The NF-κB/Rel family of transcription factors induces many genes involved in immune and inflammatory responses. Mice with germline deletions of individual NF-κB/Rel subunits have different phenotypes, suggesting that the NF-κB/Rel transcription factors have different functions. We tested whether c-Rel promotes allergic asthma using a murine model of allergen-induced pulmonary inflammation and airway hyperresponsiveness. Our investigation focused on c-Rel, which is expressed in lymphoid cells and is important for lymphocyte activation. In response to allergen sensitization and challenge, c-Rel-deficient mice did not develop increases in pulmonary inflammation, bronchoalveolar lavage fluid eosinophilia, or total serum IgE. c-Rel deficiency also prevented the induction of airway hyperresponsiveness. Allergen-treated wild-type mice had increased DNA binding to an NF-κB consensus site. Chemokine expression was altered in allergen-treated c-Rel-deficient mice. Monocyte chemoattractant protein-1, which is regulated by NF-κB, was decreased in allergen-treated c-Rel-deficient mice relative to wild-type controls. The increase in NF-κB/Rel transcription factors after allergen challenge in wild-type mice and the decrease in allergen reactivity found in c-Rel-deficient mice indicate that c-Rel promotes allergic inflammation. Alteration of pulmonary chemokine expression in c-Rel-deficient mice may inhibit allergen-induced pulmonary inflammation and airway hyperresponsiveness.
Asthma is a chronic inflammatory disease in which lymphocytes play a key role. We have previously shown the importance of T lymphocyte costimulation and activation in a murine model of allergen-induced pulmonary inflammation and airway hyperresponsiveness (AHR)3 (1, 2, 3, 4). Transcription factors and the immune genes they regulate have been implicated in the pathogenesis of chronic inflammatory diseases including asthma (5, 6, 7). The NF-κB/Rel family of transcription factors is of particular interest because it induces many genes involved in immune and inflammatory responses (7, 8, 9). Several studies have linked cytokines or allergens with activation of NF-κB (10, 11, 12, 13). Inhibition of NF-κB by glucocorticoids may be an important mechanism by which steroids attenuate inflammatory diseases such as asthma (10, 14, 15).
NF-κB has been implicated in the pathogenesis of asthma, but the roles of specific NF-κB/Rel transcription factors in the induction of allergic pulmonary inflammation and AHR are not yet defined. There are multiple NF-κB/Rel family members: c-Rel, p65 (RelA); p50 and its p105 precursor; p52 and its p100 precursor; RelB; v-Rel; and the Drosophila proteins Dorsal and DIF (8, 16, 17). The NF-κB/Rel family members have distinct tissue distributions and target gene specificities (18). The phenotypes of mice lacking specific NF-κB/Rel proteins are different, underscoring that the NF-κB/Rel transcription factors do not have redundant functions (9, 19, 20, 21, 22, 23, 24, 25, 26). One study has found that p50-deficient (p50−/−) mice do not develop allergen-induced eosinophilic airway inflammation, concomitant with decreased IL-5 and eotaxin production (27). Here we analyze c-Rel-deficient (c-Rel−/−) mice, which have a phenotype distinct from that of p50−/− mice, in a model of allergen-induced pulmonary inflammation to investigate whether c-Rel promotes allergic asthma (3). We focused on c-Rel because it is expressed predominantly in lymphocytes and is necessary for normal lymphocyte activation and proliferation.
Lymphocyte development and hemopoiesis are normal in c-Rel−/− mice, but mature T lymphocytes have proliferation defects in vitro related to decreased production of cytokines such as IL-2, IL-3, and GM-CSF (9, 19, 21). In the presence of exogenous IL-2, c-Rel−/− T cells proliferate normally and are able to differentiate into functional cytotoxic and helper T cells (21). c-Rel−/− mice have impaired humoral immunity as well: c-Rel is required for the synthesis of IgG1 and IgG2a, the transduction of survival and cell cycle progression signals in B cells, and for the generation of T cell-dependent humoral responses (9, 28). B cells deficient in the C-terminal trans-activation domain of c-Rel have defects in Ig class switching, including failure to switch to IgE (26). p50−/− mice have abnormal B cell activation, proliferation, Ab production, and Ig class switching (23, 24). The defects in isotype switching and germline CH RNA expression in p50−/− B cells, however, are different from those in B cells lacking the C-terminal trans-activation domain of c-Rel (20, 24). p50−/− T cells have proliferative defects that are less well characterized than the B cell defects (23).
