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Expression of the Costimulatory Molecule B7-H2 (Inducible Costimulator Ligand) by Human Airway Epithelial Cells Shin Kurosawa, Allen C. Myers, Lieping Chen, Shengdian Wang, Jian Ni, James R. Plitt, Nicola M. Heller, Bruce S. Bochner, and Robert P. Schleimer Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland; Mayo Graduate and Medical School, Mayo Clinic, Rochester, Minnesota; and Human Genome Sciences Inc., Rockville, Maryland

Tissue structural cells are known in some situations to play a role in the presentation of antigen and in immunoregulation. We assessed the expression of B7 homologs, known to be involved in antigen presentation and lymphocyte costimulation, in human airway epithelial cells. Flow cytometry performed on the airway epithelial cell line BEAS-2B, as well as primary bronchial epithelial cells (PBEC), showed that B7-H2 was constitutively expressed on both BEAS-2B and PBEC, whereas B7-1 and B7-2 were undetectable on either epithelial cell type. B7-H2 expression was confirmed by Western blot using a specific antibody. Stimulation with various cytokines, including tumor necrosis factor-␣, interferon-␥, and interleukin-4, slightly downregulated B7-H2 expression detected by flow cytometry, but did not significantly alter the apparent level of protein as assessed by Western blotting. Northern blotting detected mRNA for B7H2 and B7-1, but not B7-2. B7-H2 was cloned from BEAS-2B cells and the sequence verified. Expression of B7-H2 mRNA was detected by real-time reverse transcriptase–polymerase chain reaction in PBEC from three independent donors. Immunohistochemical analysis of airway derived from autopsies revealed expression of B7-H2 in human airway epithelial cells. These results demonstrate that airway epithelial cells express the costimulatory molecule B7-H2, and suggest the possibility that B7-H2 may participate in antigen presentation by epithelial cells.

Bronchial epithelium lines the mucosal surface of airways, forming a mechanical barrier that separates the external environment from the internal milieu. It has long been believed that the function of epithelial cells is limited to protecting against invading microorganisms and removing them by means of the mucociliary escalator. However, growing evidence suggests that bronchial epithelium also functions in the regulation of immune responses through production of cytokine and chemokines (1–4). In addition to the production of humoral factors, bronchial epithelial cells may

(Received in original form October 1, 2002 and in revised form December 10, 2002) Address correspondence to: Dr. Robert P. Schleimer, Johns Hopkins Asthma and Allergy Center, Room 3A.62, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: [email protected] Abbreviations: antigen-presenting cells, APC; B7 homologue 2, B7-H2; B7 homologue 3, B7-H3; ethylene diamine tetraacetic acid, EDTA; glyceraldehyde 3⬘-phosphate dehydrogenase, GAPDH; Hanks’ buffered saline solution, HBSS; inducible costimulator, ICOS; interferon-␥, IFN-␥; interleukin, IL; mean fluorescence intensity, MFI; major histocompatibility complex, MHC; primary bronchial epithelial cells, PBEC; peripheral blood mononuclear cells, PBMC; phosphate-buffered saline, PBS; reverse transcriptase– polymerase chain reaction, RT-PCR; T helper, Th; tumor necrosis factor-␣, TNF-␣. Am. J. Respir. Cell Mol. Biol. Vol. 28, pp. 563–573, 2003 DOI: 10.1165/rcmb.2002-0199OC Internet address: www.atsjournals.org

have the capacity to act as antigen-presenting cells because they have been reported to express not only major histocompatibility complex (MHC) class I and class II molecules, but also costimulatory molecules (5–8). Optimal activation of T cells requires both costimulation and T cell receptor engagement. Antigen presentation in the absence of costimulation may lead to T cell anergy. Costimulatory interactions between the B7 family ligands expressed on antigen-presenting cells (APC) and their receptors on T cells play critical roles in the growth, differentiation, and death of T cells (9–13). Engagement of the T cell costimulatory receptor CD28 by its ligands B7-1 (CD80) and B7-2 (CD86) augments activation of T cells and promotes T cell survival. In contrast, binding of B7-1 or B7-2 with CTLA-4, a homolog of CD28, may inhibit T cell responses by delivering a putative negative signal (12, 14–18). Recently, another B7-like molecule of mouse origin, B7 h (B7RP-1, GL50, LICOS) and its human ortholog, B7-H2, have been identified (19–23). Parallel studies demonstrated that B7-H2 on B cells and macrophages is a ligand for the inducible costimulator (ICOS) expressed on antigen-primed T cells. Costimulatory signals via ICOS elicit T-helper cell differentiation through Th2 cytokine production such as interleukin (IL)-10 and IL-4 (24–28). In addition, another member of the human B7 family, B7-H3, was cloned very recently (29). Expression of B7-H3 protein is not detectable in peripheral blood mononuclear cells, although it is found in various normal tissues. However, B7-H3 can be induced on dendritic cells and monocytes by inflammatory cytokines. B7-H3 costimulates proliferation of both CD4⫹ and CD8⫹ T cells, enhances the induction of cytotoxic T cells, and selectively stimulates the production of a key Th1-type cytokine, interferon (IFN)-␥. Therefore, it is suggested that B7-H3 may play a role in Th1-cell differentiation and in primary cytotoxic T lymphocyte activation, although its counter-receptor is still unknown. The question of whether respiratory epithelium expresses costimulatory molecules is relevant because epithelial cells have been demonstrated to express MHC II molecules (6–8). The relevance of epithelium in immune responses during infection or in the development of Th2 type response– associated diseases such as asthma is also recognized (2, 30–34). Unfortunately, little is known about the expression of costimulatory molecules on respiratory epithelial cells. In this study, we have investigated the expression of costimulatory molecules, B7-1, B7-2, and B7-H2 on human airway epithelial cells using the airway epithelial cell line BEAS2B and primary bronchial epithelial cells (PBEC). Northern blotting detected mRNA for B7-H2 and B7-1, but not B7-2,

