INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: CD80; CD86; rhinitis; mouse; Th2

Allergic rhinitis is caused by immediate type hypersensitivity associated with expansion of the Th2 type immune response, including the production of allergen-specific immunoglobulin E (IgE) and nasal eosinophilia (1, 2). The activation, proliferation, and cytokine production of antigen-specific T cells require two distinct signals from antigen-presenting cells (APC) (3, 4). The first signal is provided by the interaction of antigen/ MHC with the T cell receptor (TcR). The second, called the costimulatory signal, is delivered by costimulatory molecules. Among these, B7, CD28, and CD152 are the most extensively characterized and appear to be the most crucial (5). Signals through CD28 costimulate interleukin-2 (IL-2) production and T cell proliferation (8). On the other hand, signals through TcR/CD3 complexes in the absence of costimulatory signals induce clonal anergy, which leads to peripheral tolerance (3, 9, 10). In addition, CD152 mediates a negative role in T cell activation and is involved in the induction of peripheral tolerance (11). CD80 and CD86 appear to differ in their ability to differentiate and maintain Th1 and Th2 cells (14). Thus, it is useful to understand the function and expression of these costimulatory molecules in the pathogenesis of allergic rhinitis.

We and others have reported that CD86 is selectively and antigen specifically upregulated in peripheral blood B cells from atopic patients (19). In addition, we found that house dust mite-specific T cells generated from the peripheral blood of patients with perennial rhinitis express both CD80 and CD86 and function through T-T cell interactions (22). More recently, we revealed that the expression of both CD80 and CD86 is increased in the nasal mucosa from perennial allergic patients compared with that from control subjects following the nasal provocation with house dust, suggesting a local amplification of allergen-specific immune responses in perennial rhinitis (23). However, the differential involvement of CD80 and CD86 in the induction and exacerbation of allergic rhinitis remains unclear, which may be, in part, due to the lack of an adequate experimental animal model.

We have recently developed a new murine model, which represents pathophysiologic characteristics of allergic rhinitis (24). In this model, repeated intranasal administration of Schistosoma mansoni egg antigen (SEA) in the absence of adjuvants induces SEA-specific IgE production, nasal eosinophilia, and IL-4 and IL-5 but not interferon-gamma (IFN-) production in response to recall stimulation with SEA by nasal mononuclear cells. In the present study, we performed the in vivo treatment with anti-CD80 and/or CD86 monoclonal antibody (mAb) either prior to or after the intranasal sensitization, which enabled us to investigate the phase-dependent contribution of these costimulatory molecules on the initiation and exacerbation of allergic rhinitis. Furthermore, we determined the expression of CD80 and CD86 molecules in response to SEA in nasal mononuclear cells from naive (unsensitized) and sensitized mice, and investigated the correlation between expression and function of these molecules.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Young adult (6-10 wk old) BALB/c female mice were purchased from Charles River Japan (Yokohama, Japan). The mice were maintained at Okayama University Medical School according to the guidelines set forth by the Okayama University Medical Area Research Committee.

Monoclonal Antibodies

Anti-CD80 mAb (clone RM80, rat IgG_2a) and anti-CD86 mAb (clone PO3, rat IgG_2b) were generated as described previously (25). Control rat IgG was purchased from Sigma (St. Louis, MO).

Sensitization of Mice and Ab Treatment

Mice were intranasally sensitized with SEA in the absence of adjuvants as described previously with a slight modification (24, 26). Mice (n = 6 per group) were sensitized with 20 µl of 5 µg SEA diluted in Dulbecco's phosphate-buffered saline (PBS) (Gibco, NY) intranasally using a microsyringe (Hamilton Co., NV) on Days 0 and 14. Three weeks after the final sensitization, mice were challenged intranasally with 1 µg SEA for 7 consecutive d.

