Expression of genes for B7-H3 and other T cell ligands by nasal epithelial cells during differentiation and activation

Bahman Saatian,1 Xiao-Ying Yu,1 Andrew P. Lane,2 Thanh Doyle,1 Vincenzo Casolaro,3 and Ernst Wm. Spannhake1

1Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health; and Departments of 2Otolaryngology - Head and Neck Surgery and 3Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Submitted 1 May 2003 ; accepted in final form 16 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial cells of the human respiratory tract express human leukocyte antigen (HLA) and the costimulatory molecules B7-1 and B7-2. Little is known, however, about the constitutive expression of genes encoding for the more recently identified members of the B7 homolog family of costimulatory molecules or about the influence of cellular differentiation and cytokines on their activity or on that of HLA or B7-1 and B7-2. Human nasal epithelial (HNE) cells were grown at the air-liquid interface (ALI) for 2 or 21 days to model in vivo conditions. Expression of genes for HLA-B and HLA-DR1 increased during mucociliary differentiation during this period and became more similar to HNE cells obtained fresh by brush biopsy from nasal turbinates. Gene transcripts for B7-H3 and B7-H2 were abundantly expressed in cells cultured at the ALI, but neither their activities nor that of B7-2 was significantly altered during differentiation. IFN-{gamma} and TNF-{alpha} upregulated mRNA encoding for both HLA molecules, but not for the B7 molecules. This study describes, for the first time, the expression of B7-H3 and B7-H2 by HNE cells and thus expands the range of potential costimulatory signals through which these cells may interact with activated mucosal T lymphocytes. In addition, the results suggest that the extent of mucociliary differentiation of cultured cells may influence this capability.

primary cell culture; human; air-liquid interface; interferon-{gamma}; tumor necrosis factor-{alpha}


CLOSE INTERACTIONS of epithelial cells of the upper and lower respiratory tract with inflammatory and other migrating cells within the mucosal regions are important in homeostasis and host defense. Respiratory epithelial cells are well adapted to engage in these interactions by virtue of their expression of a wide range of surface binding proteins (e.g., 29, 31) that permit attachments to, and potential activation of, neutrophils, T and B lymphocytes, monocytes, and mast cells. During growth and differentiation of epithelial cells as part of normal repair processes, variation in the expression of genes coding for proteins that control cell-cell interactions may play a role in modulating inflammation and host defense (7, 12, 36). The influence of growth and differentiation on the activity of relevant genes also represents an important consideration in studies of cellular interaction in which culture systems are used to model in vivo processes.

Of particular functional importance in allergic diseases of the respiratory tract are those molecules essential for the activation of memory T cells that carry out surveillance within the epithelial mucosa. Fundamental among these are the major histocompatibility complex (MHC) class I and II proteins that ligate the T cell receptor (TCR) and the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) that bind CD28 and CTLA4 on the T cell. Whereas MHC class I is constitutively expressed by almost all cells as a part of the natural immune surveillance system, MHC class II is generally expressed on cells in association with their ability to digest foreign proteins as a part of the antigen presentation process. Class II molecules have been demonstrated on epithelial cells of the upper (17, 43) and lower (17, 32, 34) airways. Although constitutive expression of this molecule is not consistently detectable on cells from unstimulated sources (49), reports indicate that expression of the class II molecule, human leukocyte antigen (HLA)-DR, is increased on epithelial cells in several airway inflammatory conditions, including on nasal epithelium in allergic rhinitis (33) and on bronchial epithelium in chronic bronchitis (30) and bronchial asthma (2, 42). Importantly, treatment of epithelial cells with interferon (IFN)-{gamma}, a cytokine released by lymphocytes and present in varying concentrations in the airways (14), has been demonstrated to significantly increase and sustain expression of HLA-DR on primary bronchial epithelial cells and cell lines (17, 26, 32, 33). Several of these studies offer evidence in allogeneic coculture systems that airway epithelial cells are capable of providing the accessory stimulation required for T cell activation (17, 34, 49). This ability was presumed to be due, at least in part, to the activity of the costimulatory molecules, B7-1 and B7-2, which are known to be sufficient in combination with MHC I or II to promote activation and/or proliferation of both CD4+ and CD8+ T cells (22, 41). Constitutive expression of B7-1 and B7-2 has been observed in nasal, airway, and alveolar epithelial cells (34, 49), and antibodies against these molecules have been demonstrated to block proliferation of T cells in coculture with MHC matched and unmatched nasal and airway epithelial cells (34). Although human airway epithelial cell-T cell interactions in a syngeneic in vitro system have yet to be investigated in detail, studies to date are strongly suggestive that such interactions may occur in the airways in vivo, as have been described in the gut (11).

