Interleukin-18 stimulates HIV-1 replication in a T-cell line.

ARTICLE

INTRODUCTION

Interleukin-18 (IL-18) was described recently as interferon-gamma inducing factor (IGIF) [1]. Apart from its function as an essential co-factor for IFN-gamma induction, several other, even more pivotal capacities of this new cytokine can be defined in the murine and human system. IL-18 was demonstrated to enhance the activity of Th1 type T-cells and NK cells [2, 3]. In addition to Th1 type cytokines the expression of TNF-alpha and GM-CSF are particularly upregulated by IL-18 [4-6].

Because of to its capacity to induce IFN-gamma as well as CC and CXC chemokines, and to enhance the Th1-type immune response, a virustatic effect of IL-18 on HIV-1 replication can be assumed. On the other hand, it was demonstrated that IL-18 acts via NFkappaB translocation [7-9]. Since HIV replication can be activated by NFkappaB binding [10], enhancement of HIV replication by IL-18 could be possible. Recently, Shapiro et al. demonstrated a stimulation of HIV-1 replication by IL-18 via nuclear translocation of NFkappaB in monocytic cells [11]. It was the goal of this study to elucidate the effect of IL-18 on HIV-1 replication in lymphatic cells.

METHODS

Expression of human IL-18

Human IL-18 cDNA was amplified by RT-PCR from 5 x 10⁶ peripheral blood mononuclear cells (PBMC) obtained from a healthy adult volunteer after informed consent. Total RNA was isolated by the RNAzol™ method as described by the manufacturer (Molecular Research Centre, Cinncinati, OH, USA). cDNA was synthesised at 42° C for 1 h using random hexadeoxynucleotide primers Superscript™ reverse transcriptase (Gibco, Karlsruhe, Germany) according to the manufacturer's instructions. IL-18 cDNA was PCR amplified as described earlier using the following primers [12]: 5'-AGC TCG GGA TCC ATG TAC TTT GGC AAG CTT GAA TCT AAA TTA TCA-3' and 5'-ACT GAA TTC CTA GTC TTC GTT TTG AAC AGT GAA CAT TAT AGA-3'. The PCR was performed with 30 cycles at three temperature steps: 1 min at 95° C, 1 min at 60° C and 1 min at 72° C.

The amplified cDNA was cloned into the BamHI and EcoRI restriction site of the pGEX4T2 vector (Pharmacia, Freiburg, Germany) to create a glutathion-S-transferase-IL-18 fusion protein. The sequence was confirmed by sequence analysis using the BigDye™ sequence kit and an ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Langen, Germany).

The recombinant IL-18 was expressed procaryotically in E. coli HB101. A preculture of E. coli HB101 harboring the IL-18 expression plasmid was incubated in 2YTA (Gibco) medium at 37° C and centrifuged at 250 rpm to reach an optimal density of A₆₀₀ of 0.6 to 0.8. Then the culture was treated with 0.1 M IPTG (Gibco) and incubated for an additional 4 1/2 h. The bacteria were harvested and lysed by sonication in 1% Triton X-100. After centrifugation at 10,000 rpm for 10 min at 4° C the supernatant was applied to a GST-affinity resin (Pharmacia). For removal of LPS, the GST eluted fusion proteins were dialysed against PBS and underwent pansorbin chromotography (Pharmacia). In addition, human IL-18 was expressed eucaryotically by the baculovirus expression system in Sf9 cells according to the protocol of Wang et al. [13].

Generation of monoclonal antibodies against IL-18

Mouse monoclonal antibodies against E. coli-expressed recombinant human IL-18 were generated as described earlier for anti-IFN-gamma receptor antibodies [14]. Briefly, mice were immunised by 3 intraperitoneal injections of 20 mug protein at 2-3 weeks intervals. The mouse with the highest Ab titer against IL-18 as determined by ELISA, was sacrificed and its spleen harvested. Mononuclear spleen cells were mixed 1:1 with myeloma cells and fused under the influence of PEG. The hybridoma clones were screened by direct ELISA and Western blot for reactivity with recombinant E. coli expressed and baculovirus-expressed IL-18, as well as a commercially available recombinant human IL-18 (IC Chemicalien, Munich, Germany). mAb were purified through affinity purification on Protein-G sepharose 4B (Pharmacia, Freiburg, Germany).

