Human Immunodeficiency Virus Type 1 gp120 ... - Journal of Virology

2 downloads 0 Views 261KB Size Report
and Institute San Gallicano,3 Rome, Italy. Received 11 March .... After a 1-h incubation at 37C, cell monolayers were washed twice and refed with fresh medium. ...... Yoshimura, T., K. Matsushima, J. J. Oppenheim, and E. J. Leonard. 1987.
JOURNAL OF VIROLOGY, Feb. 1997, p. 1591–1597 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 71, No. 2

Human Immunodeficiency Virus Type 1 gp120 Stimulates Cytomegalovirus Replication in Monocytes: Possible Role of Endogenous Interleukin-8 MARIA R. CAPOBIANCHI,1* CATERINA BARRESI,1 PAOLA BORGHI,2 SANDRA GESSANI,2 LAURA FANTUZZI,2 FRANCO AMEGLIO,3 FILIPPO BELARDELLI,2 SERGIO PAPADIA,1 1 AND FERDINANDO DIANZANI Institute of Virology Universita ` “La Sapienza,”1 Istituto Superiore di Sanita ` ,2 and Institute San Gallicano,3 Rome, Italy Received 11 March 1996/Accepted 7 November 1996

Recombinant gp120, but not other human immunodeficiency type 1 (HIV-1) structural proteins, dosedependently stimulates human cytomegalovirus (HCMV) immediate-early antigen (IEA) expression and infectious virus yield in freshly isolated normal monocytes infected with HCMV. Monoclonal antibodies (MAbs) recognizing the gp120 V3 loop, as well as V3 loop octameric multibranched peptides and antibody to galactocerebroside, but not sCD4, abrogate the gp120 stimulation of IEA expression, suggesting that the effect involves V3 loop-galactocerebroside interaction and is not mediated by CD4. Interleukin 8 (IL-8) gene expression is enhanced in monocytes treated with gp120 at the level of both mRNA and released protein. Exogenous IL-8 could replace gp120 in the stimulation of HCMV infection, while a MAb capable of neutralizing IL-8 activity abrogates the gp120-induced HCMV stimulation. These data indicate that HIV-1 glycoprotein induces stimulation of productive infection of monocytes with HCMV and that such stimulation may be mediated by the upregulation of IL-8 gene expression. This is the first evidence that HIV-1 may affect HCMV replication indirectly, via the interaction of gp120 with the monocyte membrane, in the complete absence of retroviral replication, through the stimulation of IL-8 release. Because in HIV-1-infected individuals, HCMV infection is frequently activated and the levels of circulating IL-8 are enhanced, these findings may be pathogenetically relevant.

Human cytomegalovirus (HCMV) infection is widely diffused in the human population, with over 50% of adults showing the presence of specific antibodies by 50 years of age. Recently, the use of amplified nucleic acid detection has indicated that HCMV infection is even more common than previously suggested by serology (28, 43). Usually HCMV infection is asymptomatic in people with a mature, competent immune system, remaining mostly latent after primary infection. Occasional episodes of reactivation in these subjects are often coincident with transient immunosuppression and rarely become clinically evident. Several studies have suggested that bone marrow progenitor cells, circulating neutrophils, monocytes, and lymphocytes, as well as endothelial cells, are the sites of acute or latent in vivo HCMV infection (4, 14, 17, 30, 31, 33, 41–43, 46), and infectious virus has been isolated from circulating leukocytes during active disease (4, 38). However, HCMV is one of the most frequent opportunistic agents causing severe illness in immunocompromised hosts, including human immunodeficiency virus type 1 (HIV-1)-infected patients (25, 26). HCMV clinical manifestations, often at unusual body sites, are very common in the advanced stages of HIV infection, and up to 90% of AIDS patients display signs of disseminated HCMV infection at autopsy (47). Several reports indicate that CMV and HIV can reciprocally influence (positively or negatively) each other’s expression, suggesting that the two infections may be pathogenetically

connected (22, 27, 29, 37, 40). While there is evidence that virus replication is not essential for HCMV to either stimulate or inhibit HIV expression (27, 29, 37), the need for HIV replication has not been clearly established for its effect on HCMV. In fact, conflicting results have been obtained under different experimental conditions, suggesting that HIV productive infection can either up- (40) or downmodulate HCMV expression, possibly through its surface glycoprotein, gp120 (30), whereas tat gene-coded protein is mostly stimulatory (22, 30). Therefore, it seemed interesting to further explore the effects of HIV on HCMV replication by considering additional, indirect mechanisms. To this aim, we investigated the effects of HIV-1 structural proteins, including gp120, on HCMV infection of freshly isolated normal monocytes. In fact, these cells have been shown to be one of the major sites of HCMV persistence in vivo (17, 42, 43) and have been shown to be permissive to HCMV in vitro, since they express immediateearly antigens (IEA) and produce infectious progeny after in vitro infection with either laboratory strains or fresh isolates, as well as after cocultivation with HCMV-infected cells (14, 31, 33, 41, 46). It has been found that gp120 actually potentiates HCMV replication in these cells. Since normal monocytes show increased cytokine production after exposure to HIV gp120 (5, 18, 21, 23, 45), the possibility that the gp120-induced cytokines can be involved in the upmodulation of HCMV replication has been considered.

