(CCL)20, and CCL21 Chemokine Ligand (CXCL) 12, CC Human B Cell Chemotaxis to CXC HIV Type 1 Glycoprotein 120 In

HIV Type 1 Glycoprotein 120 Inhibits Human B Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 12, CC Chemokine Ligand (CCL)20, and CCL21 This information is current as of September 24, 2021. Gamal Badr, Gwenoline Borhis, Dominique Treton, Christiane Moog, Olivier Garraud and Yolande Richard J Immunol 2005; 175:302-310; ; doi: 10.4049/jimmunol.175.1.302 http://www.jimmunol.org/content/175/1/302 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HIV Type 1 Glycoprotein 120 Inhibits Human B Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 12, CC Chemokine Ligand (CCL)20, and CCL211

Gamal Badr,* Gwenoline Borhis,* Dominique Treton,* Christiane Moog,† Olivier Garraud,‡ and Yolande Richard2*

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40–50%, whereas R5 gp120 decreased inhibition by 20%.

This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or VH3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/ CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase C␤3 and PI3K downstream from CXCR4, Downloaded from whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase ␣ 1 and 3, CD4, CXCR4, G i, and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 http://www.jimmunol.org/ gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials. The Journal of Immunology, 2005, 175: 302–310.

uman immunodeficiency virus-1 infection is associated a superantigen to conserved VH3 framework regions, activating with strong polyclonal B cell activation, increasing the and depleting these cells (17, 18). As VH3 genes are the key de- H percentage of activated B cells in the periphery (1–5), terminants of Abs specific for bacterial polysaccharide Ags, the and with strong, sustained follicular hyperplasia in secondary lym- depletion of VH3-expressing B cells may contribute to the observed phoid organs during the asymptomatic phase of the disease (6–8). increase in the incidence of secondary infections in HIV-infected pa- by guest on September 24, 2021 The B cells of HIV-infected patients spontaneously secrete Ig but tients (19). Scamurra et al. (10) recently showed in vivo that the de- are unable to mount a T cell-dependent B cell response (1, 9, 10). pletion of peripheral VH3-expressing memory B cells, but not of naive Antiretroviral therapy decreases HIV-1-driven B cell hyperactiv- B cells, in HIV-infected patients involves gp120 binding. ity, polyclonal B cell activation, and Ig production in patients (11). Lymphocyte recirculation, which is critical for effective immu- These findings strongly suggest that the sustained replication of nity, is tightly regulated by the expression of adhesion molecules HIV-1 affects differentiation in lymphoid tissue. The mechanisms and chemoattractant receptors on lymphocytes, combined with the by which HIV-1 impairs the humoral response may result from spatial and temporal expression of ligands for these receptors by a intrinsic B cell defects or dysfunctional dialogue between T and B variety of tissue cells (20–23). Human B cells express several cells. Soluble Tat and gp120, biologically active extracellular pro- chemokine receptors, including CXCR4, CXCR5, CCR6, and teins released by HIV-1-infected cells, induce intrinsic defects in CCR7, and respond to their cognate ligands CXCL12, CXCL13, human B cells (12–15). We previously showed that Tat selectively CCL20, and CCL21 and CCL19, respectively. Triggering of the inhibits the BCR-mediated proliferation of naive and memory B BCR, CD40 and the IL-4R modulates chemokine receptor expres- cells, and the production of cytokines and Ig. In contrast, Tat dou- sion and chemotaxis in B cells (24–26). Chemokines can bind to 3 ␣ bles the germinal center cell proliferation induced by CD40 mAb various pertussis toxin (PTX) -sensitive (G i) and PTX-insensi- ␣ ␣ ␣ plus IL-4 (16). The HIV-1 envelope protein gp120 may bind like tive (G q and G 15/16)G proteins, but chemotaxis is only ob- ␣ served upon activation of G i protein-coupled receptors (22). There is strong evidence that G␤␥, rather than G␣ subunits initiate *Institut National de la Santeét de la Recherche Medicale, Unite´131, Institut Paris- i Sud sur les Cytokines, Clamart, France; †Universite´ Louis Pasteur, Strasbourg, the chemotactic response (27). We recently showed that human B France; and ‡Groupe Immunite´ des Muqueuses et Agents Pathoge`nes, Faculte´de cell chemotaxis to CXCL12, CXCL13, CCL21, and CCL20 de- Me´decine, Universite´Jean Monnet, Saint-Etienne, France pends on PI3K, phospholipase C (PLC)␤3, protein kinase C Received for publication November 1, 2004. Accepted for publication April 20, 2005. (PKC), RhoA, and NF-␬B. Although activated by chemokine re- The costs of publication of this article were defrayed in part by the payment of page ceptor-ligand interactions, neither p38 MAPK nor ERK1/2 are in- charges. This article must therefore be hereby marked advertisement in accordance ␤␥ with 18 U.S.C. Section 1734 solely to indicate this fact. volved in B cell chemotaxis (28). G stimulation also activates G 1 This work was supported by grants from the Institut National de la Santeétdela Recherche Me´dicale, Universite´Paris-Sud, and Sidaction. 2 Address correspondence and reprint requests to Dr. Yolande Richard, Institut 3 Abbreviations used in this paper: PTX, pertussis toxin; PLC, phospholipase C; PKC, National de la Santeét de la Recherche Me´dicale, Unite´131, Universite´Paris- protein kinase C; MFI, mean fluorescence intensity; CD62L, L-selectin; MARCKS, myr- Sud, 32 rue des Carnets, 92 140 Clamart, France. E-mail address: yolande.richard istoylated alanine-rich C kinase substrate; M␤CD, methyl-␤-cyclodextrin; MMP, matrix @ipsc.u-psud.fr metalloproteinase; PIP2, phosphatidylinositol-4,5-bisphosphate.

