Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

54, 174-183 (I%@)

Cell Surface Antigens and Function of Monocytes and a Monocyte-like Cell Line before and after Infection with HIV’ DEAN L. MANN, *,*SUZANNEGARTNER,~FRANCESLESANE,* WILLIAM A. BLATTNER,$ ANDMIKULAS Popovrct *Immunogenetics Section, Laboratory of ViraZ Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701; iFLaboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and #Environmental Epidemiology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 The human immunodeficiency virus (HIV-l) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-I. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR. HLA-DQ, and HLA-DP increased in HIV-I infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLADP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-l-infected monocytes. o IWO Academic press. II-C.

INTRODUCTION

The human immunodeficiency virus (HIV) is the causative agent for the acquired immunodeficiency syndrome (AIDS) (1). One of the clinical hallmarks of this disease is the depletion of the subpopulation of T lymphocytes that bear the CD4 molecule which is due to the cytopathic effect of the virus on these cells. A number of studies demonstrate quite clearly that HIV is tropic for CD4 positive lymphocytes (24). More recently, it has also been shown that the attachment of HIV to the target cell is mediated through the interaction of the large envelope protein of the virus with the cellular CD4 molecule (5-8). HIV can also infect cells other than T lymphocytes. These cells all have in common the expression of the CD4 molecule and include monocytes, dendritic cells, and Langerhans’s cells in the skin (9-14). Another common feature of these cells is the expression of major histocompatibility complex (MHC) class I and class II antigens. The expression of MHC class II molecules is critical to the function of monocytes in antigen presentation. Since HIV-infected T cells show changes in expression of CD4 and MHC class II antigen, we sought to determine if similar changes occurred in HIV-infected monocytes. One measure of monocyte function is the ’ Presented as part of a symposium entitled “Monocyte October 5, 1988, Bethesda, MD. * To whom correspondence should be addressed. 174 0090-1229&O $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

Dysfunction in HIV-Infected

Individuals,”

FUNCTION

OF

HIV-INFECTED

MONOCYTES

175

ability to stimulate allogeneic cells. As with antigen presentation, this function is dependent on MHC class II antigen expression. The results of our studies examining the expression of cell surface antigens and allogeneic stimulation of HIVinfected and uninfected peripheral blood-derived monocytes and monocyte-like cell lines are reported herein. MATERIALS

AND METHODS

Cell

The U937 cell line used in these studies was originally established from a patient with histiocytic lymphoma (15). This cell line was carried in suspension culture in RPM1 1640 medium supplemented with a 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 5 kg/ml gentamicin, and 2 rn&! L-glutamine. Macrophages were isolated from peripheral blood by methods previously described (IO, 11). In brief, peripheral blood lymphocytes (PBL) were isolated from whole blood from HIV-l seronegative individuals by the Ficoll-Hypaque gradient separation technique. The lymphocytes were suspended in the above medium with 20% FCS and 10% human AB serum and added to T-25 tissue culture flasks. The flasks were incubated at 37°C in 5% CO, in air for 3-5 days after which the nonadherent cells were removed by washing with phosphate-buffered saline (PBS) free of Ca and Mg. The PBL-derived monocytes were carried in culture with the above medium with 20% FCS. Infection

The cell lines in suspension were infected with HTLV-IIIB in the following manner. The H9-HTLV-IIIB-infected cell line was irradiated with 7OOOR (137 Cs) and cocultured with the cell lines. The PBL-derived monocytes were infected as described (10, 1 I). In brief, culture fluid from HIV-infected cell cultures containing 0.5 x IO5 cpm reverse transcriptase activity per milliliter was added to the adherent cells that had been pretreated with polybreen (2 mg/ml) for 30 min. After exposure to the virus containing culture fluids for 1 hr at 37”C, the cells were washed with PBS and maintained in the culture media containing 20% FCS. The cells were monitored for infection by assays for reverse transcriptase. The percentage of cells infected was assessed using a human serum that detected the ~17, ~24, core proteins, and the gp120 large envelope of the virus on Western blots. Monoclonal