Recently, several new B7-like proteins have been identified that mediate costimulatory signals primarily to previously activated CD4+ and CD8+ cells through interaction with receptors other than CD28 or CTLA4 (4, 40, 45). One of these, B7-H1, binds to the programmed death (PD)-1 receptor (8). Experiments in which this interaction was blocked resulted in enhanced expression of IL-2 and IFN-{gamma}, suggesting that B7-H1 provides negative regulation of these cytokines in T cells (24). Another B7 homolog, B7-H2 (B7h/B7RP-1), interacts with the inducible costimulator (ICOS) receptor on T cells. Ligation of this receptor stimulates cellular proliferation and the release of IL-2 (CD8+ cells), IL-4, IL-5, IL-10 (CD4+ cells), IFN-{gamma}, TNF-{alpha}, and granulocyte/macrophage colony-stimulating factor (13, 45). In contrast, the more recently described B7-H3 molecule, which appears to interact with a receptor other than ICOS or PD-1, was described in early studies to costimulate the proliferation of both CD4+ and CD8+ cells, enhances the induction of cytotoxic T cells, and selectively stimulates IFN-{gamma} production by T cells (4). Recent studies have proposed a downregulatory function for B7-H3 (37, 38) and expanded the potential role of this ligand in antitumor immunity (39).

In the present study, we determined the constitutive expression of genes for the MHC I and II molecules, HLA-B and HLA-DR1, and the costimulatory molecules, B7-1, B7-2, and B7-H2, in human nasal epithelial cells and, for the first time, describe the presence of high levels of message coding for the newly identified B7-H3 molecule in cells of the respiratory epithelium. To evaluate the activity of genes for these molecules during growth and differentiation in vitro, we compared expression in cells obtained by brush sampling of the inferior turbinates of human volunteers with that in turbinate epithelial cells grown in culture at the air-liquid interface (ALI) for 2 days or for 21 days, to induce mucociliary differentiation. In addition, we investigated the ability of cytokines involved in cellular signaling within the epithelial mucosa to modulate the expression of these genes in the differentiated cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human subjects. Consistent with consent procedures approved by the Committee on Human Research (CHR) of the Johns Hopkins Bloomberg School of Public Health, middle or inferior nasal turbinate tissue removed from 20 patients (11 male, 9 female) undergoing elective surgery for conditions including chronic rhinosinusitis was immediately placed in saline on ice and processed within 2 h of removal. Tissues selected for use were not polypoid, infected, inflamed, or abnormal by gross inspection. In accordance with CHR-approved protocols and after obtaining informed consent, we obtained fresh samples of nasal epithelium used for immediate extraction of RNA by nasal brushing from 12 normal volunteers (10 male, 2 female) without active symptoms of allergy, inflammation, or history of asthma.

Fresh nasal epithelial cells. Nasal epithelial cells were collected by brushing of the anterolateral surface of the inferior turbinate with a Cytosoft cytology brush (Medical Packaging, Camarillo, CA). The area around the turbinate was anesthetized by topical administration of 2% Tetracaine HCl before brushing. The brushes were washed in 1.0 ml of cold 1% PBS (Biofluids, Rockville, MD), and the cell suspensions were centrifuged at 14,000 rpm for 1 min. The supernatant was discarded, and TRIzol (Invitrogen, Carlsbad, CA) was added to the pellet and mixed well. Total RNA was immediately isolated as described in RNA isolation.

Nasal epithelial cell culture. Human nasal epithelial (HNE) cells were isolated following typical procedures, as previously described (35). After overnight digestion of the tissues at 4°C in 0.01% protease Sigma type XIV (Sigma, St. Louis, MO) in Ham's F-12 medium containing penicillin (100 U/ml; GIBCO, Gaithersburg, MD), streptomycin (100 µg/ml, GIBCO), amphotericin B (2.5 µg/ml, GIBCO), and gentamicin (50 µg/ml, GIBCO), the protease was neutralized by the addition of 10% fetal calf serum (Invitrogen), and the epithelial cells were freed from the tissue by agitation and isolated by centrifugation. The washed P (passage)0 HNE cells were then seeded, at a density ≥1.5 x 104 cells/cm2, onto Vitrogen 100-coated (1:75 in sterile water; Cohesion, Palo Alto, CA) P-100 dishes in bronchial epithelium growth medium (BEGM), as described by Bernacki and colleagues (1). This modified (16) LHC basal medium (Biofluids) contained (all sources Sigma, unless otherwise indicated) insulin (5 µg/ml), hydrocortisone (0.072 µg/ml), human epidermal growth factor (25 ng/ml, Upstate Biotechnology), triiodothyronine (6.5 ng/ml), retinoic acid (5 x 10–8 M), transferrin (10 µg/ml, GIBCO), epinephrine (0.6 µg/ml), phosphoethanolamine (0.5 µM), ethanolamine (0.5 µM), bovine pituitary extract (10 µg/ml, Upstate Biotechnology), bovine serum albumin (0.5 mg/ml), calcium (0.11 mM) from CaCl2·2H20, selenium (30 nM) from NaSeO3, manganese (0.6 nM) from MnCl2·4H20, silicon (0.5 µM) from Na2SiO3·9H20, molybdenum (1.0 nM) from (NH4)6Mo7O24·4H20, vanadium (5.0 nM) from NH4VO3, nickel (0.5 nM) from NiSO4·6H2O, tin (0.5 nM) from SnCl2·2H20, zinc (3.0 µM) from ZnSO4·7H20, iron (0.15 µM) from FeSO4·7H20, magnesium (60 nM) from MgCl2·6H20, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). Upon reaching confluence, HNE cells were transferred to human type IV placental collagen (Sigma, type VI)-coated six-well Falcon filter inserts (0.4-µm pore size; Becton Dickinson, Franklin Lakes, NJ), and the P1 cells were grown to confluence with BEGM, above (1 ml) and below (2 ml) the cells. When confluent, medium was removed from above the cultures, and the medium below the inserts was changed to ALI medium consisting of LHC basal medium-DMEM-H (50:50, GIBCO) containing the same concentrations of additives as BEGM with the exception that the concentration of epidermal growth factor was reduced to 0.63 ng/ml, and amphotericin B was omitted. Each set of cultures came from a separate surgical source and was maintained at the ALI with the apical surfaces remaining free of medium for 2 days or 21 days before study. Each group of 2-day and 21-day cultures was matched, coming from a single tissue source. To confirm the epithelial cell composition of the primary cultures, we stained cells after 2 days at the ALI using a monoclonal mouse antibody against human epithelial cell-specific antigen (NCL-ESA; Vector Laboratories, Burlingame, CA) and mouse IgG1 as control. The presence of antigen on the cell surfaces was visualized with a Vectastain ABC kit with diaminobenzidine as a substrate followed by counterstaining with hematoxylin. Similarly stained cells of the WI-38 fibroblast line served as negative controls.