Mice were treated with mAbs against CD80 and/or CD86 at the time of sensitization or challenge. For treatment at the induction phase, mice received 200 µg of anti-CD80 alone, anti-CD86 alone, anti-CD80 plus anti-CD86, or control rat IgG 30 min prior to every sensitization (Figure 1A). For treatment at the effector phase, mice received 200 µg of anti-CD80, anti-CD86, anti-CD80 plus anti-CD86, or control Ab every other day during the nasal challenge (Figure 1B).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Treatment schedule. (A) Effect of anti-CD80 and/or anti-CD86 on the induction phase of allergic rhinitis. BALB/c mice received intravenously 200 µg anti-CD80 alone, anti-CD86 alone, anti-CD80 plus anti-CD86, or control rat IgG 30 min prior to priming and boosting sensitization with 5 µg SEA in the absence of adjuvants. Three weeks later, all of the mice were challenged with 1 µg SEA without antibody treatment for 7 consecutive d. Peripheral blood was collected from the tail 7 d after the boosting sensitization and 12 h after the final challenge. In addition, mice were killed 12 h after the final challenge, and heads were removed for histologic and cytokine study. (B) Effect of anti-CD80 and/or anti-CD86 on the effector phase of allergic rhinitis. BALB/c mice were primed and boosted with 5 µg SEA at 2 wk intervals. Sensitization to SEA was confirmed by the production of specific IgE 1 wk following the boosting sensitization. Three weeks after the boosting sensitization, groups of six mice received 200 µg anti-CD80, anti-CD86, anti-CD80 plus anti-CD86, or control rat IgG every other day during the nasal challenge with SEA for 7 consecutive d. Twelve hours after the final challenge, mice were bled and killed.

Ab Determination

Blood was collected from the tail 7 d after the final sensitization and 12 h after the final challenge. The levels of SEA-specific Th2-associated Ab IgG₁ and IgE were measured by ELISA as previously described (24).

Histologic Examination

Mice were killed 12 h after the final nasal challenge. The heads were removed, fixed, and decalcified. Then coronal nasal sections were stained with Luna staining, and the number of eosinophils in nasal mucosa was determined microscopically in a high-power field (10 × 40).

In Vitro Culture of Nasal Mononuclear Cells and Cytokine Determination

Twelve hours following the final nasal challenge, mice were killed, and nasal mononuclear cells were isolated and cultured in the presence or absence of SEA as previously described (26). Then IL-4, IL-5, and IFN- production by SEA-stimulated and unstimulated cells was measured (26). The detection limit for IL-4, IL-5, and IFN- in this system was 0.1 U/ml, 40 pg/ml, and 20 pg/ml, respectively.

Flow Cytometric Analysis

Nasal mononuclear cells from naive or SEA-primed mice were incubated with or without 5 µg/ml SEA at 37° C for 72 h in 5% CO₂. Then the cells were harvested, blocked, and stained with fluorescein isothiocyanate (FITC)-labeled anti-CD80 (1G10, rat IgG_2a) or anti-CD86 (GL1, rat IgG_2a) and PE-labeled anti-B220 (RA3-6B2, rat IgG_2a) as well as isotype-matched control Abs (19). The cells were washed, fixed, and analyzed with FACScan using CellQuest software (Beckton Dickinson, Mountain View, CA). All the fluorescent-conjugated Abs were purchased from Pharmingen (San Diego, CA).

Statistical Analysis

Student's unpaired t test was used to determine the significance of the values obtained. A value of p < 0.05 was considered significant. Values were given as mean ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of CD80 and/or CD86 Blockade at the Induction Phase of Allergic Rhinitis

