Molecular Immunology 46 (2009) 1905–1910
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IL-4 increases CD21-dependent infection of pulmonary alveolar epithelial type II cells by EBV Andrea P. Malizia a,b , Jim J. Egan a , Peter P. Doran b,∗ a b
Advanced Lung Disease and Lung Transplant Program, Mater Misericordiae University Hospital. 44, Eccles Street, Dublin, 7, Ireland Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street, Dublin, 7, Ireland
a r t i c l e
i n f o
Article history: Received 27 November 2008 Received in revised form 31 December 2008 Accepted 2 January 2009 Available online 4 February 2009 Keywords: Idiopathic pulmonary ﬁbrosis Alveolar epithelial cells EBV CD21 IL-4
a b s t r a c t EBV infection has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Viral infection may occur from the early or late stage in IPF development. Whether alveolar epithelial cells, AECs, normally express EBV main receptor, CD21, remains uncertain. Such situations prompted us to exploit an efﬁcient direct infection system to investigate EBV receptor repertoire in primary human AECs. Using human primary type 2 AECs, which have been grown in basal medium supplemented with 10 ng/ml Keratinocyte Growth Factor, and type 1 AECs, supplemented with Epithelial Growth Factor, both AEC lines express CD21 mRNA and protein with a signiﬁcant increase in type 2 cells. Type 2 AECs have been exposed to TGF␤1 and IL-4, whose expression is associated with IPF development. CD21 is highly expressed in type 2 AECs following IL-4 exposure. EBV bound to type 2 AECs membrane increases signiﬁcantly following pre-treatment with IL-4 (p < 0.001) and decreasing antagonizing CD21 receptor (p < 0.01). 200 g/ml G418-mediated selection of EBV-Neomycin resistant infected cells selected IL-4 pre-exposed type 2 AECs. Our study of a viral cell line model provides evidence to suggest that CD21-dependent viral entry plays a crucial role in type 2 AECs, indicative of an IL-4 response EBV infection in IPF. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Epstein-Barr Virus is predominantly a lymphotropic herpesvirus. It is the etiologic agent of most cases of infectious mononucleosis and has been associated with the development of immunoblastic lymphoma, endemic Burkitt’s lymphoma, and certain types of Hodgkin’s disease. In addition, the virus has tropism for epithelial cells; EBV causes oral hairy leukoplakia and is associated with nasopharyngeal and gastric carcinomas (Rickinson and Kieff, 2001). Studies have suggested viral infection as the cause of epithelial injury in Idiopathic Pulmonary Fibrosis (IPF) (Egan et al., 1995; Malizia et al., 2008). Tang et al. (2003) reported EBV infection in familial and sporadic IPF cases, supporting the concept that EBV is a source of chronic antigenic stimulation in IPF (Stewart et al., 1999). In surgically acquired lung tissue EBV gp340/220 and VCA, viral proteins expressed during the viral lytic phase, have been localized to a limited number of hyperplastic type 2 alveolar epithelial cells (Egan et al., 1995). The putative role of EBV in the development of IPF has been expanded by ﬁndings that the expression of EBV latent membrane protein 1 (LMP-1) in alveolar epithelial cells is associ-
∗ Corresponding author. Tel.: +35317166319. E-mail address: [email protected]
(P.P. Doran). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.01.002
ated with a poor prognosis in IPF patients (Tsukamoto et al., 2000). Of note, an antiviral oral treatment based on valacyclovir, which is a selective antiviral agent active against EBV, reduced any further decline in pulmonary function of an IPF subject whose lung tissue was positive for EBV (Tang et al., 2003). EBV receptor CD21, or complement receptor 2 (CR2), is expressed at low levels on some epithelial cells (Fingeroth et al., 1999). Studies of viral infection of epithelial cell lines have been limited because EBV does not readily infect epithelial cells in vitro (Imai et al., 1998). Two human epithelial cell lines, RHEK and HeLa, were shown to express very low levels of CD21 and, the ability to bind EBV at the cell surface was not shown to be dependent on CD21 (Birkenbach et al., 1992). However, infection of epithelial cells that have been engineered to express high levels of CD21 can be very efﬁcient (Borza et al., 2004; Li et al., 1992). Whether epithelial cells normally express CD21 in vivo remains uncertain (Young et al., 1989). Altered Th2 cytokines and TGF␤1 expression have been determined in lung tissue and associated with IPF development. Increased expression of proﬁbrotic Th2 cytokine, IL-4, has been found in IPF pulmonary epithelium (Wallace and Howie, 1999), further associated with murine gamma-herpesvirus 68 (MHV68) infection of IFN-gammaR-/- mice, a strain of mice biased to develop Th2 responses (Mora et al., 2005). TGF␤1 is consistently associated with progressive ﬁbrosis, and increased expression being related with a variety of ﬁbrotic lung diseases (Corrin et al., 1994; Sime et
A.P. Malizia et al. / Molecular Immunology 46 (2009) 1905–1910
al., 1997). Excess production of TGF␤1 by alveolar epithelial cells has been associated with the development of temporary inﬂammation, whose reparative process may be induced by trans-differentiation of alveolar epithelial type 2 cells to type 1 cells (Bhaskaran et al., 2007; Xu et al., 2003). Viral infection of alveolar epithelial type 2 cells may alter this reparative process causing severe epithelial injury. Thus, our study identiﬁes an efﬁcient infection system to investigate primary human alveolar epithelial cells receptors for EBV infection, to enable the description of the pathogenic link between EBV and IPF.
