HIV-1 gp41 binding proteins and antibodies to gp41 could inhibit enhancement of human raji cell mhc class I and II expression by gp41

MolecularImmunology,Vol. 31, No. 13, pp. 971-982, 1994 Pergamon

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HIV-l gp41 BINDING PROTEINS AND ANTIBODIES TO gp41 COULD INHIBIT ENHANCEMENT OF HUMAN RAJI CELL MHC CLASS I AND II EXPRESSION BY gp41 YING-HUA CHEN,* GUNTHER B&K,? ROLF VORNHAGEN,$ FRANZ STEINDL,~ HERMANN KATINGER~ and MANFRED P. DIERICH* 11 *Ludwig-Boltzmann Institute of AIDS Research and Institute of Hygiene, Innsbruck, Austria; TInstitute of General and Experimental Pathology, Innsbruck, Austria; SBiotest, Dreieich, Germany; and §Institute of Applied Microbiology, Vienna, Austria (Received 4 March 1994; accepted 27 April 1994) Abstract-Based on our findings, that HIV-l soluble gp41 could bind to several proteins on the human, T, B and monocyte cells independently of CD4, we examined the effect of HIV-l soluble gp41 (sgp41; Env amino acids 539-684) on surface expression of MHC I and II, ICAM- and CD21 molecules on human Raji cells. Flow cytometry (FACS) analysis demonstrated that sgp41 could selectively enhance MHC class I and II expression on Raji cells, but did not increase expression of other cell surface antigens, such as, CD21 and CD54 (ICAM-1). Soluble gp41 could also enhance MHC class I and II expression on another human B cell line, Bjab. The sgp41-dependent enhancement of the MHC class I and II expression on Raji cells is time- and dose-dependent. The sgp41 enhancement effect on the MHC antigen expression could be inhibited by the gp41-binding proteins of 45, 49 and 62 kD (isolated from Raji-lysate) which could inhibit the sgp41-binding to Raji celis. Interestingly, this sgp41-dependent enhancement of the MHC class I and II expression could also be inhibited by two mAbs to HIV-l gp41, but not by a third mAb binding to a different site on gp41. These results demonstrate that HIV-l sgp41 can selectively enhance the human Raji cell MHC class I and II antigen expression and this enhancement effect could be inhibited by the sgp41-binding proteins and anti-gp41 antibodies, and suggest that the sgp41-dependent enhancement is mediated by its binding to Raji membrane proteins of 45, 49 and 62 kD. Key words: HIV-l

gp41, gp41-binding

proteins, anti-gp41 mAbs, MHC expression enhancement.

INTRODUCTION

The human immunodeficiency virus type 1 (HIV-l) (Barre-Sinossi et al., 1983; Gallo et al., 1984; Levy et al., 1984) is known to infect human CD4+ T cells by the interaction of its envelope glycoprotein gpl20 with cell surface proteins CD4 acting as HIV-l receptors (Klatzman et al., 1984). Although binding of HIV-l gpl20 to CD4 on the target cell surface initiates viral entry, several observations suggest that other surface proteins may be important for the interaction of HIV-l with cell surfaces or for HIV-induced cell fusion (Bedinger ef al., 1988; Hildreth and Orentas, 1989). Besides via the CD4 molecules, HIV-l can infect U937 cells via Fc receptor-mediated entry (Takeda et al., 1988) and via the complement-mediated entry (Reisinger et al., 1990; Siilder et al., 1989; Thieblemont et al., 1993). We showed that HIV-l gp41 can bind to the human T cell line H9 (Chen et al., 1992), B cell line Raji (Chen et al., 1993~) and monocyte cell line U937 (Chen et al., 1993b), independently of CD4, and among human peripheral

blood mononuclear cells preferentially to B lymphocytes and monocytes (Chen et al., 1993~). Five cellular molecules appear to be involved in this binding (Chen ef al., 1992, 1993a, b). Investigating binding of the immunosuppressive peptide of gp41, others have found binding to human cell surface proteins (Qureshi et al., 1990; Henderson and Qureshi, 1993). In this study, we examined the effect of HIV-l gp41 on expression of cell surface molecules. To our surprise, we found a selective enhancement of MHC class I and II antigen expression and this enhancement could be inhibited by gp41-binding proteins and anti-gp41 antibodies. MATERIALS

