Proinflammatory and Th2-Derived Cytokines Modulate CD40-Mediated Expression of Inflammatory Mediators in Airway Epithelia: Implications for the Role of Epithelial CD40 in Airway Inflammation

The molecule CD40 is a member of the TNFR family, which includes the TNF receptor, nerve growth factor receptor, and Fas. It is a 50-kDa integral membrane glycoprotein that was identified originally on B lymphocytes and demonstrated to play a central role in the regulation of humoral and cell-mediated immunity (reviewed in Ref. 1). For example, CD40 engagement of B cells has been shown to up-regulate the expression of B7 (CD80), ICAM-1, CD23, and lymphotoxin α (LTα) (2). Moreover, the importance of CD40 in the regulation of immune responses is underscored by the observation that interruption of the CD40-CD40 ligand (CD40L)² interaction halts the development and progression of several autoimmune diseases, including experimental encephalomyelitis, collagen-induced arthritis, and lupus, as well as responses to transplantation Ags observed in graft-vs-host disease (reviewed in Ref. 2). CD40L is a type II transmembrane protein classified as a member of the TNF family. It is expressed on activated CD4⁺ T cells, activated CD8⁺ T cells, eosinophils, mast cells, basophils, NK cells, and activated dendritic cells (1).

Airway epithelial cells form a continuous pseudostratified layer in the lung, creating a tight barrier that protects underlying tissue from the external environment. As such, airway epithelial cells have been described classically as barrier cells that are involved in homeostasis; these cells respond to a variety of environmental stimuli, resulting in the alteration of their cellular functions such as ion transport and movement of airway secretions. Recent evidence, however, suggests that airway epithelial cells might also act as immune effector cells in response to noxious endogenous or exogenous stimuli. Several studies have shown that airway epithelial cells express and secrete various immune molecules, such as lipid mediators, oxygen radicals, adhesion molecules, and a wide variety of cytokines, including chemokines (reviewed in Ref. 3). Through the expression and production of these immune molecules, the epithelium is now thought to be important in the initiation and exacerbation of inflammatory responses within the airway.

T lymphocytes play a major role in the pathogenesis of allergic airway disease, including asthma-associated inflammation. Elevated numbers of activated T cells have been observed in the bronchoalveolar lavage (BAL) fluid and bronchial tissue of asthmatic patients; the majority of these T cells are CD4⁺ (reviewed in Ref. 4). CD4⁺ T cells are categorized into Th1 and Th2 subsets with respect to their lymphokine production. Specifically, Th1 cells produce IL-2, TNF-β, and IFN-γ and are important for the development of cell-mediated immunity. In contrast, Th2 cells express IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 and are essential in the generation of humoral immune responses, including IgE production, as well as in the induction of eosinophilia. Because IgE synthesis and eosinophilia are hallmarks of allergic airway disease, it has been postulated that asthma-associated inflammation is mediated by a Th2 response.

Many reports have indicated that interactions between CD40 and its ligand, CD40L, control the development of humoral and cell-mediated immune responses. In particular, studies utilizing CD40 or CD40L knockout mice have suggested that CD40 ligation promotes inflammatory responses within the airway (5, 6, 7). Despite this observation, it is unclear which resident cell type(s) of the airway is critical in facilitating CD40-mediated airway inflammation. Because epithelial cells have been implicated in airway inflammatory responses, it is likely that cell-cell interactions between CD40-responsive airway epithelial cells and leukocytes that express CD40L, including T lymphocytes, eosinophils, and mast cells, can initiate and/or exacerbate lung inflammation. Therefore, the present study examined the expression of CD40 on airway epithelial cells and its role in amplifying inflammatory responses within the lung. Results described herein demonstrate that airway epithelial cells express CD40 and that cross-linking of CD40 up-regulates the expression of inflammatory mediators, including the chemokines IL-8, RANTES, and monocyte chemoattractant protein (MCP-1) and the adhesion molecule ICAM-1. Moreover, these results demonstrate that the proinflammatory cytokines TNF-α and IL-1β and the Th2-derived cytokines IL-4 and IL-13 modulate the positive effects of CD40 engagement on chemokine and adhesion molecule expression in these cells.

