CELLULAR
IMMUNOLOGY
126,233-238
(1990)
SHORT COMMUNICATION Signal Transduction Mechanisms of la Induction in 6 Cells by lnterleukin 4 and lmmunoglobulin Receptors TOMOFXJMI
ASHIDA,
KOH-ICHI
KUBO,
ISAO KAWABATA,
MAKOTO
KATAGIRI,
MAMI OGIMOTO,* AND HIDETAKA YAKURA*T’ Department of Pathology, Asahikawa Medical College, Asahikawa 078, Japan; and *Tokyo Metropolitan Institutefor Neurosciences, Fuchu, Tokyo 183, Japan Received October 10, 1989; accepted November 7, 1989 Molecular mechanisms of signal transduction through receptors for interleukin 4 (IL-4) are still largely unknown. To elucidate the second messenger(s) of IL-4 action in mature B cells, we performed blocking experiments with inhibitors of various aspects of cellular responses, using Ia-inducing activity of IL-4 as a readout system. In the event, only agents that are shown to inhibit calcium ion (Ca*‘) release from intracellular stores, such as 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) and ryanodine, could block the IL-4 action in a dosedependent fashion. These results suggest that the process leading to the final expression of IL-4 action may be mediated, at some point, by the release of Ca*+ from intracellular stores. In the parallel experiments with antiimmunoglobulin (Ig) antibody, we found that amiloride, an inhibitor of Na+/H+ pump, blocks the Ia induction by anti-IgM antibody. Thus the Na+/H+ exchange system activated by anti-Ig antibody may be present in mature B cells. o 1990 Academic PRESS,I~C.
INTRODUCTION IL-4* has a wide array of biological activities on a variety of cell lineages. For example, it acts on resting B cells to increase cell size and Ia expression or synergistically with anti-Ig antibody to induce B cell proliferation, or acts on lipopolysaccharide (LPS)-activated B cells to induce IgG 1 and IgE antibody production. Now its effects extend to activation and differentiation of T cells, macrophages, mast cell lines, or precursors of erythroid, megakaryocyte and granulocyte series cells (reviewed in Ref. (1)). Various attempts to elucidate the molecular mechanisms of the signaling events through IL-4 receptor have been made (2-5). However, the nature of the second messengers generated upon the binding of IL-4 to its receptor is largely unknown ’ To whom correspondence should be addressed at TMIN, 2-6 Musashidai, Fuchu, Tokyo 183, Japan. * Abbreviations used: Ca*‘, calcium ion; IL-4, interleukin 4; mAb, monoclonal antibody(ies); MFI, mean fluorescence intensity; PK-C, protein kinase C TMB-8,3,4,Strimethoxybenzoic acid 8-(diethylamino)octyl ester.
233 0008-8749190
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
SHORT COMMUNICATION
except for the results that neither activation of protein kinase C (PK-C) nor mobilization of Ca*+ is implicated in the process (2-5). In this study, we tried to identify the second messenger of IL-4 action by blocking experiments with a battery of inhibitors of cellular responses. We selected the induction of Ia antigen expression as a readout system because this is a response of short duration and is accomplished solely by IL-4. The results showed that only agents known to inhibit Cazf release from intracellular stores could inhibit the induction of Ia expression by IL-4. In accord with previous reports, Ca*+ channel blockers or PKC inhibitors did not have any effects. It is therefore likely that although the binding of IL-4 to its receptor does not mobilize Ca*+ from the extracellular milieu, it may induce the release of Ca*+, albeit in a minute amount, from intracellular stores. From a parallel set of experiments in which Ia induction by anti-Ig antibody was examined, it was suggested that there exists Na+/H+ exchange activity induced by anti-Ig antibody in mature B cells. MATERIALS
AND METHODS
