TNF Receptor-Associated Factor 1 Expressed in Resident Lung Cells Is Required for the Development of Allergic Lung Inflammation

Mice

BALB/c WT mice were purchased from Charles River Breeding Laboratories. DO11.10 mice hemizygous for the TCR transgene were purchased from The Jackson Laboratory (25). TRAF1^−/− mice (19) were backcrossed onto a BALB/c background for eight generations. Mice were maintained in a specific pathogen-free environment, according to animal protocols approved by Institutional Animal Care and Use Committee of Children’s Hospital and the CBR Institute for Biomedical Research (Boston, MA).

T cell transfer and allergic airway inflammation

OVA-specific CD4⁺ T cells were derived as previously described (26). Briefly, WT mice were immunized i.p. with 50 μg of OVA/1 mg of alum in 0.9% sterile saline. Seven days after sensitization, splenocytes were purified and cultured in complete HL-1 medium (BioWhittaker) for 4 days in the presence of 200 μg/ml LPS-free OVA (Worthington Biochemical). CD4⁺ T cells were purified using MACS columns (Miltenyi Biotec) and transferred i.v. at 2 × 10⁶ cells/mouse into naive recipients. Control mice received PBS vehicle. Starting the next day, mice were exposed daily for 7 days to OVA aerosol (10 mg/ml) in 0.9% normal saline solution for 30 min. Obstruction of airflow and lung inflammation were assessed 24 h after the last nebulization. AHR was determined by measuring enhanced pause (Penh) in conscious, unrestrained mice following graded doses of methacholine using whole body plethysmography (Buxco Electronics). Enhanced pause results were analyzed using two-way ANOVA. A value for p < 0.05 was considered statistically significant. Collection and evaluation of bronchoalveolar lavage (BAL) fluids and lung homogenates was done as described in (24). Cytokine concentrations were measured by ELISA as described (21). BAL cells were stained with CD4-FITC (RM4-5), CD8-PE (53-6.7), CD11b-PE (M1/70), CD11c-PerCP (N418), and MHC class II (M5/114.15.2) allophycocyanin (BD Biosciences). CFSE labeling of purified CD4⁺ T cells was done as previously described (21). Real-time RT-PCR analysis was performed as described previously (24). The ICAM-1 primers were purchased from Applied Biosystems. The fold induction of gene expression was calculated as the ratio of gene expression in T cell-transferred mice to PBS-treated mice.

Lung histology and immunohistochemistry

For histological analysis, lungs were inflated with 1.0 ml of 10% formalin instilled through a tracheostomy tube and embedded in paraffin. Multiple 4-μM sections were stained with diastase periodic acid-Schiff and H&E. For immunohistochemical analysis, 5-μm sections of lungs were embedded in Tissue-Tek OCT Compound (Sakura Finetek), fixed in acetone, washed in PBS, and incubated with 3% rabbit serum in PBS to prevent nonspecific binding. ICAM-1 expression was detected with the hamster IgG clone 3E2B (Chemicon International) and biotinylated goat anti-hamster IgG (eBioscience). After washing with PBS, slides were incubated with VectaStain ABC reagent (Vector Laboratories), developed using diaminobenzidine substrate kit (BD Biosciences), counterstained with hematoxylin, and cover-slipped with Permount.

BrdU staining

T cell proliferation in vivo was measured by BrdU incorporation. BALB/c WT and TRAF1^−/− recipient mice were injected i.p. with 1 mg/mouse of BrdU on days 6 and 7 of OVA nebulization. On day 8, BAL cells were harvested and lymphocytes were stained with anti-BrdU, FITC-conjugated mAb (BD Biosciences) to detect proliferating lymphocytes.