Treatment of HNE cells with cytokines. Differentiated cultures of cells grown at the ALI for 21 days were treated for 24 h with medium containing combinations of recombinant human (rh) IFN-{gamma} and rhTNF-{alpha} (R&D Systems, Minneapolis, MN) in the concentrations indicated or medium alone, by addition to the apical surfaces.

RNA isolation. Total RNA was isolated from cultured HNE cells by a standard method. One milliliter of TRIzol reagent was added to each six-well culture insert, and the cells were removed by scraping. After collection of the cells and TRIzol reagent in RNase-, DNase-free tubes and thorough mixing, the tubes were allowed to stand at room temperature for 5 min. After addition of 250 µl of chloroform (Sigma-Aldrich), the tubes were vortexed for 15 s and further incubated for 2–3 min at room temperature. They were then centrifuged at 12,000 rpm for 15 min, the aqueous phase was removed, and the RNA was precipitated with isopropanol (Sigma-Aldrich), washed with 70% ethanol, air-dried, and resuspended in 30–40 µl of diethyl polycarbonate water. RNA was quantified spectrophotometrically and visualized by staining with ethidium bromide to determine RNA integrity. Absorbance ratios at 260/280 nm were >1.80 for all samples studied.

Real-time RT-PCR. One-step RT-PCR was performed in a Light-Cycler with the SYBR Green QuantiTect RT-PCR Kit (Qiagen, Valencia, CA). To optimize the program for each target, we used RNA extracted from Raji cells, which express all genes of interest. 18S (sense 5'-GTAACCCGTTGAACCCCATT-3', antisense 5'-CCATCCAATCGGTAGTAGCG-3') was used as a housekeeping gene with which to normalize expression. The reaction mix consisted of 0.8 µg total RNA (target genes) or 0.08 µg total RNA (18S RNA), 10 µl QuantiTect SYBR Green PCR, 0.2 µl QuantiTect RT Mix, 1.5 µM target primers, or 1 µM 18S rRNA primers, in a total volume of 20 µl. For all samples, reverse transcription was carried out at 50°C for 20 min, followed by cycling to 95°C for 15 min to inactive the RT enzyme and activate the Taq polymerase. The sequences, amplicon sizes, and thermal cycles of the target genes are provided in Table 1. Amplicon expression in each sample was normalized to its 18S RNA content. Consistent use of the quantities of total RNA described above (800 and 80 ng for target molecules and 18S RNA, respectively) resulted in highly reproducible real-time PCR cycle thresholds for each of the amplicons across all cell samples. The relative abundance of target mRNA in each sample was calculated as 2 raised to the negative of its threshold cycle value times 106 after being normalized to the abundance of its corresponding 18S, similarly computed [e.g., 2–(HLA-B Threshold Cycle)/2–(HLA-B 18S Threshold Cycle) x 106]. Based on the presumption of amplicon doubling with each PCR cycle, the resulting values provide a more clear depiction of the relative abundance of various target molecules than do threshold cycle numbers themselves. Primers for HLA-DR1 and HLA-B were selected from the literature (Refs. 44 and 21, respectively), and the remaining were designed by inspection of the gene sequence, keeping amplicon sizes in the 200- to 300-bp range. All primers were commercially synthesized by Invitrogen. Negative controls, consisting of reaction mixtures containing all components except target RNA, were included with each RT-PCR run. After amplification, the products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. The identity of each product was confirmed by molecular weight profile and by subsequent DNA sequence analysis of the separated products isolated from the agarose gels. To verify that the amplified products were derived from mRNA and did not represent genomic DNA contamination, representative PCR reaction mixtures for each gene were run in the absence of the RT enzyme after first being cycled to 95°C for 15 min before PCR. In the absence of reverse transcription, no PCR products were observed. The presence of mRNA for CD11c and dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN, CD209), two markers selectively expressed by immature and mature dendritic cells (DCs) of myeloid origin (6, 23), was determined using primers for these gene products, as follows: CD11c: sense 5'-CCAGCTGCAAGGGTTTACAT-3', antisense 5'-CAATTGCATAGCGGATGATG-3'; DC-SIGN: sense 5'-GAAGACTGCGCGGAATTTAG-3', antisense 5'-TCGAAGGATGGAGAGAAGGA-3'. To assess the concurrent expression at 2 days and 21 days of a gene demonstrated to follow ciliary differentiation of human airway epithelial cells, we determined the presence of dynein 9 (DNAH9) transcript using the following primers: sense 5'-GAGAACCAGGAAGTCAAGGAATG-3' and antisense 5'-GCTGGACCTGCTGACTCAGA-3' (3).


