies employing {beta}2-microglobulin knockout mice (24) or in vivo depletion of NK1.1 cells (25), which demonstrate that CD8+ and NK1.1+ T cells are dispensable for the allergen-induced Th2 response and/or pulmonary inflammation. This suggests that CD4-CD8-NK1.1-CD3+ T cells may play a complementary role in allergic diseases. Indeed, phenotypic analysis of bronchoalveolar lavage (BAL) and lung tissue T cells in OVA-induced lung inflammation revealed that almost 25% of the T cells were CD4-CD8-NK1.1-TCR{alpha}{beta}+ (26).

Patients with HIV demonstrated a sustained overproduction of IgE and hypereosinophilia despite a significant reduction or absence of CD4+ T cells (27, 28). An expanded population of CD4-CD8- T cells (double-negative (DN) T cells), which elaborated Th2-type cytokines with apoptotic properties for eosinophils, was detected in these patients. Several other cases of eosinophilia, including hypereosinophilic syndrome, were accompanied by proliferation of peripheral blood DN T cells that produce IL-5 (29, 30).

We have shown previously that DQ6/CD4+ and DQ8/CD4+ transgenic (tg) mice develop pulmonary eosinophilia associated with lung tissue damage and AHR in response to an intranasal (i.n.) challenge with short ragweed allergenic extract (SRW) (31). An allergen-specific response in tg mice was mediated by HLA-DQ-restricted CD4+ T cells. In this study we addressed the role of CD4+ T cells in HLA-DQ-restricted allergic asthma. We show that HLA-DQ6-restricted DN CD4-CD8-TCR{alpha}{beta}+ T cells in CD4-deficient tg mice are able to generate a mixed Th1/Th2 type of response to SRW, resulting in strong pulmonary eosinophilia but weak AHR. In contrast, HLA-DQ8 tg mice lacking CD4 do not develop eosinophilia. These findings provide the rationale for developing strategies to target specific HLA-DQ molecules to effectively control allergic asthmatic conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The production and characterization of tg mice expressing human DQ6 (HLA-DQA1*0103 and HLA-DQB1*0601) and DQ8 (HLA-DQA1*0301 and HLA-DQB1*0302) genes in mice deficient in endogenous MHC class II molecules (H-2A{beta}null) have been described previously (32, 33). DQ/CD4+ tg mice were mated to mice deficient in CD4 (CD4null), provided by Dr. T. W. Mak (University of Toronto, Toronto, Canada) (34, 35). The offspring were screened for the presence of DQ a and b transgenes (32, 33), the absence of wild-type (CD4+/+) and heterozygous (CD4+/-) CD4, and the presence of CD4null gene (neomycin phosphotransferase gene (neo) by PCR of PBMC) (34). The expression of DQ, A{alpha}b, A{beta}b, E{beta}, CD8, CD4, TCR{alpha}{beta}, and TCR{gamma}{delta} molecules was monitored by flow cytometric analyses of PBMC and spleen mononuclear cells (MNC) using corresponding mAb, IVD12, 7-16-17, 25-5-16, Y-17, 53-6.72, GK1.5, H57-597, and GL3, respectively. The DN A{beta}nullCD4null mice served as a control for these studies. The DQ/CD4null and DQ/CD4+ mice used in these studies have similar background genes, because they are siblings derived from the same mating line. Mice were bred and maintained in the pathogen-free immunogenetics mouse colony at the Mayo Clinic (Rochester, MN).

Ag preparation and immunization protocol

Age- and sex-matched mice were injected i.p. with 40 µg of short ragweed, Ambrosia artemisiifolia, extract (Antigen Laboratories, Liberty, MS) adsorbed to 1 mg of aluminum hydroxide (Sigma-Aldrich, St. Louis, MO) in 0.5 ml of sterile endotoxin-free PBS on days 0 and 7 (31). On day 14 mice were challenged i.n. twice (6 h apart) with 120 µg/50 µl extract in the nostrils under light anesthesia. For anesthesia, 4 µg/0.2 ml or 6 µg/0.3 ml per mouse of avertin solution was used (31). Control mice were injected with an equal volume of aluminum hydroxide suspension in PBS and challenged i.n. with PBS alone. Forty-eight hours after challenge mice were euthanized with 20 µg/ml/mouse of avertin solution, and BAL fluids were withdrawn from mouse lungs.

BAL characterization

BAL fluids were collected, cytospin preparations were made and stained by Giemsa, and cell differentials were enumerated as described previously (31).

Eosinophil peroxidase (EPO) levels were measured as previously described (36). Human EPO (provided by J. Checkel and G. J. Gleich, Allergic Diseases Research Laboratory, Mayo Clinic) was used as a standard for these assays. Briefly, 100 µl of sample or serial standard dilutions were pipetted in duplicate into 96-well Immulon 1 plate (Dynatech, Chantilly, VA). One hundred microliters of substrate solution consisting of 1 mM hydrogen peroxide and 0.1 mM O-phenylenediamine (both from Sigma-Aldrich) in 50 mM Tris-HCl buffer (pH 8) was added to wells. The plates were incubated at 37°C for 30 min; the reaction was stopped by the addition of 50 µl of 4 M sulfuric acid. The absorbance was measured at 490 nm with a microtiter autoreader (Bio-Rad, Pleasanton, CA). The sensitivity of the assay was 1 ng/ml.

