Differential HIV replication and HIV-induced interferon production in mononuclear phagocytes: relationship to cell maturation

(~) INSTITUTPASTEUR/ELsEVIER Paris 1991

Res. Viro!. 1991, 142, 353-361

Differential HIV replication and HIV-induced interferon production in mononuclear phagocytes: relationship to cell maturation P. Mosborg-Petersen (]), F.D. Toth (1)(2), V. Zachar O)(3), J.A. Villadsen (!), N. NCrskov-Lauritsen 0), G. Aboagye-Mathiesen 0), J.-C. Chermann (4) and P. Ebbesen O)(*) ro Department o f Virus and Cancer, The Danish Cancer Society, Gustav Wieds Vej 10, DK-8000 Aarhus C (Denmark), ~2) Institute o f Microbiology, Medical University, H-4012 Debrecen (Hungary), lz) Institute o f Virology, Siovak Academy o f Sciences, Bratislava (Czechoslovakia), and ¢4) Unitd des Recherches sur ies R~troviruses et Maladies associ~es, U322 de I'INSERM, Marseille (France}

SUMMARY W e have investigated the replication of human immunodeficiency virus (HIV) and HIVinduced interferon (IFN) production in human mononuclear phagocytes at 2 different stages of in vitro maturation. Blood monocytes and monocyte-dedved macrophages from 6 healthy, HIV-seronegative donors were challenged with HIVI,,, s and HIV2Ro D. Freshly separated monocytes produced IFN when inoculated with both HIV types. In these cultures, an inverse correlation was observed between the amount of IFN production and the rate of HIV replication. In contrast to the monocytas, 5-day-old monocyte-dedved macrophages did not produce IFN when challenged with HIV, but a significant replication of HIVI,.B and HIV2Ro D was found in a;i cultures.

Key-words: HIV1; HIV2, Monocyte, Macrophage, Interferon, Replication, i~aturation; Correlation, Cell cultures, Stages.

INTRODUCTION

The human immunodeficiencyvirus (HIV) is the causative agent in the acquired immunodeficiency syndrome (AIDS) (Barr6-Sinoussi ei aL, 1983; Popovic et aL, 1984). The primary viral targets are the CD4-positive T lymphocytes, on which HIV has a cytopathic effect (Klatzman et al., !984; Dalgleish et aL, 1984). However, HIV

can also infect mononuclear phagocytes from different sources (Ho et al., 1986; Salahuddin et al., 1986; Chermann, !990). Factors that influence the rate o f virus replication in mononuclear phagocytes are of interest, because they are assumed to play an important role in the pathogenesis of AIDS (Gartner et al., 1986). In vitro HIV replication is determined by the degree of maturation of mononuclear phago-

Submitted March 6, 1991, accepted July 23, 1991. (*) Correspondenceand reprintsrequestto Dr. PeterEbbesen,The DanishCancerSociety,Departmentof Virusand Cancer,Gustav Wieds Vej 10, DK-8000Aarhus C, Denmark.

354

P. M O S B O R G - P E T E R S E N E T AL.

cytes to be differentiated in order to support productive infection (Valentin et al., 1990). Although not definitely proven, current data suggest that the replication of HIV in ceils of monocyte/macrophage lineage may be regulated by inducible interferon (IFN). Exogenous IFN is described as suppressing HIV replication in the cultures of mononuclear phagocytes (Gendelman et al., 1990a). Neutralization o f IFN activity enhances the expression o f HIVl-specific proteins in promonocytic cell line U937 (Mace et al., 1989). Furthermore, recent studies have shown that the monocyte-derived macrophages which underwent differentiation for 5-10 days in in vitro cultivation did not respond with IFN production to infection with HIV1 (Gendelman et al., 1990b; Kornbluth et al., 1990b). However, the capacity for production in non-differentiated blood monocytes has not previously been investigated. Moreover, no studies have yet investigated the ability of HIV2 to induce IFN production in mononuclear phagocytes. In order to asses how the maturation stage of blood mononuclear phagocytes is reflected in the IFN response to HIV infection, we investigated the correlation between replication of HIVI and HIV2 and the induced IFN levels. The target cells comprised freshly separated blood m o n o c y t e s and .~v.=,~,~j v.~=u~.,L = v ~ , u = = a = ~ . l O l . / l l a g ~ ; b , CUlLI v a t u u in vitro for 5 days.