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Table 1. Primer pairs, amplicon sizes, and thermal cycling conditions for real-time RT-PCR

 
Histochemical staining. Slides of cells obtained by nasal brushing were processed for standard Cytospin preparation using 40 µl of suspended cells removed from the original 1.0-ml total suspension volume and stained, following fixation in ethanol, with Papanicolaou stain. Cells on filters at 2 days and 21 days underwent a standard preparation scheme for electron microscopy: cells were fixed in situ with 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed with 1% osmium tetroxide, dehydrated through graded ethanols, and infiltrated and embedded in Epon 812. Sections (0.5–1.0 µm) were stained with 1% toluidine blue in 1% sodium borate.

Flow cytometry. Flow cytometric analysis was performed with an Epics Elite flow cytometer (Beckman/Counter, Miami Lakes, FL) equipped with a 488-nm argon laser. The instrument was prepared for use in accordance with the manufacturer's recommended procedures. Ten thousand cells were analyzed in a "live gate" established by light scatter and 7-amino-actinomycin D dye exclusion. The percentage of cells staining positively for the epithelial cell-specific cell surface antigen Ber-EP4 (20) was obtained after correction for background events and/or nonspecific binding using appropriate controls.

Statistical analyses. Data are presented as means ± SE of replicate determinations, as indicated. Statistical comparisons between groups were made by t-test or, if the data were not normally distributed, by the Mann-Whitney rank sum test. Multiple comparisons were made on normally distributed data by one-way ANOVA or ANOVA on ranks if normality criteria were not satisfied. Post hoc testing was done by Dunnett's or Dunn's methods, respectively. Statistical analyses were carried out with SigmaStat Statistical software (Jandel Scientific, San Rafael, CA). P values < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of genes in freshly prepared HNE cells obtained by nasal brush biopsy. To examine the baseline pattern of immune molecule expression in HNE cells under in vivo conditions, we took brush biopsies from the inferior nasal turbinates of volunteers with no active, or history of chronic, nasal inflammation. Cells obtained in this manner are predominantly ciliated columnar epithelial in morphology, with the presence of infiltrating leukocytes observed to a minimal extent (Fig. 1A). Real-time PCR amplification demonstrated the presence of message for both MHC molecules, HLA-B and HLA-DR, and the costimulatory molecules of interest, B7-H3, B7-H2, B7-2, and B7-1 (Fig. 2). With the exclusion of one low value of HLA-DR, the range of intersubject expression levels for each of the genes was fairly consistent, encompassing a 20- to 24-fold difference between highest and lowest expression in all cases (Fig. 3). Because the efficiency of the PCR reaction may be somewhat different for each of the target molecules, mean values for the relative abundance of different target molecules in the cells cannot be directly compared. Verified product from the B7-1 gene, considered to be inducible and often not constitutively expressed, was detected in cells from all subjects but appeared to be in least abundance in the brush biopsy samples.



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Fig. 1. A: representative sample of human nasal epithelial (HNE) cells obtained by brush biopsy of the inferior nasal turbinate. Cells were prepared by Cytospin and stained with Papanicolaou (x40 magnification). B and C: cross sections of HNE cells grown on cell culture inserts at the air-liquid interface (ALI) for 2 and 21 days, respectively. The porous membrane on which the cells were grown is seen below. Toluidine blue stain (x40 magnification). Bars represent 20 µm.

 


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Fig. 2. Expression of genes for human leukocyte antigen (HLA) and costimulatory molecules in HNE cells obtained by brush biopsy. Gel shows representative gene products and associated 18S RNA amplified during real-time PCR from 1 of 8 healthy subjects. The identity of PCR products in all experiments was verified by sequence analysis of extracted bands.