IL-4, IL-5, IL-13, IFN-{gamma}, and TNF-{alpha} contents in BAL fluids were measured by sandwich ELISA. Mini-kits for TNF-{alpha} (Genzyme, Cambridge, MA), IFN-{gamma}, and IL-4 (BD PharMingen, San Diego, CA) were used according to the instructions provided. IL-5 content was measured using a mini-kit from Endogen (Cambridge, MA). Anti-IL-13 mAb, biotinylated anti-IL-13 mAb (both from R&D Systems, Minneapolis, MN), and rIL-13 (BD PharMingen) were used in IL-13-specific ELISA. BAL samples were diluted 1/1 with dilution buffer before applying to plates. The sensitivities were: IL-4, 15 pg/ml; and IL-5, IL-13, IFN-{gamma}, and TNF-{alpha}, 10 pg/ml.

Histochemistry

In separate experiments the trachea and lungs were removed from euthanized mice (n = 4–5 per group) 48 h after allergen challenge. Sections 4-mm thick were prepared as described previously using 10% formalin for fixation and paraffin for embedding, and then were stained with H&E. Evaluation of histologic alterations was performed with three samples for each mouse in a double blind study.

Airway hyperreactivity

AHR in PBS- or SRW-sensitized mice was measured 48 h after the last i.n. challenge by recording respiratory pressure curves by whole body plethysmography (model PLY 3211; Buxco Electronics, Sharon, CT) in response to the increasing doses of inhaled methacholine (Sigma-Aldrich) as described previously (37). In the plethysmograph, mice were exposed for 3 min to nebulized PBS and subsequently increasing concentrations of nebulized methacholine in PBS. After each nebulization, recordings were taken for 3 min. Values of PenH were calculated by BioSystem XA software with the following equation: PenH = (Te/RT - 1) x (Pef/Pif), where Te is the expiration time, RT is the relaxation time, Pef is the peak expiratory flow, and Pif is the peak inspiratory flow. The PenH values were expressed for each methacholine concentration as percentage of baseline PenH values following PBS exposure.

In vivo Ab administration

Anti-CD8 (53-6.72) and anti-NK1.1 (PK136) mAb were purified from supernatants of hybridoma cells, resuspended in PBS at a concentration of 1 mg/ml, and stored at -70°C. To deplete CD8 or NK1.1+ cells, mice were injected 1 day before priming with SRW and every 5 days thereafter (including 24 h before i.n. challenge) with 200 µg of the corresponding mAb i.p. Control mice were injected with the same amount of rat (clone R35-95; BD PharMingen) or mouse (clone UPC10; Sigma-Aldrich) IgGa isotype control Abs. The efficacy of depletion was monitored by flow cytometric analysis of spleen MNC at the end of the experimental period (day 2 after SRW challenge). The measurements for animals exhibiting >1.0% NK1.1+ or CD8+ cells at the end of the experiment were excluded from the study.

Anti-CD1d mAb (clone 1B1; BD PharMingen) was administered i.v. (50 µg/mouse/day) at the same time intervals. The isotype-matched Ab, rat IgG2b (clone R35-38; BD PharMingen), was used as the control Ab. Purified anti-DQ mAb (IVD12) or isotype-matched control IgG1 (MOPC-21; Sigma-Aldrich) was used at a concentration of 200 µg/200 µl/mouse by i.p. injections 24 h before and after each SRW application. All reagents for in vivo applications were routinely tested for endotoxin contamination using the timed gel formation endotoxin kit (Sigma-Aldrich) according to the manufacturer’s instruction. No contamination was observed.

CD4+ T cell reconstitution

Spleen, inguinal, lumbar, axillary, and mesenteric lymph nodes were harvested from SRW-sensitized DQ6/CD4+ mice on day 11 of the immunization protocol. The tissues were minced and dispersed into single-cell suspension, and erythrocytes were lysed as described previously (38). Cells were washed, counted, resuspended in complete RPMI 1640, and cultured for 48 h in the presence of 200 µg/ml SRW/107 cells. Purified T cells were obtained using mouse T cell enrichment columns (R&D Systems). The CD4+ subset of T cells was purified using the MACS beads system and separation columns LS+/VS+ (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instruction. CD4- cells (nonbound) were also collected. The purity of the enriched T cell population and CD4- and CD4+ T cell fractions was determined by flow cytometry. The final populations of CD3+ T cells and CD4+ T cells were >96% pure. Either CD4- cells or CD4+ T cells were transferred as described previously (39) to naive DQ6/CD4null mice i.v. (2 x 106 cells/mouse). Five days later mice were challenged i.n. with SRW, and AHR was measured 48 h later. At the conclusion of the experiments mice were bled and analyzed for the presence of CD4+ T cells using the appropriate conjugated mAb and flow cytometry. Analysis of PBMC revealed that the percentage of cells expressing CD4 in recipient mice ranged from 3.8 to 5.7%.

Serum cytokine, total IgE, and allergen-specific Ab levels

Cytokine contents in preimmune (day 0) and immune (day 16) sera were measured by sandwich ELISA as described for BAL fluids with two modifications: plasma samples were diluted 1/4 with dilution buffer before applying to the plates, and serial dilutions of standards were prepared using a dilution buffer containing 25% normal mouse serum. The sensitivity was 30 pg/ml.