Experiments were performed with the human airway epithelial cell line 9HTEo⁻ (tracheal; a gift from Dr. Dieter Gruenert, University of California (San Francisco, CA (8)). The 9HTEo⁻ cell line was cultured in LHC-8 medium (Biofluids, Rockville, MD) containing 5% FCS, 1% penicillin-streptomycin, and 0.2% Fungizone. Cells were grown at 37°C in a 5% CO₂ environment and on Vitrogen 100 (Collagen, Palo Alto, CA)-coated flasks; Vitrogen 100 contains collagen types I and IV.

Lungs from BALB/c mice were excised, quick-frozen in liquid nitrogen to preserve Ag immunoreactivity, and sectioned. Frozen sections of the upper airway were then fixed in 3% formaldehyde (TEM grade, Tousimis, Rockville, MD) and rinsed in PBS, and nonspecific sites were blocked in PBS containing 1% BSA. Samples were stained with a rat anti-mouse CD40 Ab (IC-10, 10 μg/ml, PharMingen, San Diego, CA) or rat IgG (10 μg/ml, Sigma, St. Louis, MO) as a negative control for 1 h at room temperature. Samples were then rinsed in PBS and incubated with a mouse anti-rat Ig secondary conjugated to rhodamine fluorochrome (Molecular Probes, Eugene, OR) for 1 h at room temperature. Samples were then rinsed with PBS, mounted in a solution containing 0.1% p-phenylenediamine (Sigma), and analyzed via confocal microscopy.

To examine CD40 surface expression, airway epithelial cells were cultured with TNF-α, IL-1β, IL-4, or IL-13 at the concentrations and time points indicated. After culture, cells were lifted with HBSS (Ca²⁺ and Mg²⁺ free) containing 10% FCS and 0.05 M EDTA, washed twice with 1× PBS containing 0.2% BSA, and then stained with a mouse anti-human anti-CD40 mAb (10 μg/ml; G28.5, a gift from Dr. Randolph Noelle, Dartmouth Medical School, Lebanon, NH) for 30 min on ice. Parallel samples were stained with mouse IgG1κ immunoglobulin (10 μg/ml, Sigma) as an isotype-matched Ab control. After incubation, cells were washed as above and then incubated with the secondary Ab, FITC-conjugated goat anti-mouse F(ab′)₂ IgG1 (diluted 1:100, BioSource, Camarillo, CA) for 30 min on ice. Cells were again washed as above, resuspended in 1× PBS containing 0.2% BSA, and analyzed via flow cytometry (Becton Dickinson, FACScalibur; University of Alabama at Birmingham Core Facility).

To analyze IL-8, MCP-1, and RANTES protein expression, cells were cultured in the presence and absence of soluble CD40L (sCD40L; mCD8hgp39, a gift from Dr. R. J. Noelle) with or without TNF-α, IL-1β, IL-4, or IL-13 (R&D Systems, Minneapolis, MN) at the concentrations indicated for 18 h at 37°C. After culture, supernatants were harvested and prepared for ELISA of IL-8, MCP-1, or RANTES protein content (BioSource); cells were harvested and counted to account for differences in cell number. ELISAs were performed according to the manufacturer’s protocol (limits of detection, <3 pg/ml).

To analyze ICAM-1 and VCAM-1 surface protein expression, cells were cultured in the presence and absence of sCD40L with or without TNF-α, IL-1β, IL-4, or IL-13 (R&D Systems) at the concentrations indicated for 18 h at 37°C. After culture, cells were harvested and analyzed via flow cytometry as described above for CD40 detection with the exception that mAbs directed against ICAM-1 or VCAM-1 were utilized (R&D Systems).

Data are expressed as the mean ± SD of replicate determinations as indicated. Statistical significance was determined by ANOVA. p ≤ 0.05 was considered significant.

To examine CD40 expression in primary airway epithelial cells, immunohistochemical analysis of BALB/c mouse lung tissue was performed. Mouse lung tissue was utilized for this analysis due to: 1) the difficulty in obtaining normal human lung tissue; and 2) the potential for phenotypic changes of primary cells cultured in vitro. Therefore, lungs from BALB/c mice were excised, quick-frozen in liquid nitrogen to preserve Ag immunoreactivity, and sectioned. Frozen sections of the upper airway were then stained with either a mAb directed against murine CD40 or an appropriate negative control. As shown in Fig. 1, epithelial cells lining the bronchioles of the upper airway stained brightly for CD40 (Fig. 1,A), whereas negative control samples displayed little cross-reactivity (Fig. 1 B).