MATERIALS

AND METHODS

Cell cultures

Peripheral blood mononuclear cells (PBMC) obtained from freshly prepared buffy coats from six healthy, HiV seronegative donors were isolated on a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). PBMC were resuspended in RPMI-1640 medium and fractionated by centrifugation on discontinuous density gradient of "Percoll" (Pharmacia) (Ulmer and Flad, 1979). Cells recovered from the interface of 45 % Percoil were > 85 0/0 pure

FACS FITC IFN PBMC

= = = =

fluorescence-activated cell sorter. fluorescein isothyocyanate. interferon. peripheral blood mononuclear ceil.

monocytes, as determined by morphological criteria after panoptic Giemsa staining and by staining for non-specific esterase. To further enrich for monocytes, contaminating lymphocytes were removed by immunomagnetic technique (Funderud et al., 1990). Cells were incubated with paramagnetic beads coupled with monoclonal antibodies. "Dynabeads M-450 Pan-T" and "Dynabeads M-450 Pan-B" (Dynal, Oslo, Norway) were used. The cells coated with the beads were removed using a magnetic separator. The employed ratio of Dynabeads to the target cells was 30/1. The whole procedure was done at 4°C. The resulting monocyte population was > 95 % pure. Cell viability as determined by trypan blue exclusion was > 95 V0. All monocyte/macrophage cultures were grown in RPMI-1640 medium (Gibco Ltd., Paisley, UK) supplemented with 0.29 mg/ml glutamine, 100 units/ml penicillin, 100 ttg/ml streptomycin, 5 % human AB pooled serum and 15 % heat-inactivated foetal calf serum (Gibco). Viruses and infection of mononuclear phagocytes

The HIV1 strain IIIB and the HIV2 strain ROD (both obtained from Dr. P. Nara, NCI, Frederick Cancer Research Facility, Frederick, MD, USA) were grown in H9 cells. Virus s[ocks were titrated by syncytium-formingassay in CEM cells (Nara et ai., 1987). The population of monocytes was infected in triplicate with HIV immediately after separation and seeding in 25-cm2 plastic tissue culture flask (Nunc, Roskilde, Denmark). The population characterized as monocyte-derivedmacrophages was also infected in triplicate with HIV after 5-day cultivation of monocytes in 25-cm2 plastic tissue culture flask. The cell number infected was 5 x l 0 6. The inoculation was carried out at the multiplicity of infection 1 for 3 h. The cells were then washed four times and cultured at a concentration of 1 × 106 cells/ml until assayed. Flow cytometry

Multiple differentiation antigens expressed on mononuclear phagocytes were estimated using flow cytometry. Freshly separated monocytes were washed and then diluted to 107 cells/ml in PBS with 1 % heat-inactivated pooled human AB serum (PBSS). Small aliquots (100 ~1) were then incubated for 30 min at 4°C with 15 g.l FITC- or phycoerythrin-

PBS PBSS RT

= = =

phosphate-buffered saline. PBS with h u m a n AB serum. reverse transcriptase.

