Introduction

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Allergic asthma can be characterized by reversible airway obstruction, elevated levels of immunoglobulin E (IgE), chronic airway inflammation and airway hyperresponsiveness to bronchoconstrictor stimuli. The allergic inflammatory infiltrate in the airway tissue predominantly consists of eosinophils and CD4⁺ T-lymphocytes (1). It is now widely accepted that type 2 T-helper (Th2) lymphocytes which produce a limited set of cytokines including interleukin-3 (IL-3), IL-4, IL-5, and granulocyte-macrophage colony-stimulating factor play an important role in the initiation and effector phase of allergic asthma (1). At present, glucocorticoids are the most effective drugs in the treatment of asthma to reduce the inflammatory component and hyperresponsiveness (2, 3). However, glucocorticoids are not very selective and affect a whole range of inflammatory and non-inflammatory cells (2, 3). Therefore, a more selective inhibitor may be more desirable. The T-lymphocyte may be an important target cell for drug therapy since it confers the specificity of the allergic response through the engagement of the TCR with the peptide-MHC (major histocompatibility complex) complex. Activation of T-lymphocytes requires two signals: a specific signal generated by the peptide-MHC complex and a costimulatory signal provided by adhesion molecules on the antigen-presenting cell (APC) (4, 5). Various costimulatory interactions have been identified of which CD28/ CTLA4: B7-1/B7-2 interaction is the most important (5). Blockade of T-lymphocyte costimulation during antigenic stimulation in vitro has been shown to induce clonal anergy or incomplete T-cell activation and may therefore be valuable in the treatment of T-lymphocyte-mediated disease processes (4, 5).

Recently, we developed a murine model of allergic asthma in which mice are sensitized with ovalbumin without the use of an adjuvant (6). Repeated exposure of sensitized mice to ovalbumin aerosol induced airway inflammation characterized by eosinophil infiltration in lung tissue, trachea, and bronchoalveolar lavage (BAL) fluid and development of airway hyperresponsiveness to methacholine and serotonin (7). In the present study, we demonstrate that in a mouse model of allergic asthma treatment with mCTLA4-IgG completely inhibits airway eosinophilia and hyperresponsiveness and attenuates upregulation of IgE levels when given during the time of ovalbumin inhalation.

	Materials and Methods

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Sensitization and Challenge

Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Specified pathogen-free male BALB/c mice (6-8 wk) were obtained from the Central Animal Laboratory, Utrecht, The Netherlands. The mice were housed in macrolon cages and provided with food and water ad libitum. Active sensitization was performed by 7 intraperitoneal injections of 10 µg ovalbumin (grade V) in 0.5 ml pyrogen-free saline on alternate days (1 injection per day). This sensitization procedure has been shown to induce high titres of total IgE antibodies in serum of which 80% was ovalbumin-specific IgE (6). After sensitization, the animals were divided in four groups of six animals. Two groups were treated with mCTLA4-IgG and two groups were treated with control antibody. For each treatment, one group was exposed to 8 ovalbumin (2 mg/ml) aerosols, on consecutive days (1 aerosol per day); the other group was exposed to saline aerosols. The aerosol was generated with an ultrasonic nebulizer (DeVilbiss, particle size 3-5 µm) connected to a Plexiglas exposure chamber (5 l). Exposure was performed for 5 min and in groups of a maximum of 6 animals.

