The cytosolic free calcium in anti-μ-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores

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<ul><li><p>Eur. J . Immunol. 1987.17: 1323-1328 Free cytosolic CazC in human B cell activation 1323 </p><p>Bernard Dugas', Alphonse Calenda' , Jean-Frangois Delfraissy', Aim6 VazquezA, Jean-Francois Bach' and Pierre Galanaud' </p><p>Roussel-UCLAF', Romainville, INSERM U 25' and INSERM U 131, Paris and Department of Pathology, Brown UniversityA, Providence </p><p>The cytosolic free calcium in anti- y-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores* </p><p>The inositol phospholipid metabolism and the increase in cytosolic free Ca2+ concen- tration ([Ca2+Ii) into the cell are recognized as two important events in the anti-p- induced B cell activation. The anti+ stimulation caused the [3H]inositol incorporation and also a rapid increase in [Ca2+Ii from 85 nM to 285 nM. This signal returned to baseline a few minutes after stimulation. By using the fluorescent indicator quin-2 we demonstrated that this [Ca2+Ii uptake was derived part from extracellular medium and part from intracellular stores. Both EGTA (a calcium chelator) and TMB.8 (a drug which interferes with Ca" sequestration by smooth endoplasmic retiulum) partially suppressed the intracellular Ca2+ uptake and were fully inhibitory when added together. The role of Ca2+ from intracellular stores may also be evidenced in calcium- free experiments, or in permeabilized experiments using exogenous inositol 1,4,5- trisphosphate (IP3, the putative mobilizer of intracellular Ca"). Preventing the increase in [Ca2+Ii also prevents the apparition of early activation markers. These results are consistent with the hypothesis that the Ca2+ increase in B cells stimulated by anti-y is caused by the generation of IP3 during the phosphatidyl-inositol metabol- ism and also by the entry of extracellular Ca2' through the plasma membrane. </p><p>1 Introduction </p><p>Under appropriate conditions anti-y antibodies (Ab) can mimic the effects of antigen and activate B cells [l-61. Anti-y Ab cause resting B cells (Go stage) to enter and to progress through the GI phase of the cell cycle [7], increase the expres- sion of class I1 major histocompatibility complex and induce the membrane depolarization [8-111. Previous studies indicate that lymphocyte activation depends on the ability of cell sur- face IgM to trigger an increase in free cytoplasmic Ca2+ ions [ 12-16], phosphatidylinositol metabolism (leading to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) produc- tion) [ 17-20] and to induce the phospholipid/Ca2+-dependent protein kinase C (PK-C) activation [21-251. It has been dem- onstrated that to drive B cells from Go to G1, PK-C activity and extracellular and/or intracellular sources of Ca2+ would act synergistically [26-291. The early steps (Go to Gl) of B cell activation can be characterized by several intracytoplasmic changes leading to RNA and protein synthesis, expression of early activation markers and to cell size enlargement [30-351. Furthermore, there is conflicting data concerning the origin of Ca2+ (intra and/or extracelular) in mitogen (T or B)-induced [Ca2+Ii increase. Some studies implicate the extracellular Ca2+ [36, 371, others the Ca2+ derived from intracellular sources [38, 391 and others implicate both of the sources [40, 411. These discrepancies in results are not easily explained, but the </p><p>[I 61931 </p><p>* This work was supported by Grants from CNRN and Roussel- UCLAF. </p><p>Correspondence: Bernard Dugas, Departement des Biotechnologies, Roussel-UCLAF, 111 route de Noisy, F-93230 Romainville, France </p><p>Abbreviatons: IP,: Inositol 1,4,5-trisphosphate IP,: Inositol 1,4- bisphosphate PI: Inositol 1-phosphate [Ca*'],: Free cytosolic cal- cium concentration TMB-8: 8-(Diethy1amino)octyl 3,4,5-trimethoxy- benzoate