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HIV-1 gp41 binding to human T- and B-lymphocytes and monocytes is modulated by phorbol myristate acetate (PMA)
Immunology Letters 50 (1996) 161-166
HIV-l gp41 binding to human T- and B-lymphocytes and monocytes is modulated by phorbol myristate acetate (PMA) Ying-Hua “Institute
for Hygiene,
Chen”*b, Antje Christiansen”,
Manfred
P. Dierich”,”
University Innsbruck, and Ludwig-Boltzmann-Institute jbr AIDS Research, Fritz-Pregl-Strasse bGroup of Immunology, Department of Biology, Tsinghua University, Beijing, China
3. 6010 Innsbruck,
Austria
Received 8 December 1995; revised 1 March 1996; accepted 4 March 1996
Abstract HIV-l gp41 independently of CD4 binds to human T cells, B cells and monocytic cells. Since PMA downmodulates CD4 (HIV receptor) expression and inhibits HIV-l dependent syncytia formation, we wanted to examine whether PMA could affect gp41 binding protein expression on human cells. The strong binding of HIV-1 recombinant soluble gp41 (rsgp41; Env aa539-684) to monocytes (CD14+) and B-lymphocytes (CD19+) and B lymphoblastoid cells (Raji) could be clearly decreased by treating the cells with PMA for 48 h, while the weak binding to T lymphocytes was slightly increased by this treatment. The PMA inhibitoryand enhancing-effects could be avoided by pretreatment with staurosporine (protein kinase C inhibitor). The PMA treatment of Raji and U937 (monocytic) cells resulted in a 50-60% decrease of gp41 binding proteins (gp41bps) detectable in cell lysates of these cells in comparison with lysates of buffer-treated cells, while in the case of H9-cells PMA treatment resulted in an increase of available gp4lbps by about 35% in comparison with buffer-treated H9. Staurosporine pre-treatment could prevent these effects of PMA. Further studies of rsgp41-eluates from these buffer-treated or PMA-treated cells demonstrated that PMA modulated mainly expression of rsgp4lbps of 37, 45, 50 and 62 kDa. These results indicate that PMA exerts different effects on human T, B and monocytic cells. Production by and expression on cells of HIV- 1 gp41 bps appear to depend on protein kinase C, supporting that the four proteins on human cells may act as receptor proteins for HIV-l gp41. Keywords:
HIV-l gp41 binding; Modulation;
PMA
1. Introduction The human immunodeficiency virus type 1 (HIV-l) after binding by the envelope protein gp120 to its cellular receptor CD4 enters susceptible cells through a reaction involving HIV-l gp41 [l-3]. It has been
demonstrated by us and others that HIV-l gp41, independently of CD4, binds to several proteins on human T, B and monocytic cell lines [4-81. This binding could be partially blocked by gp41 peptides (aa581-605 and 641-675) [4]. The importance of these two sites is underscored by the fact that env peptides aa560-597 and aa637-669 and a monoclonal antibody against aa662-667 (HIV- 1 BH 10 isolate) could neutralize HIV1 infection [9- 111, and an amino acid change in gp41 (Env aa666) resulted in escape from neutralization by *Corresponding 5072870.
author. Tel.: +43 512 5073401; fax: +43 512
0165-2478/96/$12.00 0 1996 Elsevier Science B.V. All rights reserved PII SO165-2478(96)02533-3
this anti-gp41 antibody [12]. Since the HIV receptor CD4 on the cell surface could be down-modulated by PMA-treatment [13- 151, we wanted to determine the effect of PMA on HIV-l gp41 binding protein expression on human lymphocytes and monocytes.
2. Materials and methods 2.1. Isolation of peripheral blood lymphocytes and monocytes
Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adult volunteers by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were cultured in RPM1 1640 medium supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 fig/ml streptomycin (Sera-lab Ltd., Vienna, Austria). After
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et al. I Immunology
incubation for 1 h at 37°C the non-adherent and adherent cells were isolated. The non-adherent cells contained 2% monocyte (CD14+), and the adherent cells contained at least 95% monocytes (CD14+). The nonadherent cells were analysed as lymphocyte-population and the adherent cells were by lymphocyte-gate, analysed as monocytes-population by monocyte-gate. The T-lymphocytes were purified from human peripheral blood lymphocytes by the rosetting technique (described in Ref. [27]).
Letters
50 (1996)
161-166
USA) by lymphocyte-gate or monocyte-gate. By two color flow cytometric analysis, cells (PBMC or PBL) were incubated at first with monoclonal mouse antiCD19 antibody, then with FITC-conjugated sheep F(ab’),-antibody to mouse IgG and IgM for 30 min at 4”C, and after washing with PBS/BSA, cells were incubated with rsgp41-biotin for 30 min at 4°C. After washing, cells were incubated with R-phycoerythrinconjugated ExtrAvidin. Cells were washed, fixed with 1% paraformaldehyde in PBS and analysed on a FACScan under the lymphocyte- or monocyte-gate.
