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Inositol 1,4,5-trisphosphate- and arachidonic acid-induced calcium mobilization in T and B lymphocytes
CELLULAR
IMMUNOLOGY
126,245-254
(1990)
lnositol 1,4,5TrisphosphateCalcium Mobilization
and Arachidonic Acid-Induced in T and B Lymphocytes’
Jo& CORADO,'FRAN~OISELE DEIST, CLAUDE GRISCELLI, AND ALAIN FISCHER Groupe de Recherched’Irnmunologie et de Rhumatologie Pediatriques, INSERM U 132, H6pital Necker-Enfants Malades, 149. rue de S&es, 75743 Paris Cidex 15, France Received June 26. 1989; accepted October 30, 1989 Inositol triphosphate (IP3) formation and increase in intracytoplasmic calcium are mediators of signal transduction in lymphocytes. It has been proposed that IP3 induces Ca2+release from intracellular stores. It is in order to study the relationship between these two events that we have analyzed the effect of IP3 addition on Ca’+ mobilization in permeabilized resting T and B lymphocytes, EBV-B lymphocytes, and HTLV 1-T lymphocytes. IP3 induces a rapid and significant release of Ca2+ from the endoplasmic reticulum in a dose-dependent manner. Ca2’ release is more sensitive to IP3 addition in cycling cells (EBV-B lymphocytes and HTLVI-T lymphocytes) than in resting T and B lymphocytes. Arachidonic acid (AA) induces Ca2+release from the endoplasmic reticulum (ER) in a manner similar to that of IP3. Neither component has an effect on Ca2+accumulated in mitochondria, and they have no additive effectssuggesting that they act on a similar Ca2+pool. These results directly demonstrate that in T and B human lymphocytes IP3 mobilizes Ca2+from ER as in other cellular systems and that other potential second messengers,namely AA, could play a significant role in the internal mobilization of calcium during T and B lymphocyte activation. o 1990 Academic PXS, IK.
INTRODUCTION Signal transduction in lymphocytes is associatedwith an increase in intracytoplasmic calcium and with inositol trisphosphate (IP3) formation as a product of phosphoinositol4,5 biphosphate (PIP2) breakdown (l-6). The magnitude and pattern of the IP3 response appears to vary according to lymphocyte subsetsand to the activation signals (7, 8). An increase in intracellular calcium concentration depends on two sources: calcium liberation from intracellular stores and calcium influx from extracellular spacesthrough Ca2+channels (7, 9, 10). The relationship between IP3 formation and calcium mobilization in lymphocytes has been studied by different experimental approaches. In human T cell lines (9) and murine B cell lines (7), exogenous IP3 addition led to calcium release from the endoplasmic reticulum as shown by using permeabilized cells. However, Bijsterbosch and Klauss have reported that IP3 formation and an increase of intracytoplasmic calcium in murine B cells may ’ This work is supported by INSERM and Fondation “Gran Mariscal Ayacucho.” ’ To whom correspondence should be addressed. 245 0008-8749/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
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not be related because Con A activation caused an increase in cytoplasmic calcium with minimal IP3 formation (11). It has been proposed that other messengersare required for cellular activation. For instance, there is much evidence to suggestthat arachidonic acid may regulate Ca2+ fluxes in different cells and play a role as a mediator in signal transduction (12, 13). Studies with digitonin permeabilized pancreas islets have shown that arachidonic acid (AA) induces a rapid and significant releaseof Ca*+ from the endoplasmic reticulum ( 12). In view of the above observations, we have studied both the effects of IP3 and arachidonic acid on Ca2+ mobilization in human resting and preactivated T and B lymphocytes by using a permeabilized cell system. MATERIALS AND METHODS Cell Preparations Peripheral blood from healthy donors was collected in preservative-free heparin. Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Hypaque gradient (Pharmacia-Fine Chemical, Uppsala, Sweden) (d 0.77) (14). T lymphocyte-enriched preparations (E+) were obtained by rosetting PBMC with neuraminidase (Behring Werke, Marburg, Germany; 250 U/O.5 X lo9 erythrocytes)-treated sheep red blood cells. Ef cells were recovered after centrifugation on a Ficoll-Hypaque gradient and red cells were lysed by using Gey’s solution as previously reported ( 15). Resting human B cells were prepared as follows. Dense B cells were isolated from tonsils. After squeezing the tonsils, adherent cells were removed by adhesion to culture plastic dishes. Nonadherent cells were depleted of T lymphocytes by rosette formation with neuraminidase sheep erythrocytes ( 15). Nonrosetting cells, (E-) recovered after sedimentation over a Ficoll-Hypaque gradient, were further separated on a discontinuous Percoll gradient in order to purify dense B lymphocytes (16). Cells recovered at the interface between the 50% and the 60% v/v Percoll concentration were collected. The population thus obtained contained 86% sIg-positive cells, and all cells were in Go phase of the cell cycle as determined by the study of the respective content of DNA and RNA as estimated with acridine orange technique, the mean cell volume, and the absence of activation antigen expression (17, 18, 19). In addition, dense B cell populations did not proliferate in response to rIL2 and to low molecular weight B cell growth factor (LMW-BCGF), these lymphokines being able to allow preactivated B cells to proliferate ( 17,20). An HTLVl-T cell line was obtained from a child with a smoldering T-cell lymphoma due to HTLVl (2 1). B cell lines were obtained by PBMC infection with the B-95 line supernatant (EBV-producing Marmoset cells) as described elsewhere (22). Cell Permeabilization and Ca2+Accumulation Assays The cell permeabilization technique was performed according to a modification of the method described by Ransom (7). After isolation, the cells were pelleted and then resuspended at lo7 cells/ml in a calcium-absent medium containing: KCI, 130 mM; NaCI, 5 mM; MgC&, 5 mM; Na2HP04, 10 mM; KH2P04, 1.4 mM; Diisopropylflu-
CALCIUM
MOBILIZATION
247
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a
A l(h
1
ld30 Time
FIG. I. Kinetic of calcium accumulation in permeabilized T cells. Accumulation was performed with %a’+ alone (660 ti) (A), with 45Ca2+(660 nM) + MgATP (5 mM) (O), and with 45Ca2+(660 nM) + IMCA + MgATP (5 mM) (m). Data represent means f 1 SD of three experiments.