DIFFERENTIAL HIV REPLICA TION AND INTERFERON PRODUCTION

conjugated monoclonal antibodies. The adherent 5-day-old monocyte-derived macrophages were detached from the culture flask by 0.02 % EDTA in PBS at 4°C for 15 min. Cells were washed and diluted in PBSS before incubation with the monoclonal antibody as described for fresh monocytes. We used monoclonal antibodies against the following surface antigens: CD16 (FcR III) (F7523), CD4 (F766), CD14 (monocyte/macrophage marker) (R864), HLA-DR (F817), CD71 (transferrin receptor) (F829), CD33 (promonocyte cell marker) (F832). All monoclonal antibodies were purchased from DaKoPatts (Copenhagen, Denmark) except the anti-CD-16 antibody (Becton Dickinson, Mountain View, CA, USA). Control antibody used was FITC-conjugated mouse IgG (X927) and phycoerytl~rin-conjugated mouse igG (X928) (Dako-Patts). After two final washes with PBS, cells were fixed ii 1 % paraformaldehyde and analysed in a FACS analyser (BectonDickinson).

355

RESULTS H u m a n peripheral blood monocytes appeared to mature after 5 days o f in vitro cultivation and developed features characteristic for macrophages as estimated b y the evaluation o f different surface markers. The results in figure 1 show

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Interferon assay To eliminate infectious virus, IFN samples were filtered through 50-nm pore size cellulose nitrate filters (Sartorius GmbH, Gottingen, Germany). IFN activity was determined using inhibition of vesicular stomatitis virus plaque formation in WISH cells (Toth et al., 1990). The highest dilution of the titrated sample causing 50 % protection of ceils was considered as titre. An internal laboratory human IFN ~tand~rrl uaac ;r~,~l,,A~,.4 . . . ; * K . . . L . titration. This internal standard had been calibrated against a human reference IFN0c (69/19) prepared by the National Institute for Biological Standards and Control (London, UK). All IFN titres were corrected to this standard. Antibody neutralization of IFN antiviral activity was performed by incubating IFN samples for 1 h at 37°C with a 10-fold excess of horse immune serum to human IFN~, [3 and T (BoehringerMannheim GmbH, Mannheim, Germany) and then assaying the samples for the presence of residual IFN activity. The pH stability of the IFN activity was tested by treating the samples at pH = 2 for 24 h and then redialysing them to neutra!Jty prior to assaying.

HIV production Viral production in mononuclear phagocytes was determined by assaying the reverse transcriptase (RT) activity in the supernatant of the cell cultures. Samples were taken at 2, 5 and 7 days after viral challenge, and then every 3rd day. The RT assay was performed according to standard procedure (Rey et al., 1984).

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Fluorescence Intensity Fig. i. FACS profiles of the expression of different surface antigens on freshly separated (day 0) and 5-day old (day 5) in vitro cultured blood monocytes. Purified blood monocytes were isolated as described (see "Materials and Methods"). Cells were assayed for membrane-bound fluorescence signal in FACS immediately after separation or after 5 days of in vitro cultivation in 5 % AB human serum and 15 % foetal calf serum. The FACS profiles (--) illustrate the relative fluorescence intensity of the cells after staining with the monoclonal antibody; dotted lines show staining with control antibody.

356

P. MOSBORG-PETERSEN ET AL.

that freshly isolated monocytes were negative for Fc receptor class III (CDI6) expression, in contrast to monocyte-derived macrophages which were positive. Comparison of monocytes and macrophages also shows a major increase in the expression of H L A - D R and a minor increase in the transferrin (CD71) receptors. A decrease was found in the expression of CD33 (promonocyte cell marker) and CD4 receptor. No difference was observed in the expression of COl4. The size of the monocytes increased during cultivation as documented by the increase of the forward light scatter by the flow cytometric analysis and by light microscopic examination (data not shown). The phenotypic changes found are in agreement with the data on in vitro maturation of the monocytes (Landmann et aL, 1988; Andreesen et aL, 1990). To investigate the relationship between the in vitro maturation stage of blood mononuclear phagocytes and their ability to produce IFN af-

ter infection with HIV, samples of supernatant fluid were collected from each culture at 8-h intervals for 5 days and assayed for IFN. In the IFN-producing cultures, no appreciable differences were found in the kinetics of IFN induction. Namely, after an initial lag of a few hours, the IFN titres rase sharply, reaching a plateau 24 h later, and stayed constant for 48 h. The IFN titres then gradually decreased to undetectable levels until the end of the 4th day after infection (data not shown). The 24-h maximal IFN levels are shown in figure 2. It is seen that HIVinduced IFN production depended on the differentiation stage of the cells. Five-day old monocyte-derived macrophage cultures released no detectable IFN after infection with either H I V l m B or HIV2Ro D. However, freshly separated monocytes produced IFN, but an interdonor variation was apparent. All monocyte cultures infected with HIVIIIIB produced IFN. On the contrary, only 4 of 6 HIV2Roi:infected

