Enhancement of HIV-1 gp41 binding to raji cells by pwm, lps, interferon-γ and interleukin-6

0161-58~/93 S6.00 + 0.00

~olec~ar Immu~oIogy,Vol. 30, NO. 17, pp. 1583-1586, 1993 Printed in Great Britain.

PergamonPressLtci

ENHANCEMENT OF HIV-l gp41 BINDING TO RAJI CELLS BY PWM, LPS, INTERFERON-y AND INTERLEUKIN-6 YING-HUA CHEN,* SABINE OPITZ,~ GUENTHERB&X,$ FRANZ STEINDL,~ HERMANNKATINGER~and MANFRED P. DIERICH*? 1) *Ludwig-Boltzmann-Institut fiir AIDS-Forschung, Innsbruck, Austria; TInstitut fiir Hygiene, Innsbruck, Austria; JInstitute of General and Ex~rimental Pathology, Innsbruck, Austria; and $Institute of Applied Microbiology, Vienna, Austria (Received

19 August 1993)

Abstract-Based on our finding that HIV-l gp4i independently of CD4 can bind to several proteins (gp41 binding protein: GBP) on the human T-cell line H9, B-cell line Raji and mono~yte-~11 line U937, we examined the effect of mitogens and cytokines on binding of gp41 to H9, Raji and U937 cells. Flow cytometry (FACS) analysis demonstrated that PWM and LPS, IFN-y and IL-6, but not Con A, IFN-a;, -b, -w and IL-2, could increase gp41 binding to Raji cells. In controls, none of the regulators (IFN-a, -/I, -y, -0, IL-2, IL-6 Con A, PWM, LPS) could modify the binding potential of H9 and U937 cells. Our data suggest that the expression of HIV-l binding proteins is subject to regulation by PWM, LPS, IFN-y and IL-6 in the case of B-cells, while on T-cells and macrophages,

the binding proteins may be constitutively expressed. Key words: HIV-1 gp41, gp41-binding-protein

expression, enhancement.

MATERIALS AND METHODS

INTRODUCTION The human immunodeficiency virus type 1 (HIV-l) (Barre-Sinoussi et al., 1983; Gallo et al., 1984; Levy et al., 1984) after binding to its cellular receptor, CD4 (Dalgleish et al., 1984; Klatzman et al., 1984; Maddon er al., 1987), enters susceptible cells through a pH-independent fusion reaction (McClure et al., 1988; Stein et al,, 1987), the details of which are not yet clearly

understood. Besides via CD4 molecules, HIV-l can infect U937 cells via Fc receptor- (Takeda et ai., 1988) or via complement-mediated entry (Reisinger et al., 1990; Solder et al., 1989; Thieblemont et al., 1993). Although binding of the virus to CD4, FcR or CR on the cell surface initiates viral entry, several obse~ations suggest that other surface proteins may be important for the interaction of HIV-l with cell surfaces or for HIV-induced cell fusion (Bedinger et al., 1988; Hildreth and Orentas, 1989). Relevant to this, we have demonstrated that HIV-l gp41 independently of CD4 can bind to the human T-cell line H9 (Chen et al., 1992), B-cell line Raji (Chen et al., 1993a) and monocyte-cell line U937 (Chen et al., 1993b), and that five cellular molecules (P37, P45, P49, P62, P92) are involved. On investigating binding of the immunosuppressive peptide of gp41, others have found binding to 45 kD and 80 kD molecules (Henderson and Qureshi, 1993). In this study, we examined whether binding of HIV-l gp41 to H9, Raji and U937 cells could be modulated by mitogens and cytokines.

JJCorrespondenceto: Professor Dr M. P. Deirich, Institut fur Hygiene, Fritz-Pregl-Strasse

3, 6010 Innsbruck,

Austria.