Whether the immune response defects observed in c-Rel−/− lymphocytes in vitro would foster alterations in an in vivo model had not been previously studied. Specifically, we tested whether loss of c-Rel would lead to inhibition of allergic disease, including AHR.
Chemokines, known to regulate both NF-κB and allergic asthma, were altered in c-Rel−/− mice. For example, monocyte chemoattractant protein-1 (MCP-1) was decreased in allergen-treated c-Rel−/− mice relative to wild-type controls. Loss of c-Rel function inhibited the allergic phenotype, including allergen-induced physiological outcomes. Our findings suggest that the NF-κB/Rel transcription factor c-Rel promotes allergic pulmonary inflammation, may regulate the expression of chemokines that are relevant to the pathogenesis of asthma, and plays a critical role in the induction of allergic AHR.
Materials and Methods
Mice deficient in c-Rel (c-Rel−/−) were generated as previously described (9, 21, 28). Male wild-type BALB/c and C57BL/6 mice (5 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained according to the guidelines of the Committee on Animals at Harvard Medical School and the Committee on Care and Use of Laboratory animals of the Institute of Laboratory Animal Resources, National Research Council.
Ag sensitization and challenge
As previously described, mice were immunized with i.p. injections of 10 μg OVA (Sigma, St. Louis, MO) and 1 mg of Al(OH)3 (alum, J. T. Baker Chemical, Phillipsburg, NJ) on days 0 and 7 and underwent aerosol treatments with 6% OVA for 20 min/day on days 14 through 20 (2, 3). Control mice received i.p. injections and aerosol treatments with PBS.
Determination of lung resistance and airway reactivity
One day after the final aerosol challenge, lung resistance and dynamic compliance were determined as previously described (2, 3, 29, 30). In brief, mice were anesthetized with i.p. injections of pentobarbital sodium (Anthony Products, Arcadia, CA). The trachea was cannulated. An internal jugular vein was cannulated with a catheter attached to a microsyringe (Hamilton, Reno, NV) and used to administer methacholine (acetyl-β-methylcholine chloride, Sigma). As previously reported, methacholine exerted no significant systemic side effects (29). Dose-response curves to methacholine were obtained by administering increasing doses of methacholine (33 to 1000 μg/kg). Each new dose of methacholine was delivered only after the pulmonary resistance had returned to baseline. In addition, the effective methacholine dose that would have resulted in a doubling of pulmonary resistance, the log ED200, was calculated by log-linear interpolation and is used as an index of AHR.
Analysis of pathology
After determination of lung resistance and airway reactivity, mice were exsanguinated by cardiac puncture. Lungs were inflated with OCT compound (Miles, Elkhart, IN), fixed, stained with hematoxylin and eosin, and examined by light microscopy.
Determination of bronchoalveolar lavage fluid (BAL) cell differential counts
Mice underwent BAL after plethysmography. PBS (1 ml) with 0.6 M EDTA was instilled into the lungs and withdrawn three times via the tracheal cannula. The total number of live cells was determined by trypan blue exclusion with a hemocytometer. Cytospins were prepared (Shandon Scientific, Cheshire, U.K.), fixed, and stained with Diff-Quick (Dade Diagnostics of PR, Aguada, PR). An investigator who was unaware of the treatment groups performed duplicate counts of 100 cells to determine the percentages of each cell type.
Determination of serum total IgE
Blood was obtained by cardiac puncture. Serum levels of total IgE were measured by an ELISA assay. Microtiter plates (Marsh Biomedical Products, Rochester, NY) were coated with anti-IgE Ab (2 μg/ml in 0.1 M NaHCO3) (PharMingen, San Diego, CA), incubated overnight at 4°C, washed three times with PBS/0.05% Tween, blocked with 3% BSA (Sigma) in PBS, and washed again. Serum, diluted 1/20 in 1% BSA/PBS, and mouse IgEκ isotype standards were added to the wells, and the samples were incubated overnight at 4°C. Plates were washed and the secondary Ab (biotin anti-mouse κ light chain, PharMingen) diluted to 2 μg/ml was added. After incubation at room temperature for 1 h, samples were washed and treated with avidin-peroxidase (Sigma). The plates were incubated at room temperature for 1 h, washed, treated with o-phenylenediamine dihydrochloride, and read at 450 nm (Model 2550, Bio-Rad, Richmond, VA). Murine serum IgE concentrations were determined by the standard curve generated by analysis of the commercial standard.