HCl DAG: Diacylglycerol PK-C: Protein kinase C PBL: Peripheral blood lymphocytes mAb: Monoclonal antibody(ies) FCS: Fetal calf serum </p><p>use of different cell sources (normal T or B cells, lymphoma cells, transformed cells, etc.) and of different sources of mito- gens may be the reason for these discrepancies. In this study we have characterized the anti-y-induced [Ca2+Ii increase in detail, defining the contributions of extra and intracellular cal- cium in this [Ca2+Ii increase. We have shown that anti-y induced early increases in IP3, inositol 1,4-bisphosphate (IP2) and inositol 1-phosphate (IP1) reflecting the role of the phos- phatidyl-inositol metabolism in human B cell activation. We have also shown that the blockade of one or both of the sources of calcium prevented the early events in human B cell activation as defined by the expression of early activation mar- kers. </p><p>2 Materials and methods </p><p>2.1 Reagents and drugs </p><p>Insolubilized rabbit anti-p Ab (Biorad, Richmond, CA) or a soluble goat anti-y Ab (Cappel Laboratories, West Chester, PA) were used at various final concentrations (similar results were obtained with these 2 preparations). EGTA (Sigma, St. Louis, MO) was prepared as a solution of 10 mM with the pH adjusted to 7.2 TMB-8 [&amp;(diethylamino)octyl 3,4,5- trimethoxybenzoate-HCl] and rotenone were provided by Roussel-UCLAF. The acetoxymethyl ester of quin-2 was purchased from Calbiochem (La Jolla, CA) and was diluted from a dimethyl sulfoxide (DMSO) stock solution. [3H]Inositol (37 MBq/ml) was purchased from Amersham France (Les Ulis, France). </p><p>2.2 B cell preparation </p><p>Peripheral blood lymphocytes (PBL) from normal donors were separated by centrifugation on Ficoll-Hypaque (Phar- macia, Uppsala, Sweden). Monocytic and natural killer cell depletion were done according to Thiele et al. [42]. Briefly, PBL were treated with 10 mM L-eucine methyl ester (Sigma) </p><p>0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/0909-1323$02.5010 </p></li><li><p>1324 </p><p>for 45 min at room temperature and washed 3 times. B cells were isolated by two cycles of rosetting with aminoethyl- isothiouronium bromide hydrobromide-treated sheep erythro- cytes. These cells contained 97% sIg+ cells, </p></li><li><p>Eur. J. Immunol. 1987.17: 1323-1328 Free cytosolic Ca2+ in human B cell activation 1325 </p><p>these mAb was analyzed by indirect immunofluorescence using a fluorescein-conjugated goat anti-mouse Ab. Fluores- cence was evaluated by fluorescence microscopy. These studies were performed on 4F2- B cells (2 X lo6) cultured with anti-p (5 pglml) in the presence or in the absence of EGTA and/or TMB-8 or rotenone for 72 h. </p><p>3 Results </p><p>3.1 Anti-p induced the inositol phospholipid metabolism </p><p>Anti-p stimulation of resting B cells (4F2-) leads to an early dose-related increase in inositol phospholipid metabolism as revealed by the generation of IPI, IP2 and IP3 (Fig. 1A-C). </p><p>/ P </p><p>0 3 10 </p><p>1-J T I M E ( mi nut es) Figure 1. Effect of different concentrations of anti-p on the increases of [3H]inositol phosphates. The levels of tritiated IP3 (A), IP2 (B) and IP, (C) after the addition of various concentrations of anti-p 5 &amp;ml (0) and 1 yg/ml (V)] or of OKB7 [20 pdml (O)] are expressed in cpm. The indicated time points are the intervals from the addition of anti- body to the lysis of the cell in chloroform-methanol. The inositol phosphates were separated by anion-exchange chromatography as outlined in Sect. 2.7. Levels of [3H]inositol phosphates in the unstimu- lated cells were IP3, 547 cpm; IP,, 987 cpm; IP,, 652 cpm. These are representative of three different experiments. </p><p>5 lA1 1300 /**4 I </p><p>- Zmin </p><p>LO </p><p>-6193.21 Figure 2. Effect of different concentrations of anti-p on the fluores- cence of quin-2-loaded B cells. Cytosolic CaZ+ levels were determined