* Corresponding author. Mailing address: Institute of Virology, Viale di Porta Tiburtina 28, 00185 Rome, Italy. Phone: 39.6.4452846. Fax: 39.6.4469024. E-mail: [email protected].

Monocyte cultures. Human monocytes were isolated by Ficoll-Hypaque density gradient centrifugation from the peripheral blood of healthy donors and were separated from lymphocytes by adherence to plastic dishes as described

MATERIALS AND METHODS

1591

1592

CAPOBIANCHI ET AL.

J. VIROL.

FIG. 1. HCMV IEA expression in monocytes infected with HCMV. Fresh monocytes obtained by adherence of normal PBMCs were infected with HCMV AD169 at an MOI of 1 TCID50/cell. After 1 h of adsorption, the virus inoculum was discarded, and then the cultures were refed with complete medium. HCMV IEA expression was assessed after 1 day of incubation by staining with an FITC-MAb. Evans blue was used to counterstain the cells (original magnification, 3250). (A) HCMV-infected monocytes. (B) Uninfected monocytes.

previously (18). Cytochemical (i.e., sodium fluoride-inhibited esterase activity) and fluorescence-activated cell sorter analysis of surface markers (CD14 antigen) revealed that the adherent cell population consisted of .95% monocytes. These were seeded in multichamber plastic slides (Nunc, Inc., Naperville, Ill.) at a concentration of 105/ml in 0.5 ml of RPMI containing 20% heat-inactivated fetal calf serum. Duplicate cultures were tested for each experimental point, and results were expressed as means 6 standard errors of repeated experiments. Statistical significance was evaluated by Student’s t or x2 tests, as appropriate. Reagents. The following recombinant proteins were prepared in a baculovirus expression system: gp120 (HIV-1IIIB) and p24 and soluble CD4 (sCD4), obtained from Intracel, Cambridge, Mass. P17 was expressed in pGEX as a glutathione S-transferase fusion protein and was obtained from S. Adams, through the Medical Research Council AIDS Directed Programme Reagent Project, England. A monoclonal antibody (MAb) to gp120 (clone a70), recognizing the HIV-1IIIB V3 loop was obtained from Intracel. The multibranched peptide constructs (GPGRAF)8-mbpc and (APGRAF)8mbpc were synthesized by automated solid-phase peptide synthesis starting with Fmoc8-k4-k2-k-beta A-NovaSyn-KA resin (Calbiochem-Novabiochem, San Diego, Calif.) as previously described (48, 49). They were synthesized at the Protein and Nucleic Acid Shared Facility of the Medical College of Wisconsin, Milwaukee. The toxicity of these multibranched peptides for peripheral blood mononuclear cells (PBMCs) was very low (50% tissue culture infective dose [TCID50], .250 mg/ml) as determined in an MTT assay (13). The inhibitory effect on HIV-driven syncytium formation was tested by using chronically infected H9/ HIVIIIB and lymphoblastoid C8166 cells according to standard procedures (48). The 50% inhibitory dose (ID50) for both peptides was 0.5 to 1 mg/ml. A MAb to galactocerebrosides (Gal-cer) (clone mGalC) was obtained from Boehringer-Mannheim S.p.A., Milan, Italy. This antibody binds Gal-cer and sulfatides, but does not cross-react with glucocerebrosides, ceramide, sphingosine, and mixed brain gangliosides. The antibody was used at 10 mg/ml. Nylon filters (0.2 mm pore diameter) able to remove lipopolysaccharide were obtained through Nalgene, Rochester, N.Y. Escherichia coli-expressed recombinant human interleukin 8 (rhIL-8) and a neutralizing MAb to IL-8 (clone 6217.11) showing no cross-reactivity with rhRANTES, rhGRO alpha, rhMIP-1 alpha, rhMIP-1 beta, rmMIP-1 alpha, or rmMIP-1 beta were purchased from R&D System Europe Ltd., Abingdon, United Kingdom. Virus. HCMV AD169 (ATCC VR538) was propagated on human diploid HEL299 fibroblasts (ATCC), obtained from the Istituto Zooprofilattico, Brescia, Italy. Virus titrations were performed with the same cells by the limiting dilution method, by using four replicates for each 0.5-log-based dilution. The titration cultures were refed with fresh medium at day 6 or 7 postinfection, and the final