Electrophoretic mobility shift assays (EMSA) and supershift assays
Nuclear extracts were prepared as previously described from thoracic lymphocytes from OVA-sensitized and challenged or PBS-treated mice (31, 32). EMSA (6) were performed as previously described (32) with the NF-κB consensus sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega, Madison, WI), which was end-labeled with [γ-32P]ATP. Nuclear extracts were normalized for protein (Bio-Rad protein assay), and equal amounts were added within experiments. DNA-protein complexes were resolved in a 5% polyacrylamide gel in 45 mM Tris, 45 mM boric acid, 1 mM EDTA (0.5× TBE) and run at 11 V/cm with cooling. The gels were dried and underwent autoradiography with intensifying screens at −70°C. For competition analysis, nuclear extracts were preincubated with water or competitor DNA for 5 min at room temperature; all other components were then added, and the samples were processed as for EMSA. The competitors were homologous unlabeled NF-κB probe or a non-NF-κB oligonucleotide (CTLA4) of the same size. mAbs (0.5–1.0 μg) specific to NF-κB/Rel family members (p50, p52, p65, Rel-B, and c-Rel) or to other transcription factors (Fos, Jun, JunB, JunD) (Santa Cruz Biotechnology, Santa Cruz, CA) were used for supershift analysis.
Pulmonary chemokine mRNA analysis
Total RNA was isolated from the lungs of OVA-treated wild-type and c-Rel−/− mice and their PBS-treated controls using RNAzol. Chemokine RNA analysis was performed via the RiboQuant MultiProbe RNase protection assay (RPA) (7) System (PharMingen). RNA was hybridized with 32P-labeled mck-5 probe according to the supplier’s directions. After RNase treatment and purification, protected probes were run on a denaturing 5% polyacrylamide gel. RNA (20 μg/sample) was run in each lane. The gels were developed in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The identity of each protected fragment was established by analyzing its migration distance against a standard curve of the migration distance vs the log nucleotide length for each undigested probe. Samples were normalized to the housekeeping genes, L32 and GAPDH. Densitometry analysis was performed using ImageQuant software (Molecular Dynamics).
All data are reported as means ± SE. Data were analyzed with the JMP 3.0 statistical package (SAS Institute, Cary, NC). Parametric data were analyzed with the Tukey-Kramer test; nonparametric data were analyzed using the Wilcoxon/Kruskal-Wallis rank sum test. A p value of <0.05 was considered significant.
c-Rel-deficient mice do not develop pulmonary inflammation after OVA sensitization and aerosol challenge
To determine whether c-Rel plays a role in allergic pulmonary inflammation, pulmonary histology from c-Rel−/− and wild-type mice was analyzed. Histological sections of lungs from allergen-treated c-Rel−/− mice (Fig. 1,c) had minimal inflammation, appearing similar to PBS control wild-type mice (Fig. 1,A) and to PBS control c-Rel−/− mice (not shown). OVA-treated c-Rel−/− mice did not have peribronchial or perivascular eosinophilia. Lungs from wild-type mice developed inflammatory changes typical of our model in response to allergen treatment: peribronchial and perivascular infiltrates composed of eosinophils, neutrophils, and lymphocytes (Fig. 1 B) (2).
Concomitant with histological assessment, we analyzed airway eosinophilia, a feature of allergen-induced pulmonary inflammation. BAL fluid eosinophilia was significantly decreased in allergen-treated c-Rel−/− mice compared with wild-type mice (Fig. 2). Eosinophils were not detected in either PBS control group.
c-Rel-deficient mice have an attenuated serum IgE response to OVA sensitization and aerosol challenge
Allergen-treated c-Rel−/− mice had very low levels of serum IgE, similar to levels in c-Rel−/− PBS controls (5.9 ± 2.2 and 4.65 ± 2.1 ng/ml, respectively) (Fig. 3). In contrast, allergen-treated wild-type mice demonstrated a significant increase in total serum IgE compared with their PBS controls (56.4 ± 14.4 and 11.9 ± 3.7 ng/ml, respectively) (∗, p = 0.002). Serum IgE levels in allergen-treated c-Rel−/− mice were significantly lower than in allergen-treated wild-type mice (∗∗, p = 0.001).