To determine whether human airway epithelial cells can express CD40 in vitro, CD40 surface protein expression was examined on the 9HTEo⁻ human airway epithelial cell line directly via flow cytometry. There are limitations to using in vitro cultures of airway epithelial cells. The phenotype of an airway epithelial cell, whether immortalized or primary, may not reflect its phenotype in vivo. However, the 9HTEo⁻ cell line utilized for the studies described herein has been clearly defined as “epithelial” (8). Thus, the use of this cell line provides unambiguous results for airway epithelial expression of CD40. Flow cytometric analysis revealed that ∼100% of 9HTEo⁻ cells stained positively for CD40 expression (Fig. 2,A). The negative control HT-29, a human intestinal epithelial cell line that does not express CD40 (9), did not stain for CD40 (Fig. 2 B). The change in mean fluorescence intensity for 9HTEo⁻ cells staining positively for CD40 expression was ∼4-fold greater than controls.

Experiments were performed to determine whether the cytokines TNF-α, IL-1β, IL-4, and/or IL-13 modulated CD40 expression on airway epithelial cells in vitro. For these experiments, 9HTEo⁻ cells were cultured separately with these various cytokines at increasing concentrations, ranging from 0.1 to 100 ng/ml, and increasing time points, ranging from 18 to 72 h. As presented in Fig. 2 A, neither TNF-α nor IL-1β altered airway epithelial CD40 expression at the concentrations and time points examined. IL-4 and IL-13 also did not affect CD40 expression on airway epithelial cells (data not shown).

CD40 is known to play a role in cell-cell interactions that result in the modulation of immune responses (2). To determine whether cross-linking CD40 on airway epithelial cells induced the expression of chemokines, 9HTEo⁻ cells were cultured in the presence and absence of sCD40L and then analyzed for protein expression of the chemokines RANTES, MCP-1, and IL-8 via ELISAs. The 9HTEo⁻ cell line was utilized as a model airway epithelial cell for these studies, given that 9HTEo⁻ cells express CD40 (Fig. 2) and can be induced to express a variety of immune molecules, including chemokines and adhesion molecules (10). The chemokines IL-8, RANTES, and MCP-1 were examined in these experiments because each has been implicated in facilitating leukocyte migration into the airway lumen during an inflammatory response (reviewed in Ref. 11). In particular, MCP-1 mediates monocyte and basophil chemotaxis and activation whereas IL-8 primarily induces the migration of neutrophils. RANTES induces the chemotaxis of eosinophils, monocytes, and CD45 RO⁺ memory T lymphocytes. The data in Fig. 3 demonstrate that CD40 ligation induces chemokine expression in 9HTEo⁻ cells. Specifically, sCD40L up-regulated RANTES, IL-8, and MCP-1 expression in 9HTEo⁻ cells from undetectable levels to 0.6 ng/10⁶ cells, 1.3 ng/10⁶ cells, and 2.2 ng/10⁶ cells, respectively (Fig. 3,A). Importantly, sCD40L induced the expression of RANTES, IL-8, and MCP-1 in a dose-dependent manner; sCD40L-induced IL-8 expression is shown in Fig. 3 B as a representative example. The effects of sCD40L on chemokine expression in airway epithelial cells were blocked by a mAb directed against CD40L (TRAP1, data not shown).

The adhesion molecules ICAM-1 and VCAM-1 facilitate leukocyte migration within the lung. Specifically, ICAM-1 binds β₂ integrins expressed on a variety of cell types, including lymphocytes and neutrophils, whereas VCAM-1 binds VLA-4 found on eosinophils (reviewed in Ref. 12). To determine whether CD40 engagement modulated expression of ICAM-1 and/or VCAM-1 on airway epithelia, 9HTEo⁻ cells were cultured with or without sCD40L. Cells were then collected and analyzed for the surface expression of ICAM-1 and VCAM-1 via flow cytometry. As observed in Fig. 4,A, ∼20% of 9HTEo⁻ cells stained positively for ICAM-1 expression; however, sCD40L enhanced basal ICAM-1 expression ∼3-fold. The dose response for sCD40L-mediated induction of ICAM-1 expression is shown in Fig. 4,B. The change in mean fluorescence intensity for CD40-mediated modulation of ICAM-1 expression in 9HTEo⁻ cells was ∼2-fold greater than the respective isotype matched controls (data not shown). The effect of sCD40L on ICAM-1 expression in airway epithelial cells was blocked by a mAb directed against CD40L (TRAP1, data not shown). Fig. 4,A also demonstrates that 9HTEo⁻ cells express little or no detectable VCAM-1 on the cell surface. Importantly, CD40 engagement on 9HTEo⁻ cells did not induce the expression of VCAM-1 above basal levels (Fig. 4 A). Because VCAM-1 could be shed from the cell surface, supernatants from these cultures were analyzed for the presence of soluble VCAM-1 via ELISA; however, these analyses did not detect the presence of soluble VCAM-1 (data not shown), suggesting that sCD40L does not induce VCAM-1 surface expression on 9HTEo⁻ cells.