protein-coupled receptor kinases, which phosphorylate chemokine For in vitro culture assays, B cells (2 ϫ 106 cells/ml) were cultured in receptors, inducing their binding to ␤-arrestin (29) and their rapid RPMI 1640 (Invitrogen SARL) supplemented with 10 mM HEPES, 2 mM ␮ internalization (30). L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate, and 10% heat-inactivated FCS (complete medium) for 2 h, with We analyzed the effect of recombinant CXCR4-binding (X4), or without recombinant 10 nM gp120 (unless otherwise indicated). Pre- CCR5-binding (R5), or dual-binding (X4/R5) gp120 on the che- liminary experiments showed that B cell chemotaxis was maximally in- motactic response of human primary B cells. We found that gp120 hibited after2hofincubation with 10 nM gp120 from the various HIV-1 strongly inhibited the chemotactic response to CCL20 and CCL21, strains (data not shown). without inducing spontaneous apoptosis or impairing the expres- Flow cytometry sion and ligand-induced internalization of CCR6 and CCR7. B cell chemotaxis was inhibited more strongly by X4 and X4/R5 gp120 Chemokine receptor expression was analyzed by flow cytometry, using than by R5 gp120, consistent with CXCR4 being the more strongly PE-conjugated anti-CCR6 (clone 53103.111, IgG2b), anti-CCR5 (clone 45531.111, IgG2b), anti-CCR7 (clone 150503, IgG2a), and anti-CXCR4 expressed of these two receptors. The use of Abs blocking (clone 44717.111, IgG2b) mAbs from R&D Systems. PE-conjugated CD19 CXCR4, CCR5, or gp120 totally prevented the gp120-induced in- (IgG1) and CD95 (IgG1) mAbs and FITC- and PE-conjugated mouse iso- hibition of B cell chemotaxis whereas soluble CD4 slightly in- type-matched control IgG were purchased from BD Biosciences. FITC- creased it. In contrast to what was observed with CXCL12, we conjugated CD62L (clone FMC46, IgG2a) was purchased from Diaclone. To detect cytoplasmic CD95 expression, we treated B cells with the Cyto- found that X4 or X4/R5 gp120 did not activate the CXCR4- fix/Cytoperm kit (BD Biosciences) before staining with PE-conjugated dependent phosphorylation of PLC␤3, PI3K/AKT, ERK1/2, and CD95 mAb or mouse IgG1. A FACScan flow cytometer was used for data I␬B␣, but did induce that of p38 MAPK. The treatment of B cells acquisition, with CellQuest software (BD Biosciences) for data analysis.

with X4 or X4/R5 gp120 also blocked the CCL20- and CCL21- We gated on viable cells and analyzed 10,000 cells/sample. For each Downloaded from induced activation of PLC␤3, PI3K/AKT, ERK1/2, and I␬B␣, but marker, the threshold for positivity was defined based on nonspecific binding in the presence of the relevant control IgG. not that of p38 MAPK. X4 and X4/R5 gp120 also modulated CD95 and L-selectin (CD62L) surface expression whereas R5 Detection of apoptotic cells gp120 did not. CD95 expression increased as a result of the relo- B cells were fixed and permeabilized by incubation in 70% ice-cold ethanol calization of a cytoplasmic pool to the membrane, induced by the for at least 1 h, and washed twice in PBS. DNA was stained by incubating

␣ http://www.jimmunol.org/ triggering of CXCR4/G i complexes by gp120. The decrease in cells at 37°C for1hin40␮g/ml propidium iodide and 100 ␮g/ml DNase- CD62L surface expression was induced by the matrix metallopro- free RNase in PBS. Samples were analyzed by assessing FL2 red fluores- teinase (MMP)-mediated cleavage of L-selectin (CD62L) via a cence on a linear scale. The percentage of cells undergoing apoptosis was ␣ determined as the percentage of hypodiploid cells (sub G0/G1 peak). mechanism dependent on CD4, CXCR4/G i complexes and p38 We evaluated the sensitivity of B cells to CD95-mediated apoptosis, by MAPK. Therefore, gp120 strongly inhibits B cell chemotaxis and incubating medium- and gp120-treated B cells (2 ϫ 106 cells/ml) for 24 h interactions with endothelial surfaces, and may be involved in de- at 37°C with 1 ␮g/ml B-G27 (IgG2a, Diaclone) or mouse IgG2a as a creasing the efficiency of humoral responses in HIV-infected control. B cells were then collected and the percentage of cells undergoing patients. apoptosis was determined as described. Blocking of CD62L shedding and increase in CD95 expression by guest on September 24, 2021 Materials and Methods by various inhibitors Reagents B cells were incubated with 5 ␮g/ml AMD3100, 1 ␮M herbimycin A (a tyrosine kinase inhibitor), 10 ␮M SB 203580 (p38 MAPK inhibitor), 100 Soluble CD4–183 from Pharmacia, recombinant gp120 proteins from Chi- ng/ml PTX, 10 ␮g/ml brefeldin A (specifically blocking protein translo- ron or the Division of AIDS (National Institute of Allergy and Infectious cation from endoplasmic reticulum to Golgi apparatus) (all from Calbio- Diseases, Bethesda, MD), and anti-gp120 mAb were provided by the Eu- chem) or DMSO for 1 h before incubation with medium or 10 nM gp120 ropean Union Programme of European vaccine against AIDS (EVA)/Med- for 2 h. We assessed CD62L and CD95 expression by flow cytometry. ical Research Council centralized facility for AIDS reagents, National In- For experiments with MMP inhibitors, B cells were cultured with 500 stitute for Biological Standards and Control, United Kingdom (Grant ␮M EDTA, 10 ␮M GM-6001 (inhibitor of MMP-1, -2, -3, -8, and -9), QLK2-CT-1999-00609 and GP828102). Recombinant gp120 proteins were MMP-III inhibitor (inhibitor of MMP-1, -2, -3, -7, and -13), MMP-2/9 obtained from the R5 (SF162, BaL), X4 (MN, IIIB), and X4/R5 (SF2) inhibitor (all from Calbiochem) or DMSO before incubation with medium HIV-1 strains. Preliminary experiments showed that only gp120SF162 con- or 10 nM gp120 for 2 h. We assessed CD62L expression by flow tained endotoxins (data not shown). We therefore assessed the effects of ␮ cytometry. gp120SF162 in the presence of 10 g/ml polymyxin B throughout this work. The 2G12 mAb (IgG1) is directed against a conformational epitope of In vitro chemotaxis assay gp120 and neutralizes a wide range of R5 isolates, as well as gp120 IIIB, MN, and SF2. The 447-52D (IgG3) mAb recognizes the G protein-coupled The chemotaxis assay was performed in 24-well plates (Costar) carrying receptor sequence at the apex of the V3 loop and neutralizes numerous transwell permeable supports, with a 5-␮m polycarbonate membrane. As- laboratory strains and clinical isolates. Neutralizing 2F5 mAb (IgG1), di- says were performed in prewarmed migration buffer (RPMI 1640 plus 10 rected against gp41 protein from various laboratory strains and clinical mM HEPES and 1% FCS). Migration buffer (600 ␮l) containing no che- isolates, was used as a control. Blocking anti-CCR5 (2D7, IgG1) mAb and mokine, 250 ng/ml (CXCL12, CCL21), or 500 ng/ml (CCL20) chemokine mouse IgG1 and IgG2b were purchased from BD Biosciences. Nonblock- (all from R&D Systems) was added to the lower chamber and B cells were ing anti-CCR5 mAb (45531.111, IgG2b) was obtained from R&D Systems. loaded onto the inserts at a density of 0.3 ϫ 106 cells/100 ␮l. Plates were The CXCR4 antagonist, AMD3100, was purchased from Calbiochem incubated for3hat37°C, and the number of cells migrating into the lower (VWR International) and used at a concentration of 5 ␮g/ml. chamber was determined by flow cytometry. Cells from the lower chamber were centrifuged and fixed in 300 ␮lof1ϫ PBS, 1% formaldehyde and B cell preparation and culture counted with the FACScan apparatus for 60 s, gating on forward and side light scatter to exclude cell debris. The number of live cells was compared Total B cells were obtained from palatine tonsils as previously described with a 100% migration control in which 100 ␮l of cell suspension (0.3 ϫ (31). After one cycle of rosette formation, residual T cells and monocytes 106 cells) was treated in the same manner. The percentage of cells migrat- were removed with CD2- and CD14-coated magnetic beads (Dynabeads ing to medium without chemokine was subtracted from the percentage of M-450; Dynal Biotech). The total B cell population was then depleted of cells migrating to the medium with chemokines, to calculate the percentage CD38ϩ germinal center B cells by Percoll gradient separation, as previ- of specific migration. ously described (32). The resulting B cell population, including only naive In some experiments, B cells were treated with 1 ␮M herbimycin A, 100 and memory B cells, is referred to herein as “B cells” and is comprised of ng/ml PTX, 10 ␮M SB203580, or medium for 1 h before culturing with 98 Ϯ 6% CD19ϩ,93Ϯ 3% CD44ϩ,6Ϯ 3% CD38ϩ, and 2 Ϯ 3% CD3ϩ medium or 10 nM X4 or X4/R5 gp120 for 2 h. Cells were then washed and (n ϭ 25). The viability of these cells was consistently higher than 98%. assayed for chemotaxis. 304 gp120 HIV-1 ENVELOPE IMPAIRS HUMAN B CELL CHEMOTAXIS