c-Rel-deficient mice do not develop airway hyperresponsiveness after OVA sensitization and aerosol challenge
To determine whether loss of c-Rel influences allergen-induced physiological outcomes, we measured AHR which we have previously shown to be significantly increased after allergen treatment (2, 3). Previous analyses of specific NF-κB/Rel subunits after allergen challenge have not reported measurements of AHR. Allergen-treated c-Rel−/− mice did not develop AHR. Importantly, their pulmonary resistance in response to methacholine was not significantly different from PBS control c-Rel−/− mice (Fig. 4). In contrast, allergen-treated wild-type mice developed significant increases in pulmonary resistance at all doses of methacholine compared with PBS control wild-type mice. The pulmonary resistance of allergen-treated wild-type mice was significantly higher than similarly treated c-Rel−/− mice at each dose of methacholine, as expressed by the difference between the log ED200 for each group (p < 0.0001).
NF-κB/Rel is induced after allergen sensitization and challenge in wild-type mice
To test the hypothesis that NF-κB/Rel transcription factors are induced in allergen-treated mice without a germline deletion of c-Rel, EMSA analysis was performed. There was an increase in binding to an NF-κB consensus sequence by nuclear extracts of thoracic lymphocytes from allergen-treated wild-type mice compared with PBS controls (Fig. 5,A, lanes 1 and 2). To analyze binding specificity, competition assays with homologous unlabeled NF-κB oligonucleotide showed a decrease in binding (Fig. 5,A, lanes 3 and 4), which was not paralleled by competition with a non-NF-κB oligonucleotide (Fig. 5,A, lanes 5 and 6). When nuclear extracts of thoracic lymphocytes from allergen-treated mice were incubated with mAbs to the different NF-κB/Rel family members, supershifted bands were detected with the use of Abs to c-Rel, p50, and p65 but not to p52, Rel-B, or non-NF-κB/Rel transcription factors (Fig. 5 B). These results confirm that NF-κB/Rel transcription factors, including c-Rel, are induced in our OVA model of allergic pulmonary inflammation and AHR.
Pulmonary chemokine mRNA expression is altered in c-Rel-deficient mice
Chemokines are a superfamily of structurally related cytokines that promote the adhesion, chemotaxis, and activation of multiple cell types in inflammatory states (33). Because NF-κB/Rel transcription factors induce proinflammatory cytokines, we investigated whether loss of c-Rel alters the chemokine mRNA profile in the lungs of allergen-exposed mice. RNA was isolated from the lungs of allergen-treated wild-type and c-Rel−/− mice as well as their PBS controls. The lungs were harvested 24 h after the final aerosol challenge. RPA analysis demonstrated that allergen treatment led to up-regulation of the chemokines macrophage-inflammatory protein-2 (1.9-fold), IFN-γ-inducible protein-10 (3.3 fold), and monocyte chemoattractant protein-1 (MCP-1) (2.5-fold) in wild-type mice (Fig. 6, lanes 1 and 2). c-Rel−/− mice had a different pattern of chemokine mRNA expression. Although macrophage-inflammatory protein-2 was up-regulated in allergen-treated c-Rel−/− mice (1.3-fold), IFN-γ-inducible protein-10 was expressed constitutively (Fig. 6, lanes 3 and 4). Interestingly, MCP-1 was not up-regulated in allergen-treated c-Rel−/− mice (Fig. 6, lanes 3 and 4). MCP-1 has previously been implicated in asthma; Abs that neutralize MCP-1 decrease allergen-induced AHR and pulmonary inflammation in a murine model (34). Taken together, these data suggest that c-Rel−/− mice do not have global defects in pulmonary chemokine mRNA expression and that their blunted response to allergen may be due in part to chemokine modulation.
The NF-κB/Rel family of transcription factors regulates the expression of multiple genes that have been implicated in immune and inflammatory responses, including asthma (8, 16). Analyzing the effects of germline deletions or mutations of specific NF-κB/Rel subunits will elucidate their roles in various inflammatory diseases, including asthma. The present study demonstrates that absence of c-Rel is associated not only with significant attenuation of allergen-induced pulmonary and BAL eosinophilia but also with inhibition of total IgE and the physiological correlate of allergic airway inflammation, AHR. p50−/− mice do not develop eosinophilic airway inflammation in response to allergen sensitization and challenge, concomitant with a lack of IL-5 and eotaxin production; total serum IgE and AHR were not examined (27). The phenotype and immune system defects of c-Rel−/− mice are distinct from those of p50−/− mice (23, 24).
