Introduction

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Asthma is a chronic respiratory disease with characteristic pathophysiologic features including eosinophilic and lymphocytic peribronchial inflammation, bronchial smooth-muscle hypertrophy and hyperreactivity, and typical clinical features of episodic dyspnea with an obstructive pattern on pulmonary function testing (1, 2). Numerous genetic and environmental factors are thought to play a role in the pathogenesis of the disease (3, 4). A number of cell types and molecular mediators, including lymphocytes, mast cells, eosinophils (EOSs), immunoglobulin (Ig) E, and cytokines, are thought to contribute to the pathophysiology of asthma.

Abundant evidence exists that T cells have an important role in asthma pathogenesis. In murine models of asthma, passive transfer of T cells confers sensitivity to allergen and eosinophilic inflammation (5, 6). Allergen-stimulated T cell- deficient mice fail to develop airway hyperresponsiveness (AHR) (7). In several mouse asthma models, pharmacologic ablation of T cells, neutralization of T-cell growth factors, or interference with T-cell stimulatory pathways has abrogated asthma-like pathophysiology, including eotaxin expression and subsequent EOS influx (7, 10).

CD40/CD40 ligand (CD40L) interaction is important in a number of the cellular interactions that give rise to inflammatory responses (13). After allergen encounter, responding T cells express CD40L and can then engage CD40 on B cells, monocytes, dendritic cells, and epithelial cells. In the case of B cells, the CD40/CD40L interaction is critical for activation and for the induction of isotype switching (17). Binding of CD40 on monocytes results in the production of proinflammatory cytokines and nitric oxide (18, 19). Lymphocytes from CD40-deficient (CD40 /) mice are inefficient at inducing interleukin (IL)-12 production by macrophages, and consequently these animals are deficient in the generation of T-helper 1 T-cell responses critical in the induction of tissue inflammation (20, 21). CD40/ CD40L signaling may be bidirectional. It has been reported that stimulation of CD40L on the surface of T cells by CD40 results in their activation (22).

To examine the role of the CD40/CD40L interaction for the development of allergic pulmonary inflammation and bronchial hyperresponsiveness (BHR), we subjected CD40 / mice or mice deficient in CD40L (CD40L /) to repeated challenge with Aspergillus fumigatus (Af ). IgE levels, bronchoalveolar lavage (BAL) fluid (BALF) cytology, pulmonary histology, and bronchial responsiveness were evaluated in the treated mice. As expected, CD40 / and CD40L / animals were unable to mount IgE responses. However, both CD40 / and CD40L / mice sensitized with Af developed striking BALF eosinophilia and the extent of pulmonary inflammation was the same in mutant and wild-type (WT) mice. Mice deficient in CD40 also developed BHR; in contrast, CD40L / mice did not develop a significant increase in bronchial responsiveness after Af stimulation. The CD40/CD40L interaction thus is required for IgE responses but unnecessary for EOS influx and inflammation. CD40L, but not CD40, appears to be required for the induction of BHR.

	Materials and Methods

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Reagents and Mice

A mixture of culture filtrate and mycelial extracts of Af (kind gift of Greer Laboratories, Lenoir, NC) was diluted to 2 mg/ml. CD40-deficient mice were derived as previously described and maintained on a hybrid 129/C57BL/6 background (13). CD40L-deficient mice and B6/129 F2 WT control mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in a viral antigen-free facility.

Sensitization of Mice

Mice were sensitized as previously described (23). Briefly, they were lightly anesthetized by methoxyfluorane (Metofane; Mallinckrodt Veterinary, Inc., Mundelein, IL) inhalation, and 50 µl of Aspergillus antigen (Af  ) or 50 µl of saline (NS) was applied to the left naris with the mouse in the supine position. After instillation of the Af or NS, mice were kept prone until alert. Mice were immunized three times a week for 3 wk and studied 12 h after the last dose.

Quantification of Serum IgE Levels

Serum IgE levels were determined by enzyme-linked immunosorbent assay (ELISA) as described in the PharMingen protocol, using clone R35-188 antibodies for coating and clone R35-72 antibodies for detection.