To investigate the effects of treatment with anti-CD80 and/or CD86 mAb in the induction phase, mice were treated with these mAbs at every sensitization and then SEA-specific IgE and IgG₁ production in sera at 1 wk after the final sensitization and 12 h after the final challenge was measured (Figure 1A). Antigen (Ag)-specific IgE production after sensitization in the mice treated with anti-CD80 mAb alone was significantly inhibited (Figure 2A). IgE production after the challenge was efficiently enhanced, and the treatment with anti-CD80 mAb significantly inhibited this elevation of IgE. However, treatment with anti-CD86 mAb had no effect at both time points. The treatment with a combination of both anti-CD80 and CD86 mAbs did not significantly affect IgE production more than the levels observed with anti-CD80 mAb alone. In Ag-specific IgG₁ production, treatment with either anti-CD80 or anti-CD86 mAb had little effect at both Weeks 3 and 6. However, combined treatment with anti-CD80 plus anti-CD86 mAb significantly inhibited IgG₁ production (Figure 2B).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Effect of anti-CD80 and/or anti-CD86 on the induction phase of allergic rhinitis. BALB/c mice (n = 6 per group) were intranasally sensitized with SEA in the presence of anti-CD80 and/or anti-CD86 or control rat IgG, and sera were taken 7 d after boosting sensitization (upper half ). Three weeks after the boosting sensitization, mice were challenged with SEA, and sera were taken again at the end of the nasal challenge (lower half ). Titers of SEA-specific IgE (A) and IgG1 (B) were determined by ELISA. Results show (A) the mean OD at 450 nm ± SEM of six serum samples from each group at a 1:4 dilution, and (B) the mean OD at 450 nm ± SEM of six serum samples from each group at a 1:100 dilution. Data are representative of three separate experiments. *p < 0.05; p < 0.01.

Similar results were observed in local eosinophilia by histologic examination. Treatment with anti-CD80 mAb significantly inhibited eosinophil infiltration into nasal mucosa after the challenge, whereas anti-CD86 treatment had no effect (Figure 3). The number of eosinophils infiltrating nasal septum per field (10 × 40) was 91.5 ± 12.3, 40.0 ± 10.4, 106.0 ± 11.1, and 25.5 ± 9.5 in mice treated with control IgG, anti-CD80 alone, anti-CD86 alone, and anti-CD80 plus anti-CD86, respectively (mean ± SEM, n = 6). An additional effect was not seen in the treatment with both mAbs compared with the treatment with anti-CD80 alone.

View larger version (98K): [in this window] [in a new window]   Figure 3.   Nasal eosinophilia after nasal challenge in mice treated with anti-CD80 and/ or anti-CD86 in the induction phase. BALB/c mice were treated with rat IgG (A), anti-CD80 alone (B), anti-CD86 alone (C ), or anti-CD80 and anti-CD86 (D) during priming and boosting sensitization with SEA. Following the nasal challenge with SEA without antibody treatment, mice were killed. Nasal sections were fixed and decalcified, and Luna staining was performed to detect eosinophils in the nasal mucosa.

Cytokine production from nasal mononuclear cells after the challenge was examined. Nasal lymphocytes were isolated by enzyme extraction and were stimulated with SEA for 72 h (26), and cytokine production in the culture supernatants was assessed. SEA-stimulated nasal lymphocytes from control mice produced high IL-4 and IL-5, but not IFN- (Figure 4). The treatment with anti-CD80 mAb significantly decreased production of IL-4 and IL-5 from nasal lymphocytes, whereas the treatment of anti-CD86 mAb failed to affect these productions. Interestingly, the combined treatment with anti-CD86 mAb to the anti-CD80 treatment significantly augmented the inhibitory effect on IL-4 production. The anti-CD86 mAb treatment did not further inhibit IL-5 production.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Production of IL-4 (A), IL-5 (B), and IFN- (C ) by nasal lymphocytes from BALB/c mice treated with rat IgG, anti-CD80 alone, anti-CD86 alone, or anti-CD80 and anti-CD86 during priming and boosting sensitization with SEA. Nasal lymphocytes were isolated and cultured in vitro for 72 h with SEA. Cytokines were measured by ELISA. Results show mean ± SEM of three separate experiments. *p < 0.05; p < 0.01.