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in BEAS-2B cells and PBEC, and B7-H2 was cloned from BEAS-2B. Expression of B7-H2 mRNA was detected in PBEC from three different donors. Flow cytometry showed that B7-H2 was constitutively expressed on BEAS-2B cells and PBEC, whereas B7-1 and B7-2 were undetectable on either epithelial cell type. B7-H2 expression was confirmed by Western blotting using a specific antibody. Stimulation with cytokines, including tumor necrosis factor (TNF)-␣, IFN-␥ and IL-4, slightly downregulated or had no effect on B7-H2 expression as determined by flow cytometry and Western blotting. Immunohistochemical analysis of airway derived from organ donors revealed expression of B7-H2 in airway epithelial cells. These results suggest that airway epithelial cells express the costimulatory molecule B7-H2 and support the hypothesis that antigen presentation by epithelial cells have a role in maintaining mucosal responses of T cells.

Materials and Methods Isolation of PBEC Isolation of PBEC (n ⫽ 7) was performed as previously described (35). Specimens of human bronchi were obtained from cadaver donors without respiratory disorders. Tissues were dissected and rinsed five times in Ca2⫹- and Mg2⫹-free Hanks’ balanced salt solution (HBSS) and incubated overnight at 4⬚C in 0.1% protease (Calbiochem, San Diego, CA) solution in Ham’s F12 medium containing penicillin (100 U/ml), streptomycin (100 U/ml), and fungizone (1 ␮g/ml) (GIBCO-BRL, Gaithersburg, MD). Cells were detached by a gentle jet of 20% fetal bovine serum in Ham’s F12 medium. The suspension was centrifuged at 1,000 rpm for 8 min, and the cell pellet was resuspended. The purity of PBEC cultures (⬎ 95%) was confirmed by light microscopy and immunohistochemical staining for cytokeratin, performed as previously described (36).

Culture of BEAS-2B Cells and PBEC The BEAS-2B cell line, derived from human bronchial epithelium transformed by an adenovirus 12-SV40 virus, was kindly supplied by Dr. Curtis Harris (National Cancer Inst., Bethesda, MD) (37). BEAS-2B cells were cultured in Ham’s F12/Dulbecco’s modified Eagle’s medium containing glutamine (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, and 100 ng/ml streptomycin (GIBCO-BRL). PBEC were cultured in bronchial epithelial basal medium (Clonetics, San Diego, CA) containing bovine pituitary extract, hydrocortisone, human recombinant epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, bovine serum albumin-fatty acid free, gentamicin, and amphotericin-B. BEAS-2B cells and PBEC were cultured in T-75 tissue culture flasks or 6-well culture plates uncoated (BEAS-2B) or coated with collagen (PBEC) (Vitrogen; Collagen Biomaterials, Palo Alto, CA). BEAS-2B cells were used from passages 38–43. PBEC were used only at their first passage. Cells were cultured at 37⬚C with 5% CO2 in humidified air. When cells reached 80% confluence, they were stimulated with 100 ng/ml of human recombinant TNF-␣ (R&D Systems, Minneapolis, MN), 100 ng/ml of IFN-␥ (Genzyme, Cambridge, MA), 50 ng/ml of IL-4 (R&D), TNF-␣ plus IFN-␥, or TNF-␣ plus IL-4 for 18 h (for Northern blotting and real-time polymerase chain reaction [PCR]) or 24 h (for flow cytometry and Western blotting). Cells were harvested with 0.02% trypsin/ethylenediamine tetraacetic acid (EDTA) in HBSS for BEAS-2B, or with 0.02% EDTA in HBSS for PBEC, and washed three times in HBSS, with a recovery of ⬃ 5–8 million cells/flask. The viability of both BEAS-2B cells and PBEC was consistently ⬎ 95% of the cells harvested.