One peak in the melt curve indicated no secondary, non-speciﬁc products were formed. Results have been examined by GraphPad Prism version 4.00 (GraphPad Software, San Diego California USA). The oligonucleotide primers used to amplify cDNA and DNA were:CD21 Fw 5 - CAA GGC ACA ATT CCT TGG TT; Rv 5 - CTC CAG GTG CCT CTT TCT TG. SP-C Fw 5 - CTG GTT ACC ACT GCC ACC TT; Rv 5 - CTG GCC CAG CTT AGA CGT AG. GAPDH Fw 5 -GAG TCA ACG GAT TTG GTC GT; Rv 5 -TTG ATT TTG GAG GGA TCT CG. LMP-1: Fw 5 -TGA ACA CCA CCA CGA TGA CT; Rv 5 - GTG CGC CTA GGT TTT GAG AG. BRLF1: Fw 5 -AAT TTA CAG CCG GGA GTG TG; Rv 5 -AGC CCG TCT TCT TAC CCT GT. BZLF1: Fw 5 -CCA TAC CAG GTG CCT TTT GT; Rv 5 -GAG ACT GGG AAC AGC TGA GG. Cellular DNA control Fw 5 -CTG GGG TCA GCT CTG ACA GT; Rv 5 -TGG GGA CAC CAT CTA CAG TG.
2. Materials and methods 2.1. Primary cell culture and viruses Primary human pulmonary alveolar epithelial cells were purchased from ScienCell (ScienCell Research Laboratories, CA, USA) as a mixed population of type 2 and type 1 AECs, HPAEpi (Gentry et al., 2007). HPAEpi cells have been isolated from human lung tissue, cryo-served at primary culture and delivered frozen. In the lung, type 2 AECs proliferate to provide daughter cells, some of which remain type 2 AECs and some of which differentiate to become type 1 AECs. Type 2 cells have been grown and maintained in AEpiC medium (ScienCell Research Laboratories, CA, USA) supplemented with 2% Fetal Calf Serum (FCS) and 10 ng/ml of Keratinocyte growth factor, KGF (Sigma–Aldrich, UK) (Ulich et al., 1994). To trigger AECs differentiation to type 1 cells, the frozen stock as described above was cultured in AEpiC medium, 2% FCS, supplemented with supplied Epithelial Growth Factor, EGF (ScienCell Research Laboratories, CA, USA). Type 2 AECs differentiate to type 1 cells in 5–8 days (Mason, 2006; Sato et al., 2002). To determine KGF-induced differentiation, type 2 AECs type-speciﬁc surfactant protein-C, SP-C, expression was investigated by Real Time-qPCR (Winkler et al., 2008). Recombinant Neomycin resistant and Green Fluorescent Protein (GFP)-expressing EBV B95.8 strain (Keating et al., 2006; Malizia et al., 2008) have been harvested from clariﬁed culture medium of EBV carrying A549 cells, VAAK, after induction of viral lytic cycle by 30 ng/ml 12-o-tetradecaoylphorbol-13-acetate (TPA) and 2.5 mM sodium butyrate (BA) (Sigma–Aldrich, UK). Brieﬂy, cells have been incubated with TPA/BA for 3 h at 37 ◦ C, washed three times, and RPMI1640 medium containing FCS 10% has been added. After 3 days incubation, the culture media were clariﬁed by centrifugation (1200 g) for 15 min at 4 ◦ C. The supernatant was ﬁltered through a 0.45 mm pore-size membrane and stored at −80 ◦ C until use (Maruo et al., 2001). In some experiments, 10 ng/ml TGF␤1 (Sigma–Aldrich, UK) and 20 ng/ml IL-4 (Sigma–Aldrich, UK) have been added.