score for cytopathic effect was determined by light microscope examination at day 10 postinfection. Infectious titer was calculated according to the Reed and Muench formula. Virus stocks with titers of at least 5 log TCID50/ml were used. Monocytes were infected at a multiplicity of infection (MOI) of 1 TCID50/cell. After a 1-h incubation at 378C, cell monolayers were washed twice and refed with fresh medium. The expression of IEA was determined at day 1 postinfection by direct immunofluorescence by using a fluorescein isothiocyanate-conjugated MAb (FITC-MAb) to the 76-kDa nonstructural antigen of HCMV (clone E13) from Argene (previously Biosoft), Varhiles, France. The expression of HCMV late antigens (LA) was tested at day 3 postinfection, with a specific MAb (clone SL20) from Argene in an indirect immunofluorescence assay by using an FITCantimouse immunoglobulin G antiserum from Dako, Glostrup, Denmark. Staining was performed according to the manufacturer’s instructions, and phosphatebuffered saline supplemented with 10% human serum negative for HCMV antibodies was used as a blocking solution. As a control for unspecific fluorescence, an FITC-MAb to herpes simplex virus type 1 (clone H62) from Argene, which showed no staining in HCMV-infected monocytes, was used. For each experimental condition, at least 200 cells were scored, positive cells (showing nuclear fluorescence for IEA and cytoplasmic fluorescence for LA) were counted, and the results were expressed as the percentage of positive cells. To determine the infectious virus yield, monocytes were infected as described above, washed twice after the adsorption period, and refed with fresh medium. At the indicated time points, the whole culture wells, containing the same volume of medium and number of cells for each experimental condition, were frozen and thawed twice, and the cryolysates were backtitrated on diploid HEL299 fibroblasts as described above. A fresh clinical isolate of HCMV was generated from the urine of a congenitally infected neonate. It was propagated on HEL299 fibroblasts as described above and used at passage 2. IL-8 gene expression. Culture supernatants were assayed for IL-8 content with a commercial enzyme-linked immunocapture assay (ELISA) from R&D systems (detection limit, 18 pg/ml). IL-8 mRNA expression was analyzed by reverse transcription-PCR (RTPCR). Total cellular RNA was extracted by the method of Chirgwin et al. (10). The RT-PCR was performed as described in reference 24, but with the following modification. For the RT reaction, 0.5 mg of RNA was mixed with 1 mg of oligo(dT) (12 to 18 oligomer; Pharmacia, Uppsala, Sweden) and incubated for 10 min at 658C. After cooling on ice, the mixture was incubated with RT buffer (24)–1 mM deoxynucleoside 59-triphosphates (dNTP) in the presence of 50 U of Moloney murine leukemia virus reverse transcriptase for 60 min at 378C. The cDNA obtained was amplified by using 0.1 mg of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or IL-8 in a mixture containing

VOL. 71, 1997

HCMV STIMULATION BY gp120-INDUCED IL-8 IN MONOCYTES

1593

FIG. 2. Effect of HIV-1 gp120, p24, and p17 on HCMV IEA expression by monocytes. Monocytes were exposed to various amounts of HIV-1 structural proteins for 24 h and then washed and infected with HCMV as described in the legend to Fig. 1. Cells with fluorescent nuclei were counted, and the results are expressed as the percentage of positive cells. A representative experiment is shown. P , 0.001 by the x2 test for gp120; P . 0.50 for p24 and p17.

FIG. 3. Growth curve of HCM AD169 in fresh monocytes. Monocytes were infected with HCMV and sampled at the indicated time points to determine the titer of infectious virus present in the culture cryolysates. The virus titer at day 0 represents the amount of HCMV detected in the cultures after the adsorption and subsequent washings.

200 mM dNTP and 0.5 U of Taq polymerase in PCR buffer (24). The sequences of the IL-8 primers (according to reference 9) were as follows: IL-8 sense, 59-ATTTCTGCAGCTCTGTGTGAA-39; IL-8 antisense, 59-TGAATTCTCAG CCCTCTTCAA-39. The sequence of the GAPDH primers has been previously described (24). PCR was performed in a thermal cycler (Perkin-Elmer, Monza, Italy) for 20 cycles of 40 s at 948C, 40 s at 628C, and 60 s at 428C. This low number of amplification cycles ensures the detection of mRNA bands in conditions of linear amplification, as assessed in exploratory experiments. However, a basal level of IL-8 mRNA expression can be detected by increasing the number of cycles up to 25. The reaction products were analyzed on a 2.5% agarose gel with a molecular size ladder (fX174 replicative form DNA HaeIII digest; New England Biolabs, Beverly, Mass.). The expected sizes of the amplified bands were 255 bp for IL-8 and 196 bp for GAPDH.

isolate of HCMV was similarly affected by gp120, showing 6.9% 6 1.1% IEA-positive cells in control cultures and 15.5% 6 0.5% positive cells in cultures exposed to 2 mg of gp120 per ml (P 5 0.019). gp120 filtered through membranes capable of removing lipopolysaccharide was as effective as unfiltered gp120 in stimulating HCMV IEA expression (not shown). Other HIV-1 recombinant proteins (i.e., the major capsid protein p24 and the matrix protein p17) did not affect HCMV IEA expression up to a concentration of 8 mg/ml (Fig. 2). Since the expression of IEA does not necessarily lead to productive HCMV infection, especially in blood-derived cells, and some investigators have failed to show productive infection of monocytes by HCMV laboratory strains (17, 31, 41–43), we performed exploratory experiments to determine the expression of HCMV LA and the replication curve of the AD169 HCMV strain in fresh monocytes. A representative replication curve is shown in Fig. 3, indicating that virus titer progressively increased in these cell cultures, reaching a value at day 4 about 100-fold higher than that found after the adsorption period (time zero), and slightly declined thereafter. In repeated experiments, peak virus yield was observed between days 4 and 5 postinfection, indicating that a fully productive replication cycle occurs under our experimental conditions. Furthermore, the number of cells expressing HCMV LA at day 3 postinfection was consistent with the number of cells expressing HCMV IEA early after infection, indicating that IEA-expressing cells are an effective indicator of the extent of HCMV infection in monocytes (described below). We then determined the effect of gp120 on both HCMV IEA and LA expression, as well as on the virus yield. The results, shown in Table 1, indicate that both the number of HCMV IEA- and LA-expressing cells and the HCMV infectious yield are significantly higher in gp120-stimulated cultures than in untreated cultures. Furthermore, the amount of virus that remained cell bound after the adsorption period was increased .3.5-fold in gp120-treated cultures compared with in