Cloning of Human B7-H2, B7-1, and B7-2 The complementary DNA (cDNA) sequences were derived from accession nos. AB014553 for B7-H2, M27533 for B7-1, and L25259 for B7-2 in the NCBI database. The primers for B7-H2, B7-1, and B7-2 were designed as follows: B7-H2 forward, 5⬘-GGCCCGAG GTCTCCGCCCGCACCAT-3⬘ (cDNA position 106 ⬃ 131); B7-H2 reverse, 5⬘-CATACAAGCCCCGCATGTTC-3⬘ (cDNA position 720 ⬃ 701); B7-1 forward, 5⬘-GTGGCAACGCTGTCCTGTGGT-3⬘ (cDNA position 450 ⬃ 470); B7-1 reverse, 5⬘-CCAGGAGAGGT GAGGCTC-3⬘ (cDNA position 842 ⬃ 825); B7-2 forward, 5⬘-CCA AAGCCTGAGTGAGCTAGT-3⬘ (cDNA position 247 ⬃ 267); B7-2 reverse, 5⬘-CTTAGGTTCTGGGTAACCGTG-3⬘ (cDNA position 607 ⬃ 587). The primers for B7-1 and B7-2 were designed using published cDNA sequences (38). An aliquot of total cellular RNA (1 ␮g) from BEAS-2B cells and peripheral blood mononuclear cells (PBMC) of normal subjects was reverse transcribed to cDNA in the presence of MuLV reverse transcriptase (2.5 U/ml), 1 mM each of dNTP (dATP, dCTP, dGTP, dTTP), RNase inhibitor (1 U/ ml), oligo(dT)16 (2.5 ␮M), 10⫻ PCR buffer (500 mM KCl and 100 mM Tris-HCl, pH 8.3), and MgCl2 (5 mM) to a total volume of 50 ␮l. All reagents for reverse transcriptase (RT)-PCR, except primers, were purchased from Perkin-Elmer Co. (Norwalk, CT). The RT mix was incubated at 25⬚C for 10 min and 42⬚C for 20 min (conducted in a Perkin-Elmer thermocycler) for reverse transcription, followed by 99⬚C for 5 min to inactivate the reverse transcriptase. An aliquot of the RT product was subjected to PCR in the presence of a master mix containing PCR buffer, MgCl2 (final concentration, 1.5 mM for B7-H2 and B7-1, 2.5 mM for B7-2), dNTPs (final concentration, 0.2 mM of each), AmpliTaq Gold polymerase (1 U/50 ␮l of reaction; Perkin-Elmer), and paired primers for B7-H2, B7-1, and B7-2 (0.4 ␮M of each primer) to a total volume of 50 ␮l. PCR was conducted for 30 cycles under the following conditions: denaturation at 94⬚C for 1 min, annealing at 55⬚C for 2 min, and extension at 72⬚C for 2 min. The individual cycles were performed by hot start PCR (an initial denaturation of 10 min at 94⬚C). RNA from normal PBMC, which was not reverse transcribed, was used as a negative control. A 10-␮l aliquot of the PCR product was electrophoretically separated on a 1.7% agarose gel (Sigma Chemical Co., St. Louis, MO) containing ethidium bromide (5 ␮g/ml; GIBCO BRL). Ultraviolet-induced fluorescence of ethidium bromide was used for visualization of amplified products. The correct size of the bands was determined by comparison with a DNA mass standard (1 kB DNA ladder; GIBCO BRL). The band was isolated from the agarose gel and purified using a QIA EX II Gel Extraction Kit (QIAGEN, Santa Clarita, CA). The PCR-amplified products were cloned directly into TOPO TA cloning vector, pCR2.1-TOPO (Invitrogen, San Diego, CA) as HindIII-XbaI fragments and the chemically competent cells, TOP 10 F’ (Invitrogen), were transformed with the cloning vectors according to the manufacturer’s instructions. The plasmid DNAs were purified by QIA prep Spin Miniprep Kit (QIAGEN) from the transformed competent cells and used to make the cDNA probes for Northern blotting. The DNA sequences of B7-H2, B7-1, and B7-2 were verified by the dideoxy method (Johns Hopkins DNA Analysis Facility, Baltimore, MD).

RNA Extraction and Northern Blot Analysis BEAS-2B cells and PBEC were incubated with or without cytokines for 6 or 18 h, were washed with ice-cold HBSS, and total cellular RNA was extracted using the TRIzol reagent (GIBCO BRL) according to the manufacturer’s instructions. Briefly, cells were lysed in the presence of TRIzol and chloroform. The RNA was precipitated at ⫺20⬚C in isopropanol overnight. The RNA pellet was recovered by centrifugation at 4⬚C, washed once in 70%

Kurosawa, Myers, Chen, et al.: Expression of B7-H2

ethanol, dried, and suspended in diethyl-pyrocarbonate-treated water. The resulting RNA was quantitated using spectrophotometry. The RNA was stored at ⫺70⬚C until use. Ten micrograms each of the total RNA samples were run in parallel on the same 1% agarose/6% formaldehyde gel. Particular effort was made to maintain equal loading of RNA by measurement of the RNA concentration spectrophotometrically and comparing the results with the intensity of ethidium bromide–stained 28S and 18S RNA bands observed with 1 ␮g of total RNA on a 1% agarose gel. After separation by electrophoresis, RNA samples were transferred onto a single piece of GeneScreen Plus nylon membrane (Dupont-New England Nuclear, Boston, MA) by capillary blotting overnight. After the transfer, the membrane was cross-linked and prehybridized for 45 min at 65⬚C, and then hybridized for 90 min with the indicated 32P-labeled cDNA probes using Quick Hyb hybridization solution (Stratagene, La Jolla, CA). Eighty nanograms of cDNA probes for B7-H2, B7-1, B7-2, and glyceraldehyde 3⬘-phosphate dehydrogenase (GAPDH) cDNA were labeled using the random priming method (Prime-a-Gene Labeling System; Promega, Madison, WI). This procedure yields 32P-labeled probe with comparable specific activities, and the same amount of radioactive probes (1 ⫻ 106 cpm/ml of hybridization buffer) was used in each case. After hybridization, membranes were washed with 2⫻ SSC/0.2% SDS at room temperature (three times, 15 min each time) and then at 55⬚C (three times, 15 min each time). Membranes were then exposed to x-ray film for equal exposure times. The cDNA probes for B7-H2 (615bp), B7–1 (393bp) and B7–2 (361bp) used for Northern blotting were obtained by PCR amplification using the insert-containing vectors as templates. The cDNA probe for GAPDH (housekeeping gene) used as a control was 1100bp (Clontech, Palo Alto, CA).