The amount of Ag-specific IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA was measured by ELISA as we described previously (31) using appropriate alkaline phosphatase-conjugated mAb and p-NPP substrate (both from Southern Biotechnology Associates, Birmingham, AL). Serum Ab levels were quantified by comparison to purified isotype standards (Southern Biotechnology Associates) added to each plate, using the Microplate Manager software for the Macintosh computer (Bio-Rad). The sensitivities were: IgM and IgA, -2.5 ng/ml; IgG1, IgG2a, IgG2b, and IgG3, -4 ng/ml.

An IgE-specific ELISA with matching Ab pair, R35-72 and R35-92 (BD PharMingen), was used to measure total IgE. IgE levels were quantified by comparison to purified isotype standard monoclonal anti-dinitrophenyl mouse IgE isotype Ab (Sigma-Aldrich) using Microplate Manager software for the Macintosh computer (Bio-Rad). A serum pool of SRW-immunized BALB/c mice was used in each assay as an internal laboratory positive control.

Cell proliferation and cytokine production assays

Single-cell suspensions of spleen MNC obtained from each individual mouse and pooled within an experimental group (n = 5) of peribronchial lymph node cells (PBLNC) from PBS- or SRW-treated mice were prepared 5 days after i.n. SRW challenge. Cells were incubated in 96-well microtiter plates at 10 x 105 cells/well in 0.2-ml volumes at 37°C in RPMI 1640 with supplements (31, 38) containing medium alone, 10 µg/ml Con A, or 200 µg/ml SRW. After 48 h, cell proliferation was assessed by [3H]thymidine incorporation and results were expressed as mean cpm ± SEM of triplicate wells.

For in vitro mAb blocking study culture supernatants from the cell lines producing mAb specific for HLA-DQ {alpha}-chain (IVD 12), HLA-DQ6/8 (TB1), HLA-DR (L227, cross-reacts with HLA-DQ6{beta}), H-2A{alpha}b (7-16-17), H-2A{beta}b (25-5-16), H-2E{alpha}{beta}b (Y-17), CD4 (GK1.5), and CD8 (53-6.72) were used. Twenty microliters (10 µg) of dialyzed commercial anti-CD1d mAb (BD PharMingen) per well was used to block CD1-restricted response.

For measurement of cytokine production, cells were cultured at 10 x 106 cells/well in 2-ml volumes with analogous stimuli (medium alone, Con A, or SRW). At 72 h cells were taken for IL-5 ELISPOT assay, and culture supernatants were used for analysis of IFN-{gamma}, IL-4, IL-5, IL-10, IL-12, and IL-13 proteins by ELISA.

For IL-5 ELISPOT assay, cells were counted and resuspended in complete RPMI 1640 with 10 µl/107 cells of each of the following FITC-labeled BD PharMingen’s mAb: anti-CD8, anti-pan-NK (DX5), anti-B220 (RA3-6B2), and anti-Mac1 (M1/70). After labeling, cells were washed and incubated with 50 µl/107 cells of anti-FITC-coated microbeads (Miltenyi Biotec). Nonbound DN T cells were isolated using MiniMACS depletion columns. Bead-bound CD8+NK+B220+Mac1+ cells were also collected. The purity of DN T cell fractions was determined by flow cytometry using PE-labeled anti-CD3 mAb. ELISPOT assay was conducted using plastic plates (Millipore, Bedford, MA) coated with 5 µg/ml primary Ab for IL-5 (TRFK5; eBioscience, San Diego, CA) overnight. The plates were washed, blocked with complete RPMI 1640, and Con A-stimulated, SRW-stimulated, or unstimulated DN T cells and CD8+NK+B220+Mac1+ cells were seeded in serial dilutions starting at 1 x 106 cells/100 µl/well. The plates were then incubated overnight at 37°C in 5% CO2. After washing, the plates were incubated with secondary biotinylated anti-IL-5 Ab (TRFK4; eBioscience) at 1 µg/ml for 2 h, followed by a 30-min incubation with streptavidin-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/1000 in assay diluent (eBioscience). A substrate solution containing 3-amino-9-ethylcarbazole (Sigma-Aldrich; 4 mg dissolved in 1 ml of dimethylformamide) in 14 ml of 0.1 M phosphate-citrate buffer, pH 5.0, was used for developing plates. The plates were then air-dried, and spots were counted using a dissecting microscope (MZ95; Leica, Deerfield, IL).

Isolation and stimulation of CD4-CD8- (DN) T cells

tg mice were primed with SRW, and a booster injection was given as described. Seven days later, spleen MNC suspensions in complete RPMI 1640 were prepared and pooled within each experimental group (four mice per group). Nylon wool (NW) columns were prepared and populations of enriched T cells were collected as described previously. CD8+ T cells were isolated by incubation of NW T cells with FITC-labeled anti-CD8 mAb for 30 min on ice, followed by positive selection using the anti-FITC MiniMACS system according to the manufacturer’s instructions (Miltenyi Biotec). DN cells were purified by incubating remaining T cells with FITC-labeled anti-CD4 (GK1.5), anti-NK1.1 (PK136), anti-CD14 (rmC5-3), and anti-mouse Ig (Accurate Chemical and Scientific, Westbury, NY) mAb and further depletion of CD4+, NK1.1+, CD14+, and Ig+ cells with MiniMACS system or by cell sorting (BD Biosciences, Franklin Lakes, NJ). The isolated cell populations were tested for purity by flow cytometry; the average purity was >96%.