orophosphate (DIP) (Sigma, St Louis, MO), 2 mM; BSA, 0.2%; Saponin, 50 pg/ml (Sigma, St Louis, MO) and EGTA, 1 mM (Sigma, St Louis, MO) at 37°C. After a 6min incubation, the cells were pelleted and resuspended in the same medium without saponin and DFP. The cells were then transferred to a clean tube in order to minimize any possible carryover of residual saponin. Judged by trypan blue exclusion, 90% of cells were permeabilized. The cells were washed and brought up to 5 X 106/ml in a solution containing KCl, 130 mM; NaCl, 5 mM; MgC&, 3 mM; KH2P04, 1.4 mM; Na2HP04, 10 mkf; and 660 I1M of 45Ca2+concentration (Amersham; sp activ: 28.6 mCi/mg). Cells were pretreated for IO min with inhibitors of mitochondria calcium accumulation (IMCA): oligomycin (phosphorylation inhibition), 10 PM (Sigma, St Louis, MO); Rotenone (oxidation inhibitor), 0.5 pg/ml; and dinitrophenol (uncouplant oxidative phosphorylation), 0.5 mM (Prolabo, Vitry-sur-Seine) and/or inhibitor of endoplasmic reticulum (ER) calcium accumulation (IERCA) sodium orthovanadate, 2 mM (Aldrich, Strasbourg, France). MgATP, 5 mM (Sigma, St Louis, MO) and an ATP-regenerating system consisting of creatine phosphate, 5 mM (Sigma, St Louis, MO) and creatine kinase, 15 UI/ml (Sigma, St Louis, MO) were added for a 1-hr incubation at 37°C. The induction of Ca*+ release by IP3 (Sigma, Purity: 98%), AA (Sigma, Purity: 99%, diluted in Na2C03, 0.1 M and NaCl, 0.15 M), and calcium ionophore (Sigma) was performed as follows: aliquoted permeabilized cells were preloaded with 45Ca2+ and the appropriate concentration of IP3 or/and AA or CI was added to each cell. The samples were set for determinated times (0, 20 min, 1 hr, 3 hr) starting with the sample to be terminated last. At the appropriate times the cells were diluted fourfold with ice-cold termination buffer (containing: KCl, 130 mM; NaCl, 5 mM) and immediately harvested on GF/C glass fiber filters (Whatman, England). The filters were then placed in a glass scintillation vial and radioactivity is counted after 18 hr on a
248
CORADO ET AL.
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FIG. 2. IP3 induces Ca*+ release from resting T and B lymphocytes. Cells were preloaded with 45Ca2+ (660 ti) for 1 hr in the presence of MgATP (5 mM) alone; or MgATP (5 mM) + IMCA or MgATP (5 mM) + IMCA + IP3 or MgATP (5 mA4) + IERCA or MgATP (5 mM) + IERCA + IP3 or MgATP (5 n-N) + calcium ionophore (5 PM). IP3 was added for 1 min. Calcium ionophore (10 pn,l) induced Ca2+ release is shown as control. ‘%a*+ contents were determined. (A) The data are the mean + 1 SD of five experiments performed on resting T lymphocytes. (B) The data are the mean f 1 SD ofthree experiments performed on resting B lymphocytes.
minaxi tri-carb 4000 series (Packard). The percentage of releasedcalcium was calculated as follows: 45Ca2faccumulated in presence of IMCA - 45Ca2+cell contents after IP3 and/or AA addition x 1o. 45Ca2+accumulated in the presence of IMCA RESULTS
Calcium Accumulation in Permeabilized Cells Figure 1 shows that ATP-dependent calcium accumulation in intracellular compartments of T lymphocytes was maximal after a 45min incubation when a steady state had been reached. The kinetic was similar in the presence of IMCA. Calcium accumulation in the absenceof MgATP was negligible. Similar results were observed in activated T and B lymphocytes (data not shown). Figure 2 shows that IMCA reduced calcium accumulation by 2 1% for resting T lymphocytes and 15.5% for resting B lymphocytes. The mitochondria-insensitive Ca2+ uptake presumably accounts for ATP-dependent accumulation by the endo-
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20”
1’
2’
3’
Time
FIG. 3. Kinetic of Ca2+mobilization under IP3 effect. Resting T lymphocytes (-), resting B lymphocytes (-----), HTLVI-T lymphocytes (CCGX), and EBV-B lymphocytes (orno)were preloaded with 4SCa2f(660 nM) in the presence of MgATP (5 mJ4) and IMA. IP3 (10 pLM)was added for various times and 4sCa2+ contents were determined. Data is representative of one experiment.