Pulmonary Function Measurements

Pulmonary conductance (GL) was monitored in anesthetized, ventilated mice in a whole-body plethysmograph as previously described. The output signals from eight to 10 consecutive breaths were analyzed using a computerized cross-correlation method. For measurement of methacholine (MCh) responsiveness, a series of increasing doses of MCh ranging from 3.3 to 3,300 µg/kg were each infused in a volume of 1 µl/G body weight via a jugular venous catheter over 4 to 5 s. The maximally reduced GL values measured after each Mch dose were expressed as percentages of the value measured immediately before administration of that dose.

Collection of Specimens

BAL was performed with a single aliquot of 0.8 ml of phosphate-buffered saline containing 10% fetal calf serum and 1 mM ethylenediaminetetraacetic acid, which was infused into the lungs via the tracheotomy tube and then retrieved. BALF leukocyte counts were determined using a hemocytometer. The leukocyte differential was determined by cytocentrifugation of BAL specimens followed by staining with Diff-Quik (Baxter, Miami, FL). The absolute EOS count was derived as the product of the leukocyte count and the EOS fraction on differential.

Histologic Analysis

Lungs were fixed in 10% formalin under mild vacuum. Sections, 5 µm thick, were prepared and stained with hematoxylin and eosin (H&E). A previously described grading scheme was used to assess the intensity of the inflammatory infiltrate in NS- and Af-treated animals (23). Lungs that showed no focal inflammation or peribronchial or perivascular inflammatory infiltrates were scored as grade 0. Those that showed one or two centrally located microscopic foci of inflammatory infiltrate were graded as 1. In grade 2 lungs, a dense inflammatory infiltrate was seen in a perivascular and peribronchial distribution originating in the center of the lung and extending along the vessels and bronchi into the middle third of the lung parenchyma. In grade 3 lesions, the perivascular and peribronchial infiltrates extended to the periphery of the lung approaching the visceral pleura. Grading was performed by a pathologist (M.R.) unaware of the genotype or sensitization status of the mice.

	Results

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Allergen-Driven IgE Production in WT, CD40 /, and CD40L / Mice

We and others have previously shown that intranasal application of protein extracts of the mold allergen Af elicits strong IgE responses in mice (23). Because CD40/ CD40L interaction is believed to be critical in the induction of Ig isotype switching in B cells, we sought to determine whether the IgE response would be attenuated in mutant mice. IgE levels were measured by ELISA in sera from Af- and NS-treated animals. Af treatment induced a rise from 436 to 2278 ng/ml in one group of WT B6/129 mice and from 465 to 2130 ng/ml in the second set of WT mice (Figure 1, WT/Af ). No IgE was detected in the sera of either NS- or Af-treated CD40 / or CD40L / animals (Figure 1, CD40 / Af and CD40L / Af ). Thus, CD40 and CD40L are each required for the normal IgE response to inhaled allergen.

View larger version (12K): [in this window] [in a new window]   Figure 1.   IgE levels in Af-treated mice. Serum IgE levels (ng/ml) were determined by ELISA after 3 wk of repeated Af inhalation (see MATERIALS AND METHODS). Data represent mean values ± standard error of the mean (SEM) (10 to 18 mice per group). Af-induced IgE elevations are markedly impaired in both CD40 / and CD40L / mice. (Assay limit of detection is 120 ng/ml.)

BALF Eosinophilia in Allergen-Treated WT and Mutant Mice

Because CD40/CD40L interactions have been shown to be important in the induction of a wide variety of inflammatory responses, we wished to determine their role in allergen-driven EOS-predominant inflammation of the airways. EOS influx was assessed both in BALF and in tissue sections. Af-treated WT mice had airway eosinophilia nearly three orders of magnitude higher than did NS-treated WT animals (mean 8.52 × 10⁵ versus 8.40 × 10² EOSs/ml, respectively) (Figure 2A). Similarly, CD40 / mice treated with Af had BALF eosinophilia almost three orders of magnitude greater than did CD40 / animals treated with NS (mean 1.44 × 10⁶ versus 3.88 × 10³ EOSs/ ml, respectively). Af-treated CD40L / mice also displayed a similar degree of BALF eosinophilia (Figure 2B), with a mean of 1.01 × 10⁶ in the Af group and 1.48 × 10³ EOSs/ml in NS-exposed mice.