One explanation for the blunted response of c-Rel−/− mice to allergen sensitization and aerosol challenge is their defects in lymphocyte activation and proliferation. T lymphocyte activation is an important mediator of allergic pulmonary inflammation and AHR (2, 35, 36). Interruption of T lymphocyte costimulatory signals inhibits AHR, pulmonary inflammation, total serum IgE, and Th2 cytokine production (3, 37, 38). c-Rel is not necessary for normal hemopoiesis, lymphocyte development, or T cell effector function (9); however, it controls early activation events mediated by Ag receptor signals in T lymphocytes (21). c-Rel also regulates the expression of various cytokine genes, either directly (IL-2, IL-3, GM-CSF) or indirectly (IL-5, TNF-α, IFN-γ), through its effect on T cell proliferation (9, 19, 21). Current data support the predominance of Th2 cytokines (IL-4, IL-5, IL-10, IL-13) in allergic asthma, but some studies suggest that Th1 cytokines such as IL-2, IFN-γ, and TNF-α may also promote allergic airway inflammation (39, 40). IL-3 and GM-CSF prolong eosinophil survival and are elevated in the airways of asthmatics (41, 42, 43). c-Rel thus appears to be essential for T cell activation and the production of multiple cytokines involved in the pathogenesis of allergic asthma.
We found a significant reduction in the allergen-induced rise in total serum IgE in c-Rel−/− mice compared with wild-type mice. To our knowledge, this is the first reported measurement of IgE in c-Rel-deficient mice. Previous investigators have measured the Ab response to antigenic challenge in wild-type and c-Rel−/− mice and found a significant reduction in the IgG1 response to a T cell-dependent Ag in c-Rel−/− mice, but IgE levels were not reported (9). Normal induction of CD23, the low affinity receptor for IgE in c-Rel−/− mice, has been reported (44); however, no characterization of the high affinity IgE receptor in c-Rel−/− mice has been performed. B cells deficient in the C-terminal activation domain of c-Rel have marked defects in switching to IgE despite normal levels of germline CHε RNA, suggesting an important role for c-Rel in the regulation of Ig class switching (20).
Given the significant reduction in total serum IgE in c-Rel−/− mice, we did not measure OVA-specific IgE as this would likely also have been reduced. Low levels of OVA-specific IgE in c-Rel−/− mice may have led to inadequate sensitization of mast cells and basophils in the lungs and therefore a weak response to allergen challenge; however, the role of Ag-specific Abs in murine models of allergic inflammation is unresolved. In one study, passive sensitization of mice by iv injections of anti-OVA IgE or IgG1 followed by airway challenge with OVA resulted in pulmonary eosinophilia and AHR (45). In contrast, allergen-induced AHR and pulmonary eosinophilia still develop in B cell-deficient (46), IgE-deficient (47), and mast cell-deficient (48) mice. It has been proposed that AHR and pulmonary eosinophilia may develop in murine models by mast cell-dependent or CD4+ T cell-dependent pathways (46). C57BL/6 mice, the background of the wild-type and c-Rel−/− mice used in the present study, are genetically deficient in mast cell mediators and may therefore be more dependent on T cells than on mast cells (46, 49). One possibility is that c-Rel is required for a normal IgG1/IgE response to allergen sensitization and challenge but that allergen-specific IgE is not essential for the development of AHR and pulmonary eosinophilia in our T cell-dependent model (3, 4).
The response of c-Rel−/− mice to allergen sensitization and aerosol challenge may be blunted due to differences in chemokine expression in the lungs as well. For example, MCP-1 mRNA is up-regulated in the allergen-treated wild-type mice but not in the allergen-treated c-Rel−/− mice (Fig. 6). Several studies have found elevated expression of MCP-1 in the airways or BAL fluid of allergic asthmatics (50, 51, 52, 53, 54). MCP-1 induction concomitant with NF-κB activation have been found in several models of inflammatory diseases (55, 56, 57, 58). The promoters of both the human MCP-1 gene and its murine homologue have NF-κB sites (59). In a model of allergic airway inflammation, inhibition of MCP-1 significantly decreased bronchial hyperresponsiveness, pulmonary inflammation, and the recruitment of eosinophils and T lymphocytes to the airways (34). Absence of c-Rel may prevent maximal transcription of the MCP-1 gene after allergen challenge, representing a potential mechanism for loss of the allergic phenotype in c-Rel−/− mice.