2.2. HIV-l protein Rsgp41, the outer membrane of gp41 [ 161, 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-kDa polypeptide (Env amino acids 539-684) in Escherichiu coli. The fragment was purified to homogeneity by a three-step method [17]. Using the Biotin-X-NHS Kit (Calbiochem, No.: 813193; La Jolla, California) and the biotinylation method as suggested by Calbiochem (described in Ref. [18]), rsgp41 was biotin-labeled. 2.3. Antibodies FITC-conjugated sheep F(ab’),-antibody to mouse IgG and IgM was obtained from Bio Research GmbH (Austria). FITC-conjugated Streptavidin (F422) and monoclonal mouse anti-human-CD 19 antibody (M740) were obtained from Dako (Austria). R-phycoerythrinconjugated ExtrAvidin (E-401 I) from Sigma. 2.4. Treatment of cells Cells (2 x 106) in 2 ml RPMI-1640 supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 pgg/ml streptomycin were treated with 0, 10 and 20 ng/ml PMA (phorbol 1Zmyristate 13-acetate; Sigma, P-8139; Munich, Germany) for 48 h at 37°C in a 5% CO, atmosphere. To test blockade of the PMA inhibitory effect, cells were incubated with staurosporine (Sigma, S4400) (50 and 100 nM) for 30 min at 37°C. Then PMA (IO ng/ml) was added for 48 h at 37°C in a 5% CO, atmosphere. Afterwards, cells were subjected to flow cytometry analysis. 2.5. Flow cytometry analysis Cells (PBMC or lymphocytes, 5 x 105; monocytes, 1 x 105) were incubated with 50 ~1 biotin-labeled rsgp41 for 30 min at 4°C. Cells were washed with PBS/BSA (1%) and incubated with 50 ~1 FITC-conjugated Streptavidin (1:50 in PBS) for 30 min at 4°C. After washing with PBS, cells were fixed with 1% paraformaldehyde in PBS and analysed on a FACScan (Becton-Dickinson,
2.6. Isolation of rsgp41 binding proteins from cell lysates
The procedure of rsgp41 coupling to Sepharose, cell lysate preparation, adsorption with sepharose column and Coomassie blue staining was described previously [5,6]. Briefly, using a standard method from Phamacia, rsgp41 (2 mg) was coupled to 2 ml CNBr-activated Sepharose 4B. Cell lysates were absorbed with the same rsgp41-Sepharose column. The column was washed several times and eluted using 0.5 mol/l acetic acid. After neutralization of pH the rsgp41-eluates were electrophoresed in a 9.5% SDS-PAGE under non-reducing conditions. The gels were stained with Coomassie blue.
3. Results 3.1. Rsgp41 binding to PMA-treated and bufleer-treated human peripheral blood lymphocytes by two-color flow cytometric analysis Using biotin-labeled rsgp41 and mouse monoclonal antibody to human CD19, we could show that rsgp41 bound to about 99% of B-lymphocytes (CD19 + ) from human PBMCs (Fig. lC, field rsgp41+ CD19’). Rsgp41 binding to B lymphocytes was distinctly downmodulated by PMA (Fig. lD, 10 ng/ml; same result with 20 ng/ml) compared to buffer-treated lymphocytes (Fig. 1C). Also, rsgp41 binding to human B cell line Raji was clearly decreased by PMA treatment in comparison with buffer-treated cells (Fig. lE, FACS histogram overlays). Furthermore, rsgp41 bound weakly to about 20-30% of T lymphocytes (Fig. lC, left section, i.e. field rsgp41 + CD193 and this binding was increased by PMA-treatment. This result is not easily visible in Fig. 1D although obvious from mean fluorescence values. Therefore, in separate experiments, human peripheral blood T lymphocytes were treated with buffer or PMA and then tested for rsgp41 binding. As visible in Fig. 2D buffer-treated T cells bound rsgp41 (in comparison to Fig. 2A) and this binding was increased by PMA treatment (Fig. 2E,F).
Y.H. Chen et al.
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1Immunology Letters 50 (1996) 161-166
Buffer-treated
PMA-treated
E
?kq Relative fluorescence
anti-CD1
9-Ab-binding
(FL-1
intensity
1
Fig. 1. Rsgp41 binding to PMA-treated and buffer-treated (untreated) human peripheral blood lymphocytes and B cell line Raji by flow cytometric analysis. Dot blots of two-color immunofiuorescence staining (A-D): A and B serve as controls using FITC-conjugated sheep F(ab’)z-antibody to mouse Ig applied to detect bound CDl9-antibody (FITC control) and R-phycoerythrin (PE)-conjugated ExtrAvidin applied to detect bound rsgp41 (PE-avidin control); C and D use mouse anti-human CD19 antibody plus FITC-conjugated sheep F(ab’),-antibody to mouse Ig and biotinylated rsgp41 plus R-phycoerythrin-conjugated ExtrAvidin. (A$) Buffer-treated (0 ng/ml PMA for 48 h) lymphocytes; (B,D) PMA-treated lymphocytes (10 ng/ml for 48 h). FACS-histogram (E): curve 1, rsgp41 binding to buffer-treated (0 ng/ml PMA for 48 h) Raji cells; curves 2 and 3, rsgp41 binding to PMA-treated Raji (curve 2, 10 ng/ml PMA for 48 h; curve 3, 20 ng/ml for 48 h).