View larger version (12K): [in this window] [in a new window]   Figure 2.   BALF EOS counts (eoss/ml BALF) in (A) CD40-deficient mice and (B) CD40L-deficient mice after treatment with Af ( filled circles) or NS (open circles) compared with wild-type (WT) controls. Data are shown for 10 to 18 mice per group. Each point represents one animal (some values overlap and all values < 102 are plotted at the x-axis). BALF eosinophilia is dramatically increased in the Af-treated WT animals and in mice with mutations of CD40 and CD40L.

Allergic Inflammation of the Lungs in WT, CD40 / and CD40L / Mice

The extent and anatomic distribution of allergic inflammation was further examined by histologic analysis (Figure 3). Intense inflammation was evident in the lungs not only of Af-treated WT mice but also in CD40 / and CD40L / mice subjected to allergen inhalation. The nature of the infiltrate was similar in all groups and involved an admixture of EOSs and mononuclear cells surrounding the bronchi with some epithelial damage. Some perivascular inflammation and infiltration of the air spaces were evident as well.

View larger version (155K): [in this window] [in a new window]   Figure 3.   Lung histology in NS- and Af-treated WT and mutant mice. An equally intense infiltrate of EOS and mononuclear cells is present in allergen-treated WT mice and mice deficient in CD40 and CD40L. Bronchioles and vessels are surrounded by EOSs and epithelial damage is evident in all mice. These panels depict fields representative of the histology of sections stained with H&E obtained from five mice per group.

To quantitate the degree of inflammation present in the lungs of Af-treated mice of the three genotypes, we applied a previously described scoring system (see MATERIALS AND METHODS) based upon both the intensity of the infiltrate and its anatomic extent toward the visceral pleura (23). There were no significant differences in scores obtained for Af-treated WT (mean 2.46 and 2.55 in separate experiments), CD40 / (mean 1.93), or CD40L / (mean 1.90) mice (Figure 4). Together, the histologic examinations and the BALF eosinophil counts provide strong evidence that allergen-driven recruitment of EOS to the airways can occur by CD40/CD40L-independent mechanisms.

View larger version (28K): [in this window] [in a new window]   Figure 4.   Magnitude of pulmonary eosinophilia mice after treatment with NS or Af. CD40 / (open bars), CD40L / ( filled bars), and WT mice were subjected to NS or Af inhalation. H&E-stained sections prepared as in Figure 3 were scored for intensity and anatomic extent of eosinophilic infiltration as described in detail in MATERIALS AND METHODS. The mean score ± SEM is shown for each group (n = 10-18).

Pulmonary Function Studies

The induction of bronchial inflammation and BHR by allergen appears to be linked both in human asthmatics and in mouse models of the disease. We first determined whether absence of CD40 or CD40L affects the degree of airway responsiveness to intravenous MCh in naive mice by comparing the responses of sham-sensitized WT mice with those of sham-sensitized CD40 / or CD40L / mice. We found that the lack of CD40 was associated with a reduction in responsiveness to MCh, assessed in terms of GL (P < 0.001, analysis of variance [ANOVA], Figure 5). Mice lacking CD40L exhibited smaller responses to MCh than did sham-sensitized WT mice only for the highest three doses (maximal responses, P < 0.02, t test, Figure 6). It therefore appears that the presence of CD40 and, to a lesser extent, CD40L is associated with heightened airway responsiveness even in the absence of experimental antigenic stimulation.

View larger version (16K): [in this window] [in a new window]   Figure 5.   Pulmonary conductance in WT and CD40-deficient (KO) mice after treatment with Af or NS. Data represent mean values for 14 to 18 mice per group from three separate experiments; error bars indicate SEM. Responses are expressed as percentages of baseline values measured just before each MCh dose. Treatment groups are WT NS (squares, solid line), WT Af (squares, dashed line), KO NS (circles, solid line), and KO Af (circles, dashed line). Responses were significantly greater (P < 0.001 ANOVA) in sham-exposed WT mice than in sham-exposed CD40 / mice. Responses were also significantly greater in both types of Af-treated mice than in the respective NS-treated mice (P < 0.001, ANOVA).