For cytokine production, the flat-bottom 96-well microplates (Costar, Corning, NY) were first coated with 50 µl of anti-CD3 (1 µg/ml) at 4°C overnight. Plates were washed with PBS, and then spleen MNC, NW-purified, CD8+, and DN T cells were added to the wells at 2 x 105 cells/well in triplicate. Supernatants were collected after 48 h of culture, and cytokine protein concentrations were determined by ELISA.

Statistics

Data are summarized as the mean ± SEM. To calculate significance levels between treatment groups, Student’s t test (Sigma Plot; Janssen, San Ramon, CA) was used. Differences between values were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and characterization of tg mice

To study the role of HLA-DQ6 and -DQ8 Ags and CD4 molecules in allergic asthma, we generated mice tg for HLA-DQ6 or HLA-DQ8 in the absence of mouse class II (32, 33) and mated them to the CD4 knockout mice (34, 35). The expression of DQ, CD8, and CD4 molecules was determined by flow cytometric analyses of PBMC and spleen MNC. HLA-DQ molecule was expressed on 25–40% of the PBMC population and on 30–40% of spleen MNC in DQ/CD4+ mice, and its expression was similar in their CD4-deficient counterparts. The DQ/CD4+ mice expressed 5–9.3% CD4+ T cells in peripheral blood, whereas no detectable CD4 (<0.5%) was found in PBMC from DQ/CD4null mice. CD8 molecule was expressed on 10–14% of PBMC and on 7–9% of spleen MNC in DQ/CD4+ mice, while its expression was lower in DQ/CD4null mice (5–8 and 3–5%, respectively).

Cellular composition of BAL and characterization of BAL fluids

To determine the importance of HLA-DQ6 and -DQ8 Ags and CD4 molecules for the development of Th2 responses and allergic airway inflammation, DQ/CD4null tg mice were primed, boosted, and challenged i.n. with SRW as described in Materials and Methods. DQ6/CD4+ mice, DQ8/CD4+ mice, and DN A{beta}nullCD4null counterparts were used as controls for this study. Exposure of tg mice to allergen induced a selective infiltration of eosinophils into airways of DQ6/CD4+, DQ8/CD4+, and DQ6/CD4null mice, but not in DQ8/CD4null mice, as assessed by BAL at 48 h after i.n. challenge (Fig. 1GoA). Pretreatment of DQ6/CD4null mice with anti-DQ mAb significantly reduced airway eosinophilia, but not lymphocyte infiltration. Lymphocyte differential of the BAL was increased in all groups of DQ transgenics with or without CD4 compared with A{beta}nullCD4null mice. Monocytes were the primary cell type found in BAL of A{beta}nullCD4null mice and DQ8/CD4null tg mice. In vivo depletion of NK1.1 cells did not show any effect on airway inflammation observed in DQ6/CD4null mice (Fig. 1GoB).



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  		FIGURE 1. BAL cellular composition of SRW-sensitized and challenged tg mice. A, Mice were immunized as described in Materials and Methods. BAL fluids were harvested 48 h after i.n. challenge. No eosinophils were recovered from lungs of A{beta}nullCD4null mice. Eosinophils were found to dominate the cellular infiltrate into BAL of DQ6/CD4null mice and DQ/CD4+ mice. Error bars indicate the SD of the average of each cell type counted in four high-power fields per BAL in three individual experiments with four or five mice per experiment. *, Significant differences (p < 0.05) between eosinophil levels in SRW-treated tg mice and double-knockout A{beta}nullCD4null mice; {dagger}, p < 0.05, lymphocyte levels in tg mice vs those in DQ8/CD4null and A{beta}nullCD4null mice; §, p < 0.02, anti-DQ-treated DQ6/CD4null mice vs mAb-untreated SRW-immunized counterparts. B, Anti-NK1.1 treatment has no effect on the cellular composition of BAL fluids obtained from SRW-sensitized and challenged DQ6/CD4null mice. Values shown are the mean ± SEM of four mice per group.

 
The DQ/CD4+ tg mice demonstrated substantial levels of EPO in BAL at 48 h after SRW challenge (Fig. 2GoA). Despite acute BAL eosinophilia, the EPO activity in DQ6/CD4null mice was minimal (13.55 ± 2.74 ng/ml), and it did not differ from that in DQ8/CD4null mice (13.32 ± 5.40 ng/ml). SRW treatment induced local lung IL-5 and IL-13 production in DQ/CD4+ mice (Fig. 2GoB). Measurable amounts of IL-13 were found in 2 of 10 DQ6/CD4null mice. TNF-{alpha} was found only in BAL in 2 of 10 DQ8/CD4null mice (198.44 ± 141.58 pg/ml BAL fluid). No IFN-{gamma} or IL-4 was detected in any BAL samples.



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  		FIGURE 2. EPO (A) and cytokine (B) levels in BAL fluid supernatants of tg mice. Mice were sensitized to SRW as described in Materials and Methods. BAL fluids were obtained from each group of mice (n = 4–5) at 48 h after the last i.n. challenge. Results from two independent experiments were grouped together and expressed as the mean ± SEM. A, EPO concentration in BAL was detected colorimetrically. *, p < 0.00001, DQ6/CD4+ mice vs DQ/CD4null and A{beta}nullCD4null mice. B, Levels of IFN-{gamma}, IL-4, IL-5, and IL-13 were determined by ELISA. SRW sensitization and challenge resulted in an increase in IL-5 and IL-13 contents in BAL of DQ/CD4+ mice. There was no detectable IFN-{gamma} and IL-4 in any BAL samples.