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Fig. 2. IFN production in monocyte and macrophage cultures after infection with HlVlma and HIV2aoD. IFN production was followed every 8 h until day 5 after infection. The highest peak IFN levels observed in a 24-h period (24 h-48 h after infection) are shown : freshly separated monocytes after infection with HIVImB(A) and HIV2Roo (C) and by 5-day old monocyte-derivedmacrophage cultures after infection with HIVImB (B) and HIV2RoD (D). Six monocyte and macrophage cultures from healthy donors were investigated.

357

DIFFERENTIAL HIV REPLICATION AND INTERFERON PRODUCTION

cultures released IFN. In general, H I V l m B induced 5-fold higher I F N levels than HIV2Ro o in the m o n o c y t e s . U n i n d u c e d m o n o c y t e and macrophage cultures as well as those exposed to uninfected H 9 culture supernatant were also tested for I F N p r o d u c t i o n . U n i n d u c e d m o n o c y t e cultures p r o d u c e d no or low levels o f I F N activity (table I). N o significant differences were observed b e t w e e n the I F N p r o d u c t i o n o f uninduced m o n o c y t e s and that o f mock-infected m o n o c y t e cultures. In the u n i n d u c e d or m o c k infected m o n o c y t e cultures, maximal I F N titres were reached at 24 hours after seeding. The I F N activity continued at this level for an additional

T a b l e I. IFN production in uninduced and mock-

infected monocyte cultures. Maximum IFN yields (IU/106 cells in cultures) Donor

Uninduced

Mock-infected

1

10 < 10 20 30 < i0 20

20 < 10 20 40 i0 30

2 3 4 5 6

24 h and declined quite rapidly thereafter. After 72 h, spontaneous I F N production was undetectable in all cultures (data not shown). In contrast to the monocytes, uninduced or mockinfected macrophages did not release any detectable I F N activity. The I F N produced by H I V infected or uninfected m o n o c y t e cultures was identified as an acid-stable IFNa. H I V replication in the cell cultures after infection was monitored by following the culture supernatant-associated R T activity over a period o f 36 days. W e f o u n d that both H I V l m B and HIV2goD productively infected the monocyte-derived macrophage cultures from all 6 d o n o r s (fig. 3). HIVIlIIB and H I V 2 R o D showed distinct replication characteristics in m a c r o p h a g e cultures. In macrophages infected with HIV2Ro o, the R T activity appeared earlier and reached higher peak values than in those infected with H I V l m B . In the HIV-infected m o n o c y t e cultures, an interdonor variation was observed. Only 2 o f 6 monocyte cultures showed productive infection with H I V l n m . After infection with HIV2Ro D, 4 o f 6 monocyte cultures released R T activity. All monocyte cultures in which H I V replication was found showed low RT activity during the entire observation period. In order to correlate the I F N response with H I V replication, we compared the peak IFN and

Table II. IFN production and RT activity in monocyte and macrophage cultures after infection with

tl~ Freshly separated monocytes infected with HIVlmB

t2~ Five-day-old monocyte-derived macrophages infected with HIVIma

o~ Maximal IFN titre IU/i06 cells

t4) Peak RT activity

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6

8,000

~

< 10

6

Donor

104 c p m / m l

HIVIlIIB.