In the asthmatic lung, airway epithelial cells are exposed to a variety of soluble mediators, including proinflammatory and Th2-derived cytokines, that alter their cellular activity and function. To examine the effects of proinflammatory and Th2-derived cytokines on CD40-mediated expression of the chemokines RANTES, IL-8, and MCP-1, 9HTEo⁻ cells were cultured in the presence and absence of sCD40L with and without the proinflammatory cytokines TNF-α and IL-1β or the Th2-derived cytokines IL-4 and IL-13. Chemokine expression was then monitored via ELISA. TNF-α and IL-1β were utilized in these experiments because previous studies have demonstrated that these cytokines are elevated during an inflammatory response of the airway (13) and can modulate airway epithelial expression of immune molecules (reviewed in Ref. 3). IL-4 and IL-13 were included in these experiments because they are Th2-derived cytokines that have been reported to alter the activity of airway epithelial cells (14, 15).

As demonstrated in Fig. 5, TNF-α and/or IL-1β enhanced the effects of CD40 engagement on RANTES, IL-8, and MCP-1 expression. Specifically, cells cultured with TNF-α alone expressed RANTES at ∼0.4 ng/10⁶ cells; however, TNF-α synergized with sCD40L to increase RANTES expression to 2.0 ng/10⁶ cells or ∼3- to 5-fold over that amount observed with either stimulus alone (Fig. 5,A). Similarly, cells stimulated with IL-1β alone expressed approximately 1.0 ng/10⁶ cells; yet, IL-1β synergized with sCD40L to up-regulate MCP-1 expression to nearly 7.0 ng/10⁶ cells or between 3.5- and 7-fold greater than that observed with either stimulus alone (Fig. 5,B). Interestingly, TNF-α and IL-1β each combined with sCD40L to up-regulate IL-8 expression in airway epithelial cells (Fig. 5,C). In detail, TNF-α and IL-1β alone induced IL-8 expression from undetectable levels to ∼1.0 ng/10⁶ cells and 1.0 ng/10⁶ cells, respectively (Fig. 5 C). Importantly, both TNF-α and IL-1β combined with sCD40L in a synergistic fashion to enhance CD40-mediated IL-8 expression to ∼3.7 and 3.0 ng/10⁶ cells, respectively, or between 2- and 3-fold greater than that observed with either stimulus alone. In contrast, neither IL-4 nor IL-13 induced RANTES, MCP-1, or IL-8 expression and, moreover, did not modulate the effects of CD40 cross-linking on chemokine expression in these cells (data not shown).

To further examine the effect of CD40 engagement on the sensitivity of airway epithelial cells to proinflammatory cytokines, additional experiments were performed. Specifically, 9HTEo⁻ cells were cultured in the presence and absence of sCD40L in combination with TNF-α or IL-1β at increasing concentrations. Alternatively, cells were preincubated with sCD40L for varying time periods and then exposed to TNF-α or IL-1β at a single concentration. After culture, supernatants from these cultures were harvested and examined for IL-8, RANTES, or MCP-1 protein expression via ELISA. As demonstrated in Fig. 6, ligation of CD40 increased the sensitivity of 9HTEo⁻ cells to the effects of TNF-α and IL-1β in a dose-dependent manner with regard to IL-8 expression; similar results were observed for the effects of TNF-α on RANTES expression and IL-1β on MCP-1 expression (data not shown). In contrast, priming the cells with sCD40L for 2 or 6 h before exposure with TNF-α or IL-1β did not alter the effect of either cytokine on IL-8 expression (Fig. 7); similar results were observed for RANTES and MCP-1 expression (data not shown).