F-actin polymerization assay Intracellular F-actin polymerization was assessed as previously described (31). Medium- and gp120-treated B cells (8 ϫ 106/ml) were incubated in HEPES-buffered RPMI 1640 at 37°C, with or without 250 ng/ml chemokine. At the indicated times, cells (100 ␮l) were added to 400 ␮l of assay Ϫ7 buffer containing 4 ϫ 10 M FITC-labeled phalloidin, 0.5 mg/ml L-␣- lysophosphatidylcholine (both from Sigma-Aldrich) and 4.5% formaldehyde in PBS. Fixed cells were analyzed with a FACS machine and mean ﬂuorescence intensity (MFI) was determined for each sample. The percentage change in MFI was calculated for each sample at each time point as follows: [1 Ϫ (MFI before addition of chemokine/MFI after addition of chemokine) ϫ 100]. In some experiments, B cells were pretreated with 2 mM methyl-␤- cyclodextrin (M␤CD) or medium for 1 h before incubating with medium or 10 nM gp120 for 2 h. Cells were washed and subjected to F-actin polymerization assay. Preliminary experiments showed that 2 mM was the low- est dose of M␤CD inhibiting BCR- and CD40-induced B cell proliferation by Ͼ90% (data not shown). Ligand-induced chemokine receptor internalization Medium- and gp120-treated B cells were incubated for 30 and 60 min with medium or 100 ng/ml chemokine at 37°C. Cells were washed in Downloaded from ice-cold medium and stained with PE-conjugated anti-CD19, anti-CXCR4, anti-CCR7, anti-CCR6 mAbs, and PE-conjugated mouse isotype-matched control IgG for 30 min at 4°C. Single-color immunoﬂuorescence analysis was performed on 5000 viable cells. Western blots

We compared the abilities of CXCL12 and gp120 to trigger CXCR4 by http://www.jimmunol.org/ resuspending B cells at a density of 1 ϫ 107 cells/ml in prewarmed RPMI 1640 without FCS and stimulating them for 2 min at 37°C with medium,

100 ng/ml CXCL12 or 10 nM gp120SF2. Alternatively, medium- and gp120-treated B cells were resuspended at a density of 1 ϫ 107 cells/ml in prewarmed RPMI 1640 without FCS and stimulated for 2 min at 37°C with medium, or 100 ng/ml CCL21 or CCL20. Lysates were prepared as previously described (32). Equal amounts of total cellular protein were subjected to SDS-PAGE and analyzed by Western blotting. Abs recognizing phospho-AKT (S473), AKT, phospho-ERK1/2 (T202/Y204), phospho- I␬B␣ (S32/S36), phospho-PLC␤3 (S537), PLC␤3, phospho-p38 MAPK (T180/Y182), p38 MAPK, and pan phospho-PKC (all from New England by guest on September 24, 2021 Biolabs), PKC␣, PKC␤, PKC␥,I␬B␣, or ERK1/2 (from Santa Cruz Bio- technology) were used with HRP-conjugated secondary Abs. Protein bands were detected by ECL (Supersignal Westpico Chemiluminescent Sub- strate; Perbio Sciences). The ECL signal was recorded on ECL Hyperﬁlm. Films were scanned, saved as TIFF ﬁles, and analyzed with NIH Image software. FIGURE 1. Recombinant gp120 decreases B cell chemotaxis. A–C,B cells from three independent donors were incubated for 2 h, in the presence