Construction of Murine mCTLA4-IgG Fusion Protein

The cDNA encoding the extracellular transmembrane and cytoplasmic domain of murine CTLA4 was generated by RT-PCR (GeneAmp; Perkin Elmer, Foster City, CA) using primers based on a published sequence (10) from RNA of C57BL/6 purified splenocytes cultured for 72 h with Concanavalin-A (ConA) at 2 µg/ml. The cDNA was subcloned into pGEM-5Zf⁺ vector (Promega, Madison, WI) and sequenced by dideoxynucleotide sequencing. Using another PCR primer set, an EcoRI, BsteII fragment containing the extracellular domain of mCTLA4 terminating with Ser160 (11) was subcloned into an expression vector encoding a human IgG₁ heavy chain (12) with a ECD-immunoglobulin junction containing a subtilism cleavage site (13). The fusion protein combines the ECD with the hinge and Fc regions of human IgG₁ heavy chain. The immunoadhesion fusion protein is expressed as a disulphide-linked homodimer similar to IgG₁ but lacking a C_H1 domain and light chains. The homodimer has a molecular weight of approximately 110 kD consisting of two identical 55 kD subunits. The mCTLA4-immunoglobulin chimera was transiently expressed in 293 cells (14) and purified by protein A affinity chromatography. The mCTLA4-IgG was quantified by an anti-human IgG Fc ELISA.

Binding of mCTLA4-IgG to Murine Splenocytes

Binding of mCTLA4-IgG to B7-1 on murine splenic B cells activated with lipopolysaccharide (LPS) and dBcAMP (15) was evaluated by immunofluorescence and flow cytometry. Briefly, Ficoll hypaque purified splenocytes were cultured for 48 h at 2 × 10⁶ cells/ml in RPMI 1640 medium containing 10% heat-inactivated FCS (Intergen, Purchase, NY), penicillin, streptomycin, 2 mM L-glutamine (Gibco, Gaithersburg, MD), 5 × 10⁵ M 2-mercaptoethanol and stimulated with LPS (20 µg/ml; Sigma Chemical Company, St. Louis, MO) and dibutyryl cyclic AMP (dBcAMP, 300 µg/ml). Splenocytes (10⁶ cells) were suspended in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) and 0.1% sodium azide in a volume of 100 µl and incubated for 30 min with 1 µg biotinylated mCTLA4-IgG or biotinylated rhIgG (4D5, see below) as control. Subsequently, splenocytes were incubated with 1 µg PE-conjugated anti-B220 and 0.5 µg FITC-streptavidin (Caltag, S. San Francisco, CA) for 30 min, washed twice and suspended in 200 µl buffer for FACS analysis (Becton Dickinson, San Jose, CA). Analysis was performed on B220⁺ cells. Additionally, iodinated mCTLA4-IgG was used to determine the number of binding sites on LPS and dBcAMP activated splenic B cells.

Pharmacokinetics and Treatment with Antibodies

The pharmacokinetics of mCTLA4-IgG were determined by an intravenous bolus injection of 200 µg in Balb/c mice. Murine CTLA4-IgG had a serum elimination half-life for the -phase of 55 h, which is comparable to previously reported data (16, 17). Based on the pharmacokinetic parameters, a dosing schedule that maintained serum mCTLA4-IgG levels of at least 10 µg/ml was established which is comparable to the levels described by Linsley and associates (16). The recombinant human IgG1 4D5 was used as a control for mCTLA4-IgG and is a humanized mouse IgG₁ which binds to the P185 antigen expressed on human breast tumor cells (18). Ten minutes before the first ovalbumin or saline inhalation, ovalbumin-sensitized mice were intravenously injected with 110 µl saline containing either 180 µg control antibody or mCTLA4-IgG. Ten minutes before each of the seven following ovalbumin or saline inhalations, the mice were subcutaneously injected with 90 µg of the antibodies in a volume of 55 µl. All antibodies applied were tested for contamination with endotoxin using a commercial limulus amebocyte lysate assay (Biowhittaker, Walkerville, MD) and were less than 1 endotoxin unit/mg.