Antibodies

Monoclonal antibodies detecting CD4 and CD8 antigens (OKT4A and OKTS) were obtained from Ortho Diagnostic (Ruritan, NJ). Monoclonal antibodies detecting the products of the MHC class II antigens are listed as follows. TU22 detects HLA-DQ, TU43 detects HLA-DR, and B7/21 detects HLA-DP. The latter antibody was a gift of Dr. Stephen Shaw, National Institute of Cancer (Bethesda, MD). W6/32 detects the MHC class I antigens. Analysis

of Cell Surface Antigens

Infected and noninfected

U937 cells (106) were pelleted and washed with PBS

176

MANN

ET AL.

containing 1% human serum albumin. After centrifugation, 100 ~1 of the monoclonal antibodies or the human serum was added and incubated with the cells at 37°C for 1 hr. The cells were washed with the PBS-albumin medium, repelleted, and a saturating amount of fluorosceinated goat anti-mouse or anti-human immunoglobulin (Cappel, Cochranville, PA) added. This combination was incubated for 1 hr at 4°C after which the cells were washed and analyzed for fluorescence on the FACS-II (Becton-Dickinson, Sunnyville, CA). The PBL-derived monocytes, infected and noninfected, were removed from the tissue culture flasks by extensively washing with Ca- and Mg-free PBS and placing the washed cell lines on ice for a period of 1 hr. The cells were gently dislodged with a rubber policeman and resuspended in the PBS. Cells (10”) were labeled with the monoclonal antibodies and human serum for analysis of cell surface antigens and viral proteins as described above. Ten thousand cells were analyzed for the percentage of cells reacting with the antibodies. The mean fluorescence, a measure of the relative concentration of the antigen on the cell surface, was also determined. The P-3 mouse myeloma protein and HIV-l negative human serum was used as a control for background fluorescence. Lymphocyte

Proliferation

Assays

Lymphocyte proliferation response to the U937 (infected and noninfected) was established using mixed lymphocyte cultures described in detail elsewhere (IO). Cells (2 x 105) were treated with mitomycin C (6.2 kg/ml), washed, and added to 2 x lo5 PBL obtained from individuals who had no prior history of infection with HIV-I and whose sera were negative for antibodies of this virus. The cells were cultured in RPM1 1640 medium containing 10% human AB serum, 20 mm Hepes buffer, 2 pJ4 L-glutamine (GIBCO, Grand Island, NY), and 5 t&ml gentamicin sulfate (GIBCO) for 5 days at 37°C in a humid atmosphere of 5% CO, in air. The cultures were pulsed with 0.2 &I of [methyl-3H]thymidine (Swartz/Mann, Spring Valley, NY) for 6 hr, harvested, and the incorporated radioactivity was determined by standard p-scintillation counting. Stimulator-responder combinations were chosen to ensure differences in the HLA antigen phenotypes. Stimulation of allogeneic PBL by peripheral blood macrophages, infected and noninfected, were performed as follows. Macrophages (5 x 105) were added to individual wells of a 96-well Costar plate and allowed to adhere for a period of 24 hr. The plates were spun at 1000 rpm for 10 min and the supernatant was removed. Various concentrations (2 x 106, 1 x 106, 0.5 x 106) of normal PBL were added to the individual wells in a final volume of 200 t.~l. The plates were incubated for 5 days at 37°C in a humid atmosphere with 5% CO2 in air. The plates were pulsed with 0.2 l&i/well of [methyl-3H]thymidine and incubated for 6 hr. The entire plate was harvested onto glass fiber filter disks using a Micro Culture Harvester (Brandell, Gaithersburg, MD), and the incorporated radioactivity was quantitated as described above. All experiments were performed in quadruplicate. The mean counts per minute (cpm) were calculated and the results were converted to stimulation indices by dividing the average cpms of the stimulated cells by the average cpms of unstimulated cells.