as described in Sect. 2.5. Resting B cells (A) or anti-pactivated B cells (B) were incubated with various concentrations of anti-p (1 or 5 pgl ml). These data are representative of seven different experiments. </p><p>The levels of IP3, IP2 and IPI continued to rise during the first 10 min after the addition of anti-y Ab. By contrast the OKB7 mAb, directed against the CR2 antigen, did not induce under our assay conditions, the inositol phopspholipid metabolism. </p><p>3.2 Anti-p induced [Ca2+Ii change in human B cells </p><p>When anti-y was added, at a concentration of 5 pglml, to rest- ing B cells that had been loaded with 5 y~ quin-2, [Ca2'Ii rose (Fig. 2A) to a level of 275 nM and to a level of 125 nM at the anti-y concentration of 1 pg/ml. The signal returned halfway to base line after approximatively 3 min after the anti-p had been added. In similar conditions, anti-y also induced a dose- related [Ca2+Ii change on anti-y-activated B cells (Fig. 2B; 190 nM vs. 285 nM for the resting B cells). </p><p>3.3 Source of the calcium in the anti-p-induced [Ca2+]i change </p><p>In order to determine the source of the calcium, we examined the response of the cells to anti-y after removing residual cal- cium from medium with the calcium chelator EGTA, or by treating the cells with drugs that modify calcium release from intracellular stores. As shown in Table 1 both EGTA and TMB-8 partially inhibited the [Ca2+Ii change induced by anti- p, whereas when added together were fully inhibitory. In the same experiments when rotenone (mitochondria1 inhibitor) was added the anti-y-induced [Ca2+Ii change was not signifi- cantly decreased. </p><p>3.4 Purified IP3 released Ca2+ from permeabilized B cells </p><p>We verified that purified IP3 induced the release of Ca2+ from intracellular stores in B cells. B cells were permeabilized with saponin, and the [Ca"] was measured in the suspension with </p></li><li><p>Eur. J. Immunol. 1987.17: 1323-1328 1326 </p><p>Table 1. Drug effects on cytosolic calcium levels") </p><p>B. Dugas, A. Calenda, J.-F. Delfraissy et al. </p><p>4F7- B cells stimulated Treatment [ca"], (nM) with </p><p>Medium Medium 85 Anti-p Medium 285 Anti-p EGTA 135 Anti-p TMB-8 145 Anti+ EGTA + TMB-8 YO Anti-p Rotenone 280 </p><p>Cytosolic CaZ+ levels were determined as described in Sect. 2.5. 4F2- B cells were incubated in medium alone plus or minus rotenone (1 y ~ ) , EGTA (2 mM) and/or TMB-8 (50 p ~ ) for 15 min prior to the addition of anti-p (5 pg/ml). These data are representa- tive of five different experiments. </p><p>Figure 3. Purified IP3 releases CaZ+ from saponin-permeabilized 4 F T B cells. 4F2- B cells were permeabilized with saponin as described in Sect. 2.6. The free Ca2+ was evaluated with quin-2. IP3 was added from a 100 p~ stock solution to a final concentration of 1 p~ in the absence (a) or in the presence (b) of rotenone (1 p ~ ) or TMB-8 (50 VM) (c). This preparation of IP3 did not contain CaZ+ as determined in preliminary experiments. </p><p>quin-2. As shown in Fi 3 the addition of purified IP3 resulted in an increase of [Ca Ii. This change was inhibited in the presence of TMB-8 but not in the presence of rotenone. </p><p>!i </p><p>3.5 The blockade of [Ca2+Ii change prevents the apparation of early activation markers </p><p>We then examined the effect of EGTA, TMB-8 and rotenone on the expression of early activation markers defined by the 4F2 and the 5E9 mAb. Two x lo6 4F2- B cells were cultured in the presence of anti-ln. Ab with or without EGTA (2 mM), TMB-8 (50 yM) or rotenone (1 yM). After a 3-day culture the cells were recovered and analyzed for the percentage of 4F2' and 5E9' cells (Table 1). Anti-y Ab induced a marked increase in the number of 4F2' and 5E9' cells and this was inhibited by EGTA and by TMB-8 whereas rotenone had no effect in this system. By contrast when B cells were precul- tured for 12 h in the