Our examination of pulmonary eotaxin mRNA showed that at 24 h after the final allergen challenge, there was no significant difference in pulmonary eotaxin mRNA expression between PBS and allergen-treated wild-type mice. Our results in wild-type mice differ from those of other investigators who have found that eotaxin mRNA expression peaks 3–6 h after allergen challenge and remains elevated for 24 h (27, 34, 60). Potential reasons for the different results include different allergen sensitization and challenge protocols, time points examined, and murine strains. At different time points, c-Rel−/− mice might be deficient in eotaxin production relative to wild-type mice. Nonetheless, eotaxin mRNA expression in c-Rel−/− mice is at least detectable after allergen challenge, in contrast to p50−/− mice (27).
Factors other than eotaxin mRNA expression might be responsible for the observed attenuation in eosinophilia. Decreased translation of eotaxin mRNA or defective eotaxin receptor-response coupling are two possible mechanisms that have not yet been investigated. Other chemokines and cytokines that attract eosinophils and prolong their survival in allergic inflammation may be very relevant to our model, including MCP-1, IL-3, IL-5, and GM-CSF (41, 42, 43). As already noted, MCP-1 mRNA is up-regulated in OVA-treated wild-type mice but not in OVA-treated c-Rel−/− mice. Inhibition of MCP-1 prevented pulmonary and BAL eosinophilia in response to OVA but not the expression of eotaxin mRNA in a murine model; hence, MCP-1 may regulate eosinophil recruitment to the lungs via mediators other than chemokines (34). c-Rel is required by T cells for the production of IL-3 and GM-CSF (9, 21). We postulate that the most likely mechanisms for attenuated pulmonary eosinophilia in c-Rel-deficient mice are failure to up-regulate MCP-1 expression and decreased production of IL-3 and GM-CSF.
Our EMSA analysis of thoracic lymphocytes from allergen-treated wild-type mice supports a role for NF-κB/Rel transcription factors in the response to allergen sensitization and challenge. In a previous study, increased NF-κB DNA binding was detected in the lungs of allergen-treated rats, but specific NF-κB/Rel family members were not analyzed (12). The EMSA in this report is unique in its analysis of nuclear extracts from murine thoracic lymphocytes, previously shown to have allergen-specific responsiveness after aerosolized allergen challenge (3). NF-κB/Rel is induced after allergen exposure in wild-type mice (Fig. 5,A). Supershift analysis demonstrated that c-Rel, p50, and p65 were present in the NF-κB/Rel complexes induced by allergen treatment (Fig. 5 B). In vitro stimulation of T cells from wild-type mice via the TCR and CD28 costimulatory pathway induced significant increases in NF-κB/Rel complexes over those of unstimulated controls (21). Consistent with our results, the complexes were supershifted or inhibited by Abs against p50, p65, and c-Rel. As expected, in T cells from c-Rel−/− mice, the complexes were inhibited only by Abs against p50 and p65. The in vitro experiments are relevant to our in vivo disease model in which T cell activation and CD28 costimulation are essential (3, 4).
The increase in NF-κB/Rel transcription factors after allergen treatment in normal mice and the decrease in allergen reactivity associated with loss of c-Rel function suggest that c-Rel is a mediator in the inflammatory cascade leading to allergic asthma. Absence of the c-Rel transcription factor prevents development of the allergic phenotype in our studies, supporting the notion of a critical role for NF-κB/Rel regulation of lymphocyte activation, proliferation, and cytokine production in the response to allergen sensitization and aerosolized challenge. Modulation of the expression of chemokines such as MCP-1 by c-Rel may play an essential role in the pathogenesis of allergic pulmonary inflammation. We show for the first time that an NF-κB/Rel transcription factor, c-Rel, is required for allergen-induced AHR.
We thank Drs. James Lederer, Debbie Landry, Doug Faunce, and Joan Stein-Streilin for their helpful reviews of the manuscript.
This work was supported by grants from the American Heart Association (P.W.F.), the National Institutes of Health (HL 56723 and AI 45007 to P.W.F.; CA 68155 to H.C.L.; HL 36110 to G.T.D.; and NIHAI 44085 to D.L.P.), the American Cancer Society and the March of Dimes (H.C.L.), the American Lung Association (G.T.D.), and a Partners Investigator Nesson Award (G.T.D.). P.W.F. is a Career Investigator with the American Lung Association.
Abbreviations used in this paper: AHR, airway hyperresponsiveness; MCP-1, monocyte chemoattractant protein-1; BAL, bronchoalveolar lavage; EMSA, electrophoretic mobility shift assay; RPA, RNase protection assay.