Human Cytomegalovirus Infection Induces Transcription and - NCBI

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Vol. 68, No. 9

JOURNAL OF VIROLOGY, Sept. 1994, p. 5730-5737

0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Human Cytomegalovirus Infection Induces Transcription and Secretion of Transforming Growth Factor (1 S. MICHELSON,1* J. ALCAMI,'t S.-J. KIM,2 D. DANIELPOUR,2 F. BACHELERIE,1 L. PICARD,'t C. BESSIA,1 C. PAYA,l§ AND J.-L. VIRELIZIER' Unite d'Immunologie Virale, Institut Pasteur, 75724 Paris Cedex 15, France,1 and Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 208922 Received 22 March 1994/Accepted 20 June 1994 Human cytomegalovirus (CMV) infection can elicit a transitory, but profound, immunodepression in immunocompetent individuals. Cytopathogenic destruction of CMV-infected leukocytes alone does not seem sufficient to explain this phenomenon, which suggests that immune system mediators (cytokines) may play a role in amplifying local modifications wrought by CMV infection. We reported previously that transforming growth factor gi1 (TGF-f1) stimulates CMV replication (J. Alcami, C. V. Paya, J. L. Virelizier, and S. Michelson, J. Gen. Virol. 74:269-274, 1993). Since TGF-I1 can have profound negative effects on cell growth and immune responses, we investigated the induction of TGF-I1 following CMV infection of permissive fibroblasts. TGF-p1 promoter was activated by immediate-early (IE) proteins in the absence of infection and transactivated at 5 and 9 h after infection. TGF-I1 mRNA increased during the early phase of infection, suggesting that this phenomenon is due to enhanced transcription of the TGF-I11 gene. A comparative study of the influence of CMV infection and IE protein expression on TGF-I1 promoter function in permissive cells pointed to a possible cooperative role between IE proteins and protein(s) expressed during the early phase of viral infection. Induction of TGF-I1 by CMV infection could modify infected cells individually, surrounding tissues, and systemic immune reactions to the advantage of virus replication by both upregulating CMV replication and downregulating host immune responses. Human cytomegalovirus (CMV) infection can elicit a transitory, but profound, immunodepression in immunocompetent individuals. Cytopathogenic destruction of CMV-infected leukocytes alone does not seem sufficient to explain this phenomenon, which opens up the alternative hypothesis that immunoregulatory mediators (cytokines) may play a role in amplifying local modifications wrought by CMV infection. Observations made in vivo and in vitro are consistent with the notion that CMV infections are effectively accompanied by dysregulation of cytokine production. Patients with liver transplants have high levels of tumor necrosis factor alpha (TNF-c-) during CMV disease (43). Transcripts of this cytokine are also elevated in the mucosal macrophages of patients with CMV colitis (39). In lung transplant recipients experiencing CMV pneumonitis, the levels of interleukin 6 (IL-6) transcripts and protein in bronchoalveolar lavage specimens are increased as shown by in situ hybridization and immunoenzymatic assay (ELISA), respectively (14, 15), and the levels of circulating IL-2R in sera are also elevated (16). In vitro, infection of fibroblasts induces transcriptional activation of IL-6, oncostatin melanoma growthstimulating activity, platelet-derived growth factor, TNF-a, and IL-1 (42). Infection of peripheral blood leukocytes and endothelial cells by CMV results in enhanced production of IL-13, TNF-ot, gamma interferon, and colony-stimulating factor-1