3.2. Rsgp41 binding to PMA-treated human monocytes
and bufler-treated
Human peripheral blood monocytes gave a result comparable to B cells. Rsgp41 could bind to almost all human peripheral blood monocytes (Fig. 3C in comparison to buffer-treated cells shown in Fig. 3A) and this binding could be strongly reduced by PMA treatment (Fig. 3D, 10 ng/ml PMA). Treatment with 20 ng/ml PMA gave the same result as 10 ng/ml. 3.3. Blockade of PMA inhibitory and enhancing effects by staurosporine It has been demonstrated that the PKC inhibitor staurosporine could block the PMA inhibitory effect on HIV-induced syncytium formation [14]. Here we report that down-modulation of gp4 1-binding protein expression by PMA could be avoided by staurosporine. Fig. 4A shows that rsgp41 binding to B cell line Raji (see curve 1) was reduced by PMA treatement (10 ng/ml for 48 h) (see curve 2); this PMA-dependent down-modulation could be prevented by preincubation of the Raji cells with staurosporine (50 nM for 30 min) (see curve 3). Also, in case of T-lymphocytes (Fig. 4B) PMA-induced increase of gp41-binding protein expression (see curve 2 in comparison to curve 1) could be inhibited by staurosporine (see curve 3). These results suggest that the selective effects of PMA on cell surface expression on human T-, B-lymphocytes and monocytes are mediated by protein kinase C.
3.4. Quantitative analysis of gp41 binding proteins from PMA-treated and bufler-treated cells Using rsgp41 -Sepharose column, gp41 binding proteins were isolated from cell lysates of PMAtreated and untreated human B cell line Raji, monocyte cell line U937 and T cell line H9. The amount of gp41 binding proteins on Raji and U937 cells was decreased by PMA treatment by about 50-60% in some experiments nearly completely in comparison with the buffer-treated Raji and U937, respectively, while the amount of the proteins on PMA-treated H9 cells was increased by about 35% in comparison with the buffer-treated H9 (Fig. 5). Interestingly, if Raji cells were preincubated with staurosporine for 30 min at 37”C, the effect of PMA was obviously inhibited (Fig. 5). Furthermore, analysing on SDSPAGE the banding pattern of rsgp41-eluates from cell lysates of buffer, or PMA-treated and staurosporine/PMA-treated Raji cells showed that PMA treatment preferentially reduced the protein bands of 37, 45, 50 and 62 kDa (Fig. 6, lane C) when compared with the banding pattern of buffer-treated Raji cells (Fig. 6, lane B). Staurosporine could obviously inhibit the PMA effect (Fig. 6, lane D). These results give support to our previous conclusion that HIV-l gp41 could bind to five cellular proteins, and that four of them (37, 45, 50 and 62 kDa) on human cells may act as receptor proteins for HIV-l gp41. Their expression depends on a functionally intact PKC.
Y.H. Chen et al. /Immunology Letters 50 (1996) 161-166
164
Buffer-treated
PMA-treated
PMA-treated (20 “11
Relative
fluorescence
ngimll c
intensity
Fig. 2. Rsgp41 binding to PMA-treated and buffer-treated human peripheral blood T lymphocytes by flow cytometric analysis. FACS-histogram: (A-C) FITC-conjugated avidin (control); (D-F) biotinylated rsgp41 plus FITC-conjugated avidin. (A,D) Buffer-treated T lymphocytes (0 ng/ml PMA for 48 h); (B,C,E,F) PMA-treated T lymphocytes: (B,E) 10 ng/ml for 48 h, (C,F) 20 ng/ml for 48 h).
4. Discussion
In this study, we demonstrated that production and surface expression of gp41 binding proteins by human monocytes, B-lymphocytes and B lymphoblastoid cells (Raji) could be down-modulated by PMA and pretreatment with PKC-inhibitor staurosporine could avoid this effect. However, in case of human T-lymphocytes, PMA could induce weak enhancement of production and expression of gp41 binding proteins; also this PMA enhancing effect could be prevented by staurosporine. These results suggest that PMA has selective (enhancing and inhibitory) effects on production and cell surface expression of gp41 binding proteins on human untreated (0 ngiml)
PMA-treated (10
na/ml)
T-lymphocytes, B-lymphocytes and monocytes and that these effects may be mediated by protein kinase C. An enhancing effect of PMA on H9 cells’ binding of an immunosuppressive peptide of gp41 has also been observed [7]. For B cells and monocytic cells no information has been reported. PMA effects of this kind on other molecules were collected in Table 1. They concern CD20 [24], CD23 (Fc epsilon receptor) [23], CR3 (complement receptor) [19], CD25 (IL-2R /I chain) [26], MHC class II [25] and CD4 [14,15,20-221. PMA-mediated inhibition of HIV- 1-induced syncytium formation in CD4-expressing cells was reported [13- 151. Interestingly, also in these experiments the inhibitory effect of PMA on syncytia formation and on CD4 expression was blocked by staurosporine, suggesting that it was mediated by protein kinase C [13,14]. Firestein et al. [15] reported that PMA could decrease not only HIV-linduced syncytium formation but also the production
Relative
Relative
fluorescence
intensity
Fig. 3. Rsgp41 binding to PMA-treated and buffer-treated human peripheral blood monocytes. FACS-histogram: A and B, FITC-conjugated streptavidin (control); (CD) biotinylated rsgp41 plus FITCconjugated streptavidin. (AC) Buffer-treated monocytes (0 ng/ml PMA for 48 h); (B,D) PMA-treated monocytes (10 ng/ml for 48 h).
fluorescence
intensity
Fig. 4. Staurosporine prevented the PMA enhancing effect on gp41 binding protein expression on human T lymphocytes and the PMA inhibitory effect on gp41 binding protein expression on human B cell line Raji cells. FAG-histogram overlays: curve 1, rsgp41 binding to buffer-treated cells (0 ng/ml PMA for 48 h); curve 2, rsgp41 binding to PMA-treated cells (10 ng/ml, for 48 h); curve 3, rsgp41 binding to cells that were preincubated with staurosporine (50 nM) for 30 min at 37°C and then with PMA (IO ng/ml) for 48 h at 37°C.