Cell culture

Hut78 cells were cultured in RPMI 1640, supplemented with 15% heat inactivated FCS (Gibco, Karlsruhe, FRG), 100 U/ml penicillin, 100 mug/ml streptomycin and 1 mM glutamine (Biochrom, Berlin, FRG). Cells were cultured at a concentration of 1 x 10⁵/ml in a total volume of 4 ml in T-25 tissue culture flasks (Greiner, Nürtingen, Germany).

Prior to HIV-1 infection, Hut78 cells were incubated for two days with 10 or 100 ng/ml (0.55 or 5.5 nM) E. coli or baculovirus-expressed IL-18. Supernatant of wild type baculovirus-infected Sf9 cells (wt) served as the negative control of the eucaryotically expressed IL-18.

HIV-1 infection

After a two day preincubation with IL-18, cells were infected with HIV-1 strain IIIb. Virus supernatants were derived from chronically HIV-1 IIIb infected H9 cells. The titer of HIV supernatants was determined by endpoint titration on C8166 cells as described below. Hut78 cells were infected with an MOI of 0.01 for three hours. After HIV inoculation, cells were washed three times in RPMI 1640. In experiments with the permanent presence of IL-18, the cytokine was added again. Every second day, half of the cell suspension was removed and replaced by fresh media containing IL-18. After HIV inoculation, the number of viable cells was determined every 7 days by trypan blue exclusion dye. Additionally, culture supernatants were collected. For neutralisation experiments, 5 mug/ml anti IL-18 monoclonal antibodies were added 35 days after HIV infection. After one further week, culture supernatants were harvested.

Quantitation of HIV-1 replication

The amount of infectious virus in cell culture supernatants was defined by endpoint titration on C8166 cells as described earlier [15]. Briefly, culture supernatants were diluted 1:10 ­ 1:1000 000 in log 10 steps. From each dilution step, aliquots were taken to infect C8166 cells in 8 different culture wells. After one week, the number of wells with syncytia formation was counted to calculate the virus titer in the culture supernatants. The concentration of p24 was determined by ELISA (Coulter, Krefeld, Germany).

RESULTS

Enhancement of HIV-1 replication by recombinant IL-18

To investigate the influence of IL-18 on HIV-1 replication, the human T lymphoblastic cell line Hut78 was cultured in the presence of recombinant IL-18 and infected with HIV-1 strain IIIb. HIV-1 replication was monitored weekly for up to five weeks by p24 ELISA of culture supernatants. As demonstrated in Figure 1, the addition of 100 ng/ml (5.5 nM) of E. coli-expressed IL-18 resulted in a marked increase of p24 concentrations in culture supernatants of Hut78 cells after day 14 until the end of the observation.

As demonstrated in Figure 2, the baculovirus-expressed IL-18 resulted in an even stronger enhancement of HIV-1 replication. The maximal p24 concentration was higher than 510 ng/ml/10⁶ cells compared with 300-350 ng/ml/10⁶ cells after the addition of E. coli-expressed IL-18. Additionally, the enhancement of HIV-1 replication by eucaryotically-expressed IL-18 peaked already seven days after HIV inoculation compared with approximately 3 weeks in the presence of E. coli-expressed IL-18. After day 14 post-HIV infection, p24 concentrations in IL-18 and wt (wild type baculovirus infected Sf9 cell) supernatant-treated Hut78 cells decreased slightly. Until the end of observation at day 35 the concentration of p24 was 2-10 times higher under the influence of IL-18 compared with wt supernatant (data not shown).

To further demonstrate the enhanced HIV-1 replication as an IL-18 mediated effect, neutralising anti-IL-18 mAb were added after a culture period of 5 weeks. In the presence of these neutralising antibodies, the IL-18 induced increase of p24 concentration was reduced from 224 ng/ml to 55 ng/ml within one week. Without the addition of anti-IL-18 mAb, the p24 concentration was almost unchanged (204 ng/ml, Figure 3).

The influence of E. coli-expressed IL-18 on the amount of infectious HIV was defined by endpoint titration of culture supernatants on C8166 cells. Three weeks after HIV-1 infection, the amount of infectious virus was almost three times higher in the presence of IL-18 compared with the control (4.2 x 10⁶ infectious particles per ml versus 1.5 x 10⁶, Figure 4).