2.3. Flow cytometry Fluorescence-activated cell sorting analysis for CD21 positive cells. Cells were washed in cold PBS, and single cell suspensions were incubated with PBS solution containing 20 l/106 cells FITC-conjugated mouse monoclonal anti-human CD21 antibody (BioLegend, CA, USA) or 2 l/106 cells FITC-conjugated mouse IgG1 (BioLegend, CA, USA), on ice for 20 min in the dark. Cells were washed with PBS at 350 g for 5 min, and ﬁxed in 0.5 ml PBS solution containing 1% p-formaldeide at 4 ◦ C. Fixed cells were analyzed using a FACScan ﬂow cytometer and CellQuest software (BD Biosciences). Due to the low differences in CD21 positive cells from the cell population, data were reported in a column-graph chart to improve the analysis of variances between groups. 2.4. Immunoﬂuorescence staining Cells were washed, ﬁxed in ice-cold methanol for 10 min, and then soaked in PBS containing 0.1% Bovine Serum Albumin, BSA (Sigma–Aldrich, UK) and 20% mouse serum (Sigma–Aldrich, UK) for 30 min to reduce secondary aspeciﬁc bindings. Both type 2 and type 1 cells exposed to mouse anti-human CD21-FITC conjugated for 1 h (BioLegend, CA, USA) and, after washing, counterstained with 50 mg/ml Propidium Iodide (Fluka, UK) for 10 min. Images were collected using a ProgRes Camera microscope system (JENOPTIK Laser, Germany). 2.5. Virus binding assay. The amount of virus that bound to alveolar epithelial cells has been determined by adding virus to adherent monolayers in six-well tissue culture plates on ice for 2 h, washing and scraping them into ice-cold PBS. DNA was isolated for Real Time-qPCR using a QIAamp DNA Blood Mini Kit (Qiagen, UK), as previously reported (Turk et al., 2006). In some experiments, cells were pre-incubated with 2.5 g/ml mouse monoclonal antibody anti-human CD21 (BioLegend, CA, USA) or mouse IgG1 isotype (BioLegend, CA, USA) for 1 h at 37 ◦ C. The relative amounts of virus bound per cell were measured by Real Time-qPCR for EBVLMP-1 DNA copies and compared with cellular-DNA copies control (Homo sapiens chromosome 19: 1544 bp at 5 side of transforming growth factor beta 1 gene) resulting in Ct = 2−Ct or EBV relative
2.2. Real Time quantitative PCR cDNA and DNA levels have been assayed by using a Rotorgene 3.0 Real Time PCR instrument (Corbet Research, Australia) and QuantiTect SYBR Green PCR kit (Qiagen, UK). Products have been measured by Absolute Quantiﬁcation and reported as a function of crossing time (CT), the cycle number at which PCR ampliﬁcation becomes linear. mRNA expression has been normalized to control and GAPDH expression obtaining mean fold change values or 2−Ct (Ct).
Mean fold change, Ct = 2
Ct gene −Ct GAPDH sample− Ct gene −Ct GAPDH control ±
A.P. Malizia et al. / Molecular Immunology 46 (2009) 1905–1910
EBV Relative Units/cells
Ct EBV−LMP1 −Ct DNA Chr.19 sample ±
2.6. Infection assay Primary human alveolar epithelial cells have been plated onto a six-well plate at a dilution that produced 30% conﬂuent monolayer 24 h later. Cells were incubated at 37 o C with EBV in culture media. Following 2 h of incubation, growth medium has been added and cells were re-incubated for 48 h. To select EBV-infected primary human alveolar epithelial cells, 200 g/ml G418 (Sigma–Aldrich, UK) has been added for 10 days to select out those alveolar epithelial cells that had been transfected with the EBV transcript. Two weeks after infection, cells have been trypsinized, washed in PBS and the percentage of GFP-expressing infected cells against untreated cells have been visually counted in a microscope counting chamber under UV-light (Feederle et al., 2007). 2.7. Statistical analysis The statistical signiﬁcance of our experimental ﬁndings was analyzed using Student’s t-test assuming unequal variances for Real Time-qPCR. Data with p-value less than 0.05 are considered signiﬁcant. The data shown are the representative average of at least two independent experiments performed in parallel, except where stated otherwise in the text. All data are reported with their relative standard deviation (SD) data (mean ± SD). 