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Y.H. Chen et al. I Immunology Letters 50 (1996) 161-166
-
Table 1 Selective modulation of cell surface expression of gp41 binding proteins (gp4lbp). gp41 IS-peptide binding protein (ISPbp) and other molecules on human T. B and monocytic cells by PMA
--
I
u937 -
m
n.t.
0 ng/ml PMA
n
20 ng/ml PMA
m
PMA + staurosporine
gp4lbp ISPbp CD4 CD20 CD23 CD25 CR3 MHC II 0
10
rsgp41
20
30
binding proteins
T
B
Mono
Ref.
T
1
1
I _
1
j
f
1 _
I51 171 [14.15.20-221 [241 [23] 1261 1191 [251
I
T
r r
40
(ug)
Fig. 5. Quantitative analysis of gp41 binding proteins in rsgpll-eluates from cell lysates of buffer-treated and PMA-treated Raji, U937 and H9 cells. Using the same rsgp4l-sepharose column, rsgp41 binding proteins were isolated from cell lysates of buffer-treated (0 ng/ml PMA for 48 h) and PMA-treated (20 ng/ml PMA for 48 h) cells (2 x 10’ cells), or from cell lysates of Raji cells preincubated with staurosporine (50 nM staurosporine for 30 min at 37°C) and then PMA. (n.t.. not tested).
Acknowledgements The technical help of Gabi Kienast is gratefully acknowledged. This work was supported by the Austrian FWF (project P10094-Med). The work was part of the doctoral thesis of A.C.
References of viral RNA and p24 antigen. Whether this inhibitory effect on virus production was due to the down-modulation of the surface-associated CD4 receptor or modulating an accessory component(s), for example gp41 binding proteins (second HIV receptor) as shown by our experiments, is unclear.
ABC
D
f@fblQM*W-loo
66-
-62
Fig. 6. Identification of gp41 binding proteins in rsgp41-eluates from cell lysates of buffer-, PMA- and staurosporine/PMA-treated Raji cells by Coomassie blue staining. The eluates were electrophoresed in a 9.5% SDS-PAGE under non-reducing conditions. The gels were stained with Coomassie blue. Lane B, rsgp41-eluate from cell lysates of buffer-treated Raji cells; lane C, rsgp41-eluate from PMA-treated Raji cells (20 ng/ml PMA for 48 h); lane D, rsgprll-eluate from staurosporine/PMA-treated Raji cells that were preincubated with 50 nM staurosporine for 30 min at 37°C. then with 20 ng/ml PMA for 48 h. Each lane represents an eluate of I x IO’ Raji cells. Molecular weight markers (kDa) are depicted in lane A.
[I] McClure, M.O.. Marsh, M. and Weiss, R.A. (1988) EMBO J. 7, 513. [2] Stein. B., Gowda, S., Lifson. J.. Penhallow. R.. Bensch, K. and Engelmann. E. (1987) Cell 49, 659. [3] Gallaher, W.R. (1987) Cell 50, 327. [4] Chen, Y.H., Ebenbichler, C., Vornhagen, R., Steindl, F., Katinger, H. and Dierich, M.P. (1992) AIDS 6, 533. [5] Chen. Y.H., Biick, G., Vornhagen, R.. Steindl, F., Katinger. H. and Dierich, M.P. (1993) Mol. Immunol. 30, 1159. [6] Chen, Y.H.. B&k, G., Vornhagen, R., Steindl, F., Katinger, H. and Dierich, M.P. (1993) Immunol. Lett. 37, 41. [7] Henderson, L.A. and Qureshi, M.N. (1993) J. Biol. Chem. 268. 15291. [8] Qureshi, M.N., Coy, D.H.. Carry. R.F. and Henderson, L.A. (1990) AIDS 4, 553. [91 Wild, C., Gas. T., McDanal. C., Bolognesi, D. and Matthews. T. (1992) Proc. Natl. Acad. Sci. USA 89, 10537. [lOI Jiang. S.. Lin, K., Strick, N., Neurath, A.R. (1993) Nature 365, 113. [I 11 Muster. T., Steindl. F.. Purtscher. M. (1993) J. Virol. 67, 6642. 1121Cao. J.. Bergeron, L., Helseth. E., Thali, M., Repke, H. and Sodroski. J. (1993) J. Virol. 67. 2747. u31 Chowdhury. I.H., Koydnagi, Y., Kobayashi, S. (1990) Virology 176, 126. H., Manischewitz, J., Vujcic, L.. Blumenthal, R., [I41 Gold&, Dimitrov. D.S. (1994) J. Virol. 68. 1962. iI51 Firestein. G.S.. Reifler, D., Richman, D., Gruber, H.E. (1988) Cell. Immunol. 113. 63. 1161 Modrow, S., Hahn, B.H., Shaw. G.M., Gallo, R.C., WongStaal. F., Wolf. H. (1987) J. Virol. 61. 570. P71 Vornhagen, R., Hinderer. W., Nebel-Schickel. H. (1990) Biotest Bull. 4. 91. [I81 Bayer, E.A., Wilchek, M. (1980) Methods Biochem. Anal. 26, 1. [I91 Gilbert, D., Peulve. P., Dareau, M.. Ripoche. J., Fontaine, M. (1985) Eur. J. Immunol. 15, 986.