RESULTS In a first set of experiments, we tested the effects of glycoprotein gp120 on the susceptibility of monocytes to infection with a laboratory strain (AD169) of HCMV by counting the cells expressing HCMV IEA. For this purpose, recombinant gp120 was administered to 1-day-old monocyte cultures, and after overnight incubation, the cells were washed and infected with HCMV. The expression of IEA was tested 1 day later by staining with a MAb to HCMV IEA. As shown in Fig. 1, the staining was confined to the nuclei of infected cells, as expected. Furthermore, in gp120-stimulated cultures, the number of positive cells was dose-dependently increased, being maximal at a gp120 concentration of 4 mg/ml, as shown in Fig. 2. Consistent results were obtained in repeated experiments. In fact, the frequency of HCMV IEA-expressing monocytes in unstimulated cultures was 12.0% 6 2.0% (mean 6 standard error of nine independent experiments); an about twofold stimulation was observed at a gp120 concentration of 2 mg/ml (positive cells, 23.1% 6 4.2%; mean 6 standard error of five independent experiments, P 5 0.015 by Student’s t test), and a fourfold stimulation was observed at a gp120 concentration of 4 mg/ml (positive cells, 48.9% 6 13.5%; mean 6 standard error of six independent experiments; P 5 0.001). A fresh clinical

1594

CAPOBIANCHI ET AL.

J. VIROL.

TABLE 1. Effect of gp120 on expression of HCMV IEA and LA and infectious virus yield by monocytesa % of positive cells (P) HCMV IEA

HCMV LA

HCMV yield [log TCID50/ml (P)]

17.0 6 3.0 (0.001) 46.9 6 9.1

15.0 6 4.4 (0.004) 46.5 6 7.4

2.8 6 0.2 (0.02) 3.7 6 0.2

Treatment

None gp120

a Monocytes obtained by plastic adherence were exposed to gp120 (5 mg/ml), and 1 day later, they were infected with HCMV (MOI of 1 TCID50/ml). Cell monolayers were stained for IEA and LA expression on days 1 and 3 postinfection, respectively; parallel cultures were analyzed for infectious virus yield by backtitration of culture cryolysates on day 5 postinfection, as specified in Materials and Methods. Values are means 6 standard errors of four independent experiments.

control cultures (1.9 versus 1.3 log TCID50/105 cells, respectively), suggesting that very early events in the virus replicative cycle, possibly involving virus adsorption and/or penetration, could be affected by gp120 stimulation. The membrane interactions responsible for HCMV stimulation by gp120 were explored by competition experiments with either a gp120-specific MAb or sCD4 used as the competitor. The results, shown in Table 2, indicate that the MAb to gp120 abrogated the gp120 effect on HCMV IEA expression, while sCD4 was completely uneffective. Since the anti-gp120 MAb was raised against the third variable domain of the glycoprotein (V3 loop), we explored the role of this region. Specifically, we used gp120 octameric branched-peptide constructs representing the apex of the V3 loop consensus sequence, (GPGRAF)8-mbpc (MAP-5), or a slight variant of it, (APGRAF)8-mbpc (MAP-1). MAP-5 has been previously shown to bind to Gal-cer and to block HIV-1-driven syncytium

TABLE 2. Effects of sCD4, the MAb to gp120, the octameric branched V3 loop peptides, and the MAb to Gal-cer on the gp120driven stimulation of HCMV IEA expression in monocytesa Treatment (concn [mg/ml])

HCMV IEA expression (% positive cells)

Expt 1 None MAb to gp120 (10) gp120 (2) gp120 1 MAb to gp120

13.2 6 1.8 13.1 6 2.1 21.6 6 0.6 9.70 6 1.8

Expt 2 None sCD4 (5) gp120 (5) gp120 1 sCD4

10.0 6 2.1 15.0 6 3.0 .75 .75

Expt 3 None MAP-1 (5) MAP-5 (5) MAb to Gal-cer (10) gp120 (2) gp120 1 MAP-1 gp120 1 MAP-5 gp120 1 MAb to Gal-cer

7.8 6 0.8 6.3 6 0.6 5.6 6 1.4 10.4 6 0.2 20.1 6 2.5 9.1 6 2.5 7.7 6 2.2 1.7 6 0.5

P

.0.05b 0.024c

.0.05b .0.05c

.0.05b .0.05b .0.05b 0.034c 0.019c 0.010c

a Monocytes were exposed to gp120 in the presence of each competitor at the indicated concentration. One day later, the cells were infected with HCMV as described in the legend to Table 1. HCMV IEA expression was measured on day 1 postinfection, as described in Materials and Methods, and is expressed as the mean 6 standard error of two independent experiments. b With respect to untreated monocytes. c With respect to gp120-treated monocytes.