AND METHODS

Cells

The Raji and Bjab human B cell lines which originate from a Burkitt lymphoma, were grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 2 mmol/l glutamin, 100 IU/ml penicillin and 100 pg/ml streptomycin (Sera-Lab Ltd, Vienna, Austria). HIV-l

proteins

((Author to whom correspondence should be addressed at: Soluble Institut fiir Hygiene, Fritz-Pregl-Strasse 3, 6010 Innsbruck, membrane Austria. MlMM 3,11971

gp41, the cell external portion of the transprotein gp41 (Modrow et al., 1987; Willey

978

Y.-H. CHEN et ai.

et al., 1988), was derived from clone BHIO. 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-HCI (pH 9.0) supplemented with 0.2% sodium dodecyl sulphate (SDS), 0.3 molil urea, 10% glycerol and 14 mmol/l /?-mercaptoethanol, was subjected to several dialysis cycles against 0.1 molll Tris-HCl (pH 9.0).

Antibodies Mouse anti-HLA I (TgG2a) (M736) and anti-HLA DR (IgG2a) (M704) mAbs were obtained from Dako (Dako, Vienna, Austria). Mouse anti-CD21 mAb HB-5 (IgGl) was obtained from the Institute of Hygiene (Innsbruck, Austria). The production and specifity of mAb 7F7 (mouse anti-ICAM-l/CD54, IgG2a) has been described (Schulz et al., 1988). Human anti-gp41 mAbs, 4D4, 4G2 and 1H5 (IgGl) recognize epitopes within HIV-I Env amino acids 586672 and were obtained from Dr H. Katinger (Institute of Applied Microbiology, Vienna, Austria). Fluorescein isothioacyanate (FITC)conjugated rabbit F(ab’)z anti-mouse IgG + IgM antibody was obtained from Bio Research GmbH (Kaumberg, Austria).

Absorption and elution Using standard-method from Pharmacia (Pharmacia GmbH, Vienna, Austria), sgp41 (2 mg) was coupled to 2 ml CNBr-activated sepharose 4B. Cells were lysed in lysis buffer (cells: lysis buffer = 5% vol/vol) for 1 hr at 4°C. The lysis buffer comprised of 50 mmol/l Tris, 150 mmol/i NaCl, 2 mmol/l EDTA, 0.5% Nonidet P40 (P40; pH 7.4) with 3.7 mg,/ml iodacetamide. 100 pgg/mI trypsin inhibitor, 1 mmol/l phenylmethylsulphonylfluoride (PMSF). Using low flow rate, cell lysates were run five times through a column (1.5 x 8 cm) filled with 2 ml sgp41-sepharose. The column was washed with 20 ml PBS plus 0.05% Tween 20 three times, eluted with 2 ml 0.5 mol/l acetic acid and then with 5 ml PBS plus 0.05% NaN,. Eluates were neutralized at once with 0.5 mol/l NaOH.

Isolation of sgp41 -binding proteins Raji-sgp41-eluates were subjected to eIectrophoresis in a 9.5% SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions. The protein bands were cut off and eluted for 3 hr at 100 vol in an elution instrument (DNA SUB CELL-TM, BIO-RAD, Laboratories GesmbH, Vienna, Austria). The isolated proteins were identified by Coomassie Blue staining (see Fig. 4) and Western blot analysis (Chen et al., 1993a).

Stimulation of cells and inhibition of the stimulation Cells (5 x 10’) in 2 ml RPMI-1640 supplemented with 10% FCS, 2 mmol/l glutamine, 100 H-J/ml penicillin and 1OO~g~mi streptomycin were stimulated with 5 or 15 pg/ml sgp41 or Env peptides for 24 hr or 48 hr at

Relative fluorescence

intensity

(log)

Fig. I. Selective enhancement of (1) Raji MHC class I and (2) class II expression by sgp41. FACS-histogram overlays (mapping): (A) stimulation with sgp41 (5 pggjml, 48 hr); (9) PBS (control). Cells were stained with mouse mAbs to HLA-I (1:25) (l), HLA-DR (1:25) (2). human ICAM-I (lO~~g/ml) (3), and human CD21 (1 :2) (4) and FITC-conjugated rabbit F(ab’)z anti-mouse 1gG + IgM antibody (I :40). The results shown here are from one of five experiments.

37°C in a 5% CO1 atmosphere. After stimulation procedure, cells were subjected to flow cytometry analysis. In the inhibition test of the sg~l-stimulation, sgp41 was incubated with the Raji-sgp41-eluate, sgp41-binding proteins or anti-gp41 mAbs, respectively, for 30min at RT and added to Raji cells (5 x IO’) in 2 ml cell culture medium for the stimulation.