plasmic reticulum (ER) and nonspecific binding of 45Ca2+to the intracellular matrix. This was confirmed by the reduction of calcium accumulation in the presence of sodium vanadate. These results also suggest that an intact ATP-dependent Ca2+ pump is present on the ER of the permeabilized cells. Association of IMCA and IERCA blocked 100%Ca2+uptake (data not shown). IP3-Induced Calcium Release We have tested whether or not IP3 could induce the release of calcium from ER stores. Figure 2 shows that IP3 caused a release of calcium accumulated in the ER both in T and B lymphocytes asjudged after a I-min incubation with IP3. Evidence for the ER origin of calcium releasewas provided by the fact that IP3 failed to induce Ca2+ release in the presence of sodium vanadate. As expected, calcium ionophore was able to trigger Ca2’ from both ER and mitochondria. IP3 action was rapid (Fig. 3): 20 set after IP3 addition a significant percentage of Ca2+release has already occurred. Maximal release was observed after 1 min for all
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IP3(,%M) FIG. 4. Concentration-dependence of IP3-induced Ca’+ release. Resting T lymphocytes (&I), resting B lymphocytes (0) HTVI-T lymphocytes (0), and EBV-B lymphocytes (+) were preloaded with %a’+ (660 nM) in presenceof MgATP (5 mM) and IMCA. IP3 was added at indicated concentrations for 1 min and the percentage of releasedcalcium was calculated. Data represent means f 1 SD of five (resting T cells) or three (resting B cells-EBV-B lymphocytes and HTLVI-T lymphocytes) individual determinations.
cells studied. Only in the resting B lymphocytes was calcium reaccumulation observed: this occurred 3 min after IP3 addition. There was no Ca reaccumulation in other cells. Previous results (data not shown) with different Ca2+concentrations (220 nM to 1100 nM) demonstrated that IP3 was most efficient at 660 nM. As shown in Figure 4, there was a dose-dependent IP3-induced Ca*+ releaseup to a plateau observed with 10 PM to 20 PM of IP3 concentration in T lymphocytes following a I-min incubation with IP3. A 25% release from the endoplasmic reticulum was induced with a comparable concentration of IP3 in resting T and B lymphocytes (0.9 PM and 0.5 PM, respectively). For the cell lines (EBV-B and HTLVl). A 25% release from endoplasmic reticulum was obtained with 0.06 PM and 0.07 PM, respectively. Arachidonic Acid-Induced Ca” Release Liberation of calcium accumulated in the ER was observed following incubation with AA. Figure 5 shows that AA induced into the tested cells releasedCa2+in a dose-
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100 90 80 70 60 50 40 30 20 10 0
ARACHIDONIC
ACIDQPI)
FIG. 5. Concentration-dependence of arachidonic acid-induced Ca*+ release. Resting T lymphocytes (8) resting B lymphocytes (9, and EBV-B lymphocytes (+) were preloaded with %a*+ (660 nM) in the presenceof MgATP (5 mJ4) and IMCA. Arachidonic acid was added at mentioned concentration for 1min and the percentage of releasedcalcium was calculated. Data represent means + I SD ofthree experiments.
dependent manner, steady state being achieved in resting T cells, with AA concentration higher than 10 PM. A 25% releasefrom the endoplasmic reticulum was obtained with 0.3 PM AA concentration in T lymphocytes, 0.3 PM in resting B cells, and 0.3 PM in EBV-B cell lines. AA had no effect on Ca*+ accumulated in mitochondrias (data not shown). IP3 and AA comparably triggered Ca2’ releasefrom the ER, however both agents tested at optimal concentration exerted no additional effect (Fig. 6). DISCUSSION Previous studies using various permeabilized cell preparations-i.e., exocrine pancreas (23, 24, 25), liver (26, 27) pituitary cells (28), Swiss3T3 cells (29), and human neutrophils (30)-have demonstrated that IP3 mobilizes Ca2+from an intracellular pool identified as the endoplasmic reticulum by Prentki et al. (31). In T cell lines (Jurkatt) (9) and murine B cells (7), IP3 mobilizes Ca*’ from nonmitochondrial stores. However, these cells may not be representative of the physiological events occurring in resting cell populations and there is no direct evidence regarding IP3 action on Ca*+ mobilization in human T and B lymphocytes. We herein report that following cellular permeabilization, lymphocytes organelles are functional as demonstrated by an accumulation of 45Ca2+in the presence of
CORADO ET AL.
FIG.6. Comparison of IP3- and arachidonic acid-induced Ca’+ release.Cells were preloaded with %a2 for 1 hr in the presence of MgATP (5 mM) and IMCA. IP3 (10 PM) and/or arachidonic acid (10 PM) was added for I min and the percentage of released calcium was calculated. (A) Data represent means f 1 SD of five experiments performed on resting T lymphocytes. (B) Data represent means + 1 SD of three experiments performed on resting B lymphocytes.