 


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Fig. 3. Profile and range of expression of genes in brushed cells from 8 healthy subjects and the mean value (line) for each. Abundance of mRNA for each sample, determined by real-time PCR, is represented as 2 raised to the negative of its threshold cycle value times 106 [2–(Threshold Cycle) x 106] after being normalized to its corresponding 18S. The resulting values provide an accurate depiction of the relative abundance of message for each of the target molecules and, in this figure, a view of the interindividual variation for each.

 
Comparison of expression in cells cultured at the ALI for 2 and 21 days. HNE cells isolated from surgical samples of nasal turbinate were first grown to confluence in dishes and then transferred to collagen-coated cell culture inserts. After reaching confluence, ALI medium was substituted, and the cells were grown at the ALI for 2 days or 21 days before RNA extraction. The difference in morphological characteristics between nasal cells at these two time points at the ALI is shown in Fig. 1, B and C. Two-day ALI cultures exhibited a multilayer of similar appearing, flattened cells without signs of structural differentiation. In contrast, 21 days of growth at the ALI provided adequate time for the apical layer of cells to undergo vertical elongation and differentiation into secretory cells and cells with functional cilia. Consistent with these morphological changes, expression of the axonemal DNAH9, which was nominally detectable by real-time PCR at 2 days, exhibited a marked (>2,000-fold) increase at 21 days of culture (Fig. 4). Expression of DNAH9 has been shown to be associated with epithelial ciliogenesis at luminal cell boarders during human fetal development (3).



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Fig. 4. Constitutive expression of the dynein 9 (DNAH9) gene in undifferentiated (2-day ALI) and well-differentiated (21-day ALI) HNE cells derived from 6 separate tissue donors (means ± SE). The means and SE of the abundance of mRNA are expressed as described in Fig. 3. *Significant difference (P = 0.002) in gene expression from 2-day cultures by the Mann-Whitney rank sum test.

 
Comparison of gene expression for MHC and costimulatory molecules in cultures from six separate surgical samples was carried out by real-time PCR. Cultured cells from each sample were grown at the ALI, and cultures were randomly chosen to undergo RNA extraction at the 2-day or 21-day time points. The range of molecules expressed was identical to that of the brushed cells, with the exception that PCR product for B7-1 was not observed. Summary data from the real-time PCR determinations indicate that the additional 19-day period of growth at the ALI leading to cellular differentiation was associated with significant increases in the levels of expression of message for HLA-B and HLA-DR (Fig. 5). Of these two, the HLA-DR amplification was largest, increasing by ~17-fold compared with near threefold for HLA-B. Message levels for the costimulatory molecules remained unchanged during the differentiation period. As indicated above, no verifiable mRNA encoding for B7-1 was detected by real-time PCR in any of the six sets of cultures, consistent with the absence of visible bands in gels run on randomly chosen samples of PCR product. This was in contrast to the clear detection of B7-1 message in cells obtained fresh from the turbinates by brush biopsy.



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Fig. 5. Summary real-time PCR data for HLA and costimulatory gene products from 6 sets of HNE cells (means ± SE). Each set of cultures came from a separate tissue donor, and cell cultures from each of these sets were randomly divided and grown at the ALI for 2 or 21 days (d). The abundance of mRNA is expressed as described in Fig. 3. *Significant difference (P < 0.024) in gene expression from 2-day cultures by t-test.

 
To assess the presence of contamination of the cultured and fresh cells by DCs that would also express HLA and costimulatory molecules, RNA from three 2-day and three 21-day ALI cultures and three nasal brush samples were randomly chosen and probed by real-time PCR for the presence of the DC markers CD-SIGN and CD11c. Neither marker was detected in any of the samples of cultured cells. However, both markers were detected in the brushed cell preparations, suggesting the presence of DCs in those preparations. Representative gels run with the PCR reaction products are shown in Fig. 6.



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Fig. 6. Expression of message for dendritic cell markers DC-SIGN (CD209) and CD11c in cells from lymph nodes (L. N.), cells from nasal brush (N. B.) biopsy and HNE cells cultured at the ALI for 2 and 21 days. Representative separation of products from 1 of 3 real-time PCR determinations on separate samples of the 4 cell sources.

 
Consistent with these data indicating the presence of nonepithelial cells in the brush biopsy samples, flow cytometric analysis of cellular expression of the epithelial cell-specific marker Ber-EP4 in freshly brushed samples indicated the presence of only 60% positive-staining cells. In contrast, HNE cells grown at the ALI for 21 days stained 98% positive for the epithelial cell marker with this method, a value identical to that obtained in staining cells of the homogeneous human bronchial epithelial cell line BEAS-2B (Table 2). Thus these data provide additional evidence for the homogeneous nature of the epithelial cells cultured for 21 days at the ALI.


