APMIS 100: 465-469, 1992

Interferon-gamma may enhance infection of bloodderived macrophages with HIV-1 in the presence of

HIV=positive serum

MIKLOS DEGRfi, SOLVEIG BECK and HALVOR ROLLAG

Virology section, Wilhelmsens Institute of Bacteriology, University of Oslo, Rikshospitalet, Oslo, Norway

Degre, M., Beck, S. & Rollag, H. Interferon-gamma may enhance infection of blood-derived macro- phages with HIV-1 in the presence of HIV-positive serum. APMIS 100: 465469, 1992.

HIV multiplication in blood-derived macrophages was slightly inhibited by pretreatment of cells with interferon-gamma or by preincubation of virus with serum containing antibodies against HIV. When these pretreatments were combined, the HIV titres observed a short time after infection were enhanced. This effect was blocked by antibodies against Fc receptors but not by antibodies against CD4 receptors. Interferon enhanced the expression of Fc receptors on macrophages. The results indicate that IFN-)I, in appropriate combinations with HIV-antibody-containing human serum, may enhance the rate of HIV infection of macrophages.

Key words: HIV-1 infection; interferon-gamma; Fc receptor; macrophages.

M. Degre, Kapt. Wilhelmsens Bakteriologiske Institutt, University of Oslo, Rikshospitalet, N-0027 Oslo 1, Norway.

The human immunodeficiency virus (HIV) in- fects primarily CD4 + T-helper lymphocytes and monocytes. Although it is well established that the infection is mostly mediated by the CD4 receptor, recent reports indicate that additional binding mechanisms may be involved during infection of different susceptible cell types, es- pecially monocytes/macrophages (6, 7 , 8, 14, 18), and that the virus may infect cells that normally do not express CD4 antigen (5 ) . En- hanced infection in the presence of HIV anti- bodies and complement indicates that Fc recep- tors and/or complement receptors may mediate attachment and entry of the virus into the cells (4, 6, 7 , 8, 11, 16, 18). The role of Fc receptors has been confirmed by the finding that cytome- galovirus-induced Fc receptors conferred sus- ceptibility to otherwise non-susceptible fibro- blast cells (10). We (15) and others (21,23) have

Received April 18, 199 1. Accepted October 30, 199 1.

reported earlier that interferon (IFN), especially IFN-y, enhanced the expression of Fc receptors on monocytes and macrophages. Several groups have reported that cytokines may alter produc- tion of HIV in monocytes and other cell types (2, 3, 9, 12, 22). The present report shows that IFN-y, in appropriate combinations with anti- bodies against HIV, may enhance the rate of HIV infection in these cells.

MATERIALS AND METHODS

Virus and cells The IIIB strain of HIV-I virus, originally isolated

by R. Gallo, was propagated in H9 cells. The virus preparation was kept at -70°C and quantitated by reverse transcriptase (RT) assay (1 7), infectivity ti- tration in peripheral blood lymphocytes (PBL) and by a commercially available microELISA assay for p24 antigen (Viranostika, Organon Teknika, Oss, The Netherlands). Primary human monocytes were separ- ated from buffy coats of healthy blood donors (17).

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Cells were suspended in RPMI 1640, supplemented with 15% human AB serum (HABS), gentamycin (40 pg/ml) and amphotericin B (2.5 pg/ml), and distrib- uted into 24-well Costar plates (Cambridge, Mass.) (1 ml per well, 4 x lo6 cells per ml). After one h the non-adsorbed cells were removed by washing three times with Hanks balanced salt solution (BSS). The adherent cells, ca 1.3 k lo6 per well, were incubated in RPMI 1640 supplemented with HABS, gentamycin and amphotericin. After six days HABS was replaced with 10% foetal calf serum (FCS). At least 95-97% of the cells were monocytes, ascertained by morphology, non-specific esterase staining and by immunofluor- escence staining using anti-T 11 antibodies (Dako, Copenhagen, Denmark). Viability of the cells was at least 95%, as shown by the trypan blue exclusion method, or by staining with ethidium bromide-acri- dine orange (Becton Dickinson, Mountain View, Cal- if.). The number of attached cells was quantitated every five days during cultivation by counting nuclei in acridine orange-stained preparations (1).

Reugen ts Interferon-y (IFN-y) was given to us by Genentech,

(South San Francisco, Calif.). The IFN preparation contained less than 2.0 E4 ml endotoxin, tested by the limulus amoebocyte lysate test. Monoclonal anti- bodies against CD4 receptor, anti-Leu-3a, and against Fc receptor, anti-Leu 11 b, were purchased from Becton Dickinson. An HIV-positive serum was selected from our diagnostic laboratory. This serum was positive for antibodies against all major antigens in the Western Blot test.