Flow Cytometric Analysis The monoclonal antibodies used for flow cytometry were: monoclonal antibody antihuman B7-H2 (clone 2D3, mouse IgG2b; Wang and coworkers, unpublished data) monoclonal antihuman B7-1 (mouse IgG1) (PharMingen, San Diego, CA) and monoclonal antihuman B7–2 (mouse IgG1; PharMingen). A fluorescein isothiocyanate–conjugated goat F(ab⬘)2 antimouse Ig (Biosource, Camarillo, CA) was used as the secondary antibody. Cells were grown to 80% confluence in 6-well culture plates. In the experiments assessing the effect of cytokines, cells were incubated with cytokines for 24 h. Cells were washed three times with Ca2⫹- and Mg2⫹free HBSS and treated for 20 min with Ca2⫹- and Mg2-free HBSS containing 0.02% EDTA without trypsin, and then removed from plates by repeated pipetting. For each analysis, 106 cells were incubated in 60 ␮l of phosphate-buffered saline (PBS)/0.2% BSA/ 0.1% NaN3 containing saturating concentrations of each monoclonal antibody and 4 mg/ml of human IgG (Sigma Chemicals), to reduce nonspecific binding) on ice for 30 min. The cells were washed, resuspended in saturating amounts of fluorescein isothiocyanate–conjugated goat F(ab⬘)2 antimouse IgG antibody for another 30 min, and then washed again, resuspended in the buffer, and immediately analyzed with a FACS Calibur flow cytometer (Becton Dickinson, Mountainview, CA) using CellQuest software. Fluorescence was determined on all cells for each sample after debris, dead cells, and aggregates were excluded by forward angle and side scatter gating. Mean fluorescence intensity (MFI) was compared with control staining using an irrelevant isotype-matched mouse monoclonal antibody. For each sample, at least 10,000 events were collected, and histograms were generated. Data are expressed as means ⫾ SD.

Real Time PCR Analysis of B7-H2 mRNA expression was performed using real time quantitative PCR (Taqman). In brief, an oligonucleotide probe

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was designed to anneal to the B7-H2 gene between two PCR primers. The probe was then fluorescently labeled with FAM (reporter dye) on the 5⬘ end and with TAMRA (quencher dye) on the 3⬘ end. A similar probe and PCR primers were designed for human GAPDH. The probe for this gene incorporated JOE as the reporter dye. PCR reactions were run that included the primers and probes for these two genes as well as cDNA made from BEAS-2B and PBEC. Amplification was monitored as fluorescent emission detected using the ABI PRISM Sequence Detector 7700 (Applied Biosystems, Foster City, CA). Data are displayed as the number of amplification cycles required to reach an equivalent number of B7-H2 or GAPDH mRNA copies as determined using the specific probes. BEAS-2B cells (n ⫽ 2) and PBEC (n ⫽ 3) were cultured for 18 h with or without cytokines and subjected to total RNA extraction with the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Contaminating genomic DNAs was removed using RNase-Free DNase Set Protocol (QIAGEN). Samples were subjected to reverse transcription and Real Time PCR amplification with primers specific to the human B7-H2 gene (accession no. AB014553) (forward, 5⬘-GCAAACCAGTGAGTCGAAAACC-3⬘; reverse, 5⬘-GGTGACATCAGGGCTCGGT-3⬘) along with an internal reporter probe containing FAM and TAMRA fluorochromes (5⬘-FAMCTACCACATCCCACAGAACAGCTCCTTGG-TAMRA-3⬘; Biosource) by use of the Taq Man kit (Perkin-Elmer). Reaction conditions were as follows: 95⬚C for 10 min; 40 cycles at 95⬚C for 15 s/60⬚C for 1 min. Similarly, primers were designed and used in traditional RT-PCR for other B7 homologs. Primers for B7-H1 (NCBI accession no.NM_014143) are forward (5⬘-ACTGGCATT TGCTGAACGCA) and reverse (5⬘-TACACCCCTGCATCCT GCAAT). Primers for B7-H3 (NCBI accession no. NM_025240) are forward (5⬘-AGCACTGTGGTTCTGCCTCACA) and reverse (5⬘-CACCAGCTGTTTGGTATCTGTCAG). Primers for B7-DC (NCBI accession no. AF344424) are forward (5⬘-AGGCCTCGTT CCACATACCTCA) and reverse (5⬘-GTGGCTTTAGGCGCA GAACACT). Reaction conditions were identical to those listed for real time PCR analysis.