Ovalbumin-specific IgE ELISA

Serum levels of ovalbumin-specific IgE were determined as described previously (9). In short, 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated overnight at 4°C with 2 µg/ml recombinant human FcR1-IgG fusion protein (Genentech Inc., So. San Francisco, CA) diluted in PBS. After washing with PBS supplemented with 0.05% Tween-20 (PBT) the plates were blocked with ELISA buffer (2 mM EDTA, 137 mM NaCl, 50 mM Tris, 0.5% BSA, 0.05% Tween-20, pH 7.2) and left to incubate at room temperature for 1 h. After removal of ELISA buffer, plates were incubated (room temperature, 2 h) with appropriately prediluted serum samples and serial 2-fold dilutions (starting 1:10) of an ovalbumin-specific IgE reference standard in duplicate. The standard was obtained by intraperitoneal immunization of mice with ovalbumin according to previously published methods (19) and arbitrarily assigned a value of 10,000 experimental units/ml (EU/ml) ovalbumin-specific IgE. After washing, 10 µg/ml of ovalbumin diluted in ELISA buffer was added to each well and plates were left to incubate at room temperature for 1 h. The plates were washed and subsequently incubated for a period of 1 h with horseradish peroxidase-conjugated goat anti-ovalbumin mAb which was diluted in ELISA buffer. After washing, the enzyme activity was determined by 15 min incubation at room temperature with 10 mM o-phenylenediamine and 4 mM H₂O₂ in PBS, after which the reaction was stopped by addition of 4 M H₂SO₄. The optical density was read at 492 nm using a Titertek Multiskan (Flow Labs., Irvine, UK). Serum samples were compared with the ovalbumin-IgE reference standard and values were expressed in EU/ml. Results were evaluated with an analysis of variance (ANOVA) followed by a post-hoc comparison between groups using Bonferroni.

Airway Responsiveness In Vivo

Airway responsiveness was measured in vivo 24 h after the last aerosol exposure using an air-overflow pressure method (8, 9, 20). With this method the respiratory resistance to inflation is measured. Mice were anesthetized by intraperitoneal injection of urethane (2 g/kg), and placed on a heated blanket to maintain body temperature. The trachea was cannulated and a small polyethylene catheter was placed in the jugular vein for intravenous administrations. Spontaneous breathing was suppressed by intravenous injection of tubocurarine chloride (3.3 mg/kg). When it stopped, the tracheal cannula was connected to a respiration pump (Sanders Brinie, Enschede, The Netherlands). The inflation volume of the pump was 0.8 ml per beat, of which the animal inhales approximately 0.1 ml with 190 strokes per min. During inflation pO₂ and pCO₂ levels in arterial blood remained within the physiological range. A pressure transducer (Validyne, Northridge, CA) was located between the tracheal cannula and the respiration pump in order to measure changes in the respiratory resistance to inflation. Pressure signal was recorded breath-by-breath on a Gould Brush 2400 recorder (Godart, Utrecht, The Netherlands). At time intervals of at least 4 min and after the response had returned to baseline level, doubling doses of methacholine ranging from 20 µg/kg to 640 µg/kg were administered. Concentrations of methacholine were prepared in saline and kept on ice for the duration of the experiment. The increase in air-overflow pressure was measured at its peak and expressed as percentage of the pressure obtained by clamping the trachea cannula as determined at the end of the experiment.

Bronchoalveolar Lavage

BAL was performed in the same animals that were used for airway hyperresponsiveness measurements. In pilot experiments it was observed that combining the two techniques had no effect on total number of cells derived from lavage nor on the appearance of different cell types. After completion of the dose-response curve to methacholine, the animals were lavaged 5 times through the tracheal cannula with 1-ml aliquots of pyrogen-free saline warmed to 37°C. The BAL was kept on ice until further processing. The BAL cells were washed with PBS (400 × g, 4°C, 5 min) and the pellet was resuspended in 150 µl PBS. Using a Bürker-Türk chamber, total numbers of BAL cells were counted. For differential BAL cell counts, cytospin preparations were made and stained with Diff-Quick (Merz & Dade A.G., Düdingen, Switzerland). After coding, all cytospin preparations were evaluated by one observer using oil immersion microscopy (magnification: 1,000×). Cells were identified and differentiated into mononuclear cells, neutrophils, and eosinophils by standard morphology. Per cytospin preparation, at least 200 cells were counted and the absolute number of each cell type was calculated. To evaluate differences between ovalbumin- and saline-challenged mice and the effect of treatment with mCTLA4-IgG an ANOVA was performed. For cell types with a very low number in control animals (i.e., neutrophils and eosinophils) a Poisson distribution was assumed and for differences between treatment groups a Fisher exact test was used.