3. Results and discussion 3.1. CD21 is expressed at low levels in alveolar epithelial type 2 cells. Based on a previous study reporting EBV infection of pulmonary alveolar epithelial type 2 cells in vivo in IPF patients (Egan et al., 1995), we determined the expression of the EBV receptor CD21 in primary human alveolar epithelial type 2 cells (type 2 AECs) and type 1 cells (type 1 AECs). To induce epithelial cell growth of type 2 AECs, a mixed population of primary human AECs has been exposed to 10 ng/ml KGF for 5 and 10 days. Surfactant protein-C, SP-C, transcript has been investigated in KGF-differentiated AECs in comparison with EGF treated cells to determine bioactivity of type 2 AECs. Figure 1 shows a signiﬁcant SP-C gene expression in type 2 AECs culture following 5 and 10 days 10 ng/ml KGF-induced differentiation in comparison with EGF-exposed AECs (type 1 AECs) (p < 0.001). This efﬁcient method of cell differentiation prompted us to further utilize these cells as two distinct cell lines following 10 ng/ml KGF and EGF exposure for 10 days. EBV receptor-CD21 mRNA expression has been investigated in differentiated type 1 and type 2 AECs by Real Time-qPCR. CD21 mRNA is expressed in both cell cultures in comparison with osteoblasts, showing a signiﬁcant increase in type 2 AECs (p < 0.05) (negative control, osteoblasts Ct 1.58 × 10−4 ; type 1 AECs Ct (5.3 ± 2.41) × 10−4 ; type 2 AECs Ct (9.46 ± 1.84) × 10−4 ; positive control, B-cells Ct 1) (Fig. 2A). By using a temperature of melting analysis of the ﬁnal Real Time-qPCR products, similar results to B-cell positive control were determined for AECs (type 1 AECs Tm 81.7 ◦ C, type 2 AECs 81.8 ◦ C, B-cells 81.3 ◦ C, respectively) (Fig. 2B). Having determined a slight increase in CD21 expression at transcript level, CD21 protein expression has been investigated in both type 1 and type 2 AECs by ﬂow cytometry. The proportion (R1)
+(SD2DNA Chr19 )2
of CD21 positive cells is 3.32% in type 1 AECs at basal conditions, while it increases in type 2 AECs differentiated by 10 ng/ml KGF for 10 days to 6.84% (Fig. 2C). In addition, CD21 receptor is expressed in type 2 AECs as determined by immunoﬂuorescence (Fig. 2D). Despite a major number of AECs differentiated to type 1 by exposure to EGF, type 1 AEC culture showed few large ﬂat cells identiﬁed as type 2. These AECs have slight positive intracellular signals for CD21 receptor (Fig. 2D). Thus, a representative percentage of type 2 AECs expresses CD21 receptor at both mRNA and protein levels, which suggests us to further investigate their role in EBV infection. 3.2. Antagonizing CD21 receptor in type 2 AECs reduces cellular membrane binding of EBV. Although CD21-mediated infection is not the only pathway necessary for EBV to infect epithelial cells (Hutt-Fletcher, 2007), in this study a competitive binding mechanism has been utilized by antagonizing CD21 receptor in both AECs lines. A higher number of EBV bound to type 2 AECs have been determined by virus binding assay in comparison with type 1 AECs (type 1 AECs, EBV relative units/cells Ct 0.283E-02 ± 0.045; type 2 AECs, EBV relative units/cells Ct 2.044E-02 ± 0.082; p < 0.01) (Fig. 2E). To investigate CD21-mediated viral attachment, cells were preincubated with mouse monoclonal anti-human CD21 antibody to antagonize EBV binding at the cell membrane or control IgG1 Isotype for 24 h. A signiﬁcant decrease in virus attachment results by antagonizing CD21 binding in type 2 AECs (anti-CD21 treated type 2 AECs, EBV relative units/cells Ct 1.602E-02 ± 0.311; p < 0.05) (Fig. 2E). Although at least three other possible attachment mechanisms have been proposed that involve neither gp350/220 nor CD21 (Hutt-Fletcher, 2007) and AEC bioactivity responses to mouse antiCD21 antibody binding are unknown at the moment, we suggested a CD21-based EBV attachment in AECs, mainly in type 2 cells. Inhibiting CD21-mediated binding reduced the percentage of EBV bound at the cell membrane. 3.3. Th2 cytokine, IL-4, increases CD21 expression in type 2 AECs EBV infection in IPF is associated with disease progression in a multifactor setting. The sequential process of EBV infection, which
Fig. 1. Surfactant protein-C gene expression in Keratinocyte Growth Factordifferentiated alveolar epithelial cells. Real Time-qPCR has been used to quantify Surfactant protein-C, SP-C, mRNA expression in AECs following Keratinocyte Growth Factor exposure for 5 and 10 days, in comparison with Epithelial Growth Factordifferentiated type 1 AECs. n = 4, Student’s t-test (***) p < 0.001. The error bars represent SD.