Influence of IL-18 on the infection of Hut78 cells with HIV

To determine whether IL-18 enhances HIV replication mainly by stimulating virus replication itself or by increasing the rate of HIV infection, E. coli-expressed IL-18 was added only prior to and during HIV infection. As demonstrated in Figure 5, this brief initial exposure to IL-18 resulted in a reduced p24 concentration in Hut78 supernatants during the first weeks of culture. After three weeks, the p24 concentration in the culture supernatants of the IL-18 preincubated cells increased to the same level as in the culture of control cells.

To exclude an IL-18 mediated change of CD4 expression on Hut78 cells, the effect of IL-18 on the average density of the CD4 molecule on Hut78 cells was analysed by flow cytometry. No significant changes in CD4 expression on Hut78 cells could be demonstrated (data not shown).

DISCUSSION

Since IL-18 is known to induce IFN-gamma, CC and CXC chemokines and to enhance the Th1-type immune response, a virustatic effect of IL-18 on HIV-1 replication can be assumed [4]. On the other hand, a positive effect of IL-18 on HIV replication could also be suggested due to its ability to act via NFkappaB translocation [7, 9].

In this study we demonstrated that recombinant human E. coli-expressed IL-18 is able to mediate an enhancement of HIV-1 replication in lymphatic cells. A part from an increased concentration of the HIV protein p24, even an increase of the amount of infectious HIV could be demonstrated in Hut78 culture supernatants in the presence of IL-18. In addition to E. coli-derived human IL-18, in this study we used eucaryotically expressed IL-18 to enhance HIV-1 replication in Hut78 cells. First of all, eucaryotically-expressed IL-18 was applied in this study to verify its bioactivity. This baculovirus-expressed IL-18 led to an even stronger and faster increase of HIV replication. These data suggest that the bioactivity of eucaryotically-expressed recombinant human IL-18 is at least comparable with E. coli-derived IL-18.

The IL-18-mediated enhancement of HIV replication could be blocked almost completely by IL-18-neutralising mAb. This demonstrates the enhancement of HIV replication as an IL-18-specific effect. Furthermore, it demonstrates that the stimulation of HIV-1 is not due to an effect on the process of infection of cells with HIV. Since the Hut78 cells were already inoculated with HIV five weeks earlier, the whole culture was already infected with the virus prior to the addition of the neutralising antibodies. The decrease of p24 concentration in culture supernatants must therefore be solely due to a reduced HIV replication after neutralisation of IL-18.

To further distinguish between an influence of IL-18 on the infection of cells with HIV and on HIV replication, Hut78 were cultured, prior to and during HIV-1 infection, with IL-18. In contrast to the experiments with a continuous presence of IL-18 after inoculation with HIV, the pre-incubation with IL-18 resulted in a reduced p24 concentration during the first weeks compared with the cells which were not pre-treated with IL-18. Thus, the enhanced HIV replication under the continuous presence of IL-18 cannot be due to an enhanced rate of HIV infection. Moreover, the infection of cells with HIV and/or HIV replication seemed to be inhibited after pre-incubation with IL-18. A change of the average expression density of CD4 molecules under the influence of IL-18 could be excluded as being responsible for the effect of IL-18. It has to be discussed whether a pre-incubation with IL-18 induces any factors which could mediate an inhibition of HIV replication. An induction of IFN-gamma, which is an inhibitor of HIV infection, could not be detected in the culture supernatants of Hut78 cells under the influence of IL-18 (data not shown). Since the induction of chemokines by IL-18 has been already demonstrated it has to be elucidated whether these factors are responsible for the inhibition of HIV-1 infection.

For monocytic cells, Shapiro et al. have recently demonstrated a stimulating effect of IL-18 on HIV replication in monocytic U1 cells [11]. It has to be checked whether a mediation by secondary cytokines e.g. by TNF-alpha or IL-6 via the NFkappaB pathway, as has been suggested for the U1 cells, is involved in the enhancement of HIV-1 replication in lymphatic cells as well.

Further investigations are required to elucidate whether the effects of IL-18 on HIV replication have a pathophysiological relevance for the course of HIV-1 infection and the development of AIDS.

CONCLUSION

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