Proliferation assay

At 24 h after the last nebulization, mediastinal LNs were prepared using 300 U/ml type I collagenase (Worthington Biochemical) and 100 U/ml DNase I (Sigma-Aldrich) treatment for 60 min in PBS at 37°C. After filtration through a 70-μm cell strainer, single cell suspensions were isolated by density gradient using Lympholyte-M (Accurate Chemical and Scientific) and incubated with graded concentrations of LPS-free OVA protein (Worthington Biochemical) in complete HL-1 medium (BioWhittaker). Proliferation was assessed by monitoring the incorporation of [³H]thymidine (1 μCi/well) added during the last 8 h of 72 h cultures.

DC generation and migration assay

Splenic CD4⁺ DCs were prepared from collagenase D-treated spleens using the CD4⁺ Dendritic Cell Isolation kit (Miltenyi Biotec) according to the protocol provided by the manufacturer. T cells from DO11.10 mice were purified by negative magnetic bead separation using MACS columns (Miltenyi Biotec). Purified T cells were stained with FITC-conjugated anti-CD4 Ab and PE-conjugated anti-KJ1.26 Ab and sorted on a FACSVantage SE (BD Biosciences). A total of 5 × 10³/well of KJ1.26⁺ CD4⁺ T cells and 1 × 10⁴/well of splenic CD4⁺ DCs were cultured in the presence of LPS-free OVA for 72 h.

Bone marrow DCs were generated as previously described (27). Briefly, bone marrow cells were isolated and propagated in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 20 ng/ml GM-CSF (PeproTech). On day 7, cells were stimulated with either 1 μg/ml mouse recombinant TNF (PeproTech) or 1 μg/ml LPS (Sigma-Aldrich) for 24 h. Chemotaxis assays were performed as previously described (28). Stimulated bone marrow-derived DCs were resuspended at 2.5 × 10⁶/ml in assay medium (0.1% BSA in RPMI 1640 medium). Assay medium (600 μl) without or with 200 ng/ml mouse CCL19 (PeproTech) were added per well in 24-well tissue culture plates. A Transwell culture insert (Corning Costar) with 5-μm pore size was inserted into each well, and 100 μl (2.5 × 10⁵) of the DC suspension was then added into the top chamber. The plate was incubated at 37°C for 2 h. Cells that migrated to the lower chamber were collected, counted, and analyzed by flow cytometry.

Adoptive DC transfer

For adoptive transfer experiments, in vitro-generated bone marrow-derived DCs were stimulated with 1 μg/ml TNF for 24 h. After extensive washing, 2 × 10⁶ stimulated DCs were intranasally administered to naive BALB/c female mice as previously described (29). Purified OVA-immune WT CD4⁺ T cells (2 × 10⁶ cells/mouse) were i.v. injected into the DC recipients at 24 h after DC transfer. Recipients were exposed daily for 7 days to OVA aerosol and levels of inflammation were assessed 24 h after the last nebulization.

OVA-immune T cells do not confer allergic lung inflammation to TRAF1^−/− mice

To investigate whether TRAF1 expressed in cells other than T lymphocytes might play a role in allergic lung inflammation, we used an adoptive transfer model of asthma (26). OVA-immune WT CD4⁺ T cells were i.v. infused into WT or TRAF1^−/− recipients mice. These mice were then exposed daily (for 30 min) to LPS-free OVA aerosol for 7 days and subsequently analyzed for the development of lung inflammation, mucus production, and AHR.