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Table 2. Expression of epithelial cell-specific Ber-EP4 on cells from nasal brush biopsies and on HNE cells from well-differentiated cultures as assessed by flow cytometry

 
Comparison of expression in cultured and brushed cells. We assessed the influence of cellular differentiation on constitutive gene expression for each molecule by comparing cells cultured at the ALI for 2 days and 21 days with in vivo expression, as represented by that in the freshly brushed cells. We depicted the cultured cell data as the percentage of the mean of the corresponding molecule as expressed in brushed cells, after normalizing all values to their respective 18S. These comparisons are presented in Fig. 7. As described above, only expression of HLA-B and, especially, HLA-DR increased significantly with differentiation. In both cases, abundance of gene product increased during this period to levels statistically indistinguishable from those of brushed cells. In contrast, mRNA abundance for B7-2 did not undergo observable change during differentiation and remained near 30% of in vivo values. Surprisingly, gene activity for the more recently described B7-H3 and B7-H2 molecules was 680 and 390% greater, respectively, in the 2-day ALI cells than in the brushed samples. These relationships were not significantly affected by mucociliary differentiation of the epithelial cells during the subsequent 19-day growth period at the ALI, and the abundance of mRNA for both B7 molecules remained markedly higher than in cell samples obtained by brush biopsy.



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Fig. 7. Relative constitutive expression of HLA and costimulatory genes in cells in poorly differentiated (2-day ALI) and fully differentiated (21-day ALI) cells compared with brushed cells (means ± SE); n = number of samples of cultured or biopsied cells from separate sources. *Significant difference (P < 0.05) from brushed cells, by ANOVA.

 
Influence of cytokines on gene expression in differentiated cells. The responsiveness of genes encoding for immune molecules to activation by cytokines present in normal and inflamed airway mucosal regions was determined. HNE cells obtained from five separate tissue donors were grown for 21 days at the ALI and were treated with a combination of IFN-{gamma} (100 ng/ml) and TNF-{alpha} (100 ng/ml) for 24 h before extraction. The data are summarized in Fig. 8. Message for HLA-DR and HLA-B was significantly increased by cytokine treatment, 17-fold and fourfold, respectively. In contrast, gene activity for B7-H3 and B7-2 remained unaffected by treatment at this single treatment dose and time. Although mRNA levels for B7-H2 appeared to exhibit a small increase, this change was not statistically significant. Consistent with other experiments, constitutive B7-1 message was not detected in 21-day cultures. However, B7-1 gene product was detected following cytokine treatment in cultures from two of six tissue samples (data not shown).



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Fig. 8. Effects of cytokines on gene expression in HNE cells grown at the ALI for 21 days. Cultures of cells from 6 separate sources were treated with medium containing IFN-{gamma} (100 ng/ml) and TNF-{alpha} (100 ng/ml) (Tx) or medium alone (C) for 24 h. The abundance of mRNA for each treated sample and its corresponding control are represented as described in Fig. 3. *Significant difference (P < 0.05) between treated and corresponding untreated cultures, by paired t-test.

 
To further assess the responsiveness of B7-H2 and B7-H3 genes to combined treatment with IFN-{gamma} and TNF-{alpha}, cultures of HNE cells from a separate set of five patients were grown to differentiation at the ALI and treated with a combination of these cytokines at 30, 100, or 300 ng/ml or medium alone for 24 h. As shown in Table 3, expression of both B7 family genes remained constant over this dose range. To determine whether activation of the B7-H3 or B7-H2 genes by cytokine treatment (IFN-{gamma} + TNF-{alpha} at 100 ng/ml) might become apparent at a time point earlier than 24 h, cultures derived from surgical samples from five patients were grown for 21 days at the ALI and treated for 8 or 24 h and compared with medium-treated controls. Values for the fold expression relationships were not significantly different between these three groups for B7-H3 (119 ± 34, 132 ± 28, 144 ± 21) or B7-H2 (5 ± 1, 35 ± 20, 7 ± 2) (means ± SE for control and 8 h and 24 h, respectively). The greater mean and SE values for expression of B7-H2 at 8 h resulted from a selective enhancement of expression in cells from two of the five subjects. The basis for this unique response in these two subjects at the early time point is unclear.


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Table 3. Dose-response characteristics of B7-H2 and B7-H3 to treatment with IFN-{gamma} and TNF-{alpha}

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study demonstrates that epithelial cells of the human upper respiratory tract constitutively express genes for a wide range of cell surface molecules necessary for the activation of T lymphocytes. This expression was observed both in cells freshly removed from the inferior turbinates of healthy, nonsymptomatic subjects as well as in primary cells from similar regions grown in culture under conditions simulating the in vivo epithelial interface. In addition to MHC class I and II molecules and B7-1 and B7-2, message for the recently described B7 homolog B7-H3, as well as B7-H2, was also demonstrated in HNE cells. Although detected in cells of the A549 cancer line (4), the former of these novel members of the B7 family has not previously been described in primary epithelial cells of the upper or lower respiratory tract. The presence of transcript encoding for B7-H3 in these cells significantly expands the breadth of potential interactions through which epithelial cells and T cells can communicate in the airway mucosal region.