Experimental procedure Six- or seven-day-old macrophages were pretreated

with IFN-y, usually 10 IU per ml, overnight. IFN- treated and control macrophages were inoculated with 100 p1 of HIV diluted to contain 5 x lo5 tissue culture infectious doses per ml for each well and supplemented with 1 ml medium. Samples of HIV were prior to inoculation incubated for one h at room temperature with various dilutions of an HIV- positive serum or an HIV-negative serum. The inocu- lated cultures were incubated for up to three weeks. Development of HIV infection was monitored by weekly tests for RT and p24 antigen in the super- natants, as previously described in detail (13). The samples were kept at -70°C until testing and all samples from the same experiments were tested in the same run.

Quantitation of Fc receptors was done by the method previously described (1 5), using IgG-opsoni- zed Escherichiu coli, labelled with "P-orthophos- phate. The macrophage cultures were washed and incubated in serum-free medium at +4"C with 0.5 ml of bacterial suspension, containing 5 x 10' colony- forming units, for 60 min. At the end of this period the cultures were washed four times, the cells were

dissolved by addition of 0.1 N NaOH, and the amount of attached radioactivity was measured in a Packard liquid scintillation counter. Each value was based on four to five parallels. The results were ex- pressed as counts per minute per culture (CPMM). Fc receptor-mediated attachment to IFN-y-treated macrophages was related to attachment to control untreated macrophages. Non-specific binding was tested for by non-opsonized bacteria, and was shown to be negligible under the experimental conditions applied.

RESULTS

Pretreatment of macrophages with 10 or 100 IU per ml of IFN-y had a modest but consistent influence on the development of HIV infection. HIV p24 antigen titres, measured six or seven days after inoculation, were on average reduced by 25% (range 10-45%) in cells pretreated with 10 IU of IFN-y in six repeat experiments with macrophages from six different donors. Reduc- tion of RT titres averaged 15% (range 7-35%) in four experiments. The differences became less pronounced with prolonged incubation.

The preincubation of HIV with HIV-anti- body-containing serum also initially reduced HIV multiplication. Preincubation with lop3,

and lop5 dilutions of an HIV-positive serum reduced the p24 antigen titres by 47 (range 15-63), 35 (range 11-60) and 30 (range 9-41)%, respectively, in six repeat experiments, measured six or seven days after inoculation. The effect of preincubation with HIV-positive serum was similar, although less pronounced when measured by RT activity. Also this effect diminished with prolonged incubation.

The combination of IFN-y pretreatment of macrophages with preincubation of HIV with HIV antibody-positive serum gave different re- sults. Compared with either agent alone, or compared to untreated controls the combi- nation gave enhanced HIV titres in samples taken within seven days after inoculation, meas- ured either by p24 antigen or by RT. Results of a representative experiment are shown in Table 1. Each value represents the mean of three paral- lel wells. The variation between parallels was f8% or less for each of the figures presented. Practically identical results were obtained in three repeat experiments. P24 antigen levels were 176 to 229% of the controls, and two to

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HIV GROWTH ENHANCED BY IFN-y IN MACROPHAGES

TABLE 1. Effect of IFN-y and HIV-positive serum on HIV growth in blood-derived macrophages Serum (diluted) Interferon gamma

10 IU 0 HIV + HIV -

RT P24 ag RT P24 ag Six days after inoculation 10-3 140" (110) 736b (179)" 91 (35)" 296 (87) 10-4 159 (1 25) 722 (1 76)" 68 (27)" 359 (80)" 10-5 194 (1 52)" 945 (229)" 111 (44)" 322 (71)"

10-4 115 (90) 371 (90) 240 (94) 450 (100) Control 127 41 1 255 450 14 days after inoculation 10-3 281 (101) 612 (108) 295 (84) 439 (72)"

10-5 297 (107) 690 (122)" 345 (99) 452 (74)" 10-4 250 (90) 495 (88) 370 (103) 611 (100)

Control 277 564 360 610

10-4 241 (86) 565 (100) 301 (86) 439 (74)"

~~ ~

a RT, CPMM x 10, mean of three determinations (percent of control in parentheses) p24 antigen picogram per culture (percent of control in parentheses) Significantly different from controls (p < 0.05). Macrophages were pretreated with IFN-y overnight. Virus, 5 x 10' TCID per ml, was incubated with various dilutions of anti-HIV serum for one h prior to inoculation.

three times higher compared to cultures receiv- ing either treatment alone. The differences were less pronounced when measured by RT. With prolonged incubation the effect diminished and in some experiments disappeared. This tendency was reproduced in six repeat experiments using the same virus and same antibodies, but cells from different donors. The maximal enhancing effect in these experiments varied from 15 to 350% compared to untreated controls. In two experiments the enhancing effect was only ob- served with the highest dilutions of the HIV- positive serum. Preincubation of virus with lo-* dilution of the same serum reduced HIV titres by 80% or more whether or not it was combined with IFN-y treatment.