Histologic examination of the lung tissues of OVA-challenged WT recipients, which had previously received OVA-immune T cells, showed an extensive leukocyte infiltration of peribronchial regions compared with WT mice that were injected with PBS (Fig. 1⇓A). In contrast, infiltration of leukocytes into the peribronchial regions was markedly attenuated in TRAF1^−/− recipients of OVA-immune T cells (Fig. 1⇓A). Moreover, mucus production in the bronchial epithelium in the lungs of TRAF1^−/− mice was impaired compared with production in the lungs of WT animals (Fig. 1⇓B), and the absence of airway inflammation in T cell-injected TRAF1^−/− mice was associated with attenuation of airway responsiveness to methacholine (Fig. 1⇓C). Microscopic analysis of BAL fluid from OVA-challenged WT mice, which had previously received OVA-immune T cells, revealed a dramatic increase in the number of airway white blood cells with elevations in eosinophils, neutrophils, and lymphocytes (Fig. 2⇓A). In contrast, TRAF1^−/− recipients of OVA-immune T cells did not show any evidence of increased airway recruitment by leukocytes in response to OVA inhalation (Fig. 2⇓B). Although there was a similar number of mononuclear phagocytes in the BAL of all groups of mice (Fig. 2⇓B, R3), only WT mice given OVA-immune T cells displayed significant accumulation of lymphocytes and eosinophils in the pulmonary airspace (Fig. 2⇓B, R1 and R2, respectively). Thus, Ag-immune T cells were not able to confer allergic lung inflammation or AHR to TRAF1^−/− recipients, suggesting that the absence of TRAF1 expression in the recipient mice resulted in an impaired ability of transferred allergen-specific T cells to orchestrate an inflammatory reaction in the lungs.

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FIGURE 1.

Impaired peribronchial inflammation, mucus secretion and increase in AHR in allergen-challenged TRAF1^−/− mice. WT and TRAF1^−/− (KO) recipients were i.v. injected with PBS or OVA-immune CD4⁺ T cells, then exposed to an aerosol of LPS-free OVA for 30 min daily over 7 days and analyzed 24 h after the last exposure. A, Peribronchial infiltrates were analyzed by staining of representative lung sections with H&E. B, Goblet cell hyperplasia was analyzed by staining of representative lung sections with periodic acid-Schiff reagent (PAS). C, AHR was measured by whole body plethysmography on conscious unrestrained mice exposed to the indicated doses of nebulized methacholine. Mice were adoptively immunized with OVA-immune CD4⁺ T cells (circle symbols) or control PBS (square symbols). Recipients were WT (closed symbols) and TRAF1^−/− (open symbols). Airflow obstruction is expressed as the measure of enhanced pause (Penh). Data represent mean measurement of enhanced pause ± SE (n = 4 mice/group). ∗, p < 0.05.

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FIGURE 2.

Induction of bronchial inflammation in allergen-challenged mice requires TRAF1 expression by non-T cells. A, WT and TRAF1^−/− (KO) recipients were i.v. injected with PBS (▪) or OVA-immune CD4⁺ T cells (▧) and subsequently challenged, then exposed to an aerosol of LPS-free OVA for 30 min daily over 7 days and analyzed 24 h after the last exposure as in Fig. 1⇑. The BAL fluid was removed and analyzed for the number of infiltrating white blood cells, including eosinophils, neutrophils, and lymphocytes, as described in Materials and Methods. Data represent the mean of three experiments ± SE (n = 3 mice recipients). ∗, p < 0.05, significant difference between groups of mice. B, Flow cytometry analysis of the BAL fluid cells for the presence of lymphocytes (R1), eosinophils (R2), and mononuclear phagocytes (R3) based on forward and side scatter is shown.

TRAF1 expression in cells other than T lymphocytes is required for the development of a pulmonary T cell response to allergen inhalation

Because T lymphocytes are critical for the development of allergic airway inflammation, we analyzed T cell responses in the pulmonary airspace of WT and TRAF1^−/− mice. OVA exposure of WT recipients resulted in a remarkably stronger recruitment of CD4⁺ T cells to the pulmonary airspace than did OVA challenge of TRAF1^−/− recipients (Fig. 3⇓A). Moreover, WT recipients of immune T cells accumulated a higher number of CD8⁺ T cells in the lungs than did TRAF1^−/− mice in response to OVA exposure (Fig. 3⇓A). Furthermore, T cell-injected WT mice had a significantly higher number of BrdU⁺ CD4⁺ T cells in the BAL (Fig. 3⇓B), indicating that a large proportion of CD4⁺ T cells isolated from the lungs of WT recipients with T cells specifically proliferated in response to Ag.

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FIGURE 3.