MgATP. The vesicular localization of accumulated Ca2+was verified by the utilization of calcium ionophore which induced a 100%liberation of accumulated calcium by neutralizing the ionic charge of calcium allowing its mobilization through membranes. The accumulation of 45Ca2’is maximal after 45 min incubation either in the presence of ATP only or in the presenceof mitochondria inhibitors or vanadate. Ransom et al. (7) have indicated that, in murine B cells, the mitochondria calcium pool represents 62% and the endoplasmic reticulum pool 2 1% of accumulated calcium. In our system, the mitochondria calcium pool represents 2 1%and 15.5%and the endoplasmic reticulum pool 8 1.6%and 90% of accumulated calcium for T and B cells, respectively. These differences could be due to the use of lower Ca2+concentrations than those used by Ransom et al. (7). Burgesset al. (26) have demonstrated that the mitochondria calcium pool is directly correlated to the Ca2+concentration in medium. IP3 induces a rapid release of Ca*+ stored in ER both in T and B lymphocytes, whether the cells are resting or preactivated. Ca2+cannot be releasedfrom mitochondrias. These data are very similar to those obtained in other cell systems, namely in Jurkatt cells, murine B cells, and human neutrophils (7, 9, 30). However, we did not observe Ca2+ reaccumulation after 3 min (except in resting B lymphocytes) as previously described by other authors (7, 25, 27). In various cells, the rate of Ca*+ reuptake following IP3 addition appears to correlate with IP3 degradation (25, 27).
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Assuming that Ca2+reuptake following IP3 addition is indeed due to IP3 degradation, our results demonstrate that IP3 degradation depends on experimental conditions rather than on the nature of the cellular system used. Activated cells (EBV-B lymphocytes and HTLV 1-T lymphocytes) seemto be more sensitive than resting cells. The possible relationship between the preactivation state of HTLV 1-T and EBV-B cells and these results is unclear. The percentage of calcium releasein human T and B lymphocytes using 10 /.J4 of IP3 is higher than in other cellular systems (7, 24, 25, 27, 28, 29, 30, 31, 32). These differences may be due to cell types and to experimental conditions. It is impossible to correlate the physiological IP3 concentration after lymphocyte stimulation and its effect on Ca mobilization, since no data on estimations of intracellular levels of IP3 in stimulated lymphocytes are available. Several authors (12, 13, 33) have suggestedthat arachidonic acid plays a role as a second messengerin cellular activation. We observed that AA induces releaseof Ca2+ accumulated in the ER in a manner similar to that of IP3. There are no very significant differences in the three cell types studied in the presence of AA, as observed by the 25% of Ca*’ releasein opposition to the results observed with IP3. Comparing our data with those reported by Wolf et al. ( 12) we observed that a 10 @4concentration of AA induces a calcium releasefrom the ER 2.5- to 3.5-fold higher than the one found by these authors in rat pancreatic islets. Furthermore, these authors described additive effectsbetween IP3 and AA and they have suggestedthat the targets of these two components were two different calcium pools. In contrast, our results suggestthe action of these components on a similar calcium pool. It is possible that this characteristic is specific to haematopoietic cells. In conclusion, it has been demonstrated that IP3 formation and Ca2+mobilization are highly related in human T and B lymphocytes and that arachidonic acid could play a role in transducing activation signals in these cells by inducing Ca2+mobilization. ACKNOWLEDGMENT We are grateful Ms. Danielle Bresson for her excellent secretarial assistance.
REFERENCES I. OFlynn, K., Zanders, E., Lamb, J., Beverley, P., Wallace, D., Thatan, P., Tax, W., and Linch, D., Eur. J. Immunol. 15, I, 1985. 2. Weiss, A., Imboden, J., Shoback, D., and Stobo, J., P.N.A.S. 81,4169, 1984. 3. Weiss, M., Daley, J., Hodgon, J., and Reinherz, E., P.N.A.S. 81,6836, 1984. 4. Shapiro, D., Adams, B., and Niederhbuber, J., J. Immunol. 135,2256, 1985. 5. Pozzan, T., Arslan, R., Tisen, R., and Pink, T., J. Cell. Biol. 94, 335, 1982. 6. Coggeshall, K., and Cambier, J., J. Immunol. 133,33827, 1984. 7. Ransom, J., Harris, L., and Cambier, J., J. Immunol. 137,708, 1986. 8. Imboden, J., Weyand, C., and Goronzy, J., J. Immunol. 138, 1322. 1987. 9. Imboden, J., and Stobo, J., J. Exp. Med. 161,446, 1985. 10. Kruno, M., Goronzy, J., Weyand, C., and Gardner, P., Nature (London) 323,229, 1986. 11. Bijsterbosch, M., and Klauss, G., J. Immunol. 137, 1294, 1986. 12. Wolf, B., Turk, J., Sherman, W., and McDaniel, M., J. Biol. Chem. 261,3501, 1986. 13. Volpi, M., Yassin, R., Tao, W., Molski, T., Naccache, P., and Sha’afi, R., P.N.A.S. 81,5966, 1984. 14. Boyurn, A., &and. J. Clin. Lab. Invest. (suppl. 97) 21,245, 1968. 15. Fischer, A., Durandy, A., and Griscelli, C., J. Immunol. 126, 1452, 198 I. 16. Dagg, M., and Levitt, D., Clin. Immunol. Immunoputhol. 21,39, 198 1.