 
Histopathology of lung tissue

Examination of lung tissue stained with H&E revealed strong perivascular and peribronchial eosinophilia with significant epithelial damage in SRW-challenged DQ6/CD4+ and DQ8/CD4+ tg mice (Fig. 3GoA). This was not observed in the A{beta}nullCD4null groups (Fig. 3GoB). Lungs of DQ6/CD4null mice showed multiple inflammatory infiltrates, mostly in the form of severe eosinophilic perivasculitis (Fig. 3GoC) without a considerable damage to the airway epithelium; 2 of 10 mice showed an eosinophilic pneumonia with the interstitial and parenchymal localization of these cells. In contrast, no inflammation was observed in DQ8/CD4null mice (Fig. 3GoD). Lungs of PBS-treated mice were normal (not shown). In vivo treatment with either anti-CD8 or anti-NK1.1 mAb did not show any effect on airway eosinophilia observed in DQ6/CD4null mice (Fig. 3Go, E and F, respectively) compared with mAb-untreated and isotype control rat IgG2a-treated (Fig. 3GoG) counterparts.



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  		FIGURE 3. Histopathology of lung tissue of SRW-sensitized and challenged mice. Lung tissues were stained with H&E. Histological lung sections shown are representative of three sections of lung per mouse from four to eight mice in each group. A, A diffuse eosinophilic infiltration around and in bronchi, and goblet cell hyperplasia in lungs of DQ6/CD4+ mice that contrasted with a section of lung from an A{beta}nullCD4null mouse showing no inflammation (B). C, A perivascular inflammation that is comprised predominantly of eosinophils and MNCs was observed in tissue of DQ6/CD4null mice. D, Section of lung from a DQ8/CD4null mouse showing edema around large airway vessels and the absence of inflammatory cell infiltration. The pretreatment of DQ6/CD4null mice with anti-CD8 (E) or anti-NK1.1 (F) mAb or isotype control Ab (G) did not change the magnitude of the lung tissue eosinophilic inflammation induced by SRW airway challenge.

 
Lung physiology studies

Analysis of airway responsiveness in PBS-treated and SRW-exposed tg mice has shown an absence of hyperreactivity to methacholine in SRW-sensitized and challenged A{beta}nullCD4null mice (Fig. 4Go). Allergen challenge induced strong hyperreactivity in DQ6/CD4+ mice compared with PBS-treated littermates, while it was considerably lower for DQ6/CD4null mice. Reconstitution of naive DQ6/CD4null mice with allergen-restimulated CD4+ T cells obtained from SRW-sensitized DQ6/CD4+ mice led to an increase in hyperreactivity to methacholine (50 mg/ml) challenge compared with CD4- T cell recipients and SRW-sensitized and challenged counterparts that did not undergo the cell transfer. In vivo depletion of NK1.1 or CD8 cells did not show any effect on AHR observed in DQ6/CD4null mice (not shown).



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  		FIGURE 4. Respiratory system physiology of PBS-treated and SRW-sensitized and challenged tg mice. Note the absence of lung hyperreactivity in SRW-treated A{beta}nullCD4null mice compared with that in PBS-treated counterparts. Responses to methacholine were significantly greater in SRW-treated DQ6/CD4+ mice compared with PBS-treated mice of the same genotype and allergen-sensitized DQ6/CD4null mice. Mean methacholine dose response curves of four mice per control group and from 4–12 mice per experimental group (± SEM) are shown. *, p < 0.03, SRW-treated DQ6/CD4+ mice vs either PBS-treated counterparts or SRW-treated DQ6/CD4null mice; {dagger}, p < 0.01, SRW-treated DQ6/CD4null mice vs PBS-treated counterparts. Adoptive transfer of CD4+ T cells resulted in a significant increase in the airway response to high dose (50 mg/ml) methacholine challenge ({ddagger}, p < 0.002 vs CD4- T cell recipients and the counterparts without cell transfer).

 
Cytokines and allergen-specific Ab in serum

An examination of the cytokine profile in preimmune (day 0) and immune (day 16) sera revealed that DQ6/CD4null mice generated a substantial cytokine response to SRW sensitization. Significant levels of IFN-{gamma} and IL-4 were observed in these mice (Fig. 5GoA). Sensitization and challenge with SRW induced in vivo IL-5 production in DQ6/CD4+ and DQ6/CD4null tg mice. However, the expression of CD4 significantly increased the capacity of mice to produce IL-5. All these cytokines were virtually undetectable in sera of A{beta}nullCD4null mice. Anti-NK1.1 mAb treatment did not inhibit IL-5 production in the immune sera of DQ6/CD4null mice compared with control mouse IgG2a (119.6 + 21.4 and 109.5 + 14.9 pg/ml, respectively).