Results of medium or 0.7–40 nM gp120SF2 (X4/R5) (A), gp120IIIB (X4) (B), and Recombinant gp120 inhibits human B cell chemotaxis gp120BAL (R5) (C). We then analyzed the migration of medium- and f The chemotactic response of B cells to CCL21 and CXCL12 was gp120-treated B cells toward 250 ng/ml CCL21 ( ) and 250 ng/ml CXCL12 (o). Results are expressed as the mean percentage Ϯ SD of investigated after 2 h of incubation with medium or 0.7–40 nM specifically migrating cells obtained for each donor. D–F, B cells from recombinant gp120 from various HIV-1 strains (Fig. 1, A–C). 8–15 independent donors were incubated for 2 h, in the presence of me- Maximal gp120-induced inhibition was observed at a concentra- dium or 10 nM R5, X4/R5, and X4 gp120. Medium- and gp120-treated B tion of 10 nM gp120, regardless of the chemokine and gp120 cells were then analyzed for migration to 500 ng/ml CCL20 (D), 250 ng/ml Ϯ tested. For gp120SF2, maximum inhibition was 45.2 8.2% for CCL21 (E), and 250 ng/ml CXCL12 (F). Results are expressed as the mean CCL21 (n ϭ 3) and 44 Ϯ 8.7% (n ϭ 3) for CXCL12 (Fig. 1A). For percentage Ϯ SD of specifically migrating cells obtained for each donor. Ϯ ϭ gp120IIIb, maximum inhibition was 34.7 8.3% for CCL21 (n Differences between groups were assessed with Student’s unpaired t test, p Ͻ ,ءء ;p Ͻ 0.05 ,ء .and 39.3 Ϯ 6.4% (n ϭ 3) for CXCL12 (Fig. 1B). For gp120 , and values for p Ͻ 0.05 were considered significant (3 Ͻ ءءء BAL maximum inhibition was 19 Ϯ 0.5% for CCL21 and 22 Ϯ 1.4% 0.001; and , p 0.0001. NS, Not significant. G, B cells from three for CXCL12 (Fig. 1C). Similar results were obtained for CCL20 independent donors were incubated for 2 h, in the presence of medium (0), Ϯ ϭ 10 nM gp120SF2 (X4/R5), gp120IIIb (X4), and gp120BAL (R5) (IIIB), with (data not shown). In the absence of gp120, 19 6.4% (n 8) of ␮ o f ␮ Ϯ or without 10 g/ml 2F5 ( ), 2G12 mAb ( )or1.5 g/ml 447-52D mAb B cells migrated specifically in response to CCL20, 49.6 9.9% (z). We then analyzed the migration of B cells toward 250 ng/ml CCL21. ϭ Ϯ ϭ (n 15) in response to CCL21 and 41.4 11.5% (n 11) in Results are expressed as the mean percentage Ϯ SD of specifically migrat- response to CXCL12 (Fig. 1, D–F). The chemotactic response to ing cells obtained for each donor. H, B cells from three independent donors Ϯ ϭ Ϯ ϭ Ⅺ CCL20 was inhibited by 23 18.4% (n 8), 39.9 2% (n 8), were incubated for 2 h, in the presence of medium ( ) or 10 nM gp120SF2 and 39.5 Ϯ 12.8% (n ϭ 8) using R5, X4/R5, and X4 gp120, re- (X4/R5) (3) and were then analyzed or subjected to culture for3hin spectively (Fig. 1D), whereas response to CCL21 was inhibited by migration buffer containing 1% FCS. The percentage of cells undergoing 20.1 Ϯ 13.7% (n ϭ 14), 44.9 Ϯ 10.2% (n ϭ 14), and 49.6 Ϯ apoptosis was determined as the percentage of hypodiploid cells, as indi- 15.9% (n ϭ 15), respectively (Fig. 1E). The chemotactic response cated in Materials and Methods. Results are expressed as the mean per- Ϯ to CXCL12 was decreased by 23.7 Ϯ 11.2% (n ϭ 8), 43.4 Ϯ 8.3% centage SD of cells undergoing apoptosis obtained for each donor. (n ϭ 11), and 40.0 Ϯ 14.8% (n ϭ 11) with R5, X4/R5, and X4 The Journal of Immunology 305 gp120, respectively (Fig. 1F). The addition of 10 ␮g/ml 2G12 mAb totally abolished R5 gp120-induced inhibition but only partly decreased X4 and X4/R5 gp120-induced inhibition. In contrast, 1.5 ␮g/ml 447-52D mAb totally abolished the inhibition of B cell chemotaxis induced by the gp120 molecules of all virus strains, whereas 10 ␮g/ml 2F5 mAb had no effect (Fig. 1G). B cells were cultured for 2 h with medium or 10 nM gp120 (2 h) and then for 3 h in migration buffer containing only 1% FCS (5 h). The percentage of cells undergoing apoptosis was similar in medium- and Ϯ Ϯ gp120SF2-treated B cells after 2 h (13.7 6.4% vs 14.7 7.4%, respectively) or 5 h (23.4 Ϯ 7.8% vs 25.2 Ϯ 8.7%, respectively) of culture, suggesting that gp120 does not increase spontaneous apoptosis (Fig. 1H). Similar results were obtained for X4 gp120 (data not shown). Thus, gp120 delivers signals that inhibit B cell chemotaxis through CXCR4 and, to a lesser extent, through CCR5. CCR7 and CCR6 did not bind gp120. Our data therefore suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to cross- desensitization of CCR6 and CCR7. Downloaded from Recombinant gp120 affects neither the expression nor the ligand-induced internalization of CCR6 and CCR7 B cells were stained with PE-conjugated CCR5 and CXCR4 mAbs to compare the expression of these receptors before and after incubation with recombinant gp120. CXCR4 was strongly expressed

on all B cells (MFI, Ͼ400), whereas CCR5 was expressed only at http://www.jimmunol.org/ low levels (MFI, Յ40) (Fig. 2A). A 2-h incubation with recombinant gp120SF2 induced CXCR4 internalization but did not affect the spontaneous expression of CCR6 and CCR7 on B cells (Fig. 2B). We investigated whether ligand-induced internalization, which is important for chemotaxis, was impaired by gp120. We incubated medium- and gp120SF2-treated B cells with medium, 100 ng/ml CCL21, or 100 ng/ml CCL20 for up to 60 min at 37°C. We then placed cells at 4°C and stained them with anti-CCR7 mAb (Fig. by guest on September 24, 2021 2C), anti-CCR6 mAb (Fig. 2D), or mouse IgG-PE for 30 min at