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17. Kehrl, J., Muraguchi, A., and Fauci, A., Eur. J. Immunol. 14, 115, 1984. 18. Suzuki, T., Sanders, S. K., Butler, J. L., Gartland, G. L., and Cooper. M. D., J. Immune/. 137, 1208, 1986. 19. Darzynkiewicz, Z., Sharpless,T., Staiano-Coico, L., and Melamed, M. R., Proc. Nat/. Acad. Sci. USA 77,6696, 1980. 20. Suzuki, T., and Cooper, M., J. Immunol. 134,3111, 1985. 2 1. Vilmer, E., Le Deist, F., Fischer, A., Griscelli, C., Nezelof, CH., De Prost, Y., and Prieur, M., Lancel 7, 1301, 1985. 22. Dimanche, M. T., Le Deist, F., Fischer, A., Amaout, M., Griscelli, C., and Lisowska-Grospierre, B., Eur. J. Immunol. 17,417, 1987. 23. Streb, H., Irvine, R., Berridge, M., and Schulz, I., Nature (London) 306,67, 1983. 24. Wolf, B., Comens, P., Ackermann, K., Shermann, W., and McDaniel, M., Biochem. J. 227,965, 1985. 25. Biden, T., Prentki, M., Irvine, R., Benidge, M., and Wollheim, C., Biochem. J. 223,467, 1984. 26. Burgess, W., McKinney, J., Fabiato, A., Leslie, B., and Putney, J., Jr., J. Biol. Chem. 258, 15,336, 1983. 27. Joseph, S., Thomas, A., Williams, R., Irvine, R., and Williamson, J., J. Biol. Chem. 259,3077, 1984. 28. Gershengorn, M., Geras, E., Spina Porrello, V., and Rebecchi, M., J. Biol. Chem. 259, 10,675, 1984. 29. Bet-ridge,M., Heslop, J., Irvine, R., and Brown, K., Biochem. J 222, 195, 1984. 30. Prentki, M., Wollheim, C., and Lew, P.. J. Biol. Chem. 259, 13777, 1984. 3 1. Prentki, M., Biden, T., Janjic, D., Irvine, R., Benidge, M., and Wollheim, C., Nature (London) 309, 562, 1984. 32. Irvine, R., Brown, K., and Berridge, J., Biochem. J. 221,269, 1984. 33. Hokin, L., Annu. Rev. Biochem. 54,205, 1985.
IMMUNOLOGY
126,245-254
(1990)
lnositol 1,4,5TrisphosphateCalcium Mobilization
and Arachidonic Acid-Induced in T and B Lymphocytes’
Jo& CORADO,'FRAN~OISELE DEIST, CLAUDE GRISCELLI, AND ALAIN FISCHER Groupe de Recherched’Irnmunologie et de Rhumatologie Pediatriques, INSERM U 132, H6pital Necker-Enfants Malades, 149. rue de S&es, 75743 Paris Cidex 15, France Received June 26. 1989; accepted October 30, 1989 Inositol triphosphate (IP3) formation and increase in intracytoplasmic calcium are mediators of signal transduction in lymphocytes. It has been proposed that IP3 induces Ca2+release from intracellular stores. It is in order to study the relationship between these two events that we have analyzed the effect of IP3 addition on Ca’+ mobilization in permeabilized resting T and B lymphocytes, EBV-B lymphocytes, and HTLV 1-T lymphocytes. IP3 induces a rapid and significant release of Ca2+ from the endoplasmic reticulum in a dose-dependent manner. Ca2’ release is more sensitive to IP3 addition in cycling cells (EBV-B lymphocytes and HTLVI-T lymphocytes) than in resting T and B lymphocytes. Arachidonic acid (AA) induces Ca2+release from the endoplasmic reticulum (ER) in a manner similar to that of IP3. Neither component has an effect on Ca2+accumulated in mitochondria, and they have no additive effectssuggesting that they act on a similar Ca2+pool. These results directly demonstrate that in T and B human lymphocytes IP3 mobilizes Ca2+from ER as in other cellular systems and that other potential second messengers,namely AA, could play a significant role in the internal mobilization of calcium during T and B lymphocyte activation. o 1990 Academic PXS, IK.
INTRODUCTION Signal transduction in lymphocytes is associatedwith an increase in intracytoplasmic calcium and with inositol trisphosphate (IP3) formation as a product of phosphoinositol4,5 biphosphate (PIP2) breakdown (l-6). The magnitude and pattern of the IP3 response appears to vary according to lymphocyte subsetsand to the activation signals (7, 8). An increase in intracellular calcium concentration depends on two sources: calcium liberation from intracellular stores and calcium influx from extracellular spacesthrough Ca2+channels (7, 9, 10). The relationship between IP3 formation and calcium mobilization in lymphocytes has been studied by different experimental approaches. In human T cell lines (9) and murine B cell lines (7), exogenous IP3 addition led to calcium release from the endoplasmic reticulum as shown by using permeabilized cells. However, Bijsterbosch and Klauss have reported that IP3 formation and an increase of intracytoplasmic calcium in murine B cells may ’ This work is supported by INSERM and Fondation “Gran Mariscal Ayacucho.” ’ To whom correspondence should be addressed. 245 0008-8749/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
246
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ET AL.