Respiratory tract epithelial cells are well known to be directly involved in tissue rejection following lung transplant (5, 27), indicating their participation in T cell immune regulatory processes involving the airway mucosa. The broad range of immune molecule genes expressed constitutively by HNE cells suggests the possibility of a role for cells of upper and lower airways in other immunity-associated inflammatory processes that entail the activation of CD4+ memory T cells. Studies of basal-to-apical transepithelial migration of monocytes across confluent cultures of BEAS-2B cells stimulated with IFN-{gamma} and TNF-{alpha} demonstrate that T cells of the CD45RO+ (memory) subset were preferentially active (25). In addition, it has been shown that epithelial cells of the human upper and lower respiratory tract can internalize and process antigens through pathways that lead to presentation in the context of HLA-DR and to T cell activation, providing further support to the view that these cells can play a role in local immune responses in proximal airway regions (34). This activity would be presumed to be separate from processes involved with initial presentation of antigen to naïve, CD45RA+ T cells, a domain of activity relegated to professional antigen-presenting cells, such as DCs, which takes place primarily within lymphoid tissues (19, 28).

In the present study, we focused on the expression of genes for the immune molecules of interest, recognizing that the translation of this message ultimately determines the extent to which each may be involved in cell-cell interactions leading to T cell activation. Nevertheless, constitutive and/or stimulated gene expression is a prerequisite for these cellular activities and provides a useful surrogate with which to view the influence of cellular differentiation on the state of development of the cellular machinery associated with immune interactions. In fresh cells from normal subjects, gene expression for the HLA and costimulatory molecules was relatively consistent, with levels of intersubject mRNA for each ranging ~20-fold. Comparisons of expression levels between molecules cannot be made directly due to potential differences in the kinetics of PCR amplification for the different molecules. However, with this caveat in mind, the data of Fig. 3 suggest a pattern of in vivo expression in the brushed cells from normal sources that reflects a predominant expression of class I over class II MHC molecules. In addition, there appeared to be generally higher expression of the B7 costimulatory molecule homologs than of B7-2 and B7-1. As opposed to the role of classic priming of naïve T cells attributed to these latter molecules, B7-H3 and B7-H2 have been implicated in the activation of memory CD4+ and CD8+ T cells in peripheral tissues (4, 13, 46). Given the potential of local mediators, such as IFN-{gamma} (4), to stimulate translation of these gene products to protein, the high levels of these B7 family molecules could reflect a means by which the cells could modify local proliferation and selectively modulate release of IL-10, IL-2, IFN-{gamma}, and/or other immune effectors from activated T cells in the epithelial region.

Transcripts for HLA-B, HLA-DR, and B7-1 were more abundant in cells obtained by brush biopsy than they were in undifferentiated cells grown in culture at the ALI for 2 days. Of these molecules, only expression of HLA-B and HLA-DR was significantly increased in association with cellular differentiation during the additional 19 days of culture at the ALI. This observation likely reflects a "maturing" of gene expression in concert with cellular mucociliary differentiation as has previously been reported in primary human bronchial (1) and nasal (15, 47) epithelial cells in culture. In these studies, the extent of differentiation, rather than the time in culture, was the predominant factor determining constitutive activity of the mucin genes MUC3, 5AC, 5B, and 6; aquaporins 3 and 4; and the cystic fibrosis transmembrane conductance regulator. In our study, such maturing of gene expression during the differentiation process in the HNE cells was also observed in the DNAH9 gene, which has been closely linked to the process of ciliogenesis in human airway epithelial cells during fetal development (3). In the context of epithelial differentiation, it is interesting that the relative expression of B7-H3 and B7-H2 remained similar in undifferentiated and well-differentiated states, suggesting that transcripts for these molecules may be available in differentiating cells early during epithelial repair processes. Also in contrast to the HLA molecules and the classic B7 molecules, B7-H3 and B7-H2 transcripts consistently were expressed several hundredfold higher in cultured cells than in fresh cells. The observation of this significantly greater constitutive activity in culture is interesting, although its basis remains unclear. It is possible that cytokines or other mediators, present in the nasal mucosa in vivo, normally act to repress activation of these genes or that a higher level of degradation of mRNA occurs in vivo. Conversely, some condition in the cell culture system environment may provide an adequate stimulus for gene activation above constitutive levels. Little is currently known about the transcription factor regulation of the B7 family of genes that would provide insight into the relatively high levels of mRNA observed or about the relationship between this expression and cell surface protein. Unlike undifferentiated epithelial cells grown under medium that are susceptible to detachment by use of EDTA, differentiated cells grown at the ALI require vigorous treatment with proteolytic enzymes for dispersion (9). Due to the relatively high sensitivity of external domains of the B7 homologs to proteolysis, preparation of cells in this manner for flow cytometric evaluation of protein expression has proven to be problematic. The development of methods for preparing differentiated epithelial cells attached to porous plastic inserts by frozen section may provide the opportunity to study the relationships between transcript levels and protein expression in detail and to determine the distribution of these molecules on the epithelial cellular surface.