(12, 29, 49), as well as labile suppressor factor(s) (8, 27, 45). Finally, infection of bone marrow cells increases both production of alpha interferon, gamma interferon, TNF-ao (7, 23, 32, 33, 37, 38), and IL-6 and transcription of basic fibroblast growth factor (11, 12, 22) but transiently suppresses IL-lox and - production (48). We reported previously that CMV infection of fibroblasts induces a basic fibroblast growth factor-like factor (2) which can stimulate endothelial cell proliferation, thus supporting the possibility that growth factor production induced in one cell type by the virus might affect the functions of neighboring cells. St. Jeor et al. (42) recently confirmed CMV stimulation of basic fibroblast growth factor transcription in these cells. We subsequently showed that basic fibroblast growth factor inhibits CMV replication in fibroblasts (3) and that another growth factor, transforming growth factor f1 (TGF-i1) stimulates CMV replication. Since the latter factor can have profound negative effects on cell growth and immune responses, we investigated the induction of TGF-31 following CMV infection of permissive fibroblasts. TGF-,B belongs to a family of proteins (reviewed in references 24 and 40) which can be potent negative regulators of lymphocyte function (reviewed in reference 18) and therefore play a role in inflammation and immunomodulation (reviewed in reference 47). This cytokine usually circulates in a latent form associated with ot-macroglobulin and can be activated by low pH or proteolysis (26). In the studies reported here, we examined the production of TGF-p1 following CMV infection of fibroblasts and investigated the induction of TGF-,B1 promoter activity in a variety of cell types infected with CMV and/or expressing CMV immediate-early (IE) antigens. In human fibroblasts, increased TGF-p1 production appears to be transcriptional.

* Corresponding author. Mailing address: Unite d'Immunologie Virale, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33.1) 45.68.82.64. Fax: (33.1) 45.68.89.41. t Present address: Servicio de Microbiologia, Hospital de 12 Octubre, Madrid, Spain. t Present address: Institut Cochin de Genetique Moleculaire, Paris,

France. § Present address: Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota.

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MATERIALS AND METHODS Cells. Human foreskin fibroblasts (FSF) and human astrocytoma cells (U373MG) were maintained in Dulbecco's modified medium (DME) containing 2 mM glutamine and 10 and 5% fetal calf serum, respectively. Monocytic U937 cells and lymphoblastoid T cells, J-Jhan, a subclone of the Jurkat lymphoblastoid cell line, were grown in RPMI medium supplemented with 2 mM glutamine and 5% fetal calf serum. Cells were routinely tested for mycoplasma and found to be negative. Virus. Human CMV strain Ad-169 was obtained from the American Type Culture Collection (VR-538). Stocks were grown in MRC-5 cells by infecting 80% confluent monolayers at a multiplicity of infection of 0.01 and collecting culture supernatants 8 to 10 days later. Supernatants were clarified for 10 min at 900 x g and 4°C, stored at -80°C, and titered by plaque formation under carboxymethyl cellulose. Virus stocks were mycoplasma free. Virus was inactivated by UV irradiation, which reduced its titer from 106 to 102 PFU/ml. Virus was also pelleted by centrifugation at 100,000 x g for 1.5 h at 4°C, and the pellet was washed with phosphate-buffered saline (PBS) before being suspended in DME without serum. Generation of culture supernatants and measuring of TGF,31, TNF-a, and IL-6. FSF were seeded at 6 x 104/cm2. They were infected at a multiplicity of infection of 2 to 5 with active CMV or with the same amount of UV-irradiated CMV (based on the original titer) or pelleted virus by adsorption for 30 min at 37°C. Mock-infected cultures underwent incubation for the same duration in fresh medium or medium conditioned for 3 to 4 days on uninfected fibroblasts. Cells were washed twice with PBS and refed medium without serum containing 100 ,ug of bovine serum albumin (BSA) per ml for the ELISA of TGF-,B1 or containing insulin (2.5 ,ug/ml) and transferrin (10 ,ug/ml) for bioassays of TGF-j1 and TNF-a and for ELISAs of TNF-a and IL-6. Supernatants were harvested and medium was renewed at the times indicated in Results. Supernatants were clarified at 900 x g for 10 min at 4°C and were either stored at 80°C for bioassays (see below) and ELISAs of IL-6 and TNF-a with kits from British Biotechnology or were lyophilized for testing of TGF-,B1 by ELISA as previously described (9). Bioassay of TGF-I activity. TGF-fi1 activity was measured by the inhibition of [3H]thymidine incorporation into mink lung epithelial cells (CCL64) as described by Jennings et al. (17). Latent TGF-, was activated in supernatants by being heated at 95°C for 7 min. Supernatants were tested before and after activation. Activated supernatants were also tested after neutralization of TGF-r activity with a rabbit anti-pan-TGF-P serum (British Biotechnology) (1/100) at 37°C for 30 min. Controls consisted of [3H]thymidine incorporation into cells treated with a range of concentrations of purified TGF-,B1 with and without anti-TGF-4 antibodies, cells incubated with 4 mM HCI (used to activate purified TGF-,B1), TGF-43-neutralizing serum alone (1/100), medium alone, or nonimmune rabbit serum.