FUNCTION

OF

HIV-INFECTED

177

MONOCYTES

RESULTS

Cell Surface Antigen Expression on Infected and Noninfected

Monocytes

Table 1 shows the results of the expression of the CD4, MHC class I, HLA-DQ, HLA-DR, HLA-DP, and HIV-I proteins on noninfected and infected peripheral blood-derived monocytes and the U937 cell line. The percentages of cells that demonstrated active viral replication as evidenced by surface membrane staining with the human serum were 44 and 58% for the monocytes and 70% for the U937 cell line. The CD4 antigen was essentially not detected or the infected U937 compared to 67% of the uninfected cells. The percentage of cells reacting with all MHC class II antigens increased on the infected U937 cells. The percentage of cells expressing the CD4 molecule decreased in the infected peripheral blood monocytes, however, was not completely absent. HLA-DQ expression decreased in the infected monocytes from the two different sources. HLA-DR expression was low to absent on the noninfected cells and increased to 13 and 14%, respectively. on the HIV-infected cells. HLA-DP decreased slightly in donor 1 and increased slightly in expression in donor 2 in infected monocytes compared to uninfected cells. Allogeneic

Stimulation

Figure 1 shows the results of the stimulation of lymphocytes from four different individuals by the U937-uninfected and infected cell lines. There was a slight decrease in the ability of the infected U937 line to stimulate the allogeneic cells compared to the stimulation achieved by uninfected cells. Figure 2 shows the stimulation indices of PBL from two individuals by monocytes from two donors before and after infection. Decreasing concentrations of the responding cells were used in these experiments. As observed with the HIVinfected monocyte cell lines, HIV-infected monocytes demonstrated a consistent decrease in the ability to stimulate allogeneic cells at all concentrations of responding cells, except at responding cell concentrations of 0.5 x lo6 cells in all but one of the combinations tested. The percentage of CD4+ and CD8+ cells was determined in the cell cultures of HIV-l-infected and noninfected monocytes with allogeneic lymphocytes (Table 2). The cell recovery after 7 days of culture was 80 to 86% in the cultures of noninfected monocytes and 58 to 72% in the cultures where infected monocytes were used as stimulators. CD4+ cells decreased in the cultures containing HIVl-infected monocytes relative to the percentage found in cultures with noninfected macrophages . DISCUSSION

The loss or decreased expression of the CD4 molecule on the HIV-l-infected U937 cell lines and PBL-derived monocytes is consistent with observations made on the examination of the expression of this molecule on HIV-infected T lymphocyte cell lines and peripheral blood T lymphocytes (2, 7, 17). The mechanism for this lack of expression is probably due to the interaction of the CD4 molecule with

96 97 97 97

2884 1422 352 5

31 I5

67 1

HIV-l

%

HIV-l

MFU’

4405 4138

4608 4536

4057 4218

MFU

HLA class I

ON HIV-I-INFECTED

92 94

CD4

EXPRESSION

1856 984

%b

ANTIGEN

43 29

SURFACE

u Human sera containing antibodies to ~17, ~24, and gp120 viral h Percentage of total cells fluorescent. ’ Mean fluorescent units.

Donor 1 Monocytes Monocytes Donor 2 Monocytes Monocytes Cell line u937 U937 HIV-I

CELL

proteins.

8 41

1 14

3 13

%

HLA-DR

108 300

117 1075

113 819

MFU

2 38

27 31

31 22

%

HLA-DP

PBL-DERIVED

Antigens

TABLE 1 AND NONINFECTED

20 306

2319 2817

1272 1162

MFU

16 67

55 36

36 19

%

MONOCYTES

HLA-DQ

160 1341

4017 4143

1979 744

MFU

AND U937

CELL

0.8 70

I 58

0.2 44

%

HIV-I”