Effect of CD80 and/or CD86 Blockade on the Effector Phase of Allergic Rhinitis

To investigate the involvement of CD80 and CD86 in the effector phase of allergic rhinitis, similar treatments were given at the period of SEA challenge (Figure 1B). After the nasal challenge, specific IgE and IgG₁ production was prominently elevated in the control mice. Neither treatment with anti-CD80 nor anti-CD86 mAb affected both IgE and IgG₁ production. The treatment with a combination of both mAbs substantially inhibited both IgE and IgG₁ (Figure 5).

View larger version (11K): [in this window] [in a new window]   Figure 5.   Effect of anti-CD80 and/or anti-CD86 on the effector phase of allergic rhinitis. BALB/c mice (n = 6 per group) were intranasally sensitized with SEA. Sera were taken 7 d after the boosting sensitization and confirmed the identical production of SEA-specific IgE and IgG1 among the groups. Three weeks after the boosting sensitization, mice were challenged with SEA for 7 consecutive d. During the challenge, mice were treated with intravenous application of anti-CD80 alone, anti-CD86 alone, anti-CD80 plus anti-CD86, or rat IgG every other day. Sera were taken again at the end of the nasal challenge. Titers of SEA-specific IgE (A) and IgG1 (B) were determined by ELISA. Results show (A) the mean OD at 450 nm ± SEM of six serum samples from each group at a 1:4 dilution, and (B) the mean OD at 450 nm ± SEM of six serum samples from each group at a 1:100 dilution. Data are representative of two separate experiments. *p < 0.05.

The inhibitory effect by the treatment with both mAbs at the effector phase was also confirmed by eosinophil infiltration into the nasal mucosa (Figure 6). Neither treatment with anti-CD80 nor anti-CD86 mAb showed an inhibitory effect on nasal eosinophilia. The number of eosinophils infiltrating the nasal septum per field (10 × 40) was 149.9 ± 21.4, 141.5 ± 14.4, 109.5 ± 25.0, and 57.8 ± 10.3 (mean ± SEM, n = 6) in mice treated with control IgG, anti-CD80, anti-CD86, and both mAbs, respectively.

View larger version (87K): [in this window] [in a new window]   Figure 6.   Nasal eosinophilia after the nasal challenge in mice treated with anti-CD80 and/or anti-CD86 in the effector phase. BALB/c mice were sensitized with SEA. Three weeks after the boosting sensitization, mice were treated with rat IgG (A), anti-CD80 alone (B), anti-CD86 alone (C ), or anti-CD80 and anti-CD86 (D) during the nasal challenge with SEA. Twelve hours after the final challenge, mice were sacrificed. Nasal sections were fixed and decalcified, and Luna staining was performed to detect eosinophils in the nasal mucosa.

Expression of CD80 and CD86 on Nasal Mononuclear Cells from Unsensitized and SEA-sensitized Mice

To see the expression of CD80 and CD86 on nasal mononuclear cells, the cells were isolated from unsensitized or SEA-sensitized mice, stimulated with or without 5 µg/ml SEA for 72 h, and analyzed by flow cytometry. On B220 negative nasal lymphocytes, a preferential CD80 expression was seen in unsensitized mice and this was further enhanced by SEA stimulation in vitro (Figure 7A and 7B), but not by in vivo sensitization (Figure 7E and 7F). The majority of B220 negative cells bearing CD80 expressed Mac1 (data not shown). In contrast, CD86 expression on B220 negative cells was negligible in both unsensitized and sensitized mice and the in vitro stimulation did not induce their expression. On B220 positive cells in unsensitized mice, both CD80 and CD86 were minimally expressed (Figure 7A and 7C) and the Ag stimulation in vitro failed to affect their expression on B cells (Figure 7B and 7D). In sensitized mice, however, both CD80 and CD86 expression on B220⁺ nasal B cells was slightly enhanced and significantly augmented by in vitro SEA stimulation (Figure 7E-7H).