In Vitro Effect of mCTLA4-IgG on Cytokine Synthesis

In preliminary experiments the optimal conditions for ovalbumin-induced cytokine production by parabronchial lymph node cells (PBLN) have been established. PBLN cells were isolated from ovalbumin-sensitized mice (n = 5), pooled, and subsequently a single cell suspension was prepared. After washing, PBLN cells were suspended in RPMI containing 10% heat-inactivated FCS, 1% glutamax I, 50 µg/ml gentamycine, and 5 × 10⁵ M 2-mercaptoethanol and cultured for 5 days in 96-well plates at 2 × 10⁵ cells per well in a volume of 200 µl in the absence or presence of ovalbumin (10 µg/ml). Quadruplicate cultures were performed in the presence of mCTLA4-IgG (10 µg/ml) or control antibody (rh4D5, 10 µg/ml). Cell-free supernatants were harvested and kept at 20°C until analysis. Levels of interleukin-4 and interferon- (IFN-) were determined by ELISA according to the instructions of the manufacturer (Pharmingen, San Diego, CA).

Chemicals

Ovalbumin and o-phenylenediamine were purchased from Sigma Chemical Company, urethane and methacholine from Janssen Chimica (Beerse, Belgium), tubocurarine chloride from Nogepha (Alkmaar, The Netherlands), and goat anti-ovalbumin mAb from Cappel (Durham, NC). Dibutyryl cyclic AMP was obtained from Boehringer (IN).

Data Analysis

Data are expressed as mean ± SEM and comparisons between groups were made using ANOVA unless stated otherwise. A difference was considered to be significant when P < 0.05. Statistical analyses were carried out using SPSS/ PC⁺, version 4.0.1 (SPSS Inc., Chicago, IL) or GLIM, version 4.0 (NaG Inc., Oxford, UK).

	Results

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Binding of mCTLA4-IgG to Splenocytes

B220⁺ splenocytes activated with LPS and dBcAMP for 48 h showed binding of mCTLA4-IgG compared with a matched control antibody rh4D5 (Figure 1). Unstimulated splenocytes did not show binding of mCTLA4-IgG (results not shown). Binding of iodinated mCTLA4-IgG to LPS and dBcAMP activated splenic B-lymphocytes and subsequent Scatchard analysis revealed a kD of 1.8 nM with approximately 1,100 binding sites per cell (results not shown).

View larger version (29K): [in this window] [in a new window]   Figure 1.   Binding of mCTLA4-IgG (solid line) or control IgG (rh4D5, shaded histogram) to B220+ murine splenocytes incubated for 48 h with LPS (20 µg/ml) and dBcAMP (300 µg/ml). Splenocytes (106 cells) were incubated with 1 µg anti-B220-PE and 1 µg biotinylated mCTLA4-IgG or rh4D5 (control IgG) and amplified with 0.5 µg FITC-streptavidin. Unstimulated splenocytes did not stain with CTLA4-IgG (data not shown).

Serum IgE Levels

Mice were treated with control antibody or mCTLA4-IgG after sensitization at the time of challenge. In control antibody-treated mice, ovalbumin-specific IgE levels were significantly (P < 0.01) elevated in ovalbumin challenged animals compared with saline-challenged animals (Figure 2). Murine CTLA4-IgG-treated mice showed a smaller increase in the serum levels of ovalbumin-specific IgE after ovalbumin challenge which did not reach the level of significance (P = 0.120) when compared with saline-challenged mCTLA4-IgG-treated mice. Although ovalbumin-specific IgE levels in CTLA4-IgG-treated mice challenged with ovalbumin were decreased compared with ovalbumin-challenged control antibody-treated mice, this suppression was not significant.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Levels of ovalbumin-specific IgE in experimental units/ml (EU/ml) as determined by ELISA in serum of ovalbumin-sensitized animals treated with control antibody (IgG) or mCTLA4-IgG and repeatedly challenged with saline (white bars) or ovalbumin (black bars). Results are expressed as means ± SEM of six animals. *P < 0.01 compared with saline-challenged mice and determined by ANOVA followed by post-hoc comparison using Bonferonni.