View larger version (17K): [in this window] [in a new window]   Figure 6.   GL in WT and CD40L-deficient (KO) mice after treatment with Af or NS. Data represent mean values for 10 to 12 mice per group from two separate experiments; error bars indicate SEM. Responses are expressed as percentages of baseline values measured just before each MCh dose. Treatment groups are WT NS ( filled squares, solid line), WT Af ( filled squares, dashed line), KO NS ( filled triangles, solid line), and KO Af (open triangles, dashed line). Responses were not significantly greater in sham-exposed WT mice than in sham-exposed CD40L / mice (ANOVA). Responses were significantly greater in both types of Af-treated mice than in the respective NS-treated mice (P < 0.001, ANOVA).

We also determined whether the absence of CD40 or CD40L would prevent the development of AHR after Af antigenic stimulation. Significant BHR developed in Af-exposed WT mice and a similar degree of BHR was detected in mice lacking CD40 (Figure 5, P < 0.001, ANOVA). In contrast, Af exposure in mice lacking CD40L did not induce detectable BHR (Figure 6). Thus, CD40L, but not CD40, was required for the development of BHR, even though both proteins were required for the development of an IgE response and neither protein was required for the development of antigen-induced bronchopulmonary eosinophilia.

	Discussion

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Studies in humans have identified pulmonary eosinophilia and cholinergic BHR as hallmarks of asthma. We used an established murine model of asthma in which pulmonary eosinophilia and BHR develop in response to intranasal sensitization with Af to test the hypothesis that CD40 and CD40L are essential for these responses to allergen. We found that although IgE was undetectable in serum of CD40 / and CD40L / mice, Af-sensitized CD40 / and CD40L / mice developed high levels of BALF eosinophilia and dense eosinophilic infiltrates similar to those observed in WT mice. Paradoxically, development of allergen-induced BHR was prevented by the absence of CD40L but not by the absence of CD40.

The failure of CD40 / and CD40L / mice to produce an IgE response to antigen is consistent with the previous observations of others (13) and reflects the critical roles of these molecules in Ig isotype switching to IgE. B cells must receive two signals in order to make IgE. The first is provided by the cytokines IL-4 and IL-13. The second signal is generated when CD40L, which is induced on the surface of T cells after antigen exposure, binds to CD40 on the B-cell surface. Thus, the absence of either member of this receptor-ligand pair interrupts an essential signaling event for IgE production and neither CD40 / nor CD40L / mice generate IgE in response to inhaled allergen.

In addition to stimulating Ig isotype switching to IgE, CD40 and CD40L are involved in the pathogenesis of inflammation. Several inflammatory disease processes, including murine lupus, collagen-induced arthritis, and experimental allergic encephalitis, are interrupted by blockade of the CD40/CD40L pathway, implicating this receptor- ligand pair in the pathogenesis of inflammation (26). CD40/CD40L appears to be involved in pulmonary inflammatory responses as well, including responses to hypoxic stress and radiation (29, 30). The intranasal administration of soluble CD40L leads to a neutrophilic and mononuclear pulmonary infiltrate in WT but not CD40-deficient mice (31, 32).

Two groups have previously reported pulmonary responses to allergen in CD40 / and CD40L / mice, with disparate results. Our findings, using CD40 / mice, are in agreement with those of Hogan and colleagues who found that ovalbumin (OVA)-stimulated CD40 / mice had levels of BALF eosinophilia and BHR similar to those of WT mice (33). In contrast, Lei and colleagues found that OVA-stimulated CD40L / mice developed less BALF eosinophilia than did WT mice (34); pulmonary physiology was not assayed. These authors postulated that the difference between their findings in CD40L / mice and those previously reported by Hogan for CD40 / mice might be related to dosages of antigen. Our results suggest that the levels of BALF eosinophilia are similar in the two types of knockout (KO) mice treated with the same sensitization protocol and that the levels are not significantly less than those observed in WT mice for either KO group.