View larger version (34K): [in this window] [in a new window]   Figure 7.   Expression of CD80 and CD86 on nasal mononuclear cells from unsensitized or SEA-sensitized mice. Nasal lymphocytes from unsensitized (A-D) or sensitized (E-H ) mice were incubated with (B, D, F, H ) or without (A, C, E, G) SEA for 72 h. Cells were stained with PE-conjugated anti-CD80 or CD86 mAb and FITC-conjugated anti-B220 mAb or with appropriated fluorochrome-conjugated control Ig and analyzed by flow cytometry. Values given in the upper and lower right quadrant indicate the mean percentage ± SEM of B220 positive and negative cells expressing CD80 (A, B, E, F ) and CD86 (C, D, G, H ), respectively, from six individual mice. Data are representative of two separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study revealed the differential role of CD80 and CD86 costimulatory molecules in the induction and the effector phases of allergic rhinitis in mice. In the induction phase, CD80 appears to be important for the production of specific IgE, nasal eosinophilia, and Th2 cytokine production by nasal cells. On the other hand, costimulation via either CD80 or CD86 is sufficient to exacerbate rhinitis in SEA-sensitized mice. Ligation of CD28 or CD152 with CD80 or CD86 is known to be important for the pathogenesis of allergic diseases, including allergic rhinitis in both humans and experimental animals (19, 27). Sato and coworkers demonstrated that topical administration of CTLA4-Ig during nasal challenge with ovalbumin (OVA) inhibited OVA-induced nasal symptoms, histamine hyperresponsiveness, nasal lavage eosinophilia, and serum-specific IgE against OVA in OVA-presensitized mice (27). To our knowledge, this is the first study investigating the differential roles of CD80 and CD86 on the pathogenesis of allergic rhinitis in unsensitized and sensitized mice.

Controversy surrounds the differential role of CD80 and CD86 in mediating pathogenesis of allergic asthma in an experimental murine model (16, 28). Harris and coworkers showed that blockade of the CD80 signal led to the suppression of the antigen-induced eosinophil accumulation in bronchoalveolar lavage (BAL), whereas this treatment failed to suppress specific IgE production or blood eosinophilia (28). Tsuyuki and coworkers demonstrated that anti-CD80 treatment resulted in only a 20% reduction in eosinophil infiltration, whereas anti-CD86 treatment suppressed eosinophilia by more than 80% (16). Keane-Myers and coworkers reported similar results in presensitized mice (29). Haczku and coworkers showed that anti-CD86 treatment throughout the sensitization period and airway challenge led to the abolishment of both IgE-dependent and -independent allergic airway responsiveness, production of IL-4 and IL-5 in BAL, and serum IgE and IgG₁ production (30). Interestingly, they also reported that such an inhibitory effect of anti-CD86 was not seen when the antibody was administered just prior to challenge in OVA-presensitized mice, which is controversial because of the report by Keane-Myers and coworkers (16). On the other hand, Mathur and coworkers reported that combined anti-CD80/ anti-CD86 treatment throughout the antigen challenge period fully blocked the development of allergic airways, whereas a partial reduction was observed in mice treated with either anti-CD80 or anti-CD86 alone (31). These results suggested that the differences such as the phase (induction or effector), strain of responding mice, the nature of antigen, and/or the route of antigen administration could contribute to the differential role of CD80 versus CD86 in the pathogenesis of allergic inflammation. In fact, we had observed that in murine models of contact dermatitis, CD86 plays a critical role in the initiation of primary immune responses in the skin, whereas CD80 and CD86 are not essential in the effector phase of the disease (15).