4°C. In the absence of chemokine, gp120SF2 did not affect the expression of CCR7 or CCR6. The gp120- and medium-treated cells showed similar levels of ligand-induced CCR7 and CCR6 internalization. Similar results were obtained for X4 gp120 (data not shown). FIGURE 2. Chemokine receptor expression on B cells before and after incubation with recombinant gp120. A, We analyzed the expression of Receptors involved in gp120-induced inhibition of B cell CXCR4 and CCR5 by flow cytometry before incubation. One experiment chemotaxis representative of four is shown. B, B cells were incubated for 2 h with medium or 10 nM gp120 . The expression of CCR7, CCR6, and CXCR4 In addition to CXCR4 and CCR5, gp120 can interact with CD4 SF2 in medium-treated (Ⅺ) and gp120-treated (o) B cells was analyzed by flow and VH3-expressing BCR. We therefore evaluated the effect of cytometry. The experiment was performed on four independent donors and blocking CD4 and BCR signaling before adding gp120SF2.Asex- results are expressed as the mean percentage Ϯ SD of MFI values. C and pected, PTX totally abrogated the chemotaxis of medium-treated B D, Medium-treated (Ⅺ) or gp120-treated (o) B cells were kept at 4°C for cells, whereas the pretreatment of B cells with herbimycin A did 1 h (0) or incubated for 30 or 60 min at 37°C with 100 ng/ml CCL21 (C) not reverse the medium- or gp120-induced inhibition of CCL21- or 100 ng/ml CCL20 (D). Cells were washed in ice-cold medium before mediated B cell chemotaxis (Fig. 3A). Similar results were ob- and after incubation and were stained with PE-conjugated anti-CCR7 (C), PE-conjugated anti-CCR6 (D) or control IgG Abs for 30 min at 4°C. We tained for gp120MN and CCL20-mediated B cell chemotaxis (data not shown). Thus, signaling via BCR or CD4 is not required for the used four independent donors and data are expressed as the mean percent- Ϯ gp120-mediated inhibition of B cell chemotaxis. age SD MFI values for residual surface expression. The CCR5-blocking mAb totally reversed the inhibitory effect of recombinant R5 gp120BAL, but not that of X4/R5 gp120SF2,on CCL21-mediated B cell chemotaxis (Fig. 3B). AMD3100, which 3F) B cell chemotaxis. Thus, interactions between gp120 and blocks CXCR4, abolished the inhibition of CCL21-induced che- membrane CD4 increase the inhibition of B cell chemotaxis by motaxis observed with X4 gp120MN (Fig. 3C) or X4/R5 gp120SF2 gp120, without involving CD4 signaling. The integrity of lipid (data not shown). AMD3100 also totally inhibited CXCL12-in- rafts is crucial for efficient target cell infection. We therefore elim- duced chemotaxis, independently of the presence of gp120 (Fig. inated cholesterol from B cell membranes by treatment with 2 mM 3C). This gp120-induced effect is thus mediated via CXCR4 or M␤CD before incubation with X4 and X4/R5 gp120. As previ- CCR5. The incubation of gp120 with 100 nM soluble CD4 before ously shown (33, 34), this treatment did not change chemokine its addition to B cells slightly increased its potential to inhibit receptor, CD4, or BCR expression on B cells (data not shown). CCL21- (Fig. 3D), CXL12- (Fig. 3E), and CCL20-mediated (Fig. Levels of CCL21- (Fig. 4, A and B) and CXCL12-induced (Fig. 306 gp120 HIV-1 ENVELOPE IMPAIRS HUMAN B CELL CHEMOTAXIS Downloaded from http://www.jimmunol.org/

FIGURE 4. Cholesterol depletion prevents the gp120-mediated inhibition of chemokine-induced F-actin polymerization. A and B, B cells were incubated for1hat37°C with medium (open symbols) or 2 mM M␤CD (ﬁlled symbols) and then for 2 h with medium (squares) or 10 nM X4/R5

gp120SF2 (A) or X4 gp120MN (B) (triangles). Medium- and gp120-treated B cells were tested for F-actin polymerization in response to 100 ng/ml

CCL21. Results are expressed as the percentage change in MFI, as de- by guest on September 24, 2021 scribed in Materials and Methods. One experiment representative of three is shown. C, Medium-treated (open symbols) and M␤CD-treated (ﬁlled symbols) B cells were tested for F-actin polymerization in response to 100