166 [20] Blue,
Y.H. Chen et al. / Immunology Letters 50 (1996) 161-166
M.L., Daley, J.F., Levine, H., Branton, K.R. and Schlossman. SF. (1989) J. Immunol. 142, 374. [21] Hunakova, L., Sedlak, J., Chorvath, B. (1993) Neoplasma 40, 141. [22] Touraine, J.L., Sanhadji, K.. Benard, I. (1992) Thymus 20, 239. [23] Mayumi, M., Kawabe, T., Kim, K.M., Heike, T., Katamura, K., Yodoi, J., Mikawa, H. (1988) Clin. Exp. Immunol. 71, 202.
[24] Valentine, M.A., Cotner, T., Gaur, L., Torres, R.. Clark, E.A. (1987) Proc. Natl. Acad. Sci. USA 84, 8085. [25] Sarthou, P.. Jouvain-Marche, E., Cazenave, P.A., Le-Guern. C. (1987) Eur. J. Immunol. 17, 1375. [26] Leonard, W.J., Kronke, M., Peffer, N.J., Depper, J.M., Greene, W.C. (1985) Proc. Natl. Acad. Sci. USA 82, 6281. [27] Wilhelm, M., Pechumer, H., Rank, G., Kopp, E.. Riethmuller, G., Rieber, E.P. (1986) J. Immunol. Methods 90, 89.
HIV-l gp41 binding to human T- and B-lymphocytes and monocytes is modulated by phorbol myristate acetate (PMA) Ying-Hua “Institute
for Hygiene,
Chen”*b, Antje Christiansen”,
Manfred
P. Dierich”,”
University Innsbruck, and Ludwig-Boltzmann-Institute jbr AIDS Research, Fritz-Pregl-Strasse bGroup of Immunology, Department of Biology, Tsinghua University, Beijing, China
3. 6010 Innsbruck,
Austria
Received 8 December 1995; revised 1 March 1996; accepted 4 March 1996
Abstract HIV-l gp41 independently of CD4 binds to human T cells, B cells and monocytic cells. Since PMA downmodulates CD4 (HIV receptor) expression and inhibits HIV-l dependent syncytia formation, we wanted to examine whether PMA could affect gp41 binding protein expression on human cells. The strong binding of HIV-1 recombinant soluble gp41 (rsgp41; Env aa539-684) to monocytes (CD14+) and B-lymphocytes (CD19+) and B lymphoblastoid cells (Raji) could be clearly decreased by treating the cells with PMA for 48 h, while the weak binding to T lymphocytes was slightly increased by this treatment. The PMA inhibitoryand enhancing-effects could be avoided by pretreatment with staurosporine (protein kinase C inhibitor). The PMA treatment of Raji and U937 (monocytic) cells resulted in a 50-60% decrease of gp41 binding proteins (gp41bps) detectable in cell lysates of these cells in comparison with lysates of buffer-treated cells, while in the case of H9-cells PMA treatment resulted in an increase of available gp4lbps by about 35% in comparison with buffer-treated H9. Staurosporine pre-treatment could prevent these effects of PMA. Further studies of rsgp41-eluates from these buffer-treated or PMA-treated cells demonstrated that PMA modulated mainly expression of rsgp4lbps of 37, 45, 50 and 62 kDa. These results indicate that PMA exerts different effects on human T, B and monocytic cells. Production by and expression on cells of HIV- 1 gp41 bps appear to depend on protein kinase C, supporting that the four proteins on human cells may act as receptor proteins for HIV-l gp41. Keywords:
HIV-l gp41 binding; Modulation;
PMA
1. Introduction The human immunodeficiency virus type 1 (HIV-l) after binding by the envelope protein gp120 to its cellular receptor CD4 enters susceptible cells through a reaction involving HIV-l gp41 [l-3]. It has been
demonstrated by us and others that HIV-l gp41, independently of CD4, binds to several proteins on human T, B and monocytic cell lines [4-81. This binding could be partially blocked by gp41 peptides (aa581-605 and 641-675) [4]. The importance of these two sites is underscored by the fact that env peptides aa560-597 and aa637-669 and a monoclonal antibody against aa662-667 (HIV- 1 BH 10 isolate) could neutralize HIV1 infection [9- 111, and an amino acid change in gp41 (Env aa666) resulted in escape from neutralization by *Corresponding 5072870.
author. Tel.: +43 512 5073401; fax: +43 512
0165-2478/96/$12.00 0 1996 Elsevier Science B.V. All rights reserved PII SO165-2478(96)02533-3
this anti-gp41 antibody [12]. Since the HIV receptor CD4 on the cell surface could be down-modulated by PMA-treatment [13- 151, we wanted to determine the effect of PMA on HIV-l gp41 binding protein expression on human lymphocytes and monocytes.