Ma:dmal IFN titre IU/106 cells

Peak RT activity 104 cpm/ml

Purified blood monocytesfrom 6 healthy donors were cultured in 5 °7/oAB human serum and 15 ~/ofoetal calf ~erum, and infected with HIVlatBdirectly after seeding (!) in plastic culture flasks and after 5 days of in vitro cultivation (2). (3) IFN production was followedevery 8 h until 5 days after infection. The maximal IFN titre is shown. (4) HIV replicationwas followedby analysing RT activitythrough 36 daysafter infection, Peak RT activityfound in this periodis shown.

P. M O S B O R G - P E T E R S E N E T A L.

358

RT activity in the individual monocyte and macrophage cultures infected with HIVImB (table II) and HIV2Ro b (table III). An inverse correlation was found between the amount of IFN produced and the observed RT activity in the monocyte cu!t~es. No RT activity was found in monocyte cultures responding with high IFN production (peak IFN activity > 2,500 IU/106 cells) after infection with either HIV types. In monocyte cultures responding with a low IFN

production (peak IFN activity 800-1,600 IU/106 cells), a moderate, late HIV production was observed. In HIV2RoD-infected monocyte cultures without detectable IFN production ( < I0 IU/106 cells), RT activity was detected earlier with high peak values. The monocyte-derived macrophage cultures showed no IFN production, and in all cultures HIV replication was higher than in the corresponding monocyte cultures.

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Fig. 3. RT activity in monocyte and macrophage cultures infected with HIVImB and HIV2Ro D. HIV replication was followed by measuring RT activity in the culture supernatant every third day through a period of 36 days after infection of fresh monocytes with HIVlum (A) and HIV2RoD (C) and after infection of 5-day old monocyte-derived macrophage cultures with HIVlmB (B) and HIV2Ro D (D). Six monocyte and macrophage cultures from healthy donors were investigated.

DIFFERENTIAL H I V REPLICA TION A N D INTERFERON PRODUCTION

359

Table III. IFN production and RT activity in monocyte and macrophage cultures after infection with

HIV2Roo. o) Freshly separated monocytes infected with HIV2RoD

Donor

O) Maximal IFN titre IU/106 cells

(4) Peak RT activity 104 cpm/ml

1 2 3 4 5 6

1,600 < 10 4,096 2,560 < 10 800

4 12 --6 5

~2)Five-day-old monocyte-derived macrophages infected with HIV2Ro D Maximal IFN titre IU/106 cells < < < < < <

10 10 10 10 10 10

Peak RT activity 104 cpm/ml 7 65 40 I0 17 60

See legend,table I1.

DISCUSSION

Only a small percentage of the blood m o n o cytes of HIV-infected persons harbours HIV (Schnittman et al., 1989; Braun et al., 1988). In contrast to this, isolation of HIV f r o m the tissue macrophages has been shown to be m u c h easier (Koenig et al., 1986). Mononuclear phagocytes are susceptible to HIV infection at all stages o f differentiation. However, the kinetics o f virus replication depends on the stage of maturation o f the cells when infected, and despite the fact that CD4 expression on the monocytes was lowered during in vitro maturation HIV replication was higher in monocytes infected after 7 days o f cultivation than on day 0 (Valentin et al., 1990). In our investigation we also found a decrease in the expression o f CD4 on blood monocytes when culturing for five days in vitro, but there was a higher degree o f HIV replication. This suggests that the permissiveness of the m o n o n u c l e a r phagocytic cells to HIV is not related to the density of the CD4 receptor on them. A m o n g other possible factors in differentiation-related differences in susceptibility to HIV, IFN is especially interesting because monocytes are known as p o -