LINE

41 852

29 147

14 212

MFU

5 F .

z

2

FUNCTION

OF HIV-INFECTED

179

MONOCYTES

B

m

U937

U937 HIV

Bkg

.-2 0 E

g

30

I m 00 ; 20 E :: 10

0

A

B

C

D

Responder FIG. 1. Stimulation of allogeneic lymphocytes from four individuals (A, B, C, and D) with HIVl-infected and noninfected U937 cells.

the HIV large envelope protein in the cytoplasm which inhibits or prevents the cell surface expression of CD4 (18). The mechanism whereby MHC class II antigen expression is altered in HIVl-infected cells is not clearly understood. In prior studies, we have found that both HLA-DR and CD4 antigens are substantially decreased after 15 min of exposure of cell-free virus to phytohemagglutinin-activated PBL and T cell lines (17). In these studies, the ability to detect the CD4 antigen remained low after 2 hr of virus exposure, while HLA-DR returned to higher levels of expression than that seen on the unexposed cells. The MHC class II antigens, HLA-DQ and -DP, changed variably, in some instances increasing and in other instances remaining essentially unchanged after viral infection. The results presented in this report are consistent with the observation made in the study of infected and uninfected T cells. HLADR expression increased in HIV-l-infected monocytes, while HLA-DQ and -DP expression was variable and CD4 expression decreased or was absent. One explanation for the differences in MHC class II antigen expression on the U937 cells and noninfected and infected monocytes is that a subpopulation of cells is selectively infected with HIV and persists as the predominant cell in culture. This subpopulation of cells are those that express both the CD4 and HLA-DR molecules. In the U937 cell line, low levels of MHC class II antigen expression were observed on the uninfected cell, with higher levels in the infected cell line. Thus, a subpopulation of cells that expresses these antigens may be the target for continuous productive HIV-l infection. There is evidence that subpopulations of MHC class II expressing cells are present in the U937 cell line as Gitter et al. (19) have cloned and established an MHC class II antigen expressing U937 cell line.

180

MANN

ET AL.

MONOCYTE

STIMULATOR

1

Non-Infected Infected

MONOCYTE

STIMULATOR

2

cl

Non-Infected Infected

IL-

2x105 1 x105 0.5x105

ill

2 x 105 1 x 105 0.5 x 105

Cell No. Responder A Cell No. Respondor B FIG. 2. Comparison of the ability of infected and noninfected monocytes from two different donors to stimulate allogeneic lymphocytes from two different donors. Three different concentrations of responding cells were used in these experiments. The results are recorded as stimulation index, the open bars represent the stimulation by noninfected cells, and the solid bars represent the stimulation by the infected monocyte.

In other studies, it was demonstrated that the majority of PBL-derived monocytes express HLA-DR, while HLA-DP and HLA-DQ antigens are expressed on only a portion of the monocyte populations (20). Our data suggest the possibility that the previously reported distributions of MHC class II antigens represent several populations, as the monocytes isolated by the techniques described were predominantly HLA-DQ and -DP positive. After infection with HIV-l, the percentage of cells expressing HLA-DQ decreased, while those expressing the HLA-

FUNCTION

RECOVERY

OF CD4+

LYMPHOCYTES NONINFECTED

OF

HIV-INFECTED

MONOCYTES

TABLE 2 IN MIXED LYMPHOCYTE CULTURE OF HIV-I-INFECTED MONOCYTES WITH ALLOGENEK CELLS Percentage Responder

Stimulating

cells

181

of total

cells recovered”

A

Responder

CD4+

CD8+

54 42 58 28

AND

B

CD4’

CD8’

38 44

62 40

32 48

40 48

60 42

30 50

Donor 1 Monocytes, noninfected Monocytes, infected Donor 2 Monocytes, noninfected Monocytes, infected

a After 7 days of culture of 2 x IO5 responding cells.