Northern (RNA) blot analysis. FSF were seeded and infected for various times as described above. Uninfected and infected cells were washed twice in situ with cold PBS and then scraped into PBS. Cells were lysed with 50 mM Tris (pH 8)-150 mM NaCl-10 mM MgCl2-150 mM sucrose-1% Nonidet P-40. After the elimination of nuclei, Sarkosyl was added to a final concentration of 1%. RNA was extracted once with phenol, twice with phenol-chloroform (1:1), and once with chloroform. After ethanol precipitation, RNA was resuspended in diethyl pyrocarbonate-treated H20. Ten to fifteen micrograms of each RNA sample was separated on a 1.2%

TGF-1l

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agarose (Seakam) gel containing 5.5% formaldehyde at 70 V. RNA was transferred by capillarity to nitrocellulose (Sartorius) or Hybond N+ (Amersham) under vacuum with 50 mM NaOH. Blots were hybridized as previously described (20). Plasmids. The two TGF-41 promoters (TGF-5 and TGF-16) controlling expression of the chloramphenicol acetyltransferase reporter genes have been described previously (19). The TGF-5 and TGF-16 promoters were recloned into the Hindlll site upstream of the luciferase gene. Plasmids expressing CMV IE proteins under the control of their own promoters were generous gifts of R. LaFemina (Merck Sharp & Dohme, West Point, Pa.), G. Hayward (Johns Hopkins School of Medicine, Baltimore, Md.), and C. Davrinche (Institut National de la Sante et de la Recherche Medicale, Toulouse, France) and have been described by Pizzorno et al. (31) and Davrinche et al. (10). The plasmids used were pRL45, which expresses both TEl and IE2 proteins; pMP17 and pIEl containing only IE exons 1 to 4; and pMP18 containing exons 1 to 3 and 5. All plasmids were purified by chromatography on Qiagen columns according to the manufacturers' instructions and controlled by restriction enzyme analysis. Transfection assays. Adherent cells were transfected by the calcium phosphate technique at a ratio of 1 jig of DNA per 106 cells, except for kinetic studies when 5,ug of DNA per 106 cells was used. Cultures were incubated in the presence of precipitated DNA for 5 h, washed twice with PBS, and then refed growth medium. Cells were infected 1 h after refeeding by adsorption of virus for 30 min at 37°C. Cells in suspension (U937 and J-Jhan) were transfected by incubation for 20 min at room temperature in the presence of a DEAE-dextranDNA solution at 1 ,ug of DNA per 106 cells. Then cells were diluted in growth medium, washed, and resuspended at 2.5 x 105 cells per ml. Cells were harvested 24 h after transfection. Luciferase activity was measured with a luminometer (Berthold, Elancourt, France) as previously described (35) and is expressed as relative light units per milligram of protein as determined by the Bio-Rad method. Establishment of cells stably expressing CMV IE proteins. U373MG astrocytoma cells were transfected by the calcium phosphate technique with 1 ,ug of DNA containing a 1/5 ratio of a simian virus 40 neomycin resistance gene plasmid to the IE1/2 expression plasmid per 106 cells. Forty-eight hours after transfection, cells were changed to medium containing 400 ,ug of G418 per ml. This medium was changed every 2 to 3 days until the appearance of colonies within 3 to 4 weeks. A cell line, A45, stably transfected with pRL45 was thus obtained. Immunofluorescence and Western immunoblot analysis with anti-CMV IE monoclonal antibody E13 (a generous gift of M. C. Mazeron, H6pital Lariboisiere, Paris, France) showed that A45 cells expressed IE proteins of 55, 72, and 82 kDa (results not shown). A 32P-labelled HindIl-EcoRT fragment from the insert of pRL103 (31) hybridized specifically to a 1.9to 2.1-kb band in cytoplasmic RNA extracted from A45 cells (results not shown). No immunoreactivity with monoclonal antibody E13 or RNA hybridization was found with material extracted either from the parent cell line or from a neomycinresistant cell line established in parallel (results not shown). RESULTS CMV-induced TGF-41 production. Supernatants of infected FSF and uninfected control FSF were collected and clarified at 4, 24, and 48 h after infection, and TGF-4 was measured by ELISA as previously described (9). Quantification of TGF-431 and -,B2 showed that TGF-431 was detected in ever-increasing

