Eur. J . Immunol. 1987.17: 1323-1328 Free cytosolic CazC in human B cell activation 1323
Bernard Dugas', Alphonse Calenda' , Jean-Frangois Delfraissy', Aim6 VazquezA, Jean-Francois Bach' and Pierre Galanaud'
Roussel-UCLAF', Romainville, INSERM U 25' and INSERM U 131, Paris and Department of Pathology, Brown UniversityA, Providence
The cytosolic free calcium in anti- y-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores*
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.
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 , 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
* This work was supported by Grants from CNRN and Roussel- UCLAF.
Correspondence: Bernard Dugas, Departement des Biotechnologies, Roussel-UCLAF, 111 route de Noisy, F-93230 Romainville, France
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
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.
2 Materials and methods
2.1 Reagents and drugs
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 [&(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).
2.2 B cell preparation
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. . Briefly, PBL were treated with 10 mM L-eucine methyl ester (Sigma)
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/0909-1323$02.5010
Eur. J. Immunol. 1987.17: 1323-1328 Free cytosolic Ca2+ in human B cell activation 1325
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.
3.1 Anti-p induced the inositol phospholipid metabolism
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).
0 3 10
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 &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.
5 lA1 1300 /**4 I
-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.
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.
3.2 Anti-p induced [Ca2+Ii change in human B cells
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).
3.3 Source of the calcium in the anti-p-induced [Ca2+]i change
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.
3.4 Purified IP3 released Ca2+ from permeabilized B cells
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