formation (48, 49). Exploratory experiments had shown that the two peptide constructs are equally effective in blocking syncytium formation between CD4 T-lymphoblastoid and HIV-1-infected cells (ID50, 0.5 to 1 mg/ml, data not shown). Moreover, a MAb to Gal-cer was used. The results, shown in Table 2, indicate that neither of the two peptides tested nor the MAb to Gal-cer was able per se to significantly affect HCMV IEA expression. Furthermore, both V3 loop multibranched peptides and the MAb to Gal-cer abrogated the effect of gp120. These findings suggest that the gp120 interaction with the monocyte membrane responsible for the HCMV activation is not mediated by CD4, but possibly is mediated by V3 loop binding to Gal-cer, which has been suggested as a possible alternate receptor for HIV gp120 (11, 16, 19, 39, 49). However, the data do not help define whether gp120 stimulates HCMV directly or through induction of soluble mediators. In fact, gp120 has been shown to induce a number of cytokines and products of immune activation in monocytes (5, 18, 21, 23, 45). Since it has been recently shown that a chemokine (i.e., IL-8) produced by several cell types, including monocytes (50), can upmodulate HCMV replication in fibroblasts (35), we tested whether gp120 activation of HCMV could be mediated by the induction of this chemokine. We then analyzed IL-8 gene expression at the level of both mRNA (as detected by RT-PCR) and released protein (as detected by ELISA) in monocyte cultures stimulated with gp120 compared with that in untreated cultures as follows. Monocytes were exposed (or not exposed) to 5 mg of gp120 per ml. After overnight incubation, their supernatants were assayed for IL-8 content with a commercial ELISA. With no treatment, the yield of IL-8 was 11.9 6 3.4 nl/ml; with gp120 treatment, the yield of IL-8 was 63.6 6 9.4 nl/ml (mean 6 standard error of four independent experiments; P , 0.001). The results indicate that a substantial amount of this cytokine is constitutively released by cultured monocytes (range 4.8 to 21.5 ng/ml) and that gp120 treatment results in a significant stimulation of this production, causing a more than fivefold enhancement of IL-8 release (range, 31.8 to 95.0 ng/ml). In keeping with these findings, a marked induction of the IL-8 mRNA was observed in gp120-treated compared to untreated monocytes (Fig. 4) under conditions of linear amplification (i.e., 20 amplification cycles). The IL-8 mRNA band was observed in untreated monocytes with a higher number of amplification cycles (not shown), in keeping with the constitutive expression of the chemokine under these experimental conditions. These findings suggest that endogenous IL-8 could play a role in the susceptibility of fresh monocytes to HCMV and in the gp120-driven stimulatory effect. To test this hypothesis, we used a MAb capable of neutralizing IL-8 activity. Particularly, a MAb capable of neutralizing IL-8 activity was added with gp120 to monocytes, and after overnight incubation, the cells were washed and infected with HCMV. As a positive control for HCMV stimulation by the chemokine, exogenous IL-8 was added at a concentration similar to that found in gp120-stimulated cultures (i.e., 50 ng/ml). HCMV IEA expression and infectious yield were tested on days 1 and 5, respectively. The results, shown in Fig. 5, indicate that, while IL-8 at the concentration used could replace gp120 in stimulating both HCMV IEA expression and infectious virus yield, the MAb to IL-8 abrogated the gp120-induced stimulation of both parameters. DISCUSSION These findings demonstrate that normal monocytes exposed to HIV-1 gp120 have increased sensitivity to the productive

VOL. 71, 1997

HCMV STIMULATION BY gp120-INDUCED IL-8 IN MONOCYTES

FIG. 4. Effect of gp120 on IL-8 gene expression in monocytes. Total cellular RNA was extracted from monocytes exposed to gp120 (1 mg/ml) for 6 h. RNA was analyzed for the presence of IL-8 and GAPDH mRNA by RT-PCR, as described in Materials and Methods.

infection by HCMV, since both HCMV IEA expression and LA expression, as well as infectious virus yield, are higher in gp120-stimulated monocyte cultures. HCMV stimulation seems not to be restricted to a laboratory strain of HCMV, since a fresh virus isolate also infects gp120-treated monocytes more efficiently than control cells. The stimulation appears to be specific for HIV-1 gp120. In fact, the contribution of contaminants to the observed phe-

FIG. 5. Effects of neutralizing anti-IL-8 MAb on gp120-driven HCMV stimulation in monocytes. Monocytes were exposed to gp120 (5 mg/ml) in the presence or absence of anti-IL-8 MAb (4 mg/ml) for 24 h and then infected with HCMV as described in Materials and Methods. Exogenous IL-8 (50 ng/ml) was used to establish the susceptibility of monocytes to HCMV stimulation by the chemokine. HCMV IEA expression and infectious yields were measured on days 1 and 5, respectively.