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Fig. 2. EBV binding is mediated by CD21 in type 2 AECs. (A) CD21 mRNA expression at basal conditions determined by Real Time-qPCR. Osteoblasts and B-cells Akata cell lines have been used as negative (−) and positive (+) controls. n = 4, Student’s t-test (*) p < 0.05. The error bars represent SD. (B) Temperature of melting (Tm) analysis of Real Time-qPCR products: osteoblasts (dotted line); type 1 AECs (solid line), type 2 AECs (dashed line), B-cells Akata (thin line). dF/dT = ﬂuorescence, deg. = degrees celsius. (C) Cell membrane CD21 expression in type 1 and type 2 AECs determined by ﬂow cytometry. Cells treated with FITC-conjugated mouse monoclonal anti-human CD21 or anti IgG1 Isotype have been counted on <10,000 events acquired (R1) (D) CD21 expression in type 2 and type 1 AECs treated with FITC-conjugated mouse monoclonal anti-human CD21. Cells were counterstained by Propidium Iodide producing a nuclear staining. 40× and 60× immersion oil objective pictures. (E) Cells have been incubated with aliquots of EBV in culture media and in some experiments, pre-incubated with mouse monoclonal anti human-CD21 or control mouse IgG1 Isotype antibody. Relative expression of virus bound per cell has been measured by comparative analysis of Real Time-qPCR data for EBV-LMP-1 DNA and cellular DNA. EBV relative units/cells Ct = 2−Ct . n = 4, Student’s t-test, p < 0.05 (*), p < 0.01 (**). AECs1: type 1 alveolar epithelial cells; AECs2: type 2 alveolar epithelial cells; (−) control: osteoblasts; (+) control = B-cells Akata; antiCD21: mouse monoclonal anti-human CD21 antibody.
may be primary or secondary in response to stimuli, remains uncertain due to EBV infection ﬁndings in IPF at late stage. Thus, in this study we investigated CD21 expression in type 2 AECs in response to proﬁbrotic Th2 cytokine, IL-4, and TGF␤1, which have been shown to induce IPF further at the early stage (Sime et al., 1997; Wallace and Howie, 1999; Xu et al., 2003). CD21 expression has been investigated in type 2 AECs exposed to 20 ng/ml IL-4 and 10 ng/ml TGF␤1. CD21 mRNA expression increases signiﬁcantly in type 2 AECs following 4 and 24 h 20 ng/ml IL-4 exposure (p < 0.05), in comparison with untreated cells (Fig. 3A). Cell-surface CD21 protein expression has been investigated in type 2 AECs by ﬂow cytometry following TGF␤1 and IL-4 exposure for 24 h. The proportion (R1) of CD21 positive cells in type 2 AECs is 3.10% at basal conditions and 3.14% following 10 ng/ml TGF␤1 exposure. The percentage of CD21 positive cells increases to 10.63% following exposure to 20 ng/ml IL-4 (Fig. 3B). To further investigate these data, EBV attachment has been determined in TGF␤1 and IL-4-treated cells, further by antagonizing CD21 receptor. A signiﬁcant increase in EBV binding is shown in both TGF␤1 and IL-4-treated cells (type 2 AECs, EBV relative units/cells Ct 2.727E-
02 ± 0.4; TGF␤1 exposed type 2 AECs, EBV relative units/cells Ct 4.284E-02 ± 0.44; IL-4 exposed type 2 AECs, EBV relative units/cells Ct 10.63E-02 ± 1.253; p < 0.01 and p < 0.001, respectively) (Fig. 3C). Pre-treatment with mouse anti-human CD21 for 24 h reduces signiﬁcantly viral binding to type 2 AECs surface in IL-4-exposed cells (anti-CD21 treated IL4-type 2 AECs, EBV relative units/cells Ct 4.454E-02 ± 0.255; anti-CD21 treated TGF␤1-type 2 AECs, EBV relative units/cells Ct 5.706E-02 ± 0.774, p = 0.05). These data suggest that a sufﬁcient CD21-mediated EBV entry mechanism may be shown in type 2 AECs stimulated with Th2 cytokine, IL-4. To investigate AECs infection by EBV, type 2 AECs were exposed to culture media containing recombinant EBV expressing Green Fluorescent Protein (GFP) and Neomycin resistant gene. Following infection, AECs were selected by 200 g/ml G418 exposure for 10 days at 37 o C. The number of resulted infected cells in comparison with non-infected cells replicating in parallel culture determined the efﬁciency of infection. Following exposure of type 2 AECs to EBV, G418 selects rare EBVinfected cells in culture (data not shown), suggesting that virus attachment to CD21 receptor, previously determined, is not signiﬁcantly sufﬁcient for virus entry in epithelial cells at basal conditions.