ng/ml CXCL12 (circles) or 10 nM gp120MN (stars). Results are expressed as the percentage change in MFI, as described in Materials and Methods. FIGURE 3. CXCR4 and CCR5, but not CD4 and BCR, are crucial for One experiment representative of three is shown. the gp120-mediated inhibition of B cell chemotaxis. A, B cells were incu- Ⅺ o bated with medium ( ) or 10 nM gp120SF2 (X4/R5) ( )for2h,inthe presence or absence of 1 ␮M herbimycin A. We analyzed the migration of 4C) F-actin polymerization were similar in B cells pretreated with medium- and gp120-treated B cells toward 250 ng/ml CCL21. Medium- medium and M␤CD. Treatment with X4/R5 and X4 gp120 treated cells were also incubated with 100 ng/ml PTX before chemotaxis strongly decreased CCL21-induced (or CCL20-induced, data not assay (f). Results are expressed as the percentage of speciﬁcally migrating shown) F-actin polymerization at all times tested. This inhibition cells. One experiment representative of three is shown. B, B cells were was abolished in B cells treated with M␤CD (Fig. 4, A and B). incubated for 30 min at 4°C with 10 ␮g/ml CCR5 mAb (2D7) (f), 10 Although CXCL12 and X4 gp120 interacted with CXCR4, gp120 ␮ z Ⅺ g/ml mouse IgG1 ( ) or medium ( ). They were then incubated for 2 h did not induce F-actin polymerization, even in untreated B cells with medium, 10 nM gp120 (R5) or 10 nM gp120 (X4/R5). Medi- BAL SF2 (Fig. 4C). This suggests that CXCR4/gp120 and CXCR4/CXCL12 um- and gp120-treated B cells were analyzed for migration toward 250 interactions have different signaling outcomes in B cells, with only ng/ml CCL21. Results are expressed as the percentage of speciﬁcally migrating cells. One experiment representative of three is shown. C, B cells the former totally dependent on lipid raft integrity. were incubated for 30 min at 4°C with medium or AMD3100 and then with Blockade by gp120 of chemokine-induced signaling medium or 10 nM gp120MN (X4) for 2 h. We analyzed the migration of medium- and gp120-treated B cells toward 250 ng/ml CCL21 or CXCL12. We compared the effects of CXCL12, gp120SF2 (Fig. 5, left pan-

Results are expressed as the percentage of speciﬁcally migrating cells. One els) and gp120MN (Fig. 5, right panels) on the activation of various experiment representative of three is shown. D–F, 10 nM recombinant effectors downstream from CXCR4. Unlike CXCL12, we found gp120BAL (R5), gp120SF2 (X4/R5), and gp120MN (X4) was incubated for that gp120 and gp120 did not activate ERK1/2, PLC-␤3, f Ⅺ SF2 MN 30 min with 100 nM soluble CD4 ( ) or medium ( ) and then for 2 h with AKT, and PKC (␣, ␤I, ␤II, ␦, ⑀, and n) but both CXCL12 and B cells. We analyzed the migration of medium- and gp120-treated B cells gp120 increased the phosphorylation of p38 MAPK. Weaker toward 250 ng/ml CCL21 (D), 250 ng/ml CXCL12 (E), or 500 ng/ml SF2 CCL20 (F). We conducted this experiment for three independent donors p38 MAPK activation was observed if CXCR4 was triggered by ␬ ␣ and results are expressed as the mean percentage Ϯ SD of speciﬁcally gp120 rather than CXCL12. I B was not phosphorylated upon migrating cells obtained for each donor. CXCR4 triggering by gp120SF2 or gp120MN (data not shown). Consistent with it effects on chemotaxis, gp120SF2 totally prevented the CCL21-induced phosphorylation of ERK1/2, AKT, The Journal of Immunology 307 Downloaded from http://www.jimmunol.org/

FIGURE 6. gp120 inhibits the CCL21-induced phosphorylation of ␤ f z by guest on September 24, 2021 PLC 3 and AKT. Medium-treated ( ) and gp120SF2-treated ( ) B cells were stimulated for 2 min with medium or 100 ng/ml CCL21. Cells were lysed and analyzed by Western blotting (left), as described in Materials and Methods. One experiment representative of three is shown. The phosphorylation of AKT, ERK1/2, p38 MAPK, PLC␤3, and I␬B␣ was corrected for total relevant protein on stripped blots (right). The experiment was conducted for three independent donors and results are expressed as the FIGURE 5. Comparison of gp120- and CXCL12-induced patterns of mean Ϯ SD normalized phosphorylation values. phosphorylation in B cells. Cells were stimulated for 2 min with medium,

100 ng/ml CXCL12, 10 nM gp120SF2 (left panels) or 10 nM gp120MN (right panels). Cells were lysed and analyzed by Western blotting (top), as CD95 expression by a factor of 4.1 Ϯ 1.4 (n ϭ 6) (Fig. 7B), and described in Materials and Methods. One experiment representative of decreased intracytoplasmic CD95 expression (Fig. 7C). These three is shown. The phosphorylation of AKT, ERK1/2, p38 MAPK, PLC␤3, and PKC was corrected for total relevant protein on stripped blots changes were not detected after treatment with R5 gp120 (data not (bottom). The experiment was performed on three independent donors and shown). The modulation of CD62L and CD95 expression induced results are expressed as the mean Ϯ SD normalized phosphorylation values. by gp120 was prevented by adding 1.5 ␮g/ml 447-52D mAb to gp120 protein (Fig. 7D). The incubation of B cells with 5 ␮g/ml

AMD3100 or 100 ng/ml PTX before incubation with gp120SF2 for I␬B␣, and PLC␤3 in B cells (Fig. 6). The activation of p38 MAPK 2 h totally prevented changes in surface CD62L (Fig. 7E) and in gp120-treated cells was only slightly weaker after the triggering CD95 expression (Fig. 7F). No such effect was observed with 1 of CCR7 by CCL21 than in medium-treated cells. Similar results ␮M herbimycin. Inhibitors of PI3K (wortmannin), PLC (U73122), were obtained for stimulation by CCL20 (data not shown). PKC (bisindolylmaleimide I), 3-phosphoinositide-dependent kinase-1 (SH5), MEK1/2 (PD98059), and NF-␬B nuclear transloca- Treatment with gp120 decreases CD62L expression but tion (SN50) did not prevent the decrease in CD62L expression increases CD95 expression (data not shown), whereas an inhibitor of p38 MAPK (SB203580) We compared the expression of various markers on B cells cul- totally reversed this effect (Fig. 7E). None of these inhibitors tured for 2 h with medium or with 10 nM recombinant gp120 blocked the gp120-induced increase in CD95 expression (data not (SF162, BAL, SF2, MN). Whatever the gp120 used, the expression shown), whereas treatment with brefeldin A did (Fig. 7F). Prior in- of LFA-1 (CD11a/CD18), ICAM1 (CD54), CD5, CD10, CD80, cubation of gp120 with soluble CD4 inhibited the cleavage of CD62L CD86, CD23, CD38, CD44, CD27, SIgD, TACI, BCMA, and (Fig. 7E), but not the increase in CD95 expression (data not shown). BAFF receptor was not signiﬁcantly modiﬁed (data not shown). In As L-selectin is shed from the cell surface, we incubated B cells with contrast, treatment with X4 and X4/R5 gp120 decreased L-selectin MMP inhibitors before adding gp120. The addition of 500 ␮M (CD62L) expression by 66 Ϯ 12% (n ϭ 10) (Fig. 7A), increased EDTA, 10 ␮M GM6001, or 10 ␮M MMP-III inhibitor prevented 308 gp120 HIV-1 ENVELOPE IMPAIRS HUMAN B CELL CHEMOTAXIS