The differences in maximally developed gene activity for the class I and II molecules and B7-1 and B7-2 between freshly brushed cells and cultured cells were likely due, at least in part, to the presence of DC contamination in the former preparations, as assessed by the detection of DC-SIGN and CD11c markers in those samples. The observed loss of these markers in 2-day and 21-day ALI cultures is consistent with previous studies indicating the inability of DCs to survive in culture in the absence of serum in the medium for >4–5 days (17). In the case of our cultures, the P0 HBE cells were grown in serum-free, defined medium for 7–10 days before transfer to the cell culture inserts and an additional 3–4 days before the P1 cells reached confluence and were converted to ALI conditions at day 0. Thus the data may, in fact, indicate that the higher levels of expression of HLA-B and HLA-DR achieved during cellular mucociliary differentiation of the cells at the ALI reflect maximal constitutive expression in HBE cells in the absence of the DC contribution.

Consistent with previous reports of the responsiveness of the HLA molecules, and especially HLA-DR, to activation by cytokines at both mRNA and protein levels (17, 26, 32, 33), fully differentiated HNE cells showed significantly increased transcript levels following treatment with IFN-{gamma} and TNF-{alpha}. In contrast, transcript levels for B7-2 at the 100 ng/ml concentration and for B7-H3 and B7-H2 at three concentrations bracketing the latter remained unchanged by cytokine treatment. Our B7-H2 data are consistent with a recent study that found no effect of cytokine treatment on message at 6 or 18 h in undifferentiated cultured bronchial epithelial cells similarly treated (18). Our results suggest that if these specific cytokines are effective in increasing cell surface protein expression in differentiated cells, the effect would occur through mechanisms downstream of gene transcription.

The present study demonstrates the constitutive expression by primary epithelial cells of the nasal mucosa of a broad range of B7 family genes whose products ligate receptors on T cells and modulate their activity. In addition to the classic costimulatory molecules B7-1 and B7-2 (10, 34), these cells were demonstrated, for the first time, to exhibit high levels of gene expression for the more recently described B7 family members, B7-H3 and B7-H2. These two ligands have been shown to preferentially bind activated, rather than naïve, CD4+ and CD8+ T cells and to selectively modulate their proliferative and secretory activities. Unlike the B7-H2 molecule that is known to ligate the ICOS receptor on CD8+ cells (45) and on CD4+ cells to support T helper cell 2 (Th2) receptor signaling (13, 48), the receptor for B7-H3 remains unknown. This fact has likely contributed to the current controversy regarding the nature of B7-H3 activity. Initial in vitro studies indicated that this ligand enhanced TCR-mediated T cell proliferation, stimulated the release of the Th1 cytokine IFN-{gamma} from CD4+ cells, and augmented the generation of cytotoxic T lymphocytes (4). These activities would suggest that B7-H3 might act as a counterpart to B7-H2 in regulating the Th1/Th2 axis within the tissues, reducing allergic inflammatory responses. In contrast to these earlier findings, recent data point to a downregulatory function for B7-H3 in murine CD4+ and CD8+ proliferation and indicate a significant increase in Th1, but not Th2, inflammatory processes and IFN-{gamma} release in B7-H3-deficient mice (38). Most recently, studies of human B7-H3 were unable to support a costimulatory role for this molecule in T cell proliferation and IFN-{gamma} production associated with activation of the TCR complex (37). Thus the exact nature of the regulation of T cells provided by this new B7 family member is currently unclear and may be multifaceted. At present, nothing is known about the variations in constitutive expression of B7-H3 in epithelial cells from individuals with chronic sinusitis or asthma or about the endogenous factors that might influence its expression in these conditions. Nevertheless, the abundance of its gene product observed in HNE cells in the present study suggests that the involvement of this molecule in the staging and/or modulation of T cell activity in the upper respiratory tract warrants further investigation.

In summary, the present study demonstrates the importance of mucociliary differentiation in the full expression of the MHC class I and class II genes HLA-B and HLA-DR in epithelial cells of the nasal mucosa. In contrast, expression of genes for the accessory molecules investigated were found not to be differentiation dependent and remained constant during the transition to well-developed morphology. Furthermore, the novel identification of expression of the B7 family molecules B7-H3 and B7-H2 in upper respiratory epithelial cells provides evidence for an expanded range of costimulatory/inhibitory signals potentially available to these cells with which to participate in mucosal immune responses.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Environmental Health Sciences Grants ES-09004 and ES-03819 and by a grant from the American Academy of Otolaryngology-Head and Neck Surgery (A. P. Lane).


    ACKNOWLEDGMENTS
 
We thank Kristin Macri and Christopher Merlo for subject recruitment and help in obtaining and processing nasal brush biopsy cells and Jane Dees, Brian Schofield, and Judy Coram for assistance with histochemical preparations.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. W. Spannhake, Div. of Physiology, Dept. of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 No. Wolfe St., Baltimore, MD 21205 (E-mail: espannha{at}jhsph.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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