A.P. Malizia et al. / Molecular Immunology 46 (2009) 1905–1910
Fig. 3. IL-4 increases CD21 expression in type 2 AECs. (A) CD21 mRNA expression in type 2 AECs following 10 ng/ml TGF␤1 and 20 ng/ml IL-4 exposure determined by Real Time-qPCR. n = 4, Student’s t-test, p < 0.05 (*). (B) Percentage of CD21+AECs determined by ﬂow cytometry following 10 ng/ml TGF␤1 and ng/ml IL-4 exposure for 24 h. Treated cells with FITC-conjugated mouse anti-human CD21 have been counted on <10,000 events acquired. (C) EBV bound represented as EBV-LMP-1 DNA relative unit in type 2 AECs exposed to 10 ng/ml TGF␤1 and 20 ng/ml IL-4 for 24 h, and in some experiments, pre-exposed to mouse monoclonal anti human-CD21 or mouse IgG1 Isotype antibody. EBV relative units/cells Ct = 2−Ct . n = 4, Student’s t-test, p < 0.01 (**), p < 0.001 (***). IL4: interleukin-4; TGF␤1: transforming growth factor ␤-1; antiCD21: mouse monoclonal anti-human CD21 antibody.
Fig. 4. G418 exposure selects EBV-infected IL-4-treated type 2 AECs. (A) Green Fluorescent protein (GFP)-expressing EBV-infected type 2 AECs were visually counted in a microscope counting chamber following G418 selection for 10 days. Images have been selected as representative of counting in an inverted microscope under normal and UV-light. (B) Cellular mRNA have been extracted and viral BRLF-1, BZLF-1 and LMP-1 mRNA expression determined in type 2 AECs (AEC2), EBV-infected type 2 AECs following 20 ng/ml IL-4 exposure (IL-4-treated AEC2) and EBV-infected B-cells (Akata). Ampliﬁed products have been separated in 0.8% agarose gel by electrophoresis and compared to GAPDH mRNA expression. AECs2: type 2 alveolar epithelial cells; Akata: EBV-infected B-cells.
A.P. Malizia et al. / Molecular Immunology 46 (2009) 1905–1910
Type 2 AECs have been exposed to 20 ng/ml IL-4 for 24 h, followed by EBV infection and G418-based selection to is EBV-Neomycin resistant infected cells. Both IL-4-treated- and untreated-cells have been trypsinized and counted in a microscope counting chamber under UV-light. 4% of IL-4 exposed type 2 AECs, in comparison with untreated cells, express EBV-GFP (Fig. 4A). Resulted EBV-infected type 2 AECs were incubated with standard culture media for two weeks; to investigate the transcription of the viral genome in the cell, EBV gene BRLF-1, BZLF-1 and LMP-1 expressions have been determined by PCR. BRLF-1, BZLF-1 and LMP-1 mRNA expressions are shown in G418-selected type 2 AECs in comparison with non-infected cells and EBV-infected B-cells positive control, Akata (Fig. 4B). 4. Conclusions The pathogenic sequences leading to the development of IPF are unclear; one theory is that a triggering agent or event induces an inﬂammatory reaction in the lung, perpetuates itself and causes parenchymal ﬁbrosis. One potential source for a self-perpetuating triggering event could be a chronic viral infection. Studies describing EBV infection and IPF may provide the basis for such causal link. To examine the EBV receptor repertoire in pulmonary epithelial cells, a mixed population of primary human AECs has been differentiated by KGF and EGF treatment to type 2 and type 1 cells, respectively. Although CD21 is not the only receptor utilized by EBV to infect cells, antagonizing CD21 in AECs shows a signiﬁcant decrease in EBV bound to type 2 cells only. Exposure to proﬁbrotic Th2 cytokine, IL-4, induces an increase in CD21 expression at transcript and protein levels; further, the amount of EBV bound to cell membrane reduces antagonizing CD21 receptor. To conﬁrm a CD21-mediated infection of type 2 AECs, recombinant EBV expressing GFP and resistance to neomycin have been utilized. The acquired resistance