Cells H9 cells, a CD4+ human T-cell line (kindly donated by R.C. Gallo, National Cancer Institute, Bethesda, MD, U.S.A.), Raji ceils, a human B-cell line, and U937 cells, a human monocyte-cell line, were grown in RPM1 1640 supplemented with 10% fetal calf serum (FCS), 2 mmol/l glutamin, 100 III/ml penicillin and 100 pgfml streptomycin (Sera-lab Ltd, Vienna, Austria). HIT/-l Proteins Recombinant soluble gp41, rsgp41, the external portion of the transmembrane protein gp41 (McCune et al., 1988; Veronese et al., 1985; Willey et al., 1988), was derived from clone BHlO. The restriction sites RsaI and SspI were used to clone the fragment into plasmid pSB6 to generate expression of an 18 kD polypeptide (env amino acids 539-684) in Escherichia coli. The fragment was purified to homogeneity by a three-step method (Vornhagen et al., 1990). The final preparation, in 0.1 mol/l Tris-HCl (pH 9.0) supplemented with 0.2% sodium dodecyl sulphate (SDS), 0.3 mol/l urea, 10% glycerol and 14 mmol/l P-mercaptoethanol, was subjected to several dialysis cycles against 0.1 mol/l TrisHCL (pH 9.0). Cytokines and antibodies

Recombinant human IFN-cr and IFN-o were kindly provided by Dr G. R. Adolf (Bender + Co. GmbH, Vienna, Austria); rIFN-fi and rIFN-y were kindly provided by Bioferon (Laupheim, Germany). Human IL-2 and IL-6 were obtained from Beohringer Mannheim (Vienna, Austria). Con A (Canavalin A), PWM (Poke-

1584

Y.-H. CHEN et al. stimulation

A B----j

1 wsgp41

Raji + PBS

+ anti-gp41

Ab

C-I

I

Raji + PWM

Raji + LPS

Raji + Con A

Raji + IL-2

I

Raji + IL-6

cl

50

100

Relative

150

200

fluorescence

250

300

intensity

Fig. 1. Enhancement of GBPs expression on Raji by PWM, LPS and IL-6 stimulation. Raji cells were stimulated by (A), PBS (control), (B) PWM (5 pg/ml), (C) LPS (1 pg/ml), (D) Con A (5 pgg/ml), (E) IL-2 (200 U/ml) and (F) IL-6 (200 U/ml) for 48 h and stained with sgp41 (1 pg) and MAb 4B3 (1 /*g). The fluorescence mean values shown here are from one of three experiments.

weed-Mitogen) and LPS (Lipopolysaccharide) were obtained from Sigma (Deisenhofen, Germany). The human MAb 4B3 (IgGl) recognizes an epitope within HIV-l Env amino acids 584-672. Fluorescein isothioacyanate (FITC)-conjugated rabbit-anti-human IgG (202) was obtained from Dako (Dako, Austria). Stimulation of cells Cells (5 x 105) in 2 ml RPMI-1640 supplemented with 10% FCS, 2 mmol/l glutamin, 100 IU/ml penicillin and 100 pg/ml streptomycin were stimulated with PWM, LPS, Con A, or IFN-a, -p, -y and -0, or IL-2, IL-6, for 48 h at 37°C in a 5% CO, atmosphere. Afterwards, cells were subjected to flow cytometry analysis. Flow cytometry analysis of gp41 binding to cells Cells (2 x 105) were incubated with HIV-l sgp41 in 50 ~1 phosphate-buffered saline (PBS) for 30 min at 4°C. Cells were washed with PBS supplemented with 2% FCS and incubated with a human anti-gp41 MAb 4B3 (1 pg

stimulation Wsgp41 Raji + PBS

+ anti-

Relative

fluorescence

intensity (log)

Fig. 3. EIn hancement of GBPs expression on Raji by stimulation with (I) PWM, (2) LPS, (3) IL-6 and (4) IFN-y. FACS-histogram overlays (mapping): (A) stimulated cells incubated with PBS and MAb 483 (negative control); (B) not-stimulated cells incubated with sgp41 (I pg) and MAb 4B3; and (C) stimulated cells incubated with sgp41 (1 pg) and MAb 4B3.

in 50 ~1 PBS) for 30 min at 4 C. After washing with PBS plus 2% FCS, cells were incubated with 50 ,ul FITC-conjugated rabbit-anti-human IgG (1 : 50 dilution with PBS). After washing with PBS plus 2% FCS, cells were fixed with 1% paraformaldehyde in PBS and analyzed on a FACScan (Becton-Dickinson, Sunnyvale, CA, U.S.A). RESULTS

AND DISCUSSION

HIV-l gp41 contains a conserved amino-terminal sequence that serves as a fusogenic domain (Gallaher, 1987; Hildreth and Orentas, 1989; Kowalski et al., 1987). The rsgp41 represents the gp41 cell external region (Env amino acids 539-684) without the fusogenic stretch at the amino-terminus, and can bind to the cellular binding proteins on H9, Raji and U937 cell lines (Chen et al., 1992, 1993a, b). By flow cytometry analysis, it is now