We have used the murine model of allergic rhinitis that we recently developed (24). In this model, Ag was given intranasally without the use of adjuvant, which could mimic the natural exposure of the disease. In addition, the phase-specific effect of the neutralizing Ag could be analyzed in this model (Figure 1). First, we demonstrated that anti-CD80 treatment reduced specific IgE production, and this treatment additionally suppressed nasal eosinophilia and IL-4 and IL-5 production by nasal cells subsequently after the allergen challenge (Figures 2-4). However, anti-CD86 treatment had no effect on these results. In addition, the combination of both anti-CD80 and anti-CD86 was no more effective than anti-CD80 alone, except in the production of specific IgG₁. Furthermore, flow cytometric analysis also revealed that CD80 but not CD86 was expressed in response to SEA in unsensensitized B220 negative nasal mononuclear cells (Figure 7A-7D). The majority of B220 negative cells bearing CD80 expressed Mac1, suggesting that these cells are likely to be macrophages or dendritic cells. Taken together, these results suggest that CD80 was selectively expressed in nasal cells after SEA sensitization, and this molecule was primarily required for the initiation of allergic rhinitis in this model. Kuchroo and coworkers reported that anti-CD80 but not anti-CD86 treatments prevent the induction of experimental allergic encephalomyelitis (EAE) in SJL mice, although the Th2 response was induced in this treatment (32). Our results were consistent in part with results seen in models of asthma in terms of the roles of CD80 in local eosinophilia; however, the role of CD86 appeared to be different (16, 29, 30). Thus, the primary costimulatory molecule for the induction of local eosinophilia may be different between the upper and lower airways.

On the other hand, in mice presensitized intranasally with SEA, treatment with either anti-CD80 alone or anti-CD86 alone during the allergen challenge did not inhibit the orchestration of allergic inflammation (Figures 5 and 6). These results suggest that costimulation via either CD80 or CD86 was sufficient to exacerbate allergic inflammation in this model, indicating the overlapping function for CD80 and CD86 in the effector phase. Results from flow cytometric analysis indicating that B220 positive cells expressed both CD80 and CD86 following in vitro SEA stimulation in presensitized mice (Figure 8) may support this notion. The combined treatment of both anti-CD80 and anti-CD86 could inhibit the increase of Ab production and nasal eosinophilia. However, the inhibitory effect on allergic inflammation by the combined treatment during the effector phase seems to be less than the treatment during the induction phase (Figures 2, 3, 5, and 6), which is consistent with several reports (15, 31, 33). Schweitzer and coworkers reported that using antigen-presenting cells lacking expression of both CD80 and CD86, IL-4 production by previously activated Th2 cells is B7 independent in DO11.10 TCR transgenic mice (34). These results suggest that CD80/CD86 signals were required less in the Th2-type effector phase, and other costimulatory molecules, such as the inducible costimulator (ICOS) and its ligand B7RP-1, may be involved in the effector phase (35).

Lack of costimulation is believed to induce unresponsiveness or anergy (3, 9, 10). Of note, even in the combination treatment of both Abs during the nasal challenge with SEA, the levels of serum-specific Ab was not decreased after the nasal challenge compared with levels before the nasal challenge (Figure 5). These results suggested that other costimulatory molecules may be necessary in order to downregulate the pathogenesis of ongoing allergic inflammation. In fact, it was recently found that a combination of CD40-CD154 and CD80/ CD86-CD28 blockade allogeneic unresponsiveness led to the lack of proliferation, cytotoxic activity, and IL-2, IL-5, and IL-13 production (36).

In conclusion, this study revealed the phase-dependent role of CD80 and CD86 costimulatory molecules on the initiation and exacerbation of allergic rhinitis in mice. In particular, we suggest an important role for CD80 in the initiation phase and for both CD80 and CD86 in the exacerbation phase in the disease, which correlates with the expression of the molecules in the nasal cells following antigen stimulation in vitro. These observations may provide a basis for future therapeutic approaches in the management of allergic rhinitis by inhibiting CD80/ CD86-CD28 interactions in the nose of individuals with rhinitis.