If the macrophage CD4 receptors were blocked by antibodies to CD4 receptors, HIV titres were strongly reduced (Table 2). The com- bined treatment with IFN-)I and HIV-positive serum enhanced the HIV titres in such cells. This effect could be blocked by the presence of heat-aggregated y-globulin (data not shown). Also antibodies against the Fc receptor reduced HIV titres. Addition of IFN-y and HIV-positive serum to these cells did not enhance HIV titres. These results indicate that the enhancing effect of IFN-y and HIV antibodies is mediated by the Fc receptors. To exclude the possibility of a non-specific inhibition by the monoclonal anti- body preparations due to their content of so-

dium azide, cells were treated with non-relevant monoclonal antibodies, antibodies against her- pes simplex virus glycoproteins or antibodies against early antigen of cytomegalovirus. No inhibitory effect was observed (data not shown).

Pretreatment of macrophages with IFN-y en-

TABLE 2. Influence of IFN-y and HIV-positive serum on HIV growth in blood-derived macrophages a fe r

blockinp the CD4 and Fc receDtors Treatment of cells P24 RTb

antigena Negative control < I < 1 HIV +control 530 3690 IFN-)I 429 (80) 3034 (82) HIV-pos. serum 309 (58) 2388 (64) IFN-y+HIV-pos. serum 579 (113) 3989 (109) anti-Leu 3a (anti-CD4) 147 (28) 841 (23) anti-Leu 3a+HIV-pos. serum 81 (15) 120 (3) anti-Leu 3a + IFN-y 157 (30) 610 (16) anti-Leu 3a + IFN-y + 289 (54) 1834 (50)

anti-Leu 11 b (anti-Fc) 365 (69) 2783 (75) anti-Leu 1 1 b + IFN-y 188 (35) 1879 (50) anti-Leu 1 1 b + IFN-y + 196 (37) 1960 (53)

HIV-pos. serum

HIV + serum p24 antigen picogram per well (percent of control in parentheses) RT, CPMM x 10 (percent of control in paren- theses) Macrophages were pretreated with anti- bodies against the receptors for one h. Treatment with IFN-)I and HIV-positive serum was done as in Table 1.

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TABLE 3. Effect of IFN-y pretreatment of cells on attachment of IgG-opsonized E. coli to monocyte-de-

rived macrophages Experiment Pretreatment of cells with IFN-)I

10 IU 100 IU 1 1 39a -

2 392 - 3 129 173 4 131 100 5 125 127 Mean 174+41b 133 a Percent of controls not treated with IFN-y

p < 0.001 (Wilcoxon’s test).

hanced the expression of Fc receptors. In five repeat experiments with macrophages from dif- ferent donors, both 10 and 100 IU IFN-y gave consistently increased attachment of IgG-op- sonized E. coli (Table 3), although the extent of stimulation varied from one experiment to another.

DISCUSSION

The present data show that pretreatment of blood-derived macrophages with IFN-y simul- taneously stimulates Fc receptor expression on the cell surface and the rate of HIV infection of the cells in the presence of low concentrations of human serum containing antibodies against HIV. This enhancing effect is blocked by mono- clonal antibodies against the Fc receptor but not by antibodies against the CD4 receptor. No enhancing effect was seen if the HIV-antibody- containing serum was substituted by HIV-nega- tive serum, and the effect could be blocked by heat-aggregated y-globulin. These findings indi- cate that the enhanced rate of infection is prob- ably mediated by the Fc receptors. Our obser- vations are in agreement with the previous sug- gestion that, in addition to the well-established CDCmediated uptake, HIV may be internalized in the cells, especially cells of the monocyte/ macrophage lineage, by additional mechanisms mediated by complement receptors and by Fc receptors ( 4 8 , 10, 11, 16, 18). However, other alternative mechanisms cannot be excluded completely, since IFN-y may influence several macrophage functions involved in uptake of the virus.

The finding that high concentrations of the

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same antibodies may neutralize HIV is in agree- ment with earlier reports (1 1, 18). With the con- centrations of antibodies employed in these ex- periments we did not observe enhancement of HIV infection without pretreatment of cells with IFN-y. This may apparently be at variance with previously published results (6, 7, 8, 18). How- ever, those authors have mostly used other cell types, such as the monocytogenic U937 line, and they have seen enhancement with some but not all sera, and only at certain dilutions. Therefore our observations do not necessarily contradict the previously published results.

Concerning the possible in vivo relevance of these findings, it is pertinent to point out that the cells employed in our studies (and, in fact, in most of the studies published) were blood- derived macrophages transformed during in vi- tro cultivation on solid surfaces. These target cells cannot be considered identical to the cells HIV infects under natural conditions (20).

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