Mice. C57BL/6 CrSlc (B6) and DBA/2 CrSlc mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All mice were used at 6- 14 weeks of age. Antibodies and reagents. Goat anti-mouse IgM antibody was purchased from TAGO, Inc. (Burlingame, CA). Anti-Iab and -Iad monoclonal antibodies (mAbs) were purchased from Meiji Institute of Health Science (Tokyo, Japan) and Becton-Dickinson (Mountain View, CA), respectively. Biotinylation of anti-Iab antibody was performed as previously reported (6). Inhibitors and their sources were as follows: W- 13 (Seikagaku Kogyo Co., Ltd., Tokyo, Japan), omeprazol (Yoshitomi Pharmaceutical Industries, Ltd., Osaka, Japan), amiloride HCl dihydrate (Merck Sharp & Dohme Research Lab., Rahway, NJ), ryanodine (Calbiochem Corporation, La Jolla, CA), palmitoylcarnitine, nifedipine, verapamil, and TMB-8 (all from Sigma Chemical Co., St. Louis, MO). IL-4. Purified recombinant murine IL-4 was generously provided by Dr. Steven Gillis (Immunex Corporation, Seattle, WA). One unit defined by B cell costimulatory assays with anti-IgM antibody was equivalent to about 10 pg of the purified material. Cellpreparation. B cells were prepared by eliminating T cells from spleen cells with the two cycles of complement-dependent cytotoxicity, first with mAb Thy-l.2 and then with mAb Ly- 1.2 and Ly-2.2. Immunofluorescence analysis showed that the treated population was 95% surface Ig+, 95% Lyb-2+ and 3% Thy-l+. Cell cycle analysis performed as previously described (6) revealed that 94.8% of the population was at Go phase, 1.1% at G, phase, and 2.1% at S + G2/M phase. Cell culture. B cells (106/ml) from B6 or DBA/2 mice were cultured in RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), 2-mercaptoethanol(5 X 10-j M) and 2% penicillin-streptomycin in the presence of purified recombinant IL-4 (200-400 pg/ ml) or goat anti-mouse IgM ( 10 pg/ml) for 16- I8 hr. Various blockers were included in the culture at the concentrations which had been demonstrated to cause no harmful effects on cell viability. la induction assay. FACS analysis was performed as previously described (6). Cells cultured as above were harvested and washed twice. B6 B cells were first incubated
SHORT COMMUNICATION
Fluorescence
235
Intensity
FIG. 1. Effects of inhibitors on la induction by IL-4. B cells (106/ml) from B6 mice were cultured for 18 hr without any agents (0) or with purified recombinant IL-4 (200 pg/ml) in the absence (C) or presence (*) of 25 @TMB-8 (A), 10 pM verapamil (B), 10 &fpalmitoylcamitine (C), or 10 PLLMW-13 (D). Cells were harvested, washed, and incubated first with biotinylated anti-Iab mAb and then with FITC-avidin. The treated cells were analyzed with a Shomedic CS-20.
with biotinylated anti-Iab mAb for 20 min at 4°C and then with fluorescein isothiocyanate (FITC)-conjugated avidin (TAGO, Inc.) for 20 min at 4°C. For DBA/2 B cells, incubation was performed with anti-Iad mAb followed by FITC-conjugated protein A (Pharmacia Fine Chemicals, Uppsala, Sweden). After washing, cells were analyzed with a Shomedic cell sorter CS-20 (Showa Denko, K.K., Tokyo, Japan) or a JASCO FCS- 1 cell sorter (Japan Spectroscopic Co., Ltd., Tokyo, Japan). Percentage of reduction of Ia induction was calculated from mean fluorescence intensity (MFI) as follows: 1 _ (MFI with IL-4 plus inhibitor) - (MFI with medium) x ,oo (MFI with IL-4) - (MFI with medium) I RESULTS
AND DISCUSSION
The present study was conducted to elucidate the signal transduction mechanism through IL-4 receptor on mature B cells using Ia-inducing activity of IL-4 as an indicator. Inhibitors used for the experiments included a PK-C inhibitor (palmitoylcarnitine), Ca*+ channel blockers (verapamil and nifedipine), a calmodulin antagonist (W- 13) an Na+/H+ pump inhibitor (amiloride), intracellular Ca*’ inhibitors (TMB8 and ryanodine), and a K+/H+ ATPase inhibitor (omeprazol). Figure 1 shows the representative results of blocking experiments of Ia induction in B6 B cells by IL-4. As shown in Fig. 1A, TMB-8, an inhibitor of Ca*+ release from intracellular stores, strongly inhibited Ia expression induced by IL-4, whereas all the other inhibitors, such as verapamil (Fig. 1B), palmitoylcarnitine (Fig. 1C), and W- 13
236
SHORT COMMUNICATION TABLE 1 Dose-Response of Inhibitory Effect of TMB-8 and Ryanodine on Ia Induction by IL-4O Inhibitor” TMB-8
Ryanodine
Dose (PM)
Percentage of reductionb
25.00 12.50 6.25 3.12 50.00 25.00 12.50 6.25
82+0 59k2 35k 1 19k 1 84+0 44k2 2621 9+0
a DBA/2 B cells ( 106/ml) were cultured with or without purified recombinant IL-4 (400 pg/ml) for 16 hr in the presence or absence of graded doses of TMB-8 and ryanodine. Cells, washed twice, were first incubated with anti-Iad mAb and then with FITC-protein A. The treated cells were analyzed with a JASCO FCS- 1 cell sorter. b The results are expressed as mean percentage of reduction in MFI f SEM from three separate experiments.