to G418 and expression of viral BRLF-1, BZLF-1 and LMP-1 gene identiﬁes a signiﬁcant percentage of EBV-infected cells. On the low efﬁciency of infection, which conﬁrms comparable data in vivo (Egan et al., 1995), we may speculate that two distinct sub-populations of cells with different cell bioactivity may constitute cultured type 2 AECs (Reddy et al., 2004). Exactly by which cell signaling IL-4 enhances CD21 expression is not certain. In vivo, in the early phase of IPF development, increased IL-4 expression may induces CD21 expression in pulmonary epithelial type 2 cells, as further shown in chronic viral infection of a murine model (Mora et al., 2005); a reiterative EBV infection may modulate viral-cellular host responses stimulating IL-4 signaling. Viral infection of pulmonary alveolar epithelial cells has long been viewed as a critical determinant of IPF pathogenesis. These data demonstrate important interplay between IL-4 and EBV in modulating AECs bioactivity, identifying a CD21-dependent mechanism of viral entry. Acknowledgments The authors acknowledge the role of Dr. Jonathan Dean, Centre for Research in Infectious Disease (CRID), University College Dublin, in the use of FACSscan ﬂow cytometer. This work was supported by the European Union, the Irish Lung Foundation and the Irish governments’ Programme for Research in Third Level Institutions. References Bhaskaran, M., Kolliputi, N., Wang, Y., Gou, D., Chintagari, N.R., Liu, L., 2007. Transdifferentiation of alveolar epithelial type II cells to type I cells involves autocrine signaling by transforming growth factor beta 1 through the Smad pathway. J. Biol. Chem. 6, 3968–3976.
Birkenbach, M., Tong, X., Bradbury, L.E., Tedder, T.F., Kieff, E., 1992. Characterization of an Epstein-Barr virus receptor on human epithelial cells. J. Exp. Med. 176, 1405–1414. Borza, C.M., Morgan, A.J., Turk, S.M., Hutt-Fletcher, L.M., 2004. Use of gHgL for attachment of Epstein-Barr virus to epithelial cells compromises infection. J. Virol. 78, 5007–5014. Corrin, B., Butcher, D., McAnulty, B.J., Dubois, R.M., Black, C.M., Laurent, G.J., Harrison, N.K., 1994. Immunohistochemical localization of transforming growth factorbeta 1 in the lungs of patients with systemic sclerosis, cryptogenic ﬁbrosing alveolitis and other lung disorders. Histopathology 2, 145–150. Egan, J.J., Stewart, J.P., Hasleton, P.S., Arrand, J.R., Carroll, K.B., Woodcock, A.A., 1995. Epstein-Barr virus replication within pulmonary epithelial cells in cryptogenic ﬁbrosing alveolitis. Thorax 50, 1234–1239. Feederle, R., Neuhierl, B., Bannert, H., Geletneky, K., Shannon-Lowe, C., Delecluse, H.J., 2007. Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection. Int. J. Cancer 3, 588–594. Fingeroth, J.D., Diamond, M.E., Sage, D.R., Hayman, J., Yates, J.L., 1999. CD21dependent infection of an epithelial cell line, 293, by Epstein-Barr virus. J. Virol. 73, 2115–2125. Gentry, M., Taormina, J., Pyles, R.B., Yeager, L., Kirtley, M., Popov, V.L., Klimpel, G., Eaves-Pyles, T., 2007. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection. Infect. Immun. 75, 3969–3978. Hutt-Fletcher, L.M., 2007. Epstein-Barr virus entry. J. Virol. 15, 7825–7832. Imai, S., Nishikawa, J., Takada, K., 1998. Cell-to-cell contact as an efﬁcient mode of Epstein-Barr virus infection of diverse human epithelial cells. J. Virol. 72, 4371–4378. Keating, D.T., Sadlier, D.M., Patricelli, A., Smith, S.M., Walls, D., Egan, J.J., Doran, P.P., 2006. Microarray identiﬁes ADAM family members as key responders to TGFbeta1 in alveolar epithelial cells. Respir. Res. 1, 114. Li, Q.X., Young, L.S., Niedobitek, G., Dawson, C.W., Birkenbach, M., Wang, F., Rickinson, A.B., 1992. Epstein-Barr virus infection and replication in a human epithelial cell system. Nature 356, 347–350. Malizia, A.P., Keating, D.T., Walls, D., Doran, P.P., Egan, J.J., 2008. Alveolar epithelial cell injury with EBV upregulates TGF␤1 expression. Am. J. Physiol. Lung Cell Mol. Physiol. 