2. Materials and methods 2.1. Isolation of peripheral blood lymphocytes and monocytes
Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adult volunteers by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were cultured in RPM1 1640 medium supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 fig/ml streptomycin (Sera-lab Ltd., Vienna, Austria). After
162
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et al. I Immunology
incubation for 1 h at 37°C the non-adherent and adherent cells were isolated. The non-adherent cells contained 2% monocyte (CD14+), and the adherent cells contained at least 95% monocytes (CD14+). The nonadherent cells were analysed as lymphocyte-population and the adherent cells were by lymphocyte-gate, analysed as monocytes-population by monocyte-gate. The T-lymphocytes were purified from human peripheral blood lymphocytes by the rosetting technique (described in Ref. [27]).
Letters
50 (1996)
161-166
USA) by lymphocyte-gate or monocyte-gate. By two color flow cytometric analysis, cells (PBMC or PBL) were incubated at first with monoclonal mouse antiCD19 antibody, then with FITC-conjugated sheep F(ab’),-antibody to mouse IgG and IgM for 30 min at 4”C, and after washing with PBS/BSA, cells were incubated with rsgp41-biotin for 30 min at 4°C. After washing, cells were incubated with R-phycoerythrinconjugated ExtrAvidin. Cells were washed, fixed with 1% paraformaldehyde in PBS and analysed on a FACScan under the lymphocyte- or monocyte-gate.
2.2. HIV-l protein Rsgp41, the outer membrane of gp41 [ 161, 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-kDa polypeptide (Env amino acids 539-684) in Escherichiu coli. The fragment was purified to homogeneity by a three-step method [17]. Using the Biotin-X-NHS Kit (Calbiochem, No.: 813193; La Jolla, California) and the biotinylation method as suggested by Calbiochem (described in Ref. [18]), rsgp41 was biotin-labeled. 2.3. Antibodies FITC-conjugated sheep F(ab’),-antibody to mouse IgG and IgM was obtained from Bio Research GmbH (Austria). FITC-conjugated Streptavidin (F422) and monoclonal mouse anti-human-CD 19 antibody (M740) were obtained from Dako (Austria). R-phycoerythrinconjugated ExtrAvidin (E-401 I) from Sigma. 2.4. Treatment of cells Cells (2 x 106) in 2 ml RPMI-1640 supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 pgg/ml streptomycin were treated with 0, 10 and 20 ng/ml PMA (phorbol 1Zmyristate 13-acetate; Sigma, P-8139; Munich, Germany) for 48 h at 37°C in a 5% CO, atmosphere. To test blockade of the PMA inhibitory effect, cells were incubated with staurosporine (Sigma, S4400) (50 and 100 nM) for 30 min at 37°C. Then PMA (IO ng/ml) was added for 48 h at 37°C in a 5% CO, atmosphere. Afterwards, cells were subjected to flow cytometry analysis. 2.5. Flow cytometry analysis Cells (PBMC or lymphocytes, 5 x 105; monocytes, 1 x 105) were incubated with 50 ~1 biotin-labeled rsgp41 for 30 min at 4°C. Cells were washed with PBS/BSA (1%) and incubated with 50 ~1 FITC-conjugated Streptavidin (1:50 in PBS) for 30 min at 4°C. After washing with PBS, cells were fixed with 1% paraformaldehyde in PBS and analysed on a FACScan (Becton-Dickinson,
2.6. Isolation of rsgp41 binding proteins from cell lysates
The procedure of rsgp41 coupling to Sepharose, cell lysate preparation, adsorption with sepharose column and Coomassie blue staining was described previously [5,6]. Briefly, using a standard method from Phamacia, rsgp41 (2 mg) was coupled to 2 ml CNBr-activated Sepharose 4B. Cell lysates were absorbed with the same rsgp41-Sepharose column. The column was washed several times and eluted using 0.5 mol/l acetic acid. After neutralization of pH the rsgp41-eluates were electrophoresed in a 9.5% SDS-PAGE under non-reducing conditions. The gels were stained with Coomassie blue.
3. Results 3.1. Rsgp41 binding to PMA-treated and bufleer-treated human peripheral blood lymphocytes by two-color flow cytometric analysis Using biotin-labeled rsgp41 and mouse monoclonal antibody to human CD19, we could show that rsgp41 bound to about 99% of B-lymphocytes (CD19 + ) from human PBMCs (Fig. lC, field rsgp41+ CD19’). Rsgp41 binding to B lymphocytes was distinctly downmodulated by PMA (Fig. lD, 10 ng/ml; same result with 20 ng/ml) compared to buffer-treated lymphocytes (Fig. 1C). Also, rsgp41 binding to human B cell line Raji was clearly decreased by PMA treatment in comparison with buffer-treated cells (Fig. lE, FACS histogram overlays). Furthermore, rsgp41 bound weakly to about 20-30% of T lymphocytes (Fig. lC, left section, i.e. field rsgp41 + CD193 and this binding was increased by PMA-treatment. This result is not easily visible in Fig. 1D although obvious from mean fluorescence values. Therefore, in separate experiments, human peripheral blood T lymphocytes were treated with buffer or PMA and then tested for rsgp41 binding. As visible in Fig. 2D buffer-treated T cells bound rsgp41 (in comparison to Fig. 2A) and this binding was increased by PMA treatment (Fig. 2E,F).