1595

nomenon can be ruled out, since (i) gp120 filtered to remove LPS is as active as untreated gp120; (ii) other proteins obtained in the same recombinant DNA expression system, such as sCD4 and p24, or obtained in a different expression system, such as p17, do not affect HCMV infection; and (iii) the enhancing effect is abrogated by a MAb specific for the gp120 V3 loop and by multibranched peptides representing the gp120 V3 loop conserved apical region, known to effectively inhibit other gp120 effects (48, 49), as well as by antibodies to Gal-cer, whereas sCD4 is ineffective. These results suggest that the gp120 interaction with monocytes is not mediated by CD4, but rather involves the V3 loop and Gal-cer. This type of interaction appears to be crucial for other gp120-induced effects, such as fusion (11, 48) and interferon induction (3), and has been suggested to mediate HIV infection of CD4-negative cells, such as nerve epithelial, and endothelial cells (11, 16, 19, 39, 49). In this respect, it will be of interest to compare gp120 from HIV strains with different tropisms and syncytium-forming abilities with respect to their ability to stimulate HCMV replication. The effect of gp120 on HCMV infection of monocytes appears to be mainly due to the induction of a chemokine. A key role of IL-8 in such a phenomenon is supported by the following observations. (i) Endogenous IL-8, although substantially expressed by unstimulated cells, is strongly upmodulated in monocytes exposed to gp120. (ii) Exogenous IL-8 strongly upmodulates HCMV replication in monocytes. (iii) The IL-8neutralizing MAb is able to abrogate the gp120-driven stimulation of HCMV infection. The finding that in gp120-stimulated monocytes an increased amount of HCMV remains bound to the cells after the adsorption period suggests that very early events of the virus replication cycle are the possible target of the IL-8 action under the present experimental conditions. This issue is presently under investigation, in view of the recent observation that a late open reading frame of HCMV (unique short region 28) codes for an analog of the family of the seven transmembrane domain chemokine receptors (20). Taken together, our results have a number of implications. In fact, this is the first evidence that a soluble HIV-1 structural protein (i.e., gp120) can induce HCMV stimulation in cells, such as monocytes, that may have a major role in HCMV pathogenesis (14, 17, 31, 33, 41, 43, 46). gp120 has been shown to be released into the circulation of HIV-infected subjects and is thought to have a role in the progressive immune derangement of these patients by several mechanisms, including the induction of lymphocyte- and monocyte-derived cytokines and chemokines (1–3, 5, 7, 8, 18, 21, 23, 45). Recently, it has been reported that the amount of circulating IL-8 is increased in HIV-1-infected patients (32) and that HIVinfected monocytes show enhanced IL-8 expression (36). The present findings indicate that IL-8 stimulation occurs in healthy monocytes from different donors treated with HIV-1 gp120, in the absence of any sign of HIV-1 replication. Therefore, we think that the indirect effect of HIV on HCMV replication shown here, which is transmissible at distant body sites, may have even stronger pathogenetic significance than the previously reported tat-mediated transactivation of HCMV replication (22, 30, 40), which requires simultaneous infection or rather close contact between cells individually infected by the two viruses. It is widely accepted that cytokine disregulation is involved in the pathogenetic events leading to disease progression in HIV-infected individuals. Several cytokines, including interferon alpha and gamma, tumor necrosis factor alpha, IL-1 alpha and beta, IL-6, IL-10, etc., are induced in vitro by HIV-1,

1596

CAPOBIANCHI ET AL.