treated B cells were incubated for 24 h in the presence of 1 ␮g/ml B-G27 mAb, to determine whether CD95 was functional on gp120- treated B cells (Fig. 7H). The percentage of cells undergoing apoptosis increased from 35.3 Ϯ 2% in medium-treated B cells to 58.7 Ϯ 0.6% in gp120-treated B cells. The addition of 1.5 ␮g/ml 447-52D mAb to gp120 prevented the induction of apoptosis by gp120.

Discussion Lymphocyte trafﬁcking is crucial for immune surveillance in vivo, and requires a sequence of critical adhesion events enabling naive and memory cells to recirculate rapidly from the blood to the lymphoid organs (35). Circulating naive and memory B cells express L-selectin, which mediates the ﬁrst step, adhesion to high endothelial venules, and initiates extravasion toward the lymph nodes and spleen (36). The next step is controlled by G protein-linked chemokine receptors that activate integrins (37). CXCR4, a key receptor for B cell chemotaxis, also acts as a coreceptor for HIV-1 infection. Most mature B cells express CXCR4 strongly and CD4

and CCR5 weakly at their surface (38), but are not infected by Downloaded from HIV-1 in vivo. B cell phenotype and functions are nevertheless rapidly impaired after infection, suggesting that virions or viral proteins released by infected cells directly affect B cell response. As gp120 modulates the chemotaxis of monocytes, dendritic cells, CD4, and CD8 T cells (39–41), we investigated whether recom-