tent producers of IFNa (Saksela et al., 1984) and IFNa is known to play an important role in HIV replication in mononuclear phagocytes (Kornbluth et al., 1990a). Our results are the first demonstration that iFN production can be induced by H i V i and HIV2 in freshly separated blood monocytes, but this ability disappears during in vitro cultivation of the cells for 5 days. No correlations were observed between the rates of spontaneous and H I V - i n d u c e d IFN p r o d u c t i o n . The I F N produced by HIV-infected monocytes was found to be an acid-stable IFNa. The inverse correlation found between the amount of IFN produced by monocytes and HIV released into the supernatant, suggests that endogenous IFN production plays an important role in inhibition of HIV replication in blood monocytes. Indeed, previous investigations of the role of endogenous produced IFN in the replication of retrovirus has provided indirect evidence that !FN inhibit virus replication, because neutralization o f IFN activity with anti-IFN serum enhances the infectibility of the susceptible target cells (Barr&Sinoussi et al., 1979; Mace e t a ! . , 1989). It is known that the Tat transacting

360

P. M O S B O R G - P E T E R S E N E T A L .

product o f the tat gene o f HIV can prevent the activation o f IFN-associated enzymes (Roy et al., 1990). Hence, an inverse correlation between IFN production and HIV replication m a y also be possible. One might suggest that high HIV replication represses IFN0c production, as was shown for poly(I:C) and virus-induced IFNQt production in HIV-infected monocytes (Gendelman et al., 1990b). The relationship b e t w e e n replication and the IFN-inducing effect o f HIV is still to be explored. The interdonor variation in the replicative capability o f H I V l n l B and HIV2Ro D found in the macrophage cultures raises the possibility that factors other than IFN production may also play a role in the restriction o f HIV replication in mononuclear phagocytes. O f the two HIV types tested, HIV2 strain ROD was f o u n d to be more monocytotropic, as HIV2Ro D replication (RT activity) occurred earlier and reached higher levels than that o f HIV1 strain IIIB. These findings are in agreement with evaluations o f differ~nces between HIVI and HIV2 replication in the promonocytic cell line U937 (Innocenti and Seigneurin, 1990). In conclusion, this study demonstrates that a relationship exists between the maturation stage o f blood mononuclear phagocytes, their ability to produce IFN~ when challenged with HIV, and the related kinetics o f HIV replication. Further characterization o f IFN produced by HIV-infected monocytes and investigation o f its effect on HIV replication on the molecular level may permit insight into the virus/host cell relationship in cells o f mononuclear phagocytic lineage.

Acknowledgements

This work was supported in part by grants from The Danish Research Council, The Danish AIDS Foundation, Director Jacob Madsens and Wife Olga Madsens Foundation. The excellent technical assistance of Viggo Nielsen is gratefully acknowledged.

Les differences de r6plication du VIH et la production d'interf~ron, induit par le VIH, par les monocytes-macrophages: relation avec la maturation des cellules

Le sujet de l'enqu&e 6tait la r6plication du virus de l'immunod6ficienc¢ humaine (VIH) et la production d'interf6ron (IFN), induit par le VIH, par les monocytes sanguines humaines,/l deux 6tapes diff6rentes de la maturation in vitro. Des monocytes pris sur 6 donneurs en bonne sant6 et VIH s6ron6gatifs ont 6t6 expos6es a u VIHlin B et a u VIH2Ro D, soit imm6diatement suivant la s6paration ou apr6s 5 jours de culture in vitro. Des monocytes fra/chement s6par6es ont produit de I'IFN apr6s inoculation des deux types de VIH. Dans ces cultures, une variation interdonneurs a 6t6 observ6e apr6s une corr61ation inverse entre la quantit6 d'IFN produite et les cin~tiques de la r6plication du VIH. Apr6s avoir 6t6 cultiv6s in vitro pendant 5 jours, les monocytes n'ont pas produit d'IFN apr6s exposition au VIH infectant, mais une r6plication signifiante du VIH 1111Bet du VIH2RO D a 6t6 observ6e darts toutes les cultures. Mots-cids: VIHI, VIH2, Monocyte, Macrophage, Interf6ron, R6plication, Maturation; Corr61ations, Cultures cellulaires, Etapes.

References

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DIFFERENTIAL HIV REPLICA TION AND INTERFERON PRODUCTION

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