Immunoprecipitation and Immunoblotting by Western Blot For immunoprecipitation, cells were either unstimulated, or stimulated with cytokines for 24 h and harvested as described above. The cells were resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Nonidet P-40, 1 mM PMSF, 0.2 mg/ml leupeptin). The lysates were incubated for 15 min on ice before centrifugation at 15,000 ⫻ g for 20 min at 4⬚C and the supernatant was collected as the whole cell lysate. The whole cell lysates or 1 ␮g of the control protein (human B7-H2 Fc-fusion protein with human IgG1) was incubated for 1 h with gentle rotation at 4⬚C with the monoclonal antibody antihuman B7-H2 (mouse IgG1, 1:100 dilution). Protein G-Sepharose beads (50 ␮l; Amersham, Arlington Heights, IL), equilibrated and washed in lysis buffer, were added and incubated with gentle rotation at 4⬚C overnight. The beads were washed three times in cold lysis buffer, and proteins were eluted by boiling for 5 min in SDS sample buffer, and then separated by 10% tris-glycine gradient gel electrophoresis (NOVEX, San Diego, CA) and transferred to a polyvinyllidine difluoride membrane (BIO-RAD, Hercules, CA). The membrane was blocked with 5% nonfat milk powder in TBST (50 mM Tris, 0.15 M NaCl, and 0.05% Tween 20), incubated with monoclonal antibody anti-B7-H2 in TBST with agitation at 4⬚C overnight, and then incubated with anti-mouse Ig Ab (Amersham) for 1 h after washing in TBST. After extensive further washing with TBST, chemiluminescent substrate was added (ECL Western blot detection system; Amersham), and the membrane was

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Figure 1. Flow cytometric analysis of B7-H2 expression on airway epithelial cells. BEAS-2B cells or PBEC were cultured in the presence of control medium, TNF-␣ (100 ng/ml), IFN-␥ (100 ng/ml), IL-4 (50 ng/ml), TNF-␣ plus IFN-␥, or TNF-␣ plus IL-4 for 24 h. After culture, cells were stained with a monoclonal antibody to B7-H2 (solid line and filled curve) or an isotype-matched control mAb (dotted line and open curve). Representative results of three or more independent experiments are shown.

subjected to autoradiography. Although molecular weight standards were included, the intensity of the B7-H2 bands detected was so high that the standards were not visible at the exposure time used. The figure containing this data therefore only presents the B7-H2 bands.

Immunofluorescent Staining Lungs from human organ donors were procured and shipped by The National Disease Research Institute (Philadelphia, PA). Tis-

sues were shipped on ice (4⬚C) and received 12–24 h after organ removal. Once received, 1-cm blocks of lung, having 2–6 mm diameter airways, were immediately fixed in 4% formaldehyde in PBS (4⬚C, 4 h) and then rinsed with PBS. Tissues were cryoprotected overnight in 18% sucrose in PBS, covered with mounting medium and frozen in liquid nitrogen. Sections (12 ␮m) were cut on a cryostat, collected on lysine-coated slides, and air dried. Sections were incubated with blocking solution containing 1% bovine serum albumin, 10% normal goat serum, and 0.1% Tween 20 in PBS for 1 h at room temperature, and then incubated (24 h,

Kurosawa, Myers, Chen, et al.: Expression of B7-H2

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Figure 2. Flow cytometric analysis of B7-1 expression on airway epithelial cells. BEAS-2B cells or PBEC were cultured as in Figure 1. After culture, cells were stained with monoclonal antibody to B7-1 (solid line and filled curve) or an isotype-matched control mAb (dotted line and open curve). Representative results of three or more independent experiments are shown.

4⬚C) with 1 ␮g of ICOS-Ig (human IgG1) or 1 ␮g of monoclonal antibody to human B7-H2 (mouse IgG1). Slides were washed in PBS and incubated with labeled anti-human (ICOS) or anti-mouse (B7-H2) antibodies raised in goat (Alexa 488 [fluorescein] or Alexa 594 [rhodamine]; Molecular Probes, Eugene, OR) for 2 h at room temperature. Separate slides were processed similarly but the primary antibody was excluded to evaluate nonspecific staining. Washed slides were coverslipped with anti-fade glycerol (Molecular Probes) and photographed with an epifluorescence microscope (Olympus BX 60; Olympus America Inc., Melville, NY) equipped with a filter set to allow visualization of fluorescein or rhodamine. Sections were photographed with Kodak TMY 400 film and the slides were then scanned and digitized (SprintScan 35; Polaroid Corp., Cambridge, MA) to make positive micrographs.