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  		FIGURE 5. Plasma cytokines (A), SRW-specific Igs (B), and total serum IgE (C) levels in SRW-treated tg mice. Mice were bled before the first immunization, immunized, and challenged i.n. with SRW as described in Materials and Methods and then bled 48 h after i.n. challenge. Levels of cytokines (A) and Ab (B and C) were determined by sandwich ELISA and direct ELISA. The bars represent the concentration of cytokine or Ab for pooled sera from four or five individual mice per group and are representative of two independent experiments. A, Note the significant amount of IFN-{gamma}, IL-4, and IL-5 in DQ6/CD4null mice 48 h after SRW challenge (day 16). Plasma IL-5 was detected in both CD4-deficient and CD4-sufficient DQ6 mice. B, Immunization and challenge with SRW induced production of SRW-specific IgG1 and IgG2b Abs in DQ6/CD4+ and DQ6/CD4null mice. No SRW-specific Ab was detected in A{beta}nullCD4null mice. C, An increase in total serum IgE level in the immune sera was observed in DQ6/CD4null and DQ6/CD4+ mice. *, p < 0.015; {dagger}, p < 0.007 (vs total serum IgE in preimmune sera).

 
We examined the in vivo Ig isotype of the Ab response to SRW. The kinetics and intensity of the IgM and IgG3 responses were similar among all experimental groups (not shown). Interestingly, DQ6/CD4null mice were able to generate the allergen-specific IgG1 and IgG2b responses against SRW (Fig. 5GoB), although the Ab was 3- and 6-fold lower, respectively, compared with that in DQ6/CD4+ mice. No SRW-specific IgG2a Ab was detected in either A{beta}nullCD4null or DQ6/CD4+ samples, whereas low amount of this Ab was found in DQ6/CD4null mice.

Analysis of the effects of the various transgenes on total IgE levels in sera demonstrated increased levels of IgE in SRW-sensitized and challenged DQ6/CD4null and DQ6/CD4+ mice compared withpreimmune controls (Fig. 5GoC). In contrast, A{beta}nullCD4null and DQ8/CD4null mice did not exhibit a significant increase in total serum IgE during sensitization and challenge.

Proliferation assay and cytokine production

To investigate the nature of the T cell immune response to SRW, spleen MNC and PBLNC were isolated and examined for SRW-specific proliferation. Spleen MNC and PBLNC from DQ6/CD4+ and DQ6/CD4null tg mice responded to SRW, although the response in DQ6/CD4null mice was lower (Fig. 6GoA). As shown in Fig. 6GoB, the proliferative response in these mice was blocked by purified HLA-DQ-specific mAb, showing that T cells recognize SRW in the context of HLA-DQ. There was no proliferative response to SRW in DQ8/CD4null mice. It is clear that the immune response of DQ6/CD4null mice was mediated by DN T cells, because the use of mAb with CD4, CD8, or CD1 specificity had no effect on proliferation.



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  		FIGURE 6. Spleen MNC and PBLNC (A) proliferative response to SRW and inhibition of the response with mAb (B). A, Spleens and PBLNC were harvested from mice at 48 h after i.n. challenge with SRW, and cells were cocultured in vitro with 200 µg/ml allergenic extract as described in Materials and Methods. HLA-DQ6/CD4+ and HLA-DQ6/CD4null tg mice were able to maintain SRW-specific proliferation, although the response in HLA-DQ6/CD4null mice was lower than that in HLA-DQ6/CD4+ littermates. No response was detected using cells from either A{beta}nullCD4null or HLA-DQ8/CD4null mice. Data shown are the results for one of threeindividual mouse spleen and pooled PBLNC from five mice per strain in two representative experiments. B, The in vitro response of PBLNCof HLA-DQ6/CD4null mice to SRW is mediated by DQ-restricted CD4-CD8-NK1.1- T cells. PBLNC were cocultured with SRW in the presence of the indicated mAbs. The data represent the maximum change ({Delta}) in cpm of [3H]thymidine incorporation in triplicate cultures with SRW. The values displayed are the result of pooled PBLNC from five mice per strain and are representative of two analyses.

 
Direct analysis of cytokine synthesis in spleen MNC cultures of DQ6/CD4null and DQ8/CD4null mice supports the contention that the expression of specific HLA-DQ molecules impacts on cytokine responses in this system. Stimulation of spleen MNC taken from allergen-challenged DQ6/CD4null mice with SRW for 72 h resulted in the production of IL-5, IL-10, IL-13, and IFN-{gamma} (Fig. 7GoA). None of these cytokines was detected in culture supernatant preparations obtained from A{beta}nullCD4null and DQ8/CD4null mice. All the mice tested produced significant amounts of IL-12.



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  		FIGURE 7. Cytokine concentrations in spleen MNC culture supernatant (A) and frequency of IL-5-producing cells (B). A, Single-cell suspensions of spleen MNC from PBS- or SRW-treated mice were prepared 5 days after i.n. SRW challenge. The cells were cultured with SRW, supernatants were collected at 72 h, and cytokine ELISAs were performed. Results from two representative experiments were combined. No cytokines were detected in spleen MNC culture supernatants from PBS-treated mice. B, The frequencies of IL-5-producing CD8+NK+B220+Mac1+ cells and DN T cells were determined using ELISPOT assay as described in Materials and Methods. The results are expressed as the mean number of spots per 250,000 cells/well obtained in one of two independent experiments.

 
ELISPOT assay was used to compare IL-5 production by splenocytes, DN T cells and CD8+NK+B220+Mac1+ cells from DQ6/CD4null mice in response to SRW restimulation in vitro. As shown in Fig. 7GoB, the frequencies of IL-5-producing DN T cells were 16 times higher than those of CD8+NK+B220+Mac1+ cells. The number of IL-5-producing CD8+NK+B220+Mac1+ cells was overall lower in Con A-stimulated cell cultures compared with DN T cells. Similar patterns were obtained for spleen MNC.