binant gp120 affected the B cell chemotaxis essential for a high- http://www.jimmunol.org/ quality, efficient humoral response. X4 and X4/R5 gp120 decreased, by 40–50%, not only B cell chemotaxis to CXCL12, but also that to CCL20 and CCL21, whereas neither CCR6 nor CCR7 bound gp120. The percentage of cells undergoing apoptosis was similar for medium- and gp120-treated B cells, indicating that the inhibition of B cell chemotaxis was not due to apoptosis. Consis- tent with the lower levels of CCR5 than CXCR4 on B cells, R5 gp120 decreased B cell chemotaxis only half as much as X4 or FIGURE 7. The gp120-induced down-regulation of CD62L expression X4/R5 gp120. R5 gp120 inhibited B cell chemotaxis although by guest on September 24, 2021 requires MMP and p38 MAPK. Surface CD62L (A), surface CD95 (B), and Ͻ10% of B cells migrated in response to CCL3, CCL4, and CCL5, cytoplasmic CD95 (C) expression were compared by flow cytometry on the main ligands of CCR5 (data not shown). Consistent with the medium-treated (gray histogram) or gp120MN-treated (open histogram, weak chemotactic response, the triggering of CCR5 by CCL5 ac- bold line) B cells. One experiment representative of five is shown. D, The tivated PLC␤3, PI3K/AKT, I␬B␣, and PKC␣, ␤I, ␤II, ␦, ⑀, and n modulation of surface CD62L and CD95 expression was compared by flow less strongly than did CXCL12. R5 gp120 did not activate these cytometry on medium- (Ⅺ), gp120 -(3), or gp120 plus 447-52D (u) MN MN effectors. In contrast, the triggering of CCR5 by CCL5 or R5 mAb-treated B cells. This experiment was conducted for three independent donors and results are expressed as the mean Ϯ SD MFI values. E and F, gp120 led to the activation of p38 MAPK, but not ERK1/2 (data B cells were incubated for1hat37°C with medium (0), 1 ␮M herbimycin not shown). These results suggest that CCR5 is functional. It is A (Herbi), 100 ng/ml PTX, 5 ␮g/ml AMD3100 (AMD), 10 ␮M SB203580 possible that the weak chemotaxis and cell signaling are due to the (SB), or 10 ␮g/ml brefeldin A (Bref). They were then incubated for 2 h low level of CCR5 expression on the surface of B cells. Blocking f o with medium ( ) or 10 nM gp120MN ( ). Alternatively, B cells were CXCR4 or CCR5 totally reversed the X4, X4/R5, and R5 gp120- z incubated for 2 h with soluble CD4 ( ) or soluble CD4-treated gp120MN induced inhibition of B cell chemotaxis. Similarly, blocking gp120 (Ⅺ). Cell surface expression of CD62L (E) and CD95 (F) was compared with the 447-52D mAb totally prevented the gp120-mediated in- by flow cytometry on medium- and gp120-treated B cells. One experiment hibition of B cell chemotaxis, whatever the gp120 tested. CD4 representative of three is shown. G, B cells were incubated for 2 h with signaling was dispensable for the gp120-induced inhibition of B medium (f) or 10 nM gp120 (o) in the presence of medium (0), MN cell chemotaxis, but prior incubation of gp120 with soluble CD4 DMSO, 500 ␮M EDTA, 10 ␮M MMP-2/9 inhibitor, 10 ␮M MMP-III, or 10 ␮M GM6001. Cell surface expression of CD62L was analyzed by flow enhanced its inhibitory effect. Thus, membrane CD4 molecules, cytometry. One experiment representative of three is shown. H, B cells despite being present in small numbers on B cells (42), may induce Ⅺ changes in the conformation of gp120, increasing its affinity for were incubated for 2 h, in the presence of medium ( ) or 10 nM gp120SF2 (X4/R5) (3), with or without 1.5 ␮g/ml 447-52D mAb. They were then CXCR4 (or CCR5) and its inhibition of B cell chemotaxis. In incubated for 24 h with 1 ␮g/ml B-G27 mAb and the proportion of cells in contrast, the gp120-induced inhibition of monocyte chemotaxis is apoptosis was determined. We conducted this experiment for three inde- strictly dependent on CD4 signaling (39). No decrease in CCR6 pendent donors and results are expressed as the mean percentage Ϯ SD of and CCR7 expression or impairment of CCL20- and CCL21-in- apoptotic cells obtained for each donor. duced chemokine receptor internalization accompanied the gp120- induced inhibition of B cell chemotaxis. Similarly, gp120 inhibited CXCL13-dependent chemotaxis in the absence of CXCR5 down- CD62L shedding (Fig. 7G). No such inhibition was observed with regulation or ligand-receptor internalization (data not shown). This 10 ␮M MMP-2/9 inhibitor or DMSO. These data are consistent contrasts with a previous study showing that the gp120-mediated with shedding being induced by MMP-1 and MMP-3 downstream inhibition of chemotactic response was correlated with weaker ␣ from CD4 and CXCR4/G i complexes. Medium- and gp120- chemokine receptor expression in monocytes (39). Toth et al. (43) The Journal of Immunology 309 showed that CXCR4 dimerization was involved in CXCL12- and does not induce conformational changes in CXCR4 similar to gp120-induced signaling events. Based on CXCR4-associated flu- those induced by CXCL12. The gp120-induced increase in surface ␣ orescence resonance energy transfer analysis, the authors sug- CD95 expression was totally dependent on CXCR4 and G i het- gested that gp120 might not fully reproduce signaling events nor- erotrimeric proteins and was induced by the relocalization of cy- mally induced by CXCL12. The dimerization of CXCR4 and toplasmic CD95 to the membrane by an unknown mechanism. The CCR5, leading to JAK2/3 activation, has been shown to play a role gp120-induced increase in surface CD95 expression was totally in chemotaxis in certain cells (44, 45) but, AG490, a potent inhib- prevented by anti-gp120 mAb. Such a redistribution of CD95 was itor of JAK2/3, did not affect B cell chemotaxis or its inhibition by recently reported in B lymphoma cell lines (55). Increased surface gp120 (data not shown). Thus, even if gp120 induces CXCR4 or CD95 expression was correlated with a 23% increase in the CD95- CCR5 aggregation in B cells, JAK activation is not required to mediated apoptosis of gp120-treated B cells, which was totally inhibit B cell chemotaxis. Consistent with previous data showing abolished by adding the neutralizing anti-gp120 447-52D mAb. that efficient entry of both X4 and R5 HIV-1 requires intact lipid Luciani et al. (56) recently showed that the gp120-induced sus- rafts in leukocytes (33, 46), the gp120-induced inhibition of F- ceptibility of human T cells to CD95-mediated apoptosis involves actin polymerization was abolished in cholesterol-depleted B cells, erzin activation and CD95/ezrin binding. Ezrin belongs to a family whereas the chemokine-induced response was unaffected. The of proteins connecting transmembrane proteins to the actin cy- gp120-mediated inhibition of chemokine-induced F-actin poly- toskeleton. It may therefore play a role in the gp120-mediated merization was stronger than the inhibition of chemotaxis (Ն70% increase in CD95 expression and CD95-mediated apoptosis in B vs 40–50% for X4 gp120). This weaker inhibition of chemotaxis cells. An increase in susceptibility to CD95 apoptosis may be in- is probably due to the re-expression at the membrane of gp120-free volved in the depletion of peripheral B cells reported by several Downloaded from CXCR4 during the assay as no gp120 was added to the upper groups in HIV-infected patients (3, 9, 10, 57). The gp120-induced chamber. Several studies have shown that gp120/CXCR4 and decrease in membrane CD62L expression was dependent on CD4, ␣ CXCL12/CXCR4 interactions lead to different signaling profiles in CXCR4 triggering, and on the activation of G i, p38 MAPK, and T cells and monocytes (47–49). We showed that X4 or X4/R5 its cleavage by MMP. As reported by Preece et al. (58), we showed gp120, unlike CXCL12, did not activate PLC␤3, PI3K/AKT, that collagenase (MMP-1) and stromelysin (MMP-3) were in-

ERK1/2, I␬B␣ or PKC␣, ␤I, ␤II, ␦, ⑀, and n in B cells. Never- volved in this cleavage, whereas gelatinase A (MMP-2) and B http://www.jimmunol.org/ theless, B cell stimulation with CXCL12 and X4 or X4/R5 gp120 (MMP-9) were not. The MMP-dependent cleavage of L-selectin led to p38 MAPK activation by a PTX-sensitive mechanism (data upon cell activation (59), L-selectin cross-linking (60) or CXCR4 not shown), suggesting that cell signaling downstream from triggering by HIV-1 virions (61), has been described before. It ␣ CXCR4/G i complexes is still partly functional. Similarly, the in- results from the dissociation of calmodulin from the serine-phos- hibition of CCL21- and CCL20-mediated B cell chemotaxis by phorylated cytoplasmic tail of L-selectin, leading to conforma- gp120 correlated with a blockade of the CCL20- and CCL21-me- tional changes in L-selectin exposing its cleavage site to MMP diated activation of PLC␤3, PKC, PI3K/AKT, I␬B␣ and ERK1/2, (62–64). Our data suggest that serine phoshorylation of CD62L by but not of p38 MAPK. The downstream chemokine receptor, p38 p38 MAPK, but not by PKC␣, ␤I, ␤II, ␥, ␦, and ⑀ is involved in MAPK may be phosphorylated via activation of the Sos/Ras/Rac its dissociation from calmodulin. The finding that gp120 decreases by guest on September 24, 2021 pathway by G␤␥ subunits, as previously described (50), or by CD62L expression and B cell chemotaxis reveals a novel mecha- PKC. As CXCR4 triggering by gp120 activated p38 MAPK more nism by which HIV-1 can impair the humoral B cell response. weakly than CXCL12 and did not initiate PKC activation, p38 MAPK is probably activated exclusively by the Sos/Ras/Rac path- Disclosures way after exposure to gp120. 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