Results Surface Protein Expression of Costimulatory Molecules B7-H2, B7-1, and B7-2 on Human Bronchial Epithelial Cells and Regulation by Stimulation with Cytokines To determine whether human bronchial epithelial cells can express B7-H2, B7-1, and B7-2, surface expression was examined on BEAS-2B and PBEC by flow cytometry. Both BEAS-2B cells and PBEC spontaneously and strongly expressed B7-H2 on their cell surface (Figure 1). The pattern of expression was frequently bimodal, with low and high expressing populations. On average, 72.5% ⫾ 7.0 of BEAS-2B cells and 61.1% ⫾ 25.6 of PBEC stained positively for B7-H2 expression. Surprisingly, treatment by vari-

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Figure 3. Flow cytometric analysis of B7-2 expression on airway epithelial cells. BEAS-2B cells or PBEC were cultured as in Figure 1. After culture, cells were stained with monoclonal antibody to B7–2 (solid line and filled curve) or an isotypematched control mAb (dotted line and open curve). Representative results of three or more independent experiments are shown.

ous cytokines such as TNF-␣ (100 ng/ml), IFN-␥ (100 ng/ml), IL-4 (50 ng/ml), and various combinations downregulated the surface protein expression of B7-H2 when compared with the unstimulated epithelial cells in BEAS-2B cells (% reduction of MFI: 58.3% ⫾ 5.4 with TNF-␣, 29.25% ⫾ 5.7 with IFN-␥, 63.3% ⫾ 14.5 with IL-4, 75.5% ⫾ 4.5 with TNF-␣ ⫹ IFN-␥, 79.5% ⫾ 4.2 with TNF-␣ ⫹ IL-4) and in PBEC (% reduction of MFI: 38.3% ⫾ 3.51 with TNF-␣, 62.3% ⫾ 9.1 with IFN-␥, 49.3% ⫾ 2.3 with IL-4, 89.7% ⫾ 0.6 with TNF-␣ ⫹ IFN-␥, 73.0% ⫾ 7.6 with TNF-␣ ⫹ IL-4). The reduction was due almost exclusively to a shift in the proportion of cells from the high expressing population to the low expressing population. Neither type of bronchial epithelial cells expressed B7-1 and B7-2 on their cell surface,

and cytokine stimuli failed to induce B7-1 and B7-2 expression (Figures 2 and 3). In freshly isolated peripheral blood mononuclear cells, 12.4% ⫾ 4.8, 3.9% ⫾ 3.0, and 12.7% ⫾ 5.4 stained positively for B7-H2, B7-1, and B7-2, respectively (data not shown). B7-H2, B7-1, and B7-2 mRNA Expression in Human Bronchial Epithelial Cells To determine whether the downregulation of B7-H2 by cytokines was due to reduced levels of B7-H2 mRNA, we next examined cytokine effects on expression of mRNA for B7-H2, B7-1, and B7-2 in BEAS-2B cells and PBEC by Northern blot analysis. As shown in Figure 4, both unstimulated BEAS-2B cells (A ) and PBEC (B ) spontane-

Kurosawa, Myers, Chen, et al.: Expression of B7-H2

Figure 4. B7-H2, B7–1 and B7–2 mRNA expression in (A) BEAS-2B cells and (B ) PBEC. Cells were cultured as in Figure 1 except only for 18 h, and total cellular RNA was extracted for Northern blot analysis (10 ␮g/lane). Blots were hybridized with cDNA probes for B7-H2, B7-1, B7-2, or GAPDH. A representative experiment from two different experiments is shown. Cytokine treatment for 6 h showed a similar result (data not shown).

Figure 5. Real Time PCR analysis (Taqman) of B7-H2 mRNA expression and GAPDH mRNA expression in BEAS-2B and PBEC. BEAS-2B cells (n ⫽ 2) and PBEC (n ⫽ 3) were cultured as in Figure 4 for 18 h followed by RNA extraction. Shown is the number of amplification cycles (CT ) required to reach an equivalent number of B7-H2 or GAPDH mRNA copies as determined using specific probes. Open squares, medium; open circles, TNF-␣; open triangles, IFN-␥; open diamonds, IL-4; filled circles, TNF-␣/IFN-␥; filled triangles, TNF-␣/IL-4.

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Figure 6. RT-PCR of B7 homolog expression. RNA from epithelial cell line BEAS-2B (n ⫽ 2) was isolated and reverse transcribed. A dilution of cDNA was used in a PCR reaction containing primers for four B7 homolog RNAs. The housekeeping gene GAPDH and a negative control were also included in the reaction. Amplification products were run on a 1.5% agarose gel yielding the following sizes: GAPDH, 226 bp; B7-H1, 300 bp; B7-H2, 100 bp; B7-H3, 171 bp; B7-DC, 301 bp.

ously and strongly expressed B7-H2 mRNA. B7-1 mRNA was also detected in unstimulated BEAS-2B cells and PBEC; however, the expression level of B7-1 mRNA was weak as compared with that of B7-H2 mRNA. Cytokine stimulation for 6 (data not shown) or 18 h (Figure 4) had no readily apparent effects on B7-H2 and B7-1 mRNA expression in BEAS-2B cells and PBEC, despite the downregulation of surface protein expression of B7-H2 shown in Figure 1. No B7-2 mRNA was detected in BEAS-2B cells or PBEC. Real time quantitative PCR analysis (Taqman) was performed to confirm the mRNA expression of B7-H2 in human bronchial epithelial cells using PBEC from three independent donors and in BEAS-2B cells (n ⫽ 2). In agreement with the Northern blot analysis, the Taqman data reinforced the detection of constitutive mRNA expression of B7-H2 by Northern blot in PBEC and BEAS-2B cells, and the lack of effect of cytokines (Figure 5). To extend our analysis of B7 homolog expression in airway epithelial cells, we designed primers for B7-H1, B7-H2, B7-H3, and B7-DC. Data in Figure 6 show that BEAS-2B cells express mRNA for all four of these homologs, and that in each case the amplified mRNA was of the expected size. These data suggest that BEAS-2B cells may express several different B7 homologs. In preliminary studies, we have detected B7-H3 by flow cytometry (data not shown). Detection of B7-H2 in Human Bronchial Epithelial Cells by Western Blot Analysis The observation that cytokine treatment of human bronchial epithelial cells resulted in downregulation of B7-H2, as detected by flow cytometry on the cell surface, prompted