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Buffer-treated
PMA-treated
E
?kq Relative fluorescence
anti-CD1
9-Ab-binding
(FL-1
intensity
1
Fig. 1. Rsgp41 binding to PMA-treated and buffer-treated (untreated) human peripheral blood lymphocytes and B cell line Raji by flow cytometric analysis. Dot blots of two-color immunofiuorescence staining (A-D): A and B serve as controls using FITC-conjugated sheep F(ab’)z-antibody to mouse Ig applied to detect bound CDl9-antibody (FITC control) and R-phycoerythrin (PE)-conjugated ExtrAvidin applied to detect bound rsgp41 (PE-avidin control); C and D use mouse anti-human CD19 antibody plus FITC-conjugated sheep F(ab’),-antibody to mouse Ig and biotinylated rsgp41 plus R-phycoerythrin-conjugated ExtrAvidin. (A$) Buffer-treated (0 ng/ml PMA for 48 h) lymphocytes; (B,D) PMA-treated lymphocytes (10 ng/ml for 48 h). FACS-histogram (E): curve 1, rsgp41 binding to buffer-treated (0 ng/ml PMA for 48 h) Raji cells; curves 2 and 3, rsgp41 binding to PMA-treated Raji (curve 2, 10 ng/ml PMA for 48 h; curve 3, 20 ng/ml for 48 h).
3.2. Rsgp41 binding to PMA-treated human monocytes
and bufler-treated
Human peripheral blood monocytes gave a result comparable to B cells. Rsgp41 could bind to almost all human peripheral blood monocytes (Fig. 3C in comparison to buffer-treated cells shown in Fig. 3A) and this binding could be strongly reduced by PMA treatment (Fig. 3D, 10 ng/ml PMA). Treatment with 20 ng/ml PMA gave the same result as 10 ng/ml. 3.3. Blockade of PMA inhibitory and enhancing effects by staurosporine It has been demonstrated that the PKC inhibitor staurosporine could block the PMA inhibitory effect on HIV-induced syncytium formation [14]. Here we report that down-modulation of gp4 1-binding protein expression by PMA could be avoided by staurosporine. Fig. 4A shows that rsgp41 binding to B cell line Raji (see curve 1) was reduced by PMA treatement (10 ng/ml for 48 h) (see curve 2); this PMA-dependent down-modulation could be prevented by preincubation of the Raji cells with staurosporine (50 nM for 30 min) (see curve 3). Also, in case of T-lymphocytes (Fig. 4B) PMA-induced increase of gp41-binding protein expression (see curve 2 in comparison to curve 1) could be inhibited by staurosporine (see curve 3). These results suggest that the selective effects of PMA on cell surface expression on human T-, B-lymphocytes and monocytes are mediated by protein kinase C.
3.4. Quantitative analysis of gp41 binding proteins from PMA-treated and bufler-treated cells Using rsgp41 -Sepharose column, gp41 binding proteins were isolated from cell lysates of PMAtreated and untreated human B cell line Raji, monocyte cell line U937 and T cell line H9. The amount of gp41 binding proteins on Raji and U937 cells was decreased by PMA treatment by about 50-60% in some experiments nearly completely in comparison with the buffer-treated Raji and U937, respectively, while the amount of the proteins on PMA-treated H9 cells was increased by about 35% in comparison with the buffer-treated H9 (Fig. 5). Interestingly, if Raji cells were preincubated with staurosporine for 30 min at 37”C, the effect of PMA was obviously inhibited (Fig. 5). Furthermore, analysing on SDSPAGE the banding pattern of rsgp41-eluates from cell lysates of buffer, or PMA-treated and staurosporine/PMA-treated Raji cells showed that PMA treatment preferentially reduced the protein bands of 37, 45, 50 and 62 kDa (Fig. 6, lane C) when compared with the banding pattern of buffer-treated Raji cells (Fig. 6, lane B). Staurosporine could obviously inhibit the PMA effect (Fig. 6, lane D). These results give support to our previous conclusion that HIV-l gp41 could bind to five cellular proteins, and that four of them (37, 45, 50 and 62 kDa) on human cells may act as receptor proteins for HIV-l gp41. Their expression depends on a functionally intact PKC.
Y.H. Chen et al. /Immunology Letters 50 (1996) 161-166
164
Buffer-treated
PMA-treated
PMA-treated (20 “11
Relative
fluorescence
ngimll c
intensity
Fig. 2. Rsgp41 binding to PMA-treated and buffer-treated human peripheral blood T lymphocytes by flow cytometric analysis. FACS-histogram: (A-C) FITC-conjugated avidin (control); (D-F) biotinylated rsgp41 plus FITC-conjugated avidin. (A,D) Buffer-treated T lymphocytes (0 ng/ml PMA for 48 h); (B,C,E,F) PMA-treated T lymphocytes: (B,E) 10 ng/ml for 48 h, (C,F) 20 ng/ml for 48 h).