not be related because Con A activation caused an increase in cytoplasmic calcium with minimal IP3 formation (11). It has been proposed that other messengersare required for cellular activation. For instance, there is much evidence to suggestthat arachidonic acid may regulate Ca2+ fluxes in different cells and play a role as a mediator in signal transduction (12, 13). Studies with digitonin permeabilized pancreas islets have shown that arachidonic acid (AA) induces a rapid and significant releaseof Ca*+ from the endoplasmic reticulum ( 12). In view of the above observations, we have studied both the effects of IP3 and arachidonic acid on Ca2+ mobilization in human resting and preactivated T and B lymphocytes by using a permeabilized cell system. MATERIALS AND METHODS Cell Preparations Peripheral blood from healthy donors was collected in preservative-free heparin. Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Hypaque gradient (Pharmacia-Fine Chemical, Uppsala, Sweden) (d 0.77) (14). T lymphocyte-enriched preparations (E+) were obtained by rosetting PBMC with neuraminidase (Behring Werke, Marburg, Germany; 250 U/O.5 X lo9 erythrocytes)-treated sheep red blood cells. Ef cells were recovered after centrifugation on a Ficoll-Hypaque gradient and red cells were lysed by using Gey’s solution as previously reported ( 15). Resting human B cells were prepared as follows. Dense B cells were isolated from tonsils. After squeezing the tonsils, adherent cells were removed by adhesion to culture plastic dishes. Nonadherent cells were depleted of T lymphocytes by rosette formation with neuraminidase sheep erythrocytes ( 15). Nonrosetting cells, (E-) recovered after sedimentation over a Ficoll-Hypaque gradient, were further separated on a discontinuous Percoll gradient in order to purify dense B lymphocytes (16). Cells recovered at the interface between the 50% and the 60% v/v Percoll concentration were collected. The population thus obtained contained 86% sIg-positive cells, and all cells were in Go phase of the cell cycle as determined by the study of the respective content of DNA and RNA as estimated with acridine orange technique, the mean cell volume, and the absence of activation antigen expression (17, 18, 19). In addition, dense B cell populations did not proliferate in response to rIL2 and to low molecular weight B cell growth factor (LMW-BCGF), these lymphokines being able to allow preactivated B cells to proliferate ( 17,20). An HTLVl-T cell line was obtained from a child with a smoldering T-cell lymphoma due to HTLVl (2 1). B cell lines were obtained by PBMC infection with the B-95 line supernatant (EBV-producing Marmoset cells) as described elsewhere (22). Cell Permeabilization and Ca2+Accumulation Assays The cell permeabilization technique was performed according to a modification of the method described by Ransom (7). After isolation, the cells were pelleted and then resuspended at lo7 cells/ml in a calcium-absent medium containing: KCI, 130 mM; NaCI, 5 mM; MgC&, 5 mM; Na2HP04, 10 mM; KH2P04, 1.4 mM; Diisopropylflu-
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247
IN T AND B LYMPHOCYTES
a
A l(h
1
ld30 Time
FIG. I. Kinetic of calcium accumulation in permeabilized T cells. Accumulation was performed with %a’+ alone (660 ti) (A), with 45Ca2+(660 nM) + MgATP (5 mM) (O), and with 45Ca2+(660 nM) + IMCA + MgATP (5 mM) (m). Data represent means f 1 SD of three experiments.
orophosphate (DIP) (Sigma, St Louis, MO), 2 mM; BSA, 0.2%; Saponin, 50 pg/ml (Sigma, St Louis, MO) and EGTA, 1 mM (Sigma, St Louis, MO) at 37°C. After a 6min incubation, the cells were pelleted and resuspended in the same medium without saponin and DFP. The cells were then transferred to a clean tube in order to minimize any possible carryover of residual saponin. Judged by trypan blue exclusion, 90% of cells were permeabilized. The cells were washed and brought up to 5 X 106/ml in a solution containing KCl, 130 mM; NaCl, 5 mM; MgC&, 3 mM; KH2P04, 1.4 mM; Na2HP04, 10 mkf; and 660 I1M of 45Ca2+concentration (Amersham; sp activ: 28.6 mCi/mg). Cells were pretreated for IO min with inhibitors of mitochondria calcium accumulation (IMCA): oligomycin (phosphorylation inhibition), 10 PM (Sigma, St Louis, MO); Rotenone (oxidation inhibitor), 0.5 pg/ml; and dinitrophenol (uncouplant oxidative phosphorylation), 0.5 mM (Prolabo, Vitry-sur-Seine) and/or inhibitor of endoplasmic reticulum (ER) calcium accumulation (IERCA) sodium orthovanadate, 2 mM (Aldrich, Strasbourg, France). MgATP, 5 mM (Sigma, St Louis, MO) and an ATP-regenerating system consisting of creatine phosphate, 5 mM (Sigma, St Louis, MO) and creatine kinase, 15 UI/ml (Sigma, St Louis, MO) were added for a 1-hr incubation at 37°C. The induction of Ca*+ release by IP3 (Sigma, Purity: 98%), AA (Sigma, Purity: 99%, diluted in Na2C03, 0.1 M and NaCl, 0.15 M), and calcium ionophore (Sigma) was performed as follows: aliquoted permeabilized cells were preloaded with 45Ca2+ and the appropriate concentration of IP3 or/and AA or CI was added to each cell. The samples were set for determinated times (0, 20 min, 1 hr, 3 hr) starting with the sample to be terminated last. At the appropriate times the cells were diluted fourfold with ice-cold termination buffer (containing: KCl, 130 mM; NaCl, 5 mM) and immediately harvested on GF/C glass fiber filters (Whatman, England). The filters were then placed in a glass scintillation vial and radioactivity is counted after 18 hr on a
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FIG. 2. IP3 induces Ca*+ release from resting T and B lymphocytes. Cells were preloaded with 45Ca2+ (660 ti) for 1 hr in the presence of MgATP (5 mM) alone; or MgATP (5 mM) + IMCA or MgATP (5 mM) + IMCA + IP3 or MgATP (5 mA4) + IERCA or MgATP (5 mM) + IERCA + IP3 or MgATP (5 n-N) + calcium ionophore (5 PM). IP3 was added for 1 min. Calcium ionophore (10 pn,l) induced Ca2+ release is shown as control. ‘%a*+ contents were determined. (A) The data are the mean + 1 SD of five experiments performed on resting T lymphocytes. (B) The data are the mean f 1 SD ofthree experiments performed on resting B lymphocytes.