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Figure 7. (A ) Basal expression of B7-H2 protein in BEAS-2B cells and PBEC determined by Western blot. Whole-cell proteins were extracted from unstimulated BEAS2B cells and PBEC. B7-H2 was precipitated from the extracts before Western blotting as described in Materials and Methods. The extracts and control B7-H2 protein were electrophoresed and analyzed by Western blot using the monoclonal antibody to human B7-H2. (B ) Expression of B7-H2 protein determined by Western blot. Wholecell proteins extracted from PBMC, Jurkat cells, and BEAS-2B cells previously treated with control medium, TNF-␣ (100 ng/ml) plus IFN-␥ (100 ng/ml), or TNF-␣ (100 ng/ml) plus IL-4 (50 ng/ml) for 24 h were electrophoresed and analyzed using the monoclonal antibody to human B7-H2. A single band was detected in all cases.

us to examine whether cytokine stimuli could regulate the apparent level of B7-H2 protein expression in BEAS-2B cells. We therefore immunoprecipitated B7-H2 from control cells or cells stimulated as shown in Figure 7A. Analysis using Western blotting showed strong basal expression of B7-H2 in whole cell lysates of unstimulated BEAS-2B cells and PBEC. These results were consistent with the spontaneous expression of B7-H2 on the cell surface of BEAS-2B cells and PBEC detected by flow cytometry. However, treatment of BEAS-2B cells with TNF-␣ (100 ng/ml) plus IFN-␥ (100 ng/ml), or TNF-␣ plus IL-4 (50 ng/ml), for 24 h did not markedly alter the apparent level of B7-H2 protein expression (Figure 7B). As was observed with flow cytometry, there was a trend for some of the cytokines to reduce B7-H2 (this was most apparent with the combination of TNF-␣ and IL-4; see Figures 1 and 7B). B7-H2 was also detected in control samples consisting of whole cell lysates of PBMC and the Jurkat human T cell leukemia cell line. B7-H2 Expression in Human Lung Tissue Examined by Immunofluorescent Staining Immunofluorescent staining was performed to confirm B7-H2 expression in human lung tissue. Immunohistochemical analysis of airway derived from organ donors using either a monoclonal antibody to human B7-H2 or ICOSIg, which binds specifically to B7-H2 revealed the presence of B7-H2 (Figure 8). Bright staining for B7-H2 was observed in epithelium with both reagents but not in the control.

Discussion Airway epithelial cells are responsible for maintenance of a tight barrier that protects underlying tissue from the external environment and have been described classically as barrier cells. However, recent evidence suggests that airway

epithelial cells might also act as immune effector cells in response to endogenous or exogenous stimuli (1–4, 34) and play a key role in the immunologic interaction between the lung and external environment, being the primary interface for antigenic materials in immune and inflammatory responses. Several studies have shown that airway epithelial cells express and secrete various molecules involved in inflammation and immunity, including MHC class I (5, 6), CD40 (30), inflammatory lipids (39), oxygen radicals (40), adhesion molecules (41–43), and a wide variety of cytokines and chemokines(1–4, 34). In addition, airway epithelial cells, in certain circumstances, can be considered to be nonprofessional antigen-presenting cells because these cells bear MHC class II (6–8). However, CD4⫹ T cell activation, proliferation, and cytokine production require two distinct signals from the antigen-presenting cells (14, 44). The first signal is triggered by interaction of the antigen-specific TCR with the MHC class II-peptide complex, and the second signal is derived from costimulatory molecules. The most widely studied costimulators are CD28, ICOS, and CTLA-4 expressed on T cells and their ligands B7-1 (CD80), B7-2 (CD86), and B7-H2 (ICOS ligand) expressed on several types of antigen-presenting cells, including monocyte/macrophages, dendritic cells, activated B cells, keratinocytes, and some activated T cells (9–18). When considering airway epithelial cells as potential antigen-presenting cells, it is crucial to first clarify the expression of the appropriate costimulatory molecules to better understand the mechanism by which they may regulate immunologic responses during the development of immune-mediated diseases such as allergic asthma. We demonstrate in this study that airway epithelial cells express B7-H2 but little or no B7-1 or B7-2. B7-H2 is a relatively recently recognized member of the B7 family and is expressed on B cells and macrophages

Kurosawa, Myers, Chen, et al.: Expression of B7-H2

Figure 8. Airway epithelial cells express B7-H2 in vivo. Lungs from organ donors were quick-frozen in liquid nitrogen and sectioned. Sections of human airway were analyzed by immunofluorescent staining using ICOS-Ig (A ), monoclonal antibody to B7-H2 (B ), or control (C ) followed by appropriated secondary antibody conjugated to rhodamine. Section shown as control illustrates nonspecific staining excluding the primary antibodies. Representative results of two separate experiments are shown. Airway lumen is indicated by the letter L and epithelium is indicated by the letter E.