A decreased number of T cells and impaired cytokine production in the lungs of TRAF1^−/− mice. Groups of WT and TRAF1^−/− (KO) recipients injected with PBS (▪) or OVA-immune CD4⁺ T cells (▧) were sensitized as described in Fig. 1⇑. A, The number of CD4⁺ and CD8⁺ lymphocytes (R1) in the lymphocyte forward and side scatter gate shown in Fig. 2⇑B were estimated by multiplying the percentage of each population by the total number of white blood cells in the BAL sample. B, The number of BrdU⁺ CD4⁺ T lymphocytes in BAL fluid were assessed by multiplying the percentage of BrdU⁺ cells by total number of CD4⁺ T lymphocytes in the BAL sample. The concentrations of the indicated cytokines in the BAL fluid (C) and the lungs (D) were determined by ELISA. Results are expressed as the mean of three independent experiments (n = 4 mice/group). Error bars represent the SE of the mean. ∗, p < 0.05, significant difference between TRAF1^−/− and WT recipients of OVA-immune CD4⁺ T cells are indicated. E, Flow cytometry of the inguinal LN lymphocytes from WT and TRAF1^−/− recipients. The number in each blot indicates the percentage of CFSE⁺ CD4⁺ T cells. Data are representative of two experiments (n = 3 mice/group).

The Th2 cytokines IL-4, IL-5, and IL-13 are important for the development of allergic airway inflammation, mucus secretion, and AHR (30). Analysis of cytokine concentrations in BAL fluid of adoptive T cell transfer recipients revealed elevated levels of IL-4, IL-5, and IL-13 in T cell-injected WT mice (Fig. 3⇑C). In contrast, TRAF1^−/− recipients had cytokine levels indistinguishable from control mice injected with PBS. The examination of the Th1 cytokine IFN-γ levels revealed no significant differences between all four groups of mice (Fig. 3⇑C). Importantly, adoptive transfer of OVA-immune T cells conferred a significant production of a major proinflammatory cytokine TNF in the pulmonary airspace of WT mice compared with TRAF1^−/− mice (Fig. 3⇑C). Cytokine analysis of the whole lung tissue confirmed a robust Th2 cytokine production in response to OVA inhalation occurred only in WT recipients of OVA-immune T cells (Fig. 3⇑D). Taken together, these data demonstrate that TRAF1 is necessary for the development of a T cell response in the lungs.

To eliminate the possibility that transferred OVA-immune WT T cells were rejected by TRAF1^−/− recipients, we labeled T cells with CFSE before transfer, exposed mice to aerosol OVA for 7 days, and analyzed the presence of CFSE⁺ CD4⁺ T cells in the LNs. We found a similar number of CFSE⁺ CD4⁺ T cells in the inguinal LNs of WT and TRAF1^−/− mice (Fig. 3⇑E), indicating that OVA-immune WT T cells were not rejected in TRAF1^−/− recipients.

Decreased activation and maturation of CD11c⁺CD11b⁺ DCs in TRAF1^−/− airways

CD11b⁺CD11c⁺ BAL DCs have been shown to be the most effective lung APCs for the induction of T cell activation (31). These “myeloid” DCs display a remarkably long-lived Ag-presenting function, contrasting sharply with other types APC in the lungs, which have only a transient ability to present Ag (31). Importantly, these cells appear to be essential for the development of asthma, as their in vivo depletion has been shown to abrogate the onset of airway inflammation in Ag-challenged mice (32). We analyzed APC in the BAL of WT and TRAF1^−/− mice in terms of their ability to migrate to regional lymphoid tissue and present Ag to T cells.

Although the total number of cells with forward and side scatter properties of mononuclear phagocytes (Fig. 2⇑B, R3) was similar among all four groups of mice, WT recipients of OVA-immune T cells possessed a significantly higher number of CD11b⁺CD11c⁺ DCs in the BAL in comparison with control mice injected with PBS (Fig. 4⇓A). In contrast, TRAF1^−/− recipients had no increase in the number of CD11b⁺CD11c⁺ DCs in comparison with sham WT or TRAF1^−/− mice (Fig. 4⇓A). As shown in Fig. 4⇓B, BAL CD11b⁺CD11c⁺ DCs from T cell-transferred WT but not TRAF1^−/− recipients displayed increased expression of MHC class II following exposure to Ag, indicating defective activation and maturation of DCs in the BAL of TRAF1^−/− mice.