minaxi tri-carb 4000 series (Packard). The percentage of releasedcalcium was calculated as follows: 45Ca2faccumulated in presence of IMCA - 45Ca2+cell contents after IP3 and/or AA addition x 1o. 45Ca2+accumulated in the presence of IMCA RESULTS
Calcium Accumulation in Permeabilized Cells Figure 1 shows that ATP-dependent calcium accumulation in intracellular compartments of T lymphocytes was maximal after a 45min incubation when a steady state had been reached. The kinetic was similar in the presence of IMCA. Calcium accumulation in the absenceof MgATP was negligible. Similar results were observed in activated T and B lymphocytes (data not shown). Figure 2 shows that IMCA reduced calcium accumulation by 2 1% for resting T lymphocytes and 15.5% for resting B lymphocytes. The mitochondria-insensitive Ca2+ uptake presumably accounts for ATP-dependent accumulation by the endo-
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FIG. 3. Kinetic of Ca2+mobilization under IP3 effect. Resting T lymphocytes (-), resting B lymphocytes (-----), HTLVI-T lymphocytes (CCGX), and EBV-B lymphocytes (orno)were preloaded with 4SCa2f(660 nM) in the presence of MgATP (5 mJ4) and IMA. IP3 (10 pLM)was added for various times and 4sCa2+ contents were determined. Data is representative of one experiment.
plasmic reticulum (ER) and nonspecific binding of 45Ca2+to the intracellular matrix. This was confirmed by the reduction of calcium accumulation in the presence of sodium vanadate. These results also suggest that an intact ATP-dependent Ca2+ pump is present on the ER of the permeabilized cells. Association of IMCA and IERCA blocked 100%Ca2+uptake (data not shown). IP3-Induced Calcium Release We have tested whether or not IP3 could induce the release of calcium from ER stores. Figure 2 shows that IP3 caused a release of calcium accumulated in the ER both in T and B lymphocytes asjudged after a I-min incubation with IP3. Evidence for the ER origin of calcium releasewas provided by the fact that IP3 failed to induce Ca2+ release in the presence of sodium vanadate. As expected, calcium ionophore was able to trigger Ca2’ from both ER and mitochondria. IP3 action was rapid (Fig. 3): 20 set after IP3 addition a significant percentage of Ca2+release has already occurred. Maximal release was observed after 1 min for all
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IP3(,%M) FIG. 4. Concentration-dependence of IP3-induced Ca’+ release. Resting T lymphocytes (&I), resting B lymphocytes (0) HTVI-T lymphocytes (0), and EBV-B lymphocytes (+) were preloaded with %a’+ (660 nM) in presenceof MgATP (5 mM) and IMCA. IP3 was added at indicated concentrations for 1 min and the percentage of releasedcalcium was calculated. Data represent means f 1 SD of five (resting T cells) or three (resting B cells-EBV-B lymphocytes and HTLVI-T lymphocytes) individual determinations.
cells studied. Only in the resting B lymphocytes was calcium reaccumulation observed: this occurred 3 min after IP3 addition. There was no Ca reaccumulation in other cells. Previous results (data not shown) with different Ca2+concentrations (220 nM to 1100 nM) demonstrated that IP3 was most efficient at 660 nM. As shown in Figure 4, there was a dose-dependent IP3-induced Ca*+ releaseup to a plateau observed with 10 PM to 20 PM of IP3 concentration in T lymphocytes following a I-min incubation with IP3. A 25% release from the endoplasmic reticulum was induced with a comparable concentration of IP3 in resting T and B lymphocytes (0.9 PM and 0.5 PM, respectively). For the cell lines (EBV-B and HTLVl). A 25% release from endoplasmic reticulum was obtained with 0.06 PM and 0.07 PM, respectively. Arachidonic Acid-Induced Ca” Release Liberation of calcium accumulated in the ER was observed following incubation with AA. Figure 5 shows that AA induced into the tested cells releasedCa2+in a dose-
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100 90 80 70 60 50 40 30 20 10 0
ARACHIDONIC
ACIDQPI)
FIG. 5. Concentration-dependence of arachidonic acid-induced Ca*+ release. Resting T lymphocytes (8) resting B lymphocytes (9, and EBV-B lymphocytes (+) were preloaded with %a*+ (660 nM) in the presenceof MgATP (5 mJ4) and IMCA. Arachidonic acid was added at mentioned concentration for 1min and the percentage of releasedcalcium was calculated. Data represent means + I SD ofthree experiments.