(Fig. 1D), did not have any significant effects. In the experiments shown in Table 1, the inhibitory effect of TMB-8 was further examined on DBA/2 B cells, together with ryanodine, another inhibitor of Ca2+ release from intracellular stores. The inhibition caused by TMB-8 was dose-dependent, with maximal 82% reduction in MFI at 25 &4. More importantly, ryanodine also inhibited Ia induction by IL-4 in a dose-dependent fashion (Table 1). These results are mostly consistent with a prevailing view except for the involvement of Ca*+ in the process. Accumulating evidence suggests that the binding of IL4 to its receptor does not induce an increase in inositol phospholipid metabolism, PK-C translocation, or Ca*+ mobilization in resting B cells (2-5). It was shown in Fig. 1B and by others (4) that the influence of the Ca*’ channel antagonists (nifedipine and verapamil) on IL-Cinduced Ia expression is negligible. Moreover, it has been reported that there is no increase in the concentration of intracellular free Ca2+ upon stimulation by IL-4, as measured with fluorometric reagents (2,3). It was however found that two inhibitors of intracellular Ca2+ mobilization, TMB-8 and ryanodine, inhibit the IL-4 action (Fig. 1A and Table 1). TMB-8 has been postulated to reduce Ca*+ availability by stabilizing Ca2+ binding to intracellular stores and thereby inhibit the release of Ca2+ by external stimuli in platelets, neutrophils, muscles cells, and fibroblasts (7-l 1). Ryanodine (12) is also known to inhibit the release of Ca2+ from the sarcoplasmic reticulum in muscle cells ( 13, 14). Thus, one possibility would be that the full expression of IL-4 action might be controlled, at some point, by the release of Ca*’ from intracellular stores such as endoplasmic reticulum or possibly mitochondria, the amount of which is too low to be detected by the assays used in the previous reports. It is also formally possible, albeit less likely, that these inhibitors act at different aspects of cellular responses in lymphoid cell type. In either case, TMB-8 and ryanodine, together with Lyb-2 mAb (6, 15) and interferon-y ( 16, 17), should serve as important agents which help dissect the action of IL-4 in mature B cells.
231
SHORT COMMUNICATION TABLE 2 Effects of Various Inhibitors on la Induction by Anti-IgM Antibody” Inhibitor”
Dose (FM)
Percentage of reductionb
Palmitoylcarnitine TMB-8 Ryanodine w-13 Verapamil Omeprazol
IO 25 50 10 10 25
99 96 96 38 0 0
Amiloride
50 25 6
90 85 35
’ B6 or DBA/2 B cells (106/ml) were cultured with or without goat anti-mouse IgM antibody (10 pg/ml) for 16 hr in the absence or presence of highest nontoxic or graded doses of inhibitors. la expression was analyzed as described in Fig. I and Table 1. * Mean percentage of reduction in MFI from one of three experiments.
In the experiments performed as a control (Table 2), we found that Ia induction in mature B cells mediated by anti-IgM antibody was inhibited almost completely by palmitoylcamitine, TMB-8 and ryanodine, and partially by W- 13, but not by verapamil and omeprazol. Interestingly enough, amiloride, an inhibitor of the Na+/H+ exchange system, had a strong inhibitory effect on Ia induction by anti-IgM antibody. This was also dependent on the dose of amiloride, with highest 90% reduction attained at 50 PELM(Table 2). These results suggest that the activation pathway through Ig receptor leading to the induction of Ia antigen is modified by intracytoplasmic pH and at least a part of it may be mediated by the Na+/H+ exchange system. There have been several reports indicating that Na+ fluxes play an essential role in the differentiation of a pre-B cell line, 702/3, induced by LPS or phorbol esters ( 18, 19), but none for mature B cells. This is thus the first result suggesting that the Na+/ H+ exchange system is present in mature resting B cells activated through Ig receptor. In summary, the results presented herein suggest that although IL-4 does not utilize voltage-activated calcium gates in the plasma membrane, IL-4 action leading to Ia induction in mature B cells may be mediated in part by the release of Ca*’ from intracellular stores. In addition, it was suggested that there may exist Na+/H+ exchange activity in mature resting B cells activated by anti-Ig antibody. ACKNOWLEDGMENTS We thank Dr. Steven Gillis for generously providing recombinant IL-4. We are also grateful to Dr. Kazuo Ichihara, Department of Pharmacology, Asahikawa Medical College, for his helpful discussion. This work was supported in part by grants from the