The effects of TNF-α and IL-1β as well as IL-4 and IL-13 on CD40-mediated expression of the adhesion molecules ICAM-1 and VCAM-1 in airway epithelial cells were examined. Although TNF-α and IL-1β each induced the expression of ICAM-1 between 2- and 3-fold above basal levels, neither of these stimuli modulated CD40-mediated expression of ICAM-1 (Fig. 8,A). Interestingly, IL-4 and IL-13 increased ICAM-1 expression modestly in these cells; however, both of these cytokines decreased CD40-mediated ICAM-1 expression significantly (Fig. 8,A). In contrast, TNF-α alone, but not IL-1β, IL-4, or IL-13, induced expression of VCAM-1 in airway epithelial cells (Fig. 8,B). Importantly, TNF-α synergized with sCD40L to increase VCAM-1 expression between 3- and 4-fold over that observed with either stimulus alone (Fig. 8 B).

To further examine the effect of CD40 engagement on the sensitivity of airway epithelial cells to the effects of TNF-α with regard to VCAM-1 expression, 9HTEo⁻ cells were cultured in the presence and absence of sCD40L in combination with TNF-α at increasing concentrations. In addition, cells were preincubated with sCD40L for varying time periods and then exposed to TNF-α at a single dose. After culture, cells were harvested and examined for VCAM-1 surface expression via flow cytometry. ICAM-1 expression was not monitored in these experiments because neither TNF-α nor IL-1β had a significant effect on sCD40L-induced ICAM-1 expression in 9HTEo⁻ cells (Fig. 8). As demonstrated in Fig. 9,A, ligation of CD40 increased the sensitivity of 9HTEo⁻ cells to the effects of TNF-α in a dose-dependent manner with regard to VCAM-1 expression. In contrast, priming the cells with sCD40L for 2 or 6 h before exposure with TNF-α did not alter the effect of this cytokine on VCAM-1 expression (Fig. 9 B).

The data presented herein demonstrate that airway epithelial cells, in vitro and in vivo, express CD40. Moreover, these data show that ligation of CD40 expressed on airway epithelial cells up-regulates the expression of inflammatory mediators, including the chemokines IL-8, RANTES, and MCP-1 and the adhesion molecule ICAM-1. Engagement of epithelial CD40 had no effect on VCAM-1 expression. Importantly, CD40 ligation enhanced the sensitivity of airway epithelial cells to the effects of TNF-α and/or IL-1β on expression of RANTES, MCP-1, IL-8, and VCAM-1. In contrast, neither IL-4 nor IL-13 modified the effects of CD40 engagement on the expression of RANTES, MCP-1, IL-8, or VCAM-1; however, both IL-4 and IL-13 attenuated the effects of CD40 cross-linking on ICAM-1 expression. These findings suggest that epithelial CD40 plays a role in airway inflammatory responses.

The epithelial barrier in the airway has two distinct surfaces, the apical (luminal) and the basolateral (serosal) surfaces. The apical surface is exposed to the environment directly whereas the basolateral surface is protected from the environment through the existence of tight junctions. Tight junctions facilitate selective transport of materials across the epithelial barrier and dictate sequestration of proteins made by epithelia to either the apical or the basolateral compartment. To date, there is no conclusive evidence that CD40 demonstrates a polarized pattern expression on airway epithelial cells. Our observations suggest that CD40 is expressed primarily on the apical surface; however, we can detect CD40, albeit to a lesser degree, on the basolateral surface (Fig. 1). A more definitive answer to this question is being pursued via colocalization studies and laser confocal microscopy. As stated in the Introduction, T lymphocytes play a major role in the pathogenesis of allergic airway disease. In fact, elevated numbers of activated T cells have been observed in the BAL fluid and bronchial tissue of asthmatic patients (reviewed in Ref. 4). In light of the data presented herein, we anticipate that T lymphocytes, which express CD40L on activation (1), will encounter and interact with CD40 expressed on the basolateral and/or apical surface of the airway epithelium as these cells migrate from the circulation and into the airway lumen. The consequences of such cell-cell interactions, be it at the apical and/or the basolateral surface, will trigger the epithelium to express increased amounts of chemokines and adhesion molecules and thereby contribute to the airway inflammatory response.