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TABLE 1. Detection by ELISA of TGF-131 induced by CMV infection of FSF Infection

+

A. 0-24 hrs

TGF-,B1 (pmol/ml) ata:

0-4b

424"

24-481

1.27 + 0.18 1.49 + 0.31

6.12 ± 0.54 7.64 ± 1.24

3.37 ± 0.68 14.1 ± 2.64

LT

T

2r-

FSF were mock infected or infected with the Ad-169 strain of CMV, and supernatants were collected from 0 to 4, 4 to 24, and 24 to 48 h after being washed. b Time from the refeeding of cells after being washed at the end of the viral adsorption period. Cells were infected at a multiplicity of infection of 5 with the AD-169 strain of CMV, and virus was adsorbed for 30 min at 37°C. Uninfected cells were incubated with spent medium from fibroblasts. Cells were washed twice with PBS and fed serum-free medium containing 100 ,ug of BSA per ml.

amounts in infected-cell supernatants, with four times more produced by the 24- to 48-h supernatants (Table 1). TGF-,2 production was not modified (results not shown). Cell supernatants collected 24 h postinfection were tested by bioassay before and after activation and in the presence and absence of anti-TGF-, antibody (a rabbit anti-pan-TGF-, serum at a 1/100 dilution). The representative experiment illustrated in Fig. 1 shows that CMV-induced TGF-, was detected only in heat-activated supernatants from cells infected with active virus (Fig. 1, CMV). Induction by CMV was a consistent phenomenon observed in more than six separate experiments. Supernatants of cells mock infected with conditioned medium (Fig. 1, SN) or undergoing a medium change (results not shown) and of cells infected with UV-irradiated virus (Fig. 1, UV-CMV) did not contain TGF-3 activity (Fig. 1A). The use of nonimmune rabbit sera in place of anti-TGF-1 antibodies did not affect the activities of purified TGF-,B1 or infected-cell supernatants. Virus which had been pelleted so as to remove any growth factors also induced TGF-,B (Fig. 1B). Induction in 0- to 24-h supernatants was dose dependent. The amounts of TGF-3 in 24- to 48-h supernatants were further increased (results not shown). TNF-o and IL-6 concentrations in supernatants of uninfected and infected fibroblasts. IL-6 was detected at all times in supernatants from both uninfected and infected cells (Table 2). In infected cell supernatants collected during the 0- to 4-h interval, the level of IL-6 was eightfold higher than that in supernatants from uninfected cells. IL-6 production in both types of culture increased greatly between 4 and 24 h to attain approximately the same levels in infected-cell and uninfected supernatants. Between 24 and 48 h, IL-6 levels decreased markedly, but the levels in infected-cell supernatants remained

TABLE 2. Induction of IL-6 in fibroblasts by CMV infectiona Time of conditioning (h)

0-4 4-24

24-48

IL-6 concentration (pg/ml)b Uninfected cells

CMV-infected cells

23.6 + 2.8 273.1 ± 65.2 16.4 ± 4.6

190.4 ± 1.9 383.4 ± 14.6 96.1 + 8.3

aFSF cells (three wells per point) were incubated with pelleted CMV (2 PFU per cell) for 1 h at 37°C. Cultures were washed twice with PBS and refed with DME containing transferrin (10 p.g/ml) and insulin (2.5 ,.g/ml). Supernatants were collected after 4 h of incubation, and cells were refed with the aforementioned medium. Supernatant collection and refeeding were similarly performed after 24 and 48 h of incubation. IL-6 was measured by ELISA (British Biotechnology). Pelleted virus contained 11.9 pg of IL-6 per ml. b Data are means ± standard deviations.