Bronchoalveolar Cell Infiltration

In mice treated with control antibody, ovalbumin challenge induced a significant (P < 0.05) infiltration of eosinophils into the BAL fluid compared with saline-challenged animals in which no eosinophils could be detected (Figure 3). Ovalbumin challenge of mCTLA4-IgG-treated mice did not induce a significant increase in eosinophil number compared with saline-challenged control animals (Figure 3). Furthermore, the number of eosinophils in BAL from mCTLA4-IgG-treated ovalbumin-challenged mice was significantly (P < 0.05) reduced compared with ovalbumin-challenged mice treated with control antibody. No significant differences were observed between total numbers of leukocytes, mononuclear cells, and neutrophils in BAL between the four different groups (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Total numbers of eosinophils (EO), neutrophils (NE), and mononuclear cells (MNC) in bronchoalveolar lavage of ovalbumin-sensitized animals treated with control antibody (IgG) and repeatedly challenged with saline (black bars) or ovalbumin (hatched bars) or treated with mCTLA4-IgG and repeatedly challenged with saline (white bars) or ovalbumin (dotted bars). Results are expressed as means ± SEM of six animals. *P < 0.05 compared with all other groups and determined by Fisher exact test.

Airway Responsiveness to Methacholine

In mice treated with control antibody and challenged with ovalbumin, the increase in air overflow pressure was significantly potentiated after intravenous administration of methacholine from 80 to 640 µg/kg as compared with saline-challenged control antibody-treated mice (results not shown). The potentiation amounted to 49 ± 15% at 80 µg/ kg (P < 0.05), 32 ± 9% at 160 µg/kg (P < 0.05), 15 ± 3% at 320 µg/kg (P < 0.01), and 17 ± 1% at 640 µg/kg (P < 0.01). In Figure 4 the response to 80 µg/kg methacholine and the ED₅₀ values of the methacholine dose-response curves are presented. In control antibody-treated animals, the ED₅₀ value of the methacholine dose-response curve tended to be decreased in ovalbumin versus saline-challenged animals and amounted to 90 ± 15 µg/kg and 140 ± 19 µg/kg, respectively (Figure 4). In mCTLA4-IgG-treated mice ovalbumin challenge did not potentiate the airway response to either of the methacholine doses (results not shown). The response to 80 µg/kg methacholine is shown in Figure 4. The ED₅₀ values of the methacholine dose- response curves were comparable in ovalbumin and saline-challenged animals treated with mCTLA4-IgG and amounted to 157 ± 40 µg/kg and 126 ± 16 µg/kg, respectively (Figure 4).

View larger version (6K): [in this window] [in a new window]   View larger version (6K): [in this window] [in a new window]   Figure 4.   Increase in air overflow pressure after intravenous injection of 80 µg/kg methacholine (top) and ED50 value of the methacholine dose response curve (bottom) of ovalbumin-sensitized animals treated with control antibody (A and B) or mCTLA4-IgG (C and D) and repeatedly challenged with saline (A and C) or ovalbumin (B and D). Results are expressed as means of six animals (horizontal line) with individual data points. *P < 0.05 compared with all other groups and determined by ANOVA followed by a post-hoc comparison using Bonferroni.