Characterization of DN T cells

tg mice received a priming and a boost injection with SRW as described in Materials and Methods. Seven days later PBMC and spleen MNC were isolated. Flow cytometric analysis revealed that naive DQ6/CD4null and DQ8/CD4null mice have equal number of DN T cells in PBMC and spleen MNC, amounting to 5–7% of the total cell number. The majority of DN T cells (>=80%) express TCR{alpha}{beta} (Fig. 8Go). SRW treatment induced an increase in this number in PBMC of DQ6/CD4null mice only (from 5.8 ± 0.29 to 9.62 ± 0.56%; p < 0.015, naive vs allergen-treated mice; p < 0.005, SRW-treated DQ6/CD4null mice vs DQ8/CD4null mice). A significant increase in DN cells was also observed in spleen MNC of SRW-treated DQ6/CD4null mice compared with naive control littermates (11.68 ± 1.10 and 6.76 ± 0.4%, respectively; p < 0.001) and DQ8/CD4null mice. For DQ6/CD4+ mice, the relative proportion of CD4-CD8-NK1.1-CD3+ T cells remained fairly constant for PBMC, from 2.53 ± 0.34 (naive) to 3.03 ± 0.31% (sensitized), with no significant increase for spleen MNC from 1.96 ± 0.82% in naive mice to 3.51 ± 1.19% in SRW-treated mice, respectively.



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  		FIGURE 8. Flow cytometric histogram showing surface expression levels of TCR{alpha}{beta} and TCR{gamma}{delta} on peripheral blood and spleen DN T cells in DQ6/CD4null mice. The frequency of corresponding TCR was analyzed from the gated CD3+CD4-CD8-NK1.1- cells as described in Materials and Methods.

 
Spleen MNC and NW T cells from DQ6/CD4null tg mice produce IL-4, IL-5, and IL-13 in response to anti-CD3 stimulation (Fig. 9Go). IFN-{gamma} was found only in cultures of DQ6/CD4null cells. When we compared the amounts of cytokines produced by CD8+ and DN T cells in DQ6/CD4null mice we found that DN T cells were more efficient in the production of IL-4, IL-5, and IL-13. Neither IL-5 nor IL-13 was identified in stimulated cultures of DQ8/CD4null splenocytes and NW cells. However, a small amount of IL-4 was detected, which seems to be linked to DN T cells. In DQ8/CD4+ mice, IL-5 and IL-13 production by spleen MNC or NW cells was most likely dependent on CD4+ T cells.



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  		FIGURE 9. Cytokine production by anti-CD3-stimulated cells. Spleen MNC were obtained from mice 7 days after postsecondary immunization. Isolated NW cells, CD8+ T cells, and DN T cells were cultured with immobilized anti-CD3 mAb. Supernatants were collected after 48 h, and cytokine ELISA was performed. One of two representative experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper we present the evidence that the T cell coreceptor CD4 is not necessary for the induction of a Th2-type response to allergen and resulting eosinophilic airway inflammation in HLA-DQ6 tg mice. However, it is required for immunologic and pathophysiologic features of allergic asthma, such as local lung cytokine production, eosinophil activation, resulting lung tissue damage, and robust bronchial hyperreactivity. The expression of the HLA-DQ6, but not the HLA-DQ8, molecule results in CD4-independent allergen-induced lung eosinophilia mediated by DN CD4-CD8-NK1.1-TCR{alpha}{beta}+ T cells. The differences in physicochemical characteristics of the molecular structure of DQ8 and DQ6 Ags (40), the binding and presentation of distinct peptides to T cells (38, 40, 41, 42, 43), and their distinct roles in autoimmune diseases (32, 33, 40, 41, 42, 44) may in part explain this observation. The higher affinity of the DQ6/peptide/TCR interaction (40, 44) may bypass the requirement for CD4 coreceptor for activation of DQ6-restricted T cells.

In DQ/CD4null tg mice, DN T cells are localized in the spleen, LN, and peripheral blood and were able to maintain a Th1/Th2-type response to allergen with a resulting airway eosinophilia depending on the specific DQ allele expressed. DN TCR{alpha}{beta}+ T cells is a minor population of T cells (up to 5% of total lymphocytes) that exists in thymus and peripheral lymphoid organs of normal mice (45, 46) and humans (45, 47) in addition to the major CD4+ and CD8+ T cell subsets. The role of these cells in allergic diseases is undefined regardless of evidence that these cells may initiate a Th2 response producing IL-4 (48, 49). The origin of peripheral DN T cells is still unknown. They may derive from the thymic DN T cell population, or they may develop in a thymus-independent manner (50).

We demonstrate that DQ6/CD4null tg mice were able to generate Th2 cell-dependent IgG1 and IgG2b Abs to SRW, although the amounts of these Ab were significantly lower compared with those in DQ6/CD4+ tg mice. Therefore, CD4+ T cells are necessary to provide the optimal activation of B cells and optimal humoral immune response to allergen. In addition to Th2-dependent Ab, a small amount of IFN-{gamma}-dependent IgG2a Ab was found in DQ6/CD4null mice. Total IgE is a good marker for the induction of an allergic response in mice. Its level was also increased in tg mice showing airway eosinophilia. In C57BL/6 mice lacking CD4 the in vivo Ig isotype switching from IgM to IgG occurs in response to T cell-dependent Ags (35). These mice have an expanded population of DN CD4-CD8-TCR{alpha}{beta}+ T cells in the periphery (~15% of total T cells), which help in the Ab response. In vitro proliferation studies using T cells from keyhole limpet hemocyanin-primed CD4--deficient C57BL/6 or BALB/c mice demonstrated that DN T cells recognized keyhole limpet hemocyanin in the context of MHC class II (35, 51).