Flow cytometry Cells (2 x 10’) were incubated with mouse mAbs 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 50~1 FITC-conjugated rabbit F(ab’), anti-mouse IgG + IgM (I:40 dilution with PBS). After washing with PBS plus 2% FCS, cells were fixed with 1% paraformaldehyde in PBS and analysed on a FACScan (Becton-Dickinson. Sunnyvale, California, USA). Table 1. Enhancement of Raji MHC class I and II expression by sgp41 stimulation (5 pg/ml for 0 and 48 h). Specific mean fluorescence intensities were determined by FACS analysis. Values shown here are from five experiments Mean fluorescence intensity Enhancement (X0)

Antigen

Experiment

Day 0

Day 2

MHC I

1 2 3 4 5

40 100 94 53 83

175 229 225 153 424

338 129 139 189 410

MHC II

I 2 3 4 5

64 137 114 54 135

131 313 315 112 319

105 128 176 107 136

Inhibition

of enhancement

979

of MHC expression

BCDE

A

mAbs

F

t 50

0

Relative

100

200

150

fluorescence

ZtEO

300

intensity

31Fig. 4. Identification of sgp41 binding proteins by Coomassie Blue staining. Five proteins of 37,45,49. 62 and 92 kD (F, E, D, C, B), were isolated from Rajj-sgp41-lysates. A: molecular weight markers.

I 0

50

Relative

1OO

200

150

fluorescence

250

300

intensity

Fig. 2. The sgp41-dependent enhancement of MHC class f and II expression is (1) tome-de~ndent and (2) dose-dependent. FANS-histogram overlays (mapping): (1) stimulation with 5 @g/ml sgp41 for 0 hr (control), 24 hr and 48 hr; (2) with PBS (control), 5 pg/ml and 15 pg/ml sgp41 for 24 hr. Cells were stained with mouse mAbs (1: 25) to HLA-I and HLA-DR. The results show the mean values of one from two experiments.

MHC class I and II expression turned out to be timedependent (Fig. 2-l). A dose effect on the sgp41dependent enhancement of Raji MHC class I and II expression was afso observed (Fig. 2-2) Besides, sgp4f coufd also selectively enhance MHC &ass I and If expression on another B cell line, Bjab (Fig. 3). Stimulation:

__II

~~i~~~; RESULTS Rsji+(sgpll

HIV-1 gp41 contains a conserved amino-terminal sequence that serves as a fusogenic domain (Gallaher, 1987; Kowaiski et ai., 1987). The sgp41 represents the gp41 cell external region (Env amino acids 539-684) without the fusogenic stretch at the amino-terminus. Using flow cytometry, we found that sgp41 (5 pg/ml, for 48 hr) could enhance MHC class I expression (increase fluorescence intensity by about 200%) (Fig. l-l) and MHC class II expression (increase fluorescence intensity by 130%) (Fig. 1-2) on Raji ceils but not expression of other cell surface antigens, such as ICAM- (Fig. I-3) and CD21 (Fig. I-4). Table 1 shows the results of five experiments. The sgp4~-dependent enhancement of Raji

+E45)

..I~~~_

~

0

50

Relative

100

Ruorescence

150

200

intensity

Stimulation:

2

Raji*PSS~

RItji+@gpr)l

fE45)

Raji+@rJgp41 +E49) Rajif(sgp41

+EB2)

0

100

200

Relative fluorescence

Aelative fluorescence

intensity

(log)

Fig. 3. Selective enhancement of (I) MHC class I and (2) class II expression on Bjab cells by sgp41. FACS-histogram overlays (mapping): (A) stimulation with sgp41 (5 @g/ml, 48 hr); (B) PBS (control). Cells were stained with mouse mAbs (I ~25) to HLA-I (I) and HLA-DR (2).

300

400

intensity

Fig. 5. Inhibition of the sgp41-dependent enhancement of (1) MHC class I and (2) class II expression by gp41 binding proteins. The sgp4l (10 pg, SO~1) was incubated with PBS or five gp41 binding proteins (5Oyl) (E37, E45, E49 and E62, 30 pg; E92,50 gg in 150p 1PBS) for 30 min and added to Raji cell culture (5 x IO” cells in 1.Xml medium) to stimulate for 48 hr. Bars represent the fluorescence intensity (mean) of three experiments.

Y.-H. CHEN et al.