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FIGURE 4.

Decreased activation of CD11b⁺CD11c⁺ DCs in TRAF1^−/− lungs and the absence of Ag in the draining LNs of TRAF1^−/− mice. Groups of WT and TRAF1^−/− (KO) mice injected with PBS (▪) or OVA-immune CD4⁺ T cells (▧) were sensitized as described in Fig. 1⇑. A, Flow cytometry analysis of the BAL CD11b⁺CD11c⁺ DCs. BAL-derived myeloid DCs were identified as CD11b⁺CD11c⁺ cells within the mononuclear phagocyte gate (Fig. 2⇑B, R3). The number of CD11b⁺CD11c⁺ DCs was estimated by multiplying the percentage of CD11b⁺CD11c⁺ DCs by the total number of white blood cells in the BAL. B, Maturation of DCs in the lung mucosa of sensitized mice was determined by evaluating the expression of high levels of MHC class II. Mice injected with PBS (solid histogram); or with T cells (broken histogram) are shown. C, Proliferation of cells from the draining LN of sensitized mice. Cells were isolated from mediastinal LN 24 h after the last exposure to OVA and incubated at 5 × 10⁴/well in 96-well plates. [³H]Thymidine was added during the last 8 h of culture. Results are expressed as the mean of three independent experiments (n = 3 mice), and error bars represent the SE of the mean. ∗, p < 0.05, significant difference in A and C between WT and TRAF1^−/− recipients of OVA-immune CD4⁺ T cells is indicated.

Following maturation, Ag-bearing activated DC transport Ag from the airways to the lung draining LNs, where they can present this Ag to specific T cells (33). We assessed Ag-specific responses in the draining LNs of WT and TRAF1^−/− mice and observed that mononuclear cells prepared from mediastinal LNs of T cell-injected WT mice displayed significant spontaneous proliferation even in the absence of exogenous Ag (Fig. 4⇑C). These findings are consistent with successful delivery of OVA from the lungs to the draining LNs in WT mice with adoptively transferred T cells and imply an active ongoing immune response in the lung draining LNs of these mice. In contrast, there was no proliferation in cultures from the mediastinal LNs of TRAF1^−/− mice injected with OVA-specific T cells (Fig. 4⇑C). These observations suggest that there is no ongoing immune response in the lung draining LNs of TRAF1^−/− mice.

TRAF1^−/− DCs display normal Ag-presenting function and CCL19-dependent migration in vitro

The absence of proliferation in LN cultures from TRAF1^−/− mice could be due to several reasons, including impaired Ag-presenting capability of TRAF1^−/− DCs or inability of TRAF1^−/− DCs to transport Ag to the draining LNs. To evaluate the Ag-presenting capacity of TRAF1^−/− DCs, highly purified CD4⁺ T cells from OVA TCR transgenic DO11.10 mice were cultured alone or with purified splenic CD4⁺ myeloid DCs from WT or TRAF1^−/− mice in the presence of increasing concentrations of OVA. We found that TRAF1-negative DCs supported proliferation of DO11.10 T cells to the same extent as WT DCs (Fig. 5⇓A), suggesting that without TRAF1 DCs were able to acquire, process, and present the Ag to cognate T cells. Therefore, TRAF1^−/− DCs seem to have normal Ag-presenting capacity.

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FIGURE 5.