HIV-1-infected cells, or viral soluble products such as gp120 (1–3, 5–8, 18, 21, 23, 45) and are found in the circulation of HIV-infected subjects at increased levels (12, 15, 34, 44). Many of these cytokines are potentially capable of influencing the outcome of opportunistic infections that plague HIV-infected patients, particularly those due to viral agents such as HCMV. Since IL-8 is upmodulated by HIV-1 gp120 in monocytes, leading to an increased yield of HCMV, the hypothesis that IL-8 production in vivo is also responsible for stimulation of HCMV infection should be considered, especially in HIVinfected patients, in whom virus-produced gp120 can boost IL-8 production. ACKNOWLEDGMENTS This work was partly supported by grants from the Italian Ministry of Health (IX Progetto AIDS) to F.D. and to F.B. and from the Istituto Pasteur, Fondazione Cenci-Bolognetti, to F.D. We acknowledge the Medical Research Council AIDS Directed Programme Reagent Project for providing p17 (prepared by S. Adams). REFERENCES 1. Ameglio, F., M. R. Capobianchi, C. Castilletti, P. Cordiali Fei, S. Fais, E. Trento, and F. Dianzani. 1994. Recombinant gp120 induces IL-10 in resting peripheral blood mononuclear cells: correlation with the induction of other cytokines. Clin. Exp. Immunol. 95:455–458. 2. Ankel, H., M. R. Capobianchi, C. Castilletti, and F. Dianzani. 1994. Interferon induction by HIV glycoprotein 120: role of V3 loop. Virology 205:575– 579. 3. Ankel, H., M. R. Capobianchi, F. Frezza, C. Castilletti, and F. Dianzani. 1996. Interferon induction by HIV-1: a possible role of sulfatides or related glycolipid. Virology 221:113–119. 4. Bitsch, A., H. Kirchner, R. Dupke, and G. Bein. 1993. Cytomegalovirus transcripts in peripheral blood leukocytes of actively infected transplant patients detected by reverse transcription-polymerase chain reaction. J. Infect. Dis. 167:740–743. 5. Borghi, P., L. Fantuzzi, B. Varano, S. Gessani, P. Puddu, L. Conti, M. R. Capobianchi, F. Ameglio, and F. Belardelli. 1995. Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages. J. Virol. 69:1284–1287. 6. Capobianchi, M. R., F. De Marco, P. Di Marco, and F. Dianzani. 1988. Acid-labile human interferon alpha production by peripheral blood mononuclear cells stimulated by HIV-infected cells. Arch. Virol. 99:9–19. 7. Capobianchi, M. R., H. Ankel, F. Ameglio, R. Paganelli, P. M. Pizzoli, and F. Dianzani. 1992. Recombinant glycoprotein 120 of human immunodeficiency virus is a potent interferon inducer. AIDS Res. Hum. Retroviruses 8:575–579. 8. Capobianchi, M. R., F. Ameglio, P. Cordiali Fei, C. Castilletti, F. Mercuri, S. Fais, and F. Dianzani. 1993. Coordinate induction of interferon alpha and gamma by recombinant HIV-1 glycoprotein 120. AIDS Res. Hum. Retroviruses 9:957–961. 9. Carre`, P. C., R. L. Mortenson, T. E. King, Jr., P. W. Noble, C. L. Sable, and W. H. Riches. 1991. Increased expression of the interleukin-8 gene by alveolar macrophages in idiopathic pulmonary fibrosis. J. Clin. Invest. 88:1802– 1810. 10. Chirgwin, J. M., R. Przybyla, R. J. MacDonald, and W. J. Rytter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299. 11. Cook, D. G., J. Fantini, S. L. Spitalnik, and F. Gonzales-Scarano. 1994. Binding of human immunodeficiency virus type 1 (HIV-1) gp120 to galactosylceramide (GalCer): relationship to the V3 loop. Virology 201:206–214. 12. Cordiali Fei, P., A. Massa, G. Prignano, M. Pietravalle, L. Alemanno, G. Vitelli, G. Palamara, A. Giglio, G. M. Gandolfo, G. Gentili, F. Caprilli, and F. Ameglio. 1992. Behavior of several progression markers during the HIV-ab seroconversion period. Comparison with later stages. J. Biol. Regul. Homeostatic Agents 6:57–64. 13. Denizot, F., and R. Lang. 1986. Rapid colorimetric assay for cell growth and survival. J. Immunol. Methods 89:271–277. 14. Einhorn, L., and A. Oste. 1984. Cytomegalovirus infection of human blood cells. J. Infect. Dis. 149:207–214. 15. Fan, J., H. Z. Bass, and J. L. Fahey. 1993. Elevated IFN-g and decreased IL-2 gene expression are associated with HIV infection. J. Immunol. 151: 5031–5040. 16. Fantini, J., D. G. Cook, N. Nathanson, S. L. Spitalnik, and F. GonzalesScarano. 1993. Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc. Natl. Acad. Sci. USA 90:2700–2704.