E

CL

1-

CMV B.

0-24 hrs

4-

iE(L

12-

-

0O

Mock

U.UU5

U0U4

U.U0

Multiplicity of Infection

FIG. 1. Bioassay of TGF-1 in supernatants of fibroblasts. (A) TGF-1 activity in supernatants collected at the times indicated from uninfected (SN) and infected (CMV and UV-CMV) human fibroblasts was measured by the incorporation of [3H]thymidine into mink lung epithelial cells (CCL64) according to Jennings et al. (17). The graphs show the levels of incorporation in the presence of supernatant before (white bars) and after (black bars) heat activation of TGF-3 and after neutralization of activated media with anti-pan-TGF-,B antiserum (stippled bars) when CCL64 cells were incubated with supernatant conditioned on uninfected cells (SN), cells infected with active virus (CMV), and cells infected with UV-inactivated virus (UV-CMV). All supernatants tested were collected 24 h after infection or mock infection with conditioned medium. Error bars show standard deviations. Data are representative of six experiments. (B) Dose response of TGF-p1 production to CMV infection. Fibroblasts were infected with increasing multiplicities of pelleted washed CMV (black columns). Controls were mock infected (white column) with the serum-free medium used to resuspend pelleted virus. Following adsorption or 30 min at 37°C, cells were washed and refed DME containing transferrin, insulin, and 100 ,g of BSA per ml (see Materials and Methods). Supernatants were collected 0 to 24 h after infection, clarified, and stored at -80°C. They were tested as described above in the presence and absence of anti-TGF-,B-neutralizing antibody after heat activation. Results are expressed as the differences in average counts (from three wells) of [3H]thymidine incorporation into cells in the absence and presence of TGF-,3-neutralizing antibody. Thus, each column reflects the amount of TGF bioactivity per 0.4 ml of culture supernatant tested.

higher than those in uninfected-cell supernatants. Pelleted virus used for infection contained about 11.9 pg of IL-6 per ml, which represented about 2.9 pg/ml per infectious dose used. No TNF-ot was detected by ELISA (results not shown) or bioassay (Table 3) in supernatants from uninfected or infected cells at any time. TNF-ot was detected transiently when fibroblasts were pulsed with IL-1 for 1 h as a control for the

HUMAN CMV INDUCES TGF-13l

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Time p.i. 24hr

with

UI

U I

TGF,I1

u I

TABLE 3. Induction of TNF-tX in fibroblasts by IL-2 but not by CMV infectiona

Hybridization

4hr

U I

Time after infection Uninfected (h) cells

GAPDH

FIG. 2. Northern blot analysis of FSF cytoplasmic RNA hybridized with a TGF-1I probe which recognizes a 2.5-kb mRNA and with a probe for GAPDH. RNA was extracted from uninfected (U) and infected (I) fibroblasts at 4 and 24 h after infection. Fold increase in TGF-,B1 transcription was determined by densitometric scanning and normalization with GAPDH. At 4 h postinfection, transcription of infected cells was 1.4-fold that of uninfected cells; at 24 h postinfection, it was 2.4-fold.

inducibility of TNF-t- in our cultures. The removal of IL-1 after a pulse is known to result in the rapid cessation of the secretion of TNF-ox (Table 3, uninfected cells + IL-1 at 4 to 24 h) (3a). Analysis of steady-state levels of TGF-fr1 RNA. Cytoplasmic RNA extracted from uninfected fibroblasts and from cells infected for 4 and 24 h was hybridized with a TGF-i1 probe (Fig. 2) which detects a 2.5-kb mRNA as previously described

111x

10

1

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99x

TNF-as titer Uninfected cells Uninfected cells CMV-infected + IL-1 + PMA cells

0-4 4-24