Ag presentation and transmigratory abilities of TRAF1^−/− DCs. A, Normal Ag presentation by TRAF1^−/− DCs. Splenic CD4⁺ myeloid DCs were isolated from WT or TRAF1^−/− (KO) mice and irradiated. A total of 5 × 10³/well of KJ1.26⁺ CD4⁺ T cells from DO11.10 mice and 1 × 10⁴/well of CD4⁺ DCs were cultured in the presence of OVA protein for 72 h. [³H]Thymidine incorporation over the last 8 h of culture was measured. Results are expressed as the mean of two experiments (n = 3 mice), and error bars represent the SE of the mean. B, Transmigration of bone marrow-derived myeloid DCs. Bone marrow-derived cells (BMDC) from WT (▪) and TRAF1^−/− mice (KO) (▨) were cultured with GM-CSF for 7 days to generate CD11b⁺CD11c⁺ myeloid DCs and were then matured with TNF or LPS for 24 h. For transmigration assay cells were placed in the upper chamber of Transwell inserts and the migration of MHCII^highCD11b⁺CD11c⁺ cells toward CCL19 was analyzed using FACS analysis of cells in the bottom chamber. Results of a representative experiment from four independent cell preparations are shown. ∗, p < 0.05, significant difference between WT and TRAF1^−/− DCs is indicated.

A number of previous studies have demonstrated that maturation of DCs is associated with up-regulated expression of CCR7 and their increased ability to migrate in response to the CCR7 ligands, CCL19, and CCL21 (34). To investigate the transmigration ability of TRAF1^−/− DCs in vitro, we generated DCs from bone marrow of WT and TRAF1^−/− mice by culturing cells with GM-CSF and inducing their subsequent maturation by stimulation with TNF or LPS. Analysis of the migration of TRAF1^−/− DCs revealed that TNF-matured (MHCII^highCD11b⁺CD11c⁺) TRAF1^−/− DCs have a significantly higher ability to migrate in response to CCL19 than do WT DCs (Fig. 5⇑B). In contrast, there was no difference in migratory ability between WT and TRAF1^−/− DCs matured following treatment with LPS (Fig. 5⇑B). Thus, mature TRAF1^−/− DCs have an equal, if not higher, migratory capacity when compared with WT DCs in vitro.

Adoptive transfer of DCs is able to restore the immune response in draining LNs, but is not sufficient to induce lung inflammation

To investigate whether TRAF1^−/− DCs can also function normally in vivo (e.g., migrate to regional lymphoid tissue and activate T cells), we adoptively transferred bone marrow-derived DCs into TRAF1^−/− mice 24 h before adoptive transfer of OVA-immune T cells. Following 7 days of exposure to aerosol OVA, we analyzed proliferation of LN cells in vitro. Fig. 6⇓A shows that, although LN cells from TRAF1^−/− mice injected with WT DCs proliferated to the greatest extent, LN cells from TRAF1^−/− mice injected with TRAF1^−/− DCs proliferated to the same extent as LN cells from mock DC-injected WT mice. These data indicate that bone marrow-derived TRAF1^−/− DCs, when transferred into TRAF1^−/− mice, can deliver OVA to the draining LNs and induce a T cell immune response to the same extent as WT DCs.

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FIGURE 6.

Adoptive transfer of exogenous bone marrow-derived DCs into TRAF1^−/− mice restores the LN, but not the lung immune response. Bone marrow-derived WT and TRAF1^−/− (KO) DCs were intranasally transferred into TRAF1^−/− mice 1 day before adoptive transfer of OVA-immune WT T cells and at the beginning of 7 days of OVA aerosol exposure. A, Proliferation of the mediastinal LN cells from sensitized WT (▪) and TRAF1^−/− (▧) mice transferred with or without WT and TRAF1^−/− DCs was done by exposure to OVA and incubation at 5 × 10⁴/well in 96-well plates, as described in Fig. 4⇑C. [³H]Thymidine was added during the last 8 h of culture. ∗, p < 0.05. B, The number of CD4⁺ T lymphocytes in the BAL of sensitized WT (▪) and TRAF1^−/− (▧) mice injected with WT and TRAF1^−/− DCs, or with a PBS control. C, FACS analysis of MHC class II expression on WT and TRAF1^−/− BAL myeloid DCs from representative mice as indicated. MHCII⁺CD11b⁺CD11c⁺ DCs were gated and further analyzed in D. D, Quantification of MHC class II expression on WT and TRAF1^−/− BAL myeloid DCs. The percentage of BAL MHCII⁺CD11b⁺CD11c⁺ DCs in the gated regions of the FACS histograms presented in C. Results are expressed as the mean of two independent experiments (n = 3 mice), and error bars represent the SE of the mean.