3, L451–L460. Maruo, S., Yang, L., Takada, K., 2001. Roles of Epstein-Barr virus glycoproteins gp350 and gp25 in the infection of human epithelial cells. J. Gen. Virol. 82, 2373–2383. Mason, R.J., 2006. Biology of alveolar type II cells. Respirology 11, S12–S15. Mora, A.L., Woods, C.R., Garcia, A., Xu, J., Rojas, M., Speck, S.H., Roman, J., Brigham, K.L., Stecenko, A.A., 2005. Lung infection with gamma-herpesvirus induces progressive pulmonary ﬁbrosis in Th2-biased mice. Am. J. Physiol. Lung Cell Mol. Physiol. 5, L711–721. Reddy, R., Buckley, S., Doerken, M., Barsky, L., Weinberg, K., Anderson, K.D., Warburton, D., Driscoll, B., 2004. Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells. Am. J. Physiol. Lung Cell Mol. Physiol. 4, L658–667. Rickinson, A.B., Kieff, E., 2001. Epstein-Barr virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol.2. Lippincott-Williams and Wilkins, Philadelphia, Pa, pp. 2575–2627. Sato, K., Tomioka, H., Shimizu, T., Gonda, T., Ota, F., Sano, C., 2002. Type II alveolar cells play roles in macrophage-mediated host innate resistance to pulmonary mycobacterial infections by producing proinﬂammatory cytokines. J. Infect. Dis. 185, 1139–1147. Sime, P.J., Xing, Z., Graham, F.L., Csaky, K.G., Gauldie, J., 1997. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe ﬁbrosis in rat lung. J. Clin. Invest. 4, 768–776. Stewart, J.P., Egan, J.J., Ross, A.J., Kelly, B.G., Lok, S.S., Hasleton, P.S., Woodcock, A.A., 1999. The detection of Epstein-Barr virus DNA in lung tissue from patients with idiopathic pulmonary ﬁbrosis. Am. J. Respir. Crit. Care Med. 159, 1336–1341. Tang, Y.W., Johnson, J.E., Browning, P.J., Cruz-Gervis, R.A., Davis, A., Graham, B.S., Brigham, K.L., Oates Jr., J.A., Loyd, J.E., Stecenko, A.A., 2003. Herpesvirus DNA is consistently detected in lungs of patients with idiopathic pulmonary ﬁbrosis. J. Clin. Microbiol. 41, 2633–2640. Tsukamoto, K., Hayakawa, H., Sato, A., Chida, K., Nakamura, H., Miura, K., 2000. Involvement of Epstein-Barr virus latent membrane protein 1 in disease progression in patients with idiopathic pulmonary ﬁbrosis. Thorax 55, 958–961. Turk, S.M., Jiang, R., Chesnokova, L.S., Hutt-Fletcher, L.M., 2006. Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells. J. Virol. 80, 9628–9633. Ulich, T.R., Yi, E.S., Longmuir, K., Yin, S., Biltz, R., Morris, C.F., Housley, R.M., Pierce, G.F., 1994. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J. Clin. Invest. 93, 1298–1306. Wallace, W.A., Howie, S.E., 1999. Immunoreactive interleukin 4 and interferonexpression by type II alveolar epithelial cells in interstitial lung disease. J. Pathol. 187, 475–480. Winkler, M.E., Mauritz, C., Groos, S., Kispert, A., Menke, S., Hoffmann, A., Gruh, I., Schwanke, K., Haverich, A., Martin, U., 2008. Serum-free differentiation of murine embryonic stem cells into alveolar type ii epithelial cells. Cloning Stem Cells 1, 49–64AC. Xu, Y.D., Hua, J., Mui, A., O’Connor, R., Grotendorst, G., Khalil, N., 2003. Release of biologically active TGF-beta1 by alveolar epithelial cells results in pulmonary ﬁbrosis. Am. J. Physiol. Lung Cell Mol. Physiol. 3, L527–539. Young, L.S., Dawson, C.W., Brown, K.W., Rickinson, A.B., 1989. Identiﬁcation of a human epithelial cell protein sharing an epitope with the C3d/Epstein-Barr virus receptor molecule of B-lymphocytes. Int. J. Cancer 43, 786–794.