Effect of mCTLA4-IgG on Cytokine Synthesis

In the presence of control antibody, cultures of PBLN cells isolated from ovalbumin-sensitized mice stimulated in vitro with ovalbumin produced increased levels of IL-4 (188.9 ± 25.8 pg/ml) and IFN- (22.3 ± 7.3 pg/ml) compared with cultures in the absence of ovalbumin (5.7 ± 2.9 and 12.2 ± 9.5 pg/ml, respectively) (Figure 5). In the presence of mCTLA4-IgG, the ovalbumin-induced increase in IL-4 levels were almost completely reduced (Figure 5).

View larger version (9K): [in this window] [in a new window]   Figure 5.   Levels of IL-4 (closed bars) and IFN- (hatched bars) in supernatants of parabronchial lymph node cells isolated from ovalbumin-sensitized mice (n = 5) cultured for 5 days in the presence of control antibody (A and B) or mCTLA4-IgG (C and D) and stimulated with (B and D) or without (A and C) ovalbumin. Results are presented as mean ± SEM of quadruplicate cultures and are representative of two independent experiments

	Discussion

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T-lymphocyte infiltration and activation has been clearly demonstrated in patients with allergic asthma (1). These T-lymphocytes are predominantly of the Th2 subset and are thought to contribute to the characteristic symptoms of asthma such as IgE antibody production, allergic airway inflammation and hyperresponsiveness (1, 21). The T-lymphocyte may therefore be an important target cell for drug therapy in the treatment of allergic asthma. Antigen-specific T-cell activation requires the engagement of the T-cell receptor (TCR) with antigen as well as the engagement of appropriate costimulatory molecules. The most extensively characterized pathway of costimulation has been that involving the interaction of CD28 and CTLA4 on the T cell with B7-1/B7-2 on antigen-presenting cells (4, 5, 17, 22). In the present study, we used an immunoadhesion fusion protein of murine CTLA4 and human IgG₁. To verify the binding of mCTLA4-IgG to B7, splenocytes were stimulated with LPS and dBcAMP, which has been shown to be a powerful inducer of B7 expression on splenocytes (15). It is demonstrated that splenocytes stimulated in this way bind mCTLA4-IgG but not control IgG. Furthermore, Scatchard analysis of ¹²⁵I-mCTLA4-IgG binding to splenic B-cells revealed a kD of 1.8 nM and 1,100 binding sites per cell which is comparable to literature data (15). In addition, we have observed inhibition of murine mixed lymphocyte reactions by mCTLA4-IgG (results not shown). Here, we demonstrate that in a murine model of allergic asthma after initial sensitization, blockade of B7 molecules by mCTLA4-IgG treatment at the time of antigen challenge inhibits the development of the characteristic symptoms airway eosinophilia and hyperresponsiveness. In addition, upregulation of ovalbumin-specific IgE levels in serum is attenuated by mCTLA4-IgG treatment. Inhibition of airway hyperresponsiveness and eosinophilia by blockade of B7 molecules has also been observed in other murine models using S. mansoni (23) or ovalbumin (24, 25). Both cellular infiltrates and inflammatory changes within the bronchial mucosa were markedly decreased after CTLA4-IgG treatment (24). Furthermore, Coyle and colleagues (25) demonstrated upregulation of B7-2 but not B7-1 expression on the surface of lung B-cells after ovalbumin challenge of sensitized mice. Altogether these data suggest an important role for this costimulatory pathway in animal models of airway eosinophilia and hyperresponsiveness.

Murine CTLA4-IgG binds to B7-1 (CD80) and B7-2 (CD86) expressed on APC with a much higher affinity than CD28 and thereby effectively blocks costimulation of T-lymphocytes. CD28 is constitutively expressed on T cells and is an important accessory molecule for T-lymphocyte activation and cytokine production (22). CTLA4 is expressed on activated T-cells and is thought to be a negative regulator of T-cell activation (26). Previous work has shown that CD28 costimulation plays a critical role in the activation of naive CD4⁺ T-lymphocytes and in the prevention of anergy induction (22, 29). Likewise, mCTLA4-IgG treatment has been shown to inhibit primary T-cell-dependent antibody responses, prolong allo- and xenograft survival, and reduce or prevent autoimmune disease (22, 29). At present, the role of CD28/B7 in costimulation of Th1- or Th2-mediated disease processes is still controversial.