DR increased. Since CD4 is necessary for HIV-l binding and changes in HLA-DR expression occur with infection, our results are consistent with the interpretation that monocytes expressing both of these molecules are selected for HIV-l infection. Both the HIV-infected U937 cell lines and the peripheral blood-derived monocytes demonstrated diminished ability to stimulate allogeneic cells compared to uninfected cells. This decrease did not necessarily correlate with the number of cells actively producing virus as detected by cell surface staining for viral protein. The changes in ability to stimulate may be due to relative numbers of cells expressing the different MHC class II gene products since allogeneic stimulation is dependent on differences in these antigens stimulating and responding cell population (21). The only consistent change observed in our studies was an increase in the percentage of the total cell populations expressing HLA-DR and variable changes in HLA-DP and -DQ expression depending on the type of cell that was infected. Thus, a direct correlation of MHC class II antigen expression and with the decreased ability to stimulate allogeneic cells by HIV-infected monocytes cannot be made. The decreased [3H]thymidine incorporation in the responding cells was most likely due to the depletion of total T cells and the relative decrease in the percentage of CD4+ cells at the end of the culture period. Similar results have been reported by Lewis et al. (22) where it was demonstrated that allogeneic stimulation by HIV- l-infected cells greatly enhanced virus transmission to the responding cell population with concomitant depletion of CD4+ cells. A variety of lymphokines that support T cell responses are produced by monocytes (23). Relative differences in production of these products in HIV- 1 infected cells may also account for the differences in allogeneic stimulation that were observed. The role of monocytes in HIV infection and the potential biologic significance of this infection was gradually being elucidated. HIV-infected monocytes have been demonstrated in biologic specimens (11-13). In vitro monocytes do not appear to be subject to the dramatic cytopathic effects of infection that occur in T lymphocytes. Thus, monocytes or cells of this lineage may be the reservoir for

182

MANN

ET AL.

HIV infection. Infection of monocytes and replication of the virus in these cells may in fact represent or mimic the normal immune response. The results of our studies demonstrate that HIV-l-infected monocyte cultures can present alloantigens to T cells. In other studies (manuscript in preparation), we found that infected monocyte cultures were also capable of presenting tetanus toxin and streptokinase to autologous T cells. Antigen presentation by HIV-l-infected monocytes to T cells may be an efficient mechanism of virus transmission in that CD4+ T cell activation is critical to HIV-l infection. REFERENCES 1. Popovic, M., Samgadharan, M. G., Read, E., and Gallo, R. C., Detection, isolation and continuous production of cytopathic retrovirus (HTLV-III) from patients with AIDS and pre-AIDS. Science

224, 497-500,

1984.