4. Discussion
In this study, we demonstrated that production and surface expression of gp41 binding proteins by human monocytes, B-lymphocytes and B lymphoblastoid cells (Raji) could be down-modulated by PMA and pretreatment with PKC-inhibitor staurosporine could avoid this effect. However, in case of human T-lymphocytes, PMA could induce weak enhancement of production and expression of gp41 binding proteins; also this PMA enhancing effect could be prevented by staurosporine. These results suggest that PMA has selective (enhancing and inhibitory) effects on production and cell surface expression of gp41 binding proteins on human untreated (0 ngiml)
PMA-treated (10
na/ml)
T-lymphocytes, B-lymphocytes and monocytes and that these effects may be mediated by protein kinase C. An enhancing effect of PMA on H9 cells’ binding of an immunosuppressive peptide of gp41 has also been observed [7]. For B cells and monocytic cells no information has been reported. PMA effects of this kind on other molecules were collected in Table 1. They concern CD20 [24], CD23 (Fc epsilon receptor) [23], CR3 (complement receptor) [19], CD25 (IL-2R /I chain) [26], MHC class II [25] and CD4 [14,15,20-221. PMA-mediated inhibition of HIV- 1-induced syncytium formation in CD4-expressing cells was reported [13- 151. Interestingly, also in these experiments the inhibitory effect of PMA on syncytia formation and on CD4 expression was blocked by staurosporine, suggesting that it was mediated by protein kinase C [13,14]. Firestein et al. [15] reported that PMA could decrease not only HIV-linduced syncytium formation but also the production
Relative
Relative
fluorescence
intensity
Fig. 3. Rsgp41 binding to PMA-treated and buffer-treated human peripheral blood monocytes. FACS-histogram: A and B, FITC-conjugated streptavidin (control); (CD) biotinylated rsgp41 plus FITCconjugated streptavidin. (AC) Buffer-treated monocytes (0 ng/ml PMA for 48 h); (B,D) PMA-treated monocytes (10 ng/ml for 48 h).
fluorescence
intensity
Fig. 4. Staurosporine prevented the PMA enhancing effect on gp41 binding protein expression on human T lymphocytes and the PMA inhibitory effect on gp41 binding protein expression on human B cell line Raji cells. FAG-histogram overlays: curve 1, rsgp41 binding to buffer-treated cells (0 ng/ml PMA for 48 h); curve 2, rsgp41 binding to PMA-treated cells (10 ng/ml, for 48 h); curve 3, rsgp41 binding to cells that were preincubated with staurosporine (50 nM) for 30 min at 37°C and then with PMA (IO ng/ml) for 48 h at 37°C.
165
Y.H. Chen et al. I Immunology Letters 50 (1996) 161-166
-
Table 1 Selective modulation of cell surface expression of gp41 binding proteins (gp4lbp). gp41 IS-peptide binding protein (ISPbp) and other molecules on human T. B and monocytic cells by PMA
--
I
u937 -
m
n.t.
0 ng/ml PMA
n
20 ng/ml PMA
m
PMA + staurosporine
gp4lbp ISPbp CD4 CD20 CD23 CD25 CR3 MHC II 0
10
rsgp41
20
30
binding proteins
T
B
Mono
Ref.
T
1
1
I _
1
j
f
1 _
I51 171 [14.15.20-221 [241 [23] 1261 1191 [251
I
T
r r
40
(ug)
Fig. 5. Quantitative analysis of gp41 binding proteins in rsgpll-eluates from cell lysates of buffer-treated and PMA-treated Raji, U937 and H9 cells. Using the same rsgp4l-sepharose column, rsgp41 binding proteins were isolated from cell lysates of buffer-treated (0 ng/ml PMA for 48 h) and PMA-treated (20 ng/ml PMA for 48 h) cells (2 x 10’ cells), or from cell lysates of Raji cells preincubated with staurosporine (50 nM staurosporine for 30 min at 37°C) and then PMA. (n.t.. not tested).
Acknowledgements The technical help of Gabi Kienast is gratefully acknowledged. This work was supported by the Austrian FWF (project P10094-Med). The work was part of the doctoral thesis of A.C.
References of viral RNA and p24 antigen. Whether this inhibitory effect on virus production was due to the down-modulation of the surface-associated CD4 receptor or modulating an accessory component(s), for example gp41 binding proteins (second HIV receptor) as shown by our experiments, is unclear.
ABC
D
f@fblQM*W-loo
66-
-62
Fig. 6. Identification of gp41 binding proteins in rsgp41-eluates from cell lysates of buffer-, PMA- and staurosporine/PMA-treated Raji cells by Coomassie blue staining. The eluates were electrophoresed in a 9.5% SDS-PAGE under non-reducing conditions. The gels were stained with Coomassie blue. Lane B, rsgp41-eluate from cell lysates of buffer-treated Raji cells; lane C, rsgp41-eluate from PMA-treated Raji cells (20 ng/ml PMA for 48 h); lane D, rsgprll-eluate from staurosporine/PMA-treated Raji cells that were preincubated with 50 nM staurosporine for 30 min at 37°C. then with 20 ng/ml PMA for 48 h. Each lane represents an eluate of I x IO’ Raji cells. Molecular weight markers (kDa) are depicted in lane A.
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