We believe that the discrepancy between our results and those of Lei and associates (34) regarding the role of CD40L in the generation of airway eosinophilia after allergen exposure may have arisen either from differences in the allergen used or from distinct mechanisms of antigen sensitization. The protocol of Lei and coworkers (34) involved intraperitoneal priming with OVA in the presence of the adjuvant, alum. It is possible that allergen encounter at serosal surfaces and/or in the presence of alum drives an immune response via cellular interactions that are CD40L-dependent. In contrast, Af, the antigen used in our study, elicits a vigorous hypersensitivity response after airway exposure alone. It is a significantly more potent allergen than OVA, driving 10-fold greater increases in BALF eosinophilia (unpublished observations). Thus, the generation of equivalent pulmonary eosinophilic inflammation in the absence or presence of CD40L in our study may be related either to the mucosal pathway of sensitization or to the relatively greater potency of Af.

Despite the overall similarities in IgE and eosinophilic responses to allergen between the two types of mutant mice examined, we observed two differences in the physiologic responses of CD40 / and CD40L / mice. First, the sham-stimulated CD40 /, but not the CD40L / mice, were significantly less responsive to Mch than were the WT mice. The reason for this relative hyporesponsiveness of CD40 / mice not subjected to experimental allergen exposure was not clear. It is possible that low-level antigenic stimulation by resident microflora or nonspecific irritants could maintain some level of bronchial responsiveness via a CD40-dependent mechanism. Alternatively, the expression of CD40 per se on airway epithelial cells, monocytes, and macrophages could have effects on their function even in the absence of its interaction with CD40L. Such a cell-autonomous effect on cellular function could perhaps lead to a basal rate of production of factors that enhance airway responsiveness. There is also a remote possibility which cannot be easily excluded that there were differences among the mice in the expression of genes unrelated to CD40 and CD40L. Because these mice were derived by targeted mutagenesis, and because we used large numbers of experimental mice along with equivalent numbers of mice of the appropriate genetic background, we believe it highly improbable that other genes relevant to the inflammatory response were affected.

The second difference between the physiologic responses of CD40 / and CD40L / mice was that the Af-stimulated CD40 /, but not the CD40L / mice, developed enhanced airway responsiveness compared with sham-stimulated mice. Thus, CD40L is essential for the development of antigen-induced BHR, even though it is not required in the generation of eosinophilic pulmonary inflammation. One possible explanation for the requirement for CD40L in the induction of BHR is that the CD40L molecule itself modulates the function of T cells independent of CD40 binding. An alternative explanation is that, in addition to CD40, CD40L can interact with an alternative partner on other cells, including EOSs, thereby inducing BHR. Our results using the CD40L / mice again demonstrate that an influx of EOSs into the airways may not be sufficient for the development of BHR. We have previously shown that mice of certain strains (35) develop marked airway eosinophilia with little or no shift in airway responsiveness after antigenic stimulation; perhaps those animals, like the CD40L / mice in the current study, fail to induce those EOSs to release mediators necessary for the induction of BHR.

It has recently been postulated that there are two parallel pathways that can lead from antigenic stimulation to BHR. The first involves production of antigen-specific IgE and perhaps IgG subtypes, mast cell degranulation, and release of mast cell-derived cytokines; the second involves T-lymphocyte activation via the T-cell receptor and costimulatory molecules, leading to release of T cell-derived cytokines, including IL-5, and the expansion and chemoattraction of eosinophils. Our present results are consistent with earlier findings that the first, antibody-driven, pathway is not required for the induction of BHR (23, 33) and provide evidence that CD40L, but not CD40, is essential for the induction of BHR via the second pathway.

In summary, we have found that both CD40 and CD40L are required for the production of IgE and that neither is necessary for development of pulmonary eosinophilia in response to allergen inhalation. CD40, but not CD40L, acts to enhance airway responsiveness in the absence of experimental antigenic stimulation. Induction of BHR after experimental antigen exposure requires CD40L, but not CD40. Thus, these two related molecules have differing roles in the development of the pathophysiologic features of asthma.