2. Popovic, M., Gallo, R. C., and Mann, D. L., OKT4 bearing molecule is a receptor for the human retrovirus HTLV-III. Clin. Res. 33, 56OA, 1984. 3. Dalgleish, A. G., Beverley, P. C. L., Clapham, P. R., Crawford, D. H., Greaves, M. R., and Weiss, R. A., The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312, 763-766, 1984. 4. McDougal, J. S., Nicholson, J. K. A., Cross, G. D., Cort, S. P., Kennedy, M. S., and Mawle, A. C., Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T-4) molecule: Conformation dependence, epitope mapping, antibody inhibition and potential for idiotypic mimicry. J. Xmmunol. 137, 2937-2944, 1986. 5. Sattentau, Q. J., Dagleish, A. G., Weiss, R. A., and Beverley, P. C. L., Epitopes of the CD4 antigen and HIV infection. Science 234, 1120-l 123, 1986. 6. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A., and Axel, R., The T4 gene encodes for the AIDS virus receptor and is expressed in the immune system and the brain. Ceil 47, 338-348, 1986. 7. Hoxie, J. A., Flaherty, L. E., Haggerty, B. S., and Rackowski, J. L., Infection of T4 lymphocytes by HTLV-III does not require expression of the OKT4 epitope. J. Immunol. 136, 361-363, 1986. 8. McDougal, J. S., Kennedy, M. S., Sligh, J. W., Cort, S. P., Mawle, A., and Nicholson, K. A., Binding of HTLV-IIVLAV to T4+ cells by a complex of I 10k viral protein and the T4 molecule. Science 231, 382-384, 1986. 9. Ho, D. D., Rota, T. R., and Hirsch, M. S., Infection of monocyte/macropbages by human T lymphotropic virus type III. J. Clin. Invest. 77, 1712-1715, 1986. 10. Gartner, S., Markovits, P., Markovitz, D. M., Kaplan, M. H., Gallo, R. C., and Popovic, M., The role of mononuclear phagocytes in HTLV-IIULAV infection. Science 223, 215-219, 1986. Il. Gartner, S., Markovitz, D. M., Betts, R. F., and Popovic M., Virus isolation and identification of HTLV-IIFLAV-producing cells in brain tissue from a patient with AIDS. JAMA 256, 2365-2371, 1986. 12. Koenig, S., Gendelman, H. E., Orenstein, J. M., Dalcanto, M. C., Rezeslokpour, G. H., Yungbluth, M., Janotta, F., Akosamit, A., Martin, M. A., and Fauci, A. S., Detection of macrophages in brain tissue from AIDS patients with encephalopathy. Science 223, 1089-1098, 1986. 13. Tschachler, E., Groh, V., Popovic, M., Mann, D. L., Konrad, K., Safai, B., Eron, L., Veronese, F. D., Wolff, K., and Stingle, G., Epidennal Langerhans cells: A target for HTLV-IlI/LAV infection. J. Invest. Derm., in press. 14. Stoler, M. H., Eskin, T. A., Benn, S., Angerer, R. C., and Angerer, L. M., Human T-cell lymphotropic virus type II infection of the central nervous system. JAMA 256, 2360-2364, 1986. 15. Sandstrom, C., and Nilsson, K., Establishment and characterization of a human histiocytic lymphoma cell line. Int. J. Cancer 17, 565-571, 1976. 16. Mann, D. L., LeSane, F., Popovic, M., Arthur, L. O., Robey, W. G., Blattner. W. A., and Newman, M. J., HTLV-III large envelope protein (gp120) suppresses PHA induced lymphocyte blastogenesis. .I. Zmmunol. 138, 2640-2644, 1987.

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OF

HIV-INFECTED

MONOCYTES

183

17. Mann, D. L., Read-Connale, E.. Arthur, L. O., Robey, W. G., Wemet. P., Schneider, E. M., Blattner, W. F., and Popovic. M., HLA-DR is involved in the HIV-l binding site on cells expressing MHC class II antigens. J. Zmmunol. 141, 1131-1136, 1988. 18. Hoxie, J. A., Alpers, S. D., Rackowski, J. L., Huebner, K., Haggerty, B. S., Cedarbaum, A. J.. and Reed, J. C., Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV. Science 234, 1123-l 127, 1968. 19. Gitter, B. D., Finn. 0. J., and Metzger, R. S., Cytofluorometric isolation of 1937, an la antigenbearing variant of the Ia-negative human monocyte line U937. J. Immunol. 134, 280-283, 1985. 20. Gonwa, T. A., Frost, J. P., and Karr. R. W., All human monocytes have the capability of expressing HLA-DP molecules upon stimulation with interferon-y. J. Zmmunol. 137, 519-524, 1986. 21. Jaraquemada, D., Navarrette, C., Oilier, W., Awad, J., Okoye, R., and Festenstein, H., HLADW specificities are independent of HLA-DQ, HLA-DR and other class II specificities and define a biologically important segregant series which strongly activates a functionally distinct T cell subset. Hum. Immunol. 16, 259-270, 1986. 22. Lewis, D. E., Yoffe, B., Bonsworth, C. G., Hallinger, F. B., and Rich, R., Human immunodeficiency virus-induced pathology favored by cellular transmission and activation. FASEB J. 2, 251-255, 1988. 23. Nathan, C. F., Secretary products of macrophages. J. C/in. Invest. 79, 319-326, 1987. Received September 26. 1989; accepted September 29, 1989