980

Relative

a

150

100

50

0

fluorescence

: ~sji+~es~

200

intensity

Stimulation

’

A ’

.:l:i,::::::’ :: 0

100

300

200

Relative fluorescence

400

intensity

b

Relative

fluorescence

~ntensjt~ (log)

Fig. 6. Inhibition of the sgprll-dependent enhancement of (1) MHC class I and (2) class II expression by the anti-gp41 antibodies. (a) Relative fluorescence intensity (mean); (b) FACS histogram overlays (mapping): (A) not stimulated cells; (B-E) stimulated cells (5 pgjrnl sgp41, 48 hr); (B) inhibition by PBS; (C) inhibition by mAb 4D4; (D) inhibition by mAb 4G2; (E) inhibition by mAb lH.5. The results shown here are from four experiments,

Inhibition of the sgpctl-dependent enhancement of MHC class I and ?I expression by individual sgp41-binding proteins and anti-gp41 antibodies

By SDS-PAGE, five sgp41-binding proteins of 37,45, 49,62 and 92 kD, were isolated from Raji-sgp41-eluates. The isolated proteins were identified by Coomassie Blue staining (Fig. 4) and Western blot analysis (Chen et al., 1993a). Three sg~l-binding proteins, E45 (45 kD), E49 (49 kD) and E62 (62 kD), could inhibit the sgp41dependent enhancement of Raji MHC class I expression (inhibited fluorescence intensity by about 40%, 40% and 35%, respectively) (Figs 5-1; 7-l) and class II expression (inhibited fluorescence intensity by about 30%, 40% and 20%, respectively) (Figs 5-2; 7-2). The protein E37 (37 kD) showed a very weak inhibition and protein E92 (92 kD) no inhibition. Of the three human anti-gp41 mAbs tested, two mAbs (4G2 and lHS), could inhibit

the sgp41-dependent enhancement of MHC class I and II expression (inhibited fluorescence intensity by about 50%, respectively), while the mAb 4D4 had no inhibiting effect (Figs 6, 7). DISCUSSION The present results indicate that HIV-l sgp41 can enhance MHC class I and II expression on the human B cell line Raji. The process is selective and specific because sgp41 could not increase the expression of a number of other cell surface antigens, ICAM- and CD21 (Figs 1-3, l-4), CD19 and CD23 (data not shown). Besides, sgp41 could enhance MHC class I and II expression on another human B cell line, Bjab. It is obvious that the sgp41-dependent MHC class I and II expression was mediated by its binding to Raji cell

Inhibition

981

of”en~~~cernen~ of MHC expression

v E37

E45

E49

E62

E92

404

4G2

lH5

E37

E45

E49

E62

EQ2

4D4

4G2

1H15

Fig. 7. Examination of dose-e%ct of the sgp41 binding proteins and antibodies. By a higher concentration (sgp41: inhibitor = 1: 2 mol/mol), these proteins and antibodies could not completely inhibit the sgp41-dependent MHG class I (A) and II (B) upregulation (could not completely inhibit Buorescence intensity).

membrane proteinsbecause three sgp4l~~i~di~~ proteins, 45, 49 and 62 kD, of Eve from Raji-sg~4l-el~at~ could inhibit the sg~4~-~e~n~en~ enhancement, respectively. We had ~~nl~nstrat~~ that these three proteins could also inhibit sgpll binding to Raji cells, but that the proteins of 37 and 92 kD could inhibit the binding only slightly jChen et al., 1993~). One of several possibie explanations far this event is that the proteins of 45, 49 and 62 kD, in contrast to those of 37 and 92 kD, may possess a high binding activity for HIV- 1 gp41. These

results provide further support for the assumption that the proteins, 45, 49 and 62 kD, could be the putative cellular receptor proteins for HIV-I gp4l binding. The sg~l~epende~t e~hancemcnt of MHC expression could be inhibited by the human monoclonal ant&p41 antibodies, 4G2 and fH5; ~nteresti~gly~ the mAb 4G2, but not 4D4, could also inhibit the sgp4t binding to

H9 cells (Chcn & ab., 1992). A variety of cytokines, partic~~larly IFNs, have been shown to increase MHC expression (Rosa en al., 1985; Halloran ef a/., 1986). Some viruses can up- or dozen-replate MWC expression, but the mechanisms appear to be interferonindependent (King and Kesson, 1988; Paabo et a& 1989; Maudsley and Pound, 1991). The exact mecha~ism~s~ of sgp4 1-induced enhancement of Raji MHC class I and II antigen expression has not been fully elucidated. A~knoM,led~ements---This work was supported by the Ludwig~oltzmann-Ges~ll~ha~~ and the State of Tyrol, Austria.

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CHEN

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