J. VIROL. 17. Gerna, G., D. Zipeto, E. Percivalle, M. Parea, M. G. Revello, R. Maccario, G. Peri, and G. Milanesi. 1992. Human cytomegalovirus infection of the major leukocyte subpopulations and evidence for initial viral replication in polymorphonuclear leukocytes from viremic patients. J. Infect. Dis. 166:1236– 1244. 18. Gessani, S., P. Puddu, B. Varano, P. Borghi, L. Contu, L. Fantuzzi, and F. Belardelli. 1994. Induction of beta interferon by human immunodeficiency virus type 1 and its gp120 protein in human monocytes-macrophages: role of beta interferon in restriction of virus replication. J. Virol. 68:1983–1986. 19. Harouse, J. M., R. G. Collman, and F. Gonza ´lez-Scarano. 1991. Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/39 sulfo-galactosyl ceramide-positive cell line. J. Virol. 69:7383–7390. 20. Gao, J. L., and P. M. Murphy. 1994. Human cytomegalovirus open reading frame US28 encodes a functional beta chemochine receptor. J. Biol. Chem. 269:28539–28542. 21. Glienke, W., H. Von Briesen, R. Esser, S. Muller, R. Andreesen, and H. Rubsamen-Waigmann. 1993. Expression of macrophages products after in vitro infection of human monocytes-macrophages with HIV. Res. Virol. 144:35–40. 22. Ho, W. Z., J. M. Harouse, R. F. Rando, E. Gonczol, A. Srinivasan, and S. A. Plotkin. 1990. Reciprocal enhancement of gene expression and viral replication between human cytomegalovirus and human immunodeficiency virus type 1. J. Gen. Virol. 71:97–103. 23. Hochestein, H. D., V. Natarajan, and W. L. Farrar. 1991. The HIV-1 gp120 envelope protein has the intrinsic capacity to stimulate monokine secretion. J. Immunol. 147:2892–2901. 24. Jacobsen, H., J. Mestan, S. Mittnach, and C. W. Dieffenbach. 1989. Beta interferon subtype 1 induction by tumor necrosis factor. Mol. Cell. Biol. 9:3037–3042. 25. Jacobson, M. A. 1994. Current management of cytomegalovirus disease in patients with AIDS. AIDS Res. Hum. Retroviruses 10:917–923. 26. Katlama, C. 1993. Cytomegalovirus infection in acquired immune-deficiency syndrome. J. Med. Virol. 1:128–133. 27. Koval, V., C. Clark, M. Vaishnav, S. A. Spector, and D. H. Spector. 1991. Human cytomegalovirus inhibits human immunodeficiency virus replication in cells productively infected by both viruses. J. Virol. 65:6969–6978. 28. Landini, M. P. 1993. New approaches and perspectives in cytomegalovirus diagnosis. Prog. Med. Virol. 4:157–177. 29. Lathey, J. L., D. H. Spector, and S. A. Spector. 1994. Human cytomegalovirus-mediated enhancement of human immunodeficiency virus type-1 production in monocyte-derived macrophages. Virology 199:98–104. 30. Lazzarotto, T., G. Furlini, M. C. Re, E. Ramazzotti, B. Campisi, and P. Landini. 1994. Human cytomegalovirus replication correlates with differentiation in a hematopoietic progenitor cell line and can be modulated by HIV-1. Arch. Virol. 135:13–28. 31. Maciejewski, J. P., E. E. Bruening, R. E. Donahue, S. E. Sellers, C. Carter, N. S. Young, and S. Jeor. 1993. Infection of mononucleated phagocytes with human cytomegalovirus. Virology 195:327–336. 32. Matsumoto, T., T. Miike, R. P. Nelson, W. L. Trudeau, R. F. Lockey, and J. Yodoi. 1993. Elevated serum levels of IL-8 in patients with HIV infection. Clin. Exp. Immunol. 93:149–151. 33. Minton, E. J., C. Tysoe, J. H. Sinclair, and J. G. P. Sissons. 1994. Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow. J. Virol. 68:4017–4021. 34. Mosmann, T. R. 1994. Cytokine patterns during the progression to AIDS. Science 265:193–194. 35. Murayama, T., K. Kuno, F. Jisaki, M. Obuchi, D. Sakamuro, T. Furukawa, N. Mukaida, and K. Matsushima. 1994. Enhancement of human cytomegalovirus replication in a human lung fibroblast cell line by interleukin-8. J. Virol. 68:7582–7585. 36. Ohashi, K., R. Akazawa, and M. Kurimoto. 1994. Effects of interferon-alpha on a reduced release of interleukin-8 from latently HIV-1-infected monocytic cell line U937 cells. J. Interferon Res. 14:129–132. 37. Rando, R. F., A. Srinivasan, J. Feingold, E. Gonczol, and S. Plotkin. 1990. Characterization of multiple molecular interactions between human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1). Virology 176:87–97. 38. Revello, M. G., M. Furione, M. Zavattoni, and G. Gerna. 1994. Human cytomegalovirus infection: diagnosis by antigen and DNA detection. Med. Microbiol. 5:265–276. 39. Scheglovitova, O., M. R. Capobianchi, G. Antonelli, D. Guanmu, and F. Dianzani. 1993. CD-4 positive lymphoid cells rescue HIV-1 replication from abortively infected human primary endothelial cells. Arch. Virol. 132:267– 280. 40. Skolnik, P. R., B. R. Kolstoff, and M. Hirsch. 1988. Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. J. Infect. Dis. 157:508–514. 41. Soderberg, C., S. Larsson, S. Bergstedt-Lindqvist, and E. Mo ¨ller. 1993. Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection. J. Virol. 67:3166–3175. 42. Taylor-Wiedeman, J., J. G. P. Sissons, L. K. Borysiewicz, and J. H. Sinclair.

VOL. 71, 1997

43. 44. 45.

46. 47.

HCMV STIMULATION BY gp120-INDUCED IL-8 IN MONOCYTES

1991. Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J. Gen. Virol. 72:2059–2064. Taylor-Wiedeman, J., P. Sissons, and J. Sinclair. 1994. Induction of endogenous human cytomegalovirus gene expression after differentiation of monocytes from healthy carriers. J. Virol. 68:1597–1604. Von Sydow, M., A. Sonnenberg, H. Gaines, and O. Strannegard. 1992. Interferon alpha and tumor necrosis factor alpha in serum of patients in various stages of HIV-1 infection. AIDS Res. Hum. Retroviruses 7:375–380. Wahl, L. M., M. L. Corcoran, S. W. Pyle, L. O. Arthur, A. Harel-Bella, and W. L. Arrar. 1989. Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1. Proc. Natl. Acad. Sci. USA 86:621–625. Waldman, W. J., D. A. Knight, E. H. Huang, and D. D. Sedmak. 1995. Bidirectional transmission of infectious cytomegalovirus between monocytes and vascular endothelial cells: an in vitro model. J. Infect. Dis. 171:263–272. Webster, A., J. E. McLaughlin, M. A. Johnson, V. C. Emery, and P. D.

1597

Griffiths. 1995. Use of the polymerase chain reaction to detect genomes of human immunodeficiency virus and cytomegalovirus in post-mortem tissues. J. Med. Virol. 47:23–28. 48. Yahi, N., J. Fantini, K. Mabrouk, C. Tamalet, P. De Micco, J. Van Rietschoten, H. Rochat, and J.-M. Sabatier. 1994. Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages. J. Virol. 68:5714–5720. 49. Yahi, N., J.-M. Sabatier, S. Baghdiguian, F. Gonzalez-Scarano, and J. Fantini. 1995. Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line. J. Virol. 69:320–325. 50. Yoshimura, T., K. Matsushima, J. J. Oppenheim, and E. J. Leonard. 1987. Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1). J. Immunol. 139:788–793.