I

stimulation

gp41 Ab

u937 u937 u937 U937

n sgp41

+ PBS

+ anti

gp41 Ab

+ PWM + LPS

+ Con A

u937

+ IL-2

US37

+ IL-6

US37 + IFN-alfa US37

+ IFN-beta

US37 + IFN-gamma US37 0

50

Relative

1on

fluorescence

150

200

intensity

Fig. 2. Enhancement of GBPs expression on Raji by stimulation with IFNs. Raji cells were stimulated by (A) PBS (control), (B) IFN-r (1000 U/ml), (C) IFN-b (1000 U/ml), (D) IFN-y (500 U/ml) and (E) IFN-w (1000 U/ml), for 48 h and stained with sgp41 (1 pg) and MAb 4B3 (1 pg). The fluorescence mean values shown here are from one of four experiments.

+ IFN-omega

1 0

20

Relative

40

60

fluorescence

SO

100

intensity

Fig. 4. Regulation of GBPs expression on U937 cells by stimulation with mitogenes and interleukins. U937 cells were stimulated by (A) PBS (control), (B) PWM, (C) LPS, (D) IL-2, (E) IL-6, (F) IFN-c(, (G) IFN-P, (H) IFN-;, (I) IFN-w (concentration, see Figs 1, 2) for 48 h and stained with sgp41 (1 pg) and MAb 4B3 (1 pg). Bars represent the fluorescence mean values from one of three experiments.

Enhancement

of HIV-l gp41 binding protein expression

stimulation H9 + PBS

;a

sgp41

+ anti-

gp41 Ab

H9 + PWM H9 + LPS H9+ConA HS + IL-2 H9 + IL-6

:

H9 + IFN-alla H9 + IFN-beta H9 + IFN-gamma H9 + IFN-omega 0

20

40

60

Relative fluorescence

80

100

intensity

Fig. 5. Regulation of GBPs expression on H9 cells by stimulation with mitogenes and interleukins (see Fig. 4). Values represent the fluorescence mean from one of three experiments. demonstrated that the mitogens PWM and LPS, but not Con A, could increase HIV-l gp41 binding by about 80% and 165%, respectively, on Raji cells (Figs 1 and 3); IL-6, but not IL-2, could enhance gp41 binding by about 175% (Figs 1 and 3); of the interferons IFN-cr, -b, -y and -0, only IFN-7 acted stimulatory, enhancing binding by about 70% on Raji cells (Figs 2 and 3), while binding to U937 and H9 remained unaltered after stimulation with PWM, LPS, Con A, IL-2, IL-6, IFN-c(, -/?, -y and -o (Figs 4 and 5). It has been reported that a hypercytokinaemia, including IL-6, was observed in infected individuals (Habeshaw et al., 1992). The IL-6 production by human B-lymphocytes could be induced by HIV-l (Boue et al., 1992), probably by the viral Tat protein (Levy, 1993). Besides, HIV-infection is associated with an increase in IL-6 receptor expression seen on activated B-cells and monocytes (Vander Meyden et al., 1993). PWM and LPS are mitogenes that can stimulate B-lymphocytes. Con A is a T-lymphocyte-stimulationmitogen. Interferon-y, acting as a B-cell maturation factor, can also directly induce the maturation of resting B-lymphocytes, inducing surface phenotype change and immunoglobulin secretion (Trinchieri and Perussia, 1985). Our data demonstrate that PWM and LPS and IFN-y could enhance HIV-l gp41 binding to B-cell line Raji, but not to T- and monocyte-cell lines H9 and U937 cells. The results indicate that the polyclonal activation of B-cells, a typical characteristic in HIV-infected individuals (Pahwa et al., 1985; Shirai et al., 1992), may be a mechanism inducing an increase in gp41 binding to B-cells. Whether the enhancement effects of IL-6, PWM, LPS and IFN-y, respectively, on HIV-l gp41 binding to Raji cells are important for the HIV-l pathogenesis shall be the subject of further investigation. Acknowledgements-This work was supported by the LudwigBoltzmann-Gesellschaft and the state of Tyrol, Austria. The contribution of S.O. is part of her doctoral thesis. REFERENCES Barre-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauget C., Axler-Bin C., VezinetBrun F., Rouzioux C., Rozenbaum W. and Montagnier L. (1983) Isolation of a T-lymphotropic retrovirus from a

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