Vol. 65, No. 11

JOURNAL OF VIROLOGY, Nov. 1991, p. 6277-6282

0022-538X/91/116277-06$02.00/0 Copyright C) 1991, American Society for Microbiology

Human Immunodeficiency Virus Type 1 Envelope Glycoproteins gpl20 and gpl60 Induce Interleukin-6 Production in CD4+ T-Cell Clones NAOKI OYAIZU,1 NARENDRA CHIRMULE,l YOKO OHNISHI,l VANIAMBADI S. KALYANARAMAN,2 AND SAVITA PAHWA1* Department of Pediatrics, North Shore University Hospital-Cornell University Medical College, Manhasset, New York 11030,1 and Department of Cell Biology, Advanced BioScience Laboratories Inc., Kensington, Maryland 208952

Received 8 February 1991/Accepted 1 August 1991

Polyclonal B-cell activation is a characteristic feature of AIDS and of the AIDS-related complex. Since the immunoregulatory cytokine interleukin-6 (IL-6) plays a major role in inducing B-cell differentiation, we examined the effects of native human immunodeficiency virus type 1 envelope glycoproteins gpl20 and gpl60 on IL-6 induction. In this study, we have demonstrated that both gpl20 and gpl60 have the ability to induce IL-6 mRNA and biologically active IL-6 protein secretion in peripheral blood mononuclear cells in vitro. The envelope protein preparations had no detectable endotoxin as tested by the Limulus amebocyte lysate assay, and hence we can rule out the effect of contaminating endotoxin, which is a potent inducer of IL-6 in monocyte/macrophage cell cultures. In addition, we have shown that the envelope glycoproteins act directly on CD4+-cloned T cells to induce IL-6 production in the absence of monocytes. These findings indicate that monocytes and T cells both contribute to the secretion of IL-6, which plays an important role in the pathogenesis of B-cell activation in human immunodeficiency virus infection.

Infection with the human immunodeficiency virus type 1 (HIV-1) is known to cause an intense polyclonal activation of B cells as manifested by hypergammaglobulinemia, elevated levels in serum of immune complexes and autoantibodies, increased numbers of spontaneous immunoglobulinsecreting cells, accumulation of B cells and plasma cells in the lymphoid tissue, and an elevated frequency of B-cell lymphomas (1, 5). However, the mechanism of polyclonal B-cell activation that accompanies HIV infection is not well understood. A number of soluble factors have been shown to regulate the growth and differentiation of B lymphocytes. One of these cytokines, termed interleukin-6 (IL-6), has been shown to be the major factor necessary for the terminal differentiation of activated B cells into immunoglobulin-secreting cells (12). Recently, Nakajima et al. reported that exposure of normal peripheral blood mononuclear cells (PBMCs) to HIV whole-virus preparation induces IL-6 production in vitro and that the main source of IL-6 is the monocyte (18). In addition, infection with HIV is associated with elevated IL-6 levels in plasma regardless of the presence of coinfections (2). These findings suggest that HIV plays a role in inducing increased IL-6 production, with consequent enhancement of terminal differentiation of B cells in vivo. We have previously demonstrated that crude HIV preparations induce T-cell-dependent polyclonal B-cell activation in vitro (19, 20). Furthermore, we have shown that both native and synthetic HIV envelope proteins activate B cells and induce immunoglobulin secretion (4). A recombinant fusion protein constituting env-gag region has also been reported to induce B-cell proliferation and immunoglobulin G secretion (17). In the present study, we have investigated the influence of the highly purified native HIV-1 envelope *

Corresponding author.