In our experiments, the inhibition of airway eosinophilia, hyperresponsiveness, and upregulation of IgE may be due to incomplete T-cell activation leading to anergy or decreased cytokine production. In preliminary studies, we have observed that CD4⁺ T-lymphocytes in the parabronchial lymph node of ovalbumin-sensitized mice produce IL-4 after stimulation with ovalbumin (Hofstra and associates, unpublished observations). A similar shift in type 2 cytokine production by parabronchial lymph node T-lymphocytes of ovalbumin-sensitized mice has been observed by Renz and colleagues (30). In the present study, we demonstrate that mCTLA4-IgG almost completely inhibits the in vitro IL-4 production by parabronchial lymph node cells isolated from ovalbumin-sensitized mice. No definitive conclusion can be made about the mechanism by which mCTLA4-IgG inhibits the parameters in our mouse model of allergic asthma. However, it is tempting to speculate that inhibition of T-lymphocyte activation and cytokine synthesis is involved. Interestingly, in vivo blockade of B7 molecules during antigen challenge has been shown to inhibit the antigen-induced proliferation of lymph node cells or thoracic lymphocytes and IL-4 and IL-5 production (23).

Although mCTLA4-IgG can induce clonal anergy in naive CD4⁺ T-lymphocytes in vitro, the precise mechanism of immunosuppression in vivo remains unclear (17, 22, 29). A link between in vitro anergy and in vivo tolerance has not been conclusively demonstrated. In agreement herewith, it has been shown that in vivo secondary antibody responses to T-lymphocyte-dependent antigens are not completely suppressed by mCTLA4-IgG at the time of sensitization (16, 17, 29).

Recently, we demonstrated that antibodies to IL-5 prevented the airway eosinophilia whereas antibodies to IFN- prevented the airway hyperresponsiveness in our murine model of allergic asthma (8). It was suggested that besides type 2 T-lymphocytes which produce IL-4 and IL-5, bystander T-lymphocytes are involved that produce IFN- (8). Our present observation that CTLA4-IgG inhibits airway hyperresponsiveness and eosinophilia suggests that both IL-5 and IFN- production are dependent on costimulatory molecules.

In the present study, although airway eosinophilia and hyperresponsiveness were absent after mCTLA4-IgG treatment, IgE upregulation was only partially inhibited. This suggests that memory IgE responses are less dependent on CD4⁺ T-lymphocyte-derived cytokines and cognate B-T contact (31). Furthermore, mast cells or basophils may be an additional source of cytokines for B-cell IgE production during ovalbumin challenge (32). Recently, Krinzman and associates (24) demonstrated total inhibition of serum IgE levels after CTLA4-IgG treatment which was even below the level in unchallenged ovalbumin-sensitized mice. In contrast to our results, these data suggest that CTLA4-IgG treatment rapidly decreases ongoing IgE synthesis. Previously, CTLA4-IgG has also been shown to inhibit H. polygyrus-induced elevations in serum IgE but not blood eosinophilia (33). However, these authors used a parasite infection and not a protein antigen, and investigated a primary and not a memory immune response.

In summary, we demonstrated that blockade of B7 molecules by mCTLA4-IgG treatment effectively inhibits airway eosinophilia and hyperresponsiveness and attenuates upregulation of IgE levels in a murine model of allergic asthma. Furthermore, ovalbumin-induced IL-4 production by parabronchial lymph node cells is almost completely inhibited by incubation with CTLA4-IgG in vitro. More studies are needed to explore the therapeutic potential of blocking T-lymphocyte costimulation by CTLA4-IgG treatment as a possible immunosuppressive treatment for allergic asthma.