In sharp contrast to WT mice, there was no accumulation of CD4⁺ T cells in the pulmonary airspace of TRAF1^−/− mice given either WT or TRAF1^−/− DCs (Fig. 6⇑B), despite the signs of an ongoing immune response in the draining LNs of DC-injected TRAF1^−/− mice (Fig. 6⇑A). Moreover, the expression of MHC class II on BAL CD11b⁺CD11c⁺ DCs from DC-injected TRAF1^−/− mice was not significantly different from MHC class II expression on CD11b⁺CD11c⁺ DCs from TRAF1^−/− mice without supplementary DCs (Fig. 6⇑, C and D). Taken together these results indicate that the adoptive transfer of preactivated DCs into the lungs of TRAF1^−/− mice, although sufficient to restore an immune response in the draining LNs, is not adequate to extend the inflammatory response to the airways.

Decreased expression of ICAM-1 in the airways of sensitized TRAF1^−/− mice

We have recently demonstrated that, following inhalation of aerosol LPS, TRAF1^−/− mice fail to up-regulate ICAM-1 expression in their lungs and demonstrate impaired recruitment of T lymphocytes to the pulmonary airways (24). To examine the role of ICAM-1 in TRAF1-mediated allergic lung inflammation, we analyzed expression of ICAM-1 in the airways of sensitized TRAF1^−/− mice. WT recipients of OVA-immune T cells demonstrated increased transcription of the ICAM-1 gene when compared with WT mice injected with PBS (Fig. 7⇓A). However, there was no significant induction of ICAM-1 gene transcription in the lungs of T cell-injected TRAF1^−/− mice (Fig. 7⇓A). Analysis of ICAM-1 protein expression revealed that ICAM-1 was expressed in PBS-treated WT and TRAF1^−/− mice (Fig. 7⇓B, left panels). This is consistent with previously published observations that have found significant levels of ICAM-1 in the alveolar structures and vascular endothelium of normal untreated lungs (35). Importantly, WT recipients of OVA-immune T cells displayed higher levels of ICAM-1 expression than did PBS-treated WT mice (Fig. 7⇓B). In contrast, we found no significant increase in ICAM-1 protein expression in the lungs of T cell-injected TRAF1^−/− mice compared with PBS-treated controls. Taken together with an essential role of ICAM-1 in allergic lung inflammation (36, 37, 38), our results suggest that ICAM-1 expression in the pulmonary airways might be important for the development of TRAF1-mediated allergic lung inflammation.

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FIGURE 7.

Decreased ICAM-1 expression in the lungs of TRAF1^−/− mice. Groups of WT and TRAF1^−/− (KO) mice were sensitized as described in Fig. 1⇑. A, Expression of ICAM-1 was measured by quantitative real-time PCR in the lung tissue of WT (▪) and TRAF1^−/− recipients (▧) injected with PBS or OVA-immune CD4⁺ T cells. Relative quantification of gene expression was done by the threshold comparative C_T method. The mRNA expression of the indicated molecules was normalized to that of β₂-microglobulin from the same sample. The fold induction was calculated as the ratio of gene expression for CD4⁺ T cell-transferred mice to PBS-transferred mice. Data are expressed as mean + SEM of two independent experiments, and each experiment included three mice per group. ∗, p < 0.05. B, Immunohistochemical detection of ICAM-1. Mouse lungs from sensitized mice were stained with hamster Ab 3E2B to mouse ICAM-1 as the primary Ab. Arrow indicates ICAM-1 antigenicity. Original magnification at ×100. Results are representative of four mice in each group.