glycoproteins gp120 and gpl60 in inducing IL-6 production. We demonstrate that in vitro exposure to the envelope proteins induces the expression of IL-6 mRNA and the secretion of IL-6 by normal PBMCs. Furthermore, we show here that CD4+-cloned T lymphocytes can also be induced to produce IL-6 by the HIV envelope glycoproteins. MATERIALS AND METHODS HIV-1 envelope glycoproteins. The external envelope protein, gpl20, and the unprocessed complete envelope protein, gp160, were purified from supernatants of human T-cell leukemia virus type 111451-infected 6D5451 cells (9) as described in detail previously. The molecular masses of the two proteins were 110 and 140 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting (9). For the present study, the envelope protein preparations were further examined for and found to be free of endotoxin, as tested by the Limulus amebocyte lysate assay (Sigma, St. Louis, Mo.; E-TOXATE, limit of detection of endotoxin, 0.06 EU/ml). Mannosylated bovine serum albumin (mBSA; made by diazotization of BSA with para-aminophenyl-ct-D-mannopyranoside [Sigma] as previously described [11]) was used as a control for envelope proteins. Recombinant gp120 (rgpl20) was obtained from Smith Kline & French Laboratories (King of Prussia, Pa.) and from American Bio-Technologies, Inc. (Cambridge, Mass.). Cells. Unfractioned and fractioned PBMCs and antigenspecific T-helper (Th) cell clones were utilized for these studies. PBMCs were isolated from venous blood of healthy, HIV-seronegative volunteers by Ficoll-Hypaque density gradient centrifugation. E-rosetting T cells were separated from nonrosetting cells by overnight rosette formation with neuraminidase-treated sheep erythrocytes, which was followed by Ficoll-Hypaque density gradient centrifugation. 6277

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After lysis of rosetted sheep erythrocytes, T cells from the pellet were further depleted of contaminating monocytes by two cycles of plastic petri dish adherence. These nonadherent cells were

1h

or

gpl 20 gpl 60 mBSA

greater than 97% CD2 positive and contained

less than 1% esterase-positive cells. Tetanus toxoid (Tt)- and purified protein derivative (PPD) antigen-specific T-cell clones were generated from healthy donors as described previously (3) and propagated by cyclic stimulation with the relevant antigen. Three clones used for this study, Tt2.1, PPD3.5, and Tt4.2, all expressed the phenotype CD4/CD29 (4B4); they did not require addition of exogenous IL-2 and could secrete various lymphokines upon stimulation with antigen in the presence of antigen-presenting cells. Irradiated (3,000 rads) autologous Epstein-Barr virus (EBV)immortalized B cells, designated EBV200 for Tt2.1, EBV300 for PPD3.5 and EBV400 for Tt4.2, were used as antigenpresenting cells. Culture conditions. RPMI 1640 (Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco), 2 mM L-glutamine, 100 U of penicillin G per ml, and 100 ,ug of streptomycin per ml was used for all cultures. PBMCs were precultured for 72 h to abolish spontaneous IL-6 mRNA expression (18). The T-cell clones were rested for 10 days in the absence of antigen and then cultured alone or mixed with irradiated autologous EBV-immortalized B cells in a 1:1 ratio for these experiments. Test cells at a final concentration of 106/ml were cultured with one of the following: gp120, gpl60, mBSA (used at various concentrations), 1 Fg of lipopolysaccharide (LPS) (LPS WS. typhosa 0901; Difco, Detroit, Mich.) per ml, tetanus antigen (2 ,ug/ml), PPD antigen (2 ,ug/ml), or phorbol myristate acetate (PMA; 10 ng/ml, Sigma) combined with ionomycin (1 ,uM; Calbiochem Corp., La Jolla, Calif.). Blocking studies with antibodies and soluble CD4. In certain experiments, 0.1 ,ug of gpl20 or gpl60 per ml was precultured with 1 ,ug of soluble CD4 (sCD4; Genentech Inc., San Francisco, Calif.) per ml or with a 1:100 dilution of goat anti-gpl20 or anti-gpl60 antibody (gifts from M. G. Sarngadharan, Advanced BioScience Laboratories Inc., Kensington, Md.) for 2 h at 4°C before addition to cell cultures. Quantitation of IL-6. The amount of IL-6 released in the culture supernatant was measured after 24 h with the stimuli indicated above. The IL-6 was quantitated by enzyme-linked immunosorbent assay (ELISA; Genzyme Corp., Boston, Mass.) and by [14C]thymidine incorporation of the IL-6dependent B9 hybridoma cells (a gift of G. Tosato, National Institutes of Health, Bethesda, Md.) (8). Activity of IL-6 was expressed as units per milliliter and was calculated by titrating the activity of the culture supernatants against recombinant IL-6 (Genzyme) with known concentrations (1.6 U/ml = 0.313 ng/ml). Northern (RNA) blot analysis for IL-6. Total cellular RNA was extracted from 107 cells with RNAzol (Cinna/Biotex, Friendswood, Tex.) according to the manufacturer's protocol. RNA was denatured with formaldehyde, fractioned on a 6% formaldehyde-1% agarose gel, and transferred to nylon membranes (Nytran; Schleicher & Schuell, Keene, N.H.). Hybridization was performed overnight at 42°C in the presence of formamide with 32P-labeled DNA probes for IL-6 (900-bp EcoRI-HindIII fragment of HuINF-,B2, kindly provided by P. B. Sehgal, Rockefeller University, N.Y.) (13) and for ,-actin (24). After each hybridization, RNA blots were stripped of 32P-labeled probe by incubation at 720C for in 2.5 mM Tris-HCl-0.1 mM EDTA-0.5 mM sodium same filter was used for all the hybridizations.