Previous reports indicate that bronchial epithelial cells express CD40 (16, 17). In particular, Gormand et al. (17) have reported that the cytokines TNF-α and IFN-γ increased the basal expression of CD40 on bronchial epithelial cell lines. Moreover, their data suggest that ligation of CD40 expressed on bronchial epithelial cell lines enhanced the expression of IL-6 and GM-CSF from these cells; however, CD40 engagement did not alter the sensitivity of these cells to the effects of TNF-α with regard to IL-6 and GM-CSF expression.

The data presented herein contrast with those of Gormand et al. First, although our data also demonstrate CD40 expression in both lung tissue and airway epithelial cell lines, such expression was not modulated by the cytokines TNF-α, IL-1β, IL-4, or IL-13. Second, as was similarly observed by Gormand et al., our data demonstrate that the expression of inflammatory molecules, including RANTES, IL-8, MCP-1, and ICAM-1, was increased on ligation of CD40 on airway epithelial cells. Third, as stated above, our results indicate that CD40 engagement enhances the sensitivity of airway epithelial cells to the effects of the proinflammatory cytokines TNF-α and/or IL-1β. Specifically, CD40 ligation enhanced the response of the cells to TNF-α with regard to the expression of IL-8, RANTES, and VCAM-1, as well as IL-1β, with regard to MCP-1 and IL-8 expression. Interestingly, CD40 ligation before cytokine exposure did not prime airway epithelial cells to respond to either TNF-α or IL-1β. Together, our results suggest that, as CD40L⁺ cells migrate into the lung and cross the epithelial barrier, ligation of epithelial CD40 will render the epithelium more sensitive and responsive to the effects of proinflammatory cytokines present in the local microenvironment.

In addition to airway epithelial cells, other CD40-responsive cell types in the lung have been identified. Lazaar et al. (18) reported that cross-linking CD40 on airway smooth muscle cells with CD40L up-regulates the expression of the pleiotropic cytokine IL-6. Similarly, Sempowski et al. (19) demonstrated that cross-linking CD40 on lung fibroblasts with CD40L induces the expression of IL-6 and the chemokine IL-8; expression of both of these molecules was enhanced further in the presence of IFN-γ. Moreover, Zhang et al. (20) reported that engagement of CD40 on lung fibroblasts increased PGE₂ synthesis via the induction of cyclooxygenase-2.

Recent reports suggest that CD40 plays a role in airway inflammatory responses in vivo. For example, Adawi et al. have reported that disruption of CD40-CD40L interactions blunts hyperoxic lung injury (21) and protects against radiation-induced pulmonary toxicity (22). Specifically, these authors demonstrated that mice pretreated with an Ab against CD40L protected against oxygen-induced lung injury as well as radiation-induced pneumonitis and fibrosis. Studies utilizing CD40 or CD40L knockout mice have also implicated CD40 in airway inflammatory responses in vivo. Wiley et al. (5) have shown that treatment of wild-type CD40 mice with sCD40L increased polymorphonuclear cell infiltration of the alveolar space and an accumulation of alveolar macrophages with increased Ia expression; such an increase in cell infiltration of the lungs was not observed in CD40L-treated CD40 knockout mice. Similarly, Lei et al. (7) have reported altered airway immune responses in CD40L knockout mice. These authors observed that CD40L knockout mice sensitized with OVA followed by an OVA aerosol challenge, as a model of allergic airway inflammation, displayed a reduced airway inflammatory response when compared with similarly sensitized and challenged wild-type controls. Specifically, significantly less numbers of monocytes, lymphocytes, neutrophils, and eosinophils were detected in the BAL fluid of CD40L knockout at 72 h postchallenge as compared with controls. Moreover, decreased serum levels of OVA-specific IgE and IgG1 and IL-4 and decreased BAL levels of IL-4 and TNF-α were detected in the CD40L knockout mice as compared with wild-type controls; however, similar levels of IL-5 were detected in the serum and BAL fluid of both control and knockout mice. In addition, lung endothelial cell expression of VCAM-1 in OVA-sensitized and challenged CD40L knockout mice was reduced as compared with controls.

It is evident that CD40 plays a role in lung inflammation in vivo. Identifying the CD40-responsive cells within the airway that promote inflammation is critical in understanding the mechanisms that underlie airway inflammation and, moreover, in generating novel therapies that ameliorate inflammatory diseases such as asthma.

We thank Dr. Randolph J. Noelle, Dr. Etty Benveniste, and Albert Tousson for their assistance, and Dr. Dale J. Benos, the Department of Physiology and Biophysics, and the Department of Cell Biology for their continued support.