dependent manner, steady state being achieved in resting T cells, with AA concentration higher than 10 PM. A 25% releasefrom the endoplasmic reticulum was obtained with 0.3 PM AA concentration in T lymphocytes, 0.3 PM in resting B cells, and 0.3 PM in EBV-B cell lines. AA had no effect on Ca*+ accumulated in mitochondrias (data not shown). IP3 and AA comparably triggered Ca2’ releasefrom the ER, however both agents tested at optimal concentration exerted no additional effect (Fig. 6). DISCUSSION Previous studies using various permeabilized cell preparations-i.e., exocrine pancreas (23, 24, 25), liver (26, 27) pituitary cells (28), Swiss3T3 cells (29), and human neutrophils (30)-have demonstrated that IP3 mobilizes Ca2+from an intracellular pool identified as the endoplasmic reticulum by Prentki et al. (31). In T cell lines (Jurkatt) (9) and murine B cells (7), IP3 mobilizes Ca*’ from nonmitochondrial stores. However, these cells may not be representative of the physiological events occurring in resting cell populations and there is no direct evidence regarding IP3 action on Ca*+ mobilization in human T and B lymphocytes. We herein report that following cellular permeabilization, lymphocytes organelles are functional as demonstrated by an accumulation of 45Ca2+in the presence of
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FIG.6. Comparison of IP3- and arachidonic acid-induced Ca’+ release.Cells were preloaded with %a2 for 1 hr in the presence of MgATP (5 mM) and IMCA. IP3 (10 PM) and/or arachidonic acid (10 PM) was added for I min and the percentage of released calcium was calculated. (A) Data represent means f 1 SD of five experiments performed on resting T lymphocytes. (B) Data represent means + 1 SD of three experiments performed on resting B lymphocytes.
MgATP. The vesicular localization of accumulated Ca2+was verified by the utilization of calcium ionophore which induced a 100%liberation of accumulated calcium by neutralizing the ionic charge of calcium allowing its mobilization through membranes. The accumulation of 45Ca2’is maximal after 45 min incubation either in the presence of ATP only or in the presenceof mitochondria inhibitors or vanadate. Ransom et al. (7) have indicated that, in murine B cells, the mitochondria calcium pool represents 62% and the endoplasmic reticulum pool 2 1% of accumulated calcium. In our system, the mitochondria calcium pool represents 2 1%and 15.5%and the endoplasmic reticulum pool 8 1.6%and 90% of accumulated calcium for T and B cells, respectively. These differences could be due to the use of lower Ca2+concentrations than those used by Ransom et al. (7). Burgesset al. (26) have demonstrated that the mitochondria calcium pool is directly correlated to the Ca2+concentration in medium. IP3 induces a rapid release of Ca*+ stored in ER both in T and B lymphocytes, whether the cells are resting or preactivated. Ca2+cannot be releasedfrom mitochondrias. These data are very similar to those obtained in other cell systems, namely in Jurkatt cells, murine B cells, and human neutrophils (7, 9, 30). However, we did not observe Ca2+ reaccumulation after 3 min (except in resting B lymphocytes) as previously described by other authors (7, 25, 27). In various cells, the rate of Ca*+ reuptake following IP3 addition appears to correlate with IP3 degradation (25, 27).
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Assuming that Ca2+reuptake following IP3 addition is indeed due to IP3 degradation, our results demonstrate that IP3 degradation depends on experimental conditions rather than on the nature of the cellular system used. Activated cells (EBV-B lymphocytes and HTLV 1-T lymphocytes) seemto be more sensitive than resting cells. The possible relationship between the preactivation state of HTLV 1-T and EBV-B cells and these results is unclear. The percentage of calcium releasein human T and B lymphocytes using 10 /.J4 of IP3 is higher than in other cellular systems (7, 24, 25, 27, 28, 29, 30, 31, 32). These differences may be due to cell types and to experimental conditions. It is impossible to correlate the physiological IP3 concentration after lymphocyte stimulation and its effect on Ca mobilization, since no data on estimations of intracellular levels of IP3 in stimulated lymphocytes are available. Several authors (12, 13, 33) have suggestedthat arachidonic acid plays a role as a second messengerin cellular activation. We observed that AA induces releaseof Ca2+ accumulated in the ER in a manner similar to that of IP3. There are no very significant differences in the three cell types studied in the presence of AA, as observed by the 25% of Ca*’ releasein opposition to the results observed with IP3. Comparing our data with those reported by Wolf et al. ( 12) we observed that a 10 @4concentration of AA induces a calcium releasefrom the ER 2.5- to 3.5-fold higher than the one found by these authors in rat pancreatic islets. Furthermore, these authors described additive effectsbetween IP3 and AA and they have suggestedthat the targets of these two components were two different calcium pools. In contrast, our results suggestthe action of these components on a similar calcium pool. It is possible that this characteristic is specific to haematopoietic cells. In conclusion, it has been demonstrated that IP3 formation and Ca2+mobilization are highly related in human T and B lymphocytes and that arachidonic acid could play a role in transducing activation signals in these cells by inducing Ca2+mobilization. ACKNOWLEDGMENT We are grateful Ms. Danielle Bresson for her excellent secretarial assistance.
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