PP1. The

6-

E co

J

Concentration (Ag/ml) FIG. 1. Dose response of HIV-1 envelope proteins gpl20 and gpl60 for the induction of IL-6 secretion in PBMCs. PBMCs (106 cells per ml) were precultured for 72 h in 10% FCS and subsequently treated with various concentrations of gpl20, gpl60, or mBSA. Supernatants were collected after 24 h and analyzed for IL-6 by using the B9 cell bioassay. Results represents at least three exper-

iments.

PFA fixation of cloned Th cells and EBV-immortalized B cells. In certain experiments, cloned Th cells and/or EBVimmortalized B cells were fixed with paraformaldehyde (PFA). PFA fixation was performed by culturing 2 x 106 Th cells or EBV-immortalized B cells per ml in an equal volume of prewarmed 1% PFA in Hanks balanced salt solution at 37°C for 7.5 min and then washing extensively four times with Hanks balanced salt solution.

RESULTS Induction of IL-6 in freshly isolated PBMCs by HIV envelope glycoproteins. Figure 1 shows that both gpl20 and gpl60 induce IL-6 secretion from PBMCs. The ability of gp120 and gpl60 to induce IL-6 was equivalent. Maximum induction of IL-6 was observed at concentrations of 0.01 to 0.1 ,ug/ml for both of the envelope proteins. The envelope protein-induced IL-6 secretion reached a plateau level at 24 h and was unchanged thereafter up to 96 h (data not shown). Because glycan moieties of envelope glycoprotein have a high mannose content (6), possible nonspecific effects mediated by lymphocyte mannose-binding proteins were ruled out by using mBSA as a control in these experiments. Treatment with mBSA did not induce detectable IL-6 secretion. IL-6 has been shown to be produced by a variety of cells, including T cells, B cells, monocytes, and fibroblasts (7, 12). To determine which cell population in PBMCs could be stimulated by HIV-1 envelope proteins to produce IL-6, we examined the effects of gp120 and gpl60 on purified peripheral blood T-cell fraction and non-T-cell fraction (mixture of B cells and monocytes). As shown in Table 1, both gp120 and gpl60 could induce IL-6 secretion mainly from the non-T-cell fraction. The envelope proteins could also induce detectable, albeit small levels of IL-6 secretion in culture supernatants of purified peripheral blood T cells. Pretreatment of gpl20 or gp160 with their respective antibodies could abrogate the ability of these envelope proteins to induce IL-6 secretion. Addition of anti-gpl20 or anti-gpl60 antibodies alone to the cell cultures did not induce IL-6 secretion. Stimulation with LPS produced large amounts of IL-6 secretion from unfractioned PBMCs and from the

VOL. 65,

1991

IL-6 SECRETION BY HIV-1 ENVELOPE PROTEINS

TABLE 1. Effects of HIV-1 envelope proteins on the induction of IL-6 in various cells' IL-6 secreted (U/ml) Addition (,ug/ml)

Medium mBSA (0.1) LPS (1)

gpl20 (0.1) gp120 + anti-gpl20b gpl60 (0.1) gpl60 + anti-gpl60b

Anti-gpl20 Anti-gpl60

PBMC PBMC

Purified

~~T cell

Non-T cell NnTcl