presence of anti-y Ab the addition of EGTA, TMB-8 or rotenone did not block the appearance of these activation markers (Table 2). </p><p>4 Discussion </p><p>It has previously been shown that several signals are required for B cell proliferation and differentiation [49-531. The </p><p>Table 2. Drug effect on early activation markers expressiona) </p><p>Fluorescent cells 4F2- B cells Treatment (% positive) stimulated with slg 4F2 SE9 </p><p>Medium Medium 9s &lt; 1 &lt; I Anti-p Medium 95 73 29 Anti-p EGTA 95 4 &lt; 1 Anti-p TMB-8 95 2 &lt; 1 </p><p>Anti-p Rotenonc 95 73 24 Anti-p EGTA+TMB-8 95 &lt; 1 &lt; 1 </p><p>a) 4F2- B cells were stimulated with anti-p (5 pg/ml), in the absence or in the presence of EGTA (2 mM) and/or TMB-8 (50 p ~ , or rotenone (1 p~), and then were stained with a fluorescein-conju- gated F(ab'), fragment of anti-human Ig, with 4F2 and 5E9 as described in Sect. 2.8. </p><p>Table 3. Drug effect on early activation markers expression after a 12- h preactivation of 4F2- B cells with anti-pa) </p><p>Fluorescent cells 4F2- B cells Treatment (% positive) stimulated with sIg 4F2 5EY </p><p>Medium Medium 95 &lt; 1 &lt; 1 Anti-p Medium 95 73 26 Anti-p EGTA 95 72 32 Anti-p TMB-8 95 76 22 Anti-p EGTA + TMB-8 YS 73 10 Anti-p Rotenone 95 71 23 </p><p>a) 4F2- B cells were prestimulated for 12 h with anti-p ( 5 pg/ml) and then EGTA (2 mM) and/or TMB-8 (50 p~), or Rotenone (1 p ~ ) were added to the culture until the end of the culture. The fluores- cence analysis was done after a 72-h culture as described in Sect. 2.8. </p><p>molecular events involved in the initiation of B or T cell activa- tion implicate early breakdown of phosphatidyl-inositol by phospholipase-C yielding inositol phosphates and DAG lead- ing to the PK-C activation [17-251. Physiological increases of [Ca2']i induce the translocation of PK-C from a soluble to a membrane-bound compartment [54, 551. The origin of cytosolic free calcium in mitogen-induced lymphocyte (T or B) transformation is subject to controversy. It has been recently demonstrated that to drive lymphocytes into the cell cycle, extracellular and/or intracellular calcium could be involved in the early events of the cell activation [56, 571. Different para- meters could be at the origin of this discrepancy in results: (a) different cell purification procedures, (b) the use of different type of cell activators, (c) the use of different cell sources (normal T or B cells, lymphoma cells, myeloma cells, hybridoma, transformed cells, etc.), and (d) the heterogeneity in the purified cell populations. The present study demon- strates that in an in viho system of B cell activation by anti-p sequential biochemical steps are involved in the induction of resting B cells to an activated cell. Anti-y Ab generate signifi- cant elevations in the level of IP3, IP2 and IPI. This induction of the phosphatidyl-inositol metabolism appears to be a specific effect of perturbation of the membrane IgM because a mAb directed against the CR2 antigen (OKB7) does not </p></li><li><p>Eur. J. Immunol. 1987.17: 1323-1328 Free cytosolic Ca2+ in human B cell activation 1327 </p><p>9 Monroe, J. G. and Cambier, J. C., J. Exp. Med. 1983.157: 2073. 10 Monroe, J. G. and Cambier, J. C., J. Immunol. 1983. 130: 626. 11 Cambier, J. C. and Monroe, J. G., J. Immunol. 1984. 133: 576. 12 Braun, J., Sha'afi, R. I. and Unanue, E. R., J. Cell. Biol. 1979.82: </p><p>13 Ramson, J . T. and Cambier, J. C., J. Immunol. 1986. 136: 66. 14 Ramson, J. T., Digiusto, D. L. and Cambier, J. C., J. Imrnunol. </p><p>15 Dugas, B., Vazquez, A., Delfraissy, J. F., Gerard, J. P., Rannou, </p><p>16 Paul, W. E., Mizuguchi, J., Brown, M., Nakanishi, K., Hornbeck, </p><p>17 Mishell, R. H. and Kirk, C. J., Trends Pharm. Sci. 1981.2: 86. 18 Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishiz...</p></li></ul>


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