Using spleen MNC and PBLNC from in vivo allergen-primed and challenged DQ6/CD4null mice for proliferation inhibition studies, we were able to demonstrate that the SRW-specific response is DQ6 restricted. This response was not blocked by anti-CD8 mAb. Similarly, mAb specific to CD4 and CD1 had no effect on the DQ6-dependent proliferation in CD4-deficient mice, eliminating the involvement of other cell types besides DN T cells in the immune response to SRW in these mice. The DQ/CD4+ tg mice response is dependent on DQ-restricted CD4+ T cells. Therefore, the expanded population of DN TCR{alpha}{beta}+ T cells in DQ6/CD4null mice has partially substituted for the role normally performed by the CD4+ Th cells.

Furthermore, sera and spleen MNC cytokine profiles were monitored. An absence of all cytokines tested in SRW-challenged A{beta}nullCD4null mice with a normal distribution of CD8+ T cells and NK1.1+ cells supports our hypothesis that DQ6-restricted DN T cells are responsible for elevated levels of IFN-{gamma}, IL-4, and IL-5 cytokines in the immune sera of DQ6/CD4null mice. Our ELISPOT data confirm this observation for IL-5. The fact that IFN-{gamma} and IL-4 were not detected in allergen-sensitized DQ6/CD4+ mice shows the CD4-dependent difference in T cell activation, signaling, and resulting cytokine profile. The markedly enhanced production of IFN-{gamma} was apparently suppressive (52, 53) for the disease outcome in DQ6/CD4null mice.

The ability of MHCII-restricted DN TCR{alpha}{beta}+ T cells to develop into Th cells with IFN-{gamma} production in response to Leishmania major Ags was previously demonstrated with CD4-deficient mice (54). The Toxocara canis-sensitized surface CD4-deficient mutant mice have shown an eosinophilia and Th2 response to Ag despite the absence of CD4+ cells (55). The DN T cells producing IL-4 and IL-5 cytokines were responsible for these effects. We specifically analyzed cytokine production by DN and CD8+ T cells in SRW-treated tg mice using an in vitro stimulation with immobilized anti-CD3 mAb. It was somewhat surprising to find that DN T cells in DQ6/CD4null mice retained the capacity to secrete high levels of IL-4, IL-5, and IL-13 in the face of their ability to produce large amounts of IFN-{gamma}, particularly given the established role of IFN-{gamma} as a potent suppressor of Th2 cell activity (56, 57).

Thus, different mechanisms of airway inflammatory response to SRW in CD4-deficient and CD4-sufficient groups of HLA-DQ transgenics can be observed. For DQ6/CD4+ mice, airway eosinophilia was associated with pulmonary edema, strong EPO activity, high local lung Th2 type cytokine production, epithelial hyperplasia, and shedding. In contrast, DQ6/CD4null tg mice did not show any of these characteristics of the inflammatory response despite strong BAL eosinophilia (63.67 ± 11.07% of total cell number). We also found that following SRW treatment DQ6/CD4+ tg mice demonstrated a significantly greater airway responsiveness than their DQ6/CD4null tg counterparts despite similar degrees of eosinophilia in these two lines. Transfer of purified and cultured in vitro with SRW CD4+ T cells obtained from SRW-sensitized donors into naive DQ6/CD4null mice rendered the recipients susceptible to increased AHR after allergen challenge. Therefore, the presence of eosinophils in the lungs by itself is not enough to induce strong hyperreactivity; this discordance between airway eosinophilia and AHR has been noted in several other animal models (17, 58). The recruited eosinophils need to be activated. Collectively, our experiments show a critical role for HLA-DQ polymorphism, CD4+ T cells, and DN T cells in ragweed-induced allergic disease.


   Acknowledgments
 
We thank Dr. Tak W. Mak (University of Toronto, Toronto, Canada) for providing CD4 knockout mice. We thank Julie Hanson and her staff (Immunogenetic Mouse Colony) for outstanding mouse production, James Checkel and Dr. Gerald J. Gleich (Allergic Diseases Research Laboratory) for the generous gift of the human EPO, General Hematology Laboratory staff for H&E staining of the cytospin slides, Linda McGee and her staff (Pathology Specimen Processing Laboratory) for help in preparing mouse lung histology slides, and Tomas Beito for preparing the cell hybridomas.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI14764. Back

2 Address correspondence and reprint requests to Dr. Chella S. David, Department of Immunology, Mayo Clinic, Rochester, MN 55905. E-mail address: david.chella{at}mayo.edu Back

3 Abbreviations used in this paper: AHR, airway hyperreactivity; BAL, bronchoalveolar lavage; DN, double-negative; EPO, eosinophil peroxidase; i.n., intranasal(ly); MNC, mononuclear cell; NW, nylon wool; PBLNC, peribronchial lymph node cell; SRW, short ragweed allergenic extract; tg, transgenic. Back

Received for publication August 22, 2000. Accepted for publication November 7, 2001.


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 Introduction
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 Results
 Discussion
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