(19–23). B7-H2 binds its receptor, ICOS, expressed on antigen-primed T cells such as activated T cells and memory T cells (24–28). Recent studies indicate that the ICOS/ B7-H2 pathway delivers the costimulatory signals for IL-4 and IL-10 production, T cell proliferation, and CD40 ligand upregulation (24–28, 45). In addition, growing data suggest that ICOS/B7-H2 costimulation contributes to Th2-associ-

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ated inflammatory responses not only through the regulation of IL-4, but also by providing a signal for upregulation of chemokine receptors. Gonzalo and coworkers reported that blocking the ICOS/B7-H2 interaction using ICOS-Ig and/or a neutralizing ICOS monoclonal antibody attenuated T cell expansion, Th2 cytokine production, eosinophilic inflammation, and Th2-associated expression of chemokine receptors such as CCR3, CCR4, and CCR8 in allergic lung inflammation (46). On the other hand, studies using mice genetically lacking ICOS indicate that a lack of ICOS results in severely deficient T cell–dependent B cell responses, including impairments in germinal center formation, immunoglobulin class switching, and production of allergy-mediating IgE (45, 47, 48). Therefore, it has been speculated that the ICOS/B7-H2 pathway may play an important role ¨ zkaynak and colleagues (49) in T cell–B cell interactions. O and Rottman and coworkers (50) showed that blockade of ICOS signaling led to increased heart allograft acceptance and prevented experimental autoimmune encephalomyelitis. These studies clearly provide evidence that ICOS may regulate not only Th2 responses, but also Th1 responses. Flow cytometric evaluation of epithelial cells revealed that B7-H2 was constitutively and strongly expressed on both BEAS-2B and PBEC, whereas B7-1 and B7-2 were undetectable on either epithelial cell type. B7-H2 expression was confirmed by Western blot using a specific antibody. Stimulation with various cytokines, including TNF-␣, IFN-␥, and IL-4, downregulated B7-H2 expression detected by flow cytometry. This was most clear when TNF-␣ and IL-4 were used in combination. This effect was present but less apparent in studies of Western blotting (Figure 7). This discrepancy between flow cytometry and Western blotting may reflect the presence of an intracellular pool of B7-H2. Northern blotting detected mRNA for B7-H2 and B7-1, but not B7-2, and the identity of B7-H2 was confirmed by cloning and sequencing the BEAS-2B–derived cDNA. Expression of B7-H2 mRNA was detected in PBEC from three independent donors. Immunohistochemical analysis of airway derived from autopsies revealed expression of B7-H2 in airway epithelial cells. Taken together, these results unequivocally establish that epithelial cells express B7-H2. Our findings would support the hypothesis that airway epithelial cells may play a role as antigen-presenting cells during the development of airway and inflammatory immune responses. Despite a report that B7-1 and B7-2 are induced and expressed at significant levels in primary bronchial cells and A549, a type II respiratory cell line (5), we failed to detect expression of either B7-1 or B7-2 in our studies. In agreement with our findings, Cunningham and colleagues showed that human alveolar epithelial cells fail to express B7-1 and B7-2 (51). A recent study by Wahl and coworkers demonstrated B7-H2 expression on renal tubular epithelial cells, suggesting that B7-H2 expression on epithelium may not be limited to the airways (52). ICOS predominates over CD28 in regulating cytokine production from recently activated T cells, but not from naive T cells where CD28-dependent signaling predominates (27). In light of the data presented here, we speculate that activated T lymphocytes expressing ICOS may encounter and interact with B7-H2 expressed on airway epithelial

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cells, resulting in maintenance of activation in the airways in the presence of continued antigen exposure. Our results showed that BEAS-2B and PBEC do not express B7-1 and B7-2. This finding further suggests that B7-H2 may provide a primary costimulatory signal on airway epithelial cells. There is increasing evidence that allergic asthma, a chronic airway inflammatory disease characterized by reversible airway obstruction, hyperreactivity, and lymphocyte/eosinophil recruitment, is driven and maintained by chronically activated T cells with a Th2 phenotype. These T cells promote the activation and recruitment of B cells and eosinophils and regulate the Ig class switch to the development of antigen-specific IgE responses (53–56). Although there is as yet no evidence that B7-H2 expressed on airway epithelial cells can productively interact with ICOS on T lymphocytes in vivo or in vitro, identification of B7-H2 expressing resident cells within the airway tract other than alveolar macrophages and dendritic cells expands the possible mechanisms that underlie T cell activation in airway inflammation. Acknowledgments: The authors thank Ms. Bonnie Hebden for excellent assistance in the preparation of this manuscript. This work was supported by National Institutes of Health Grants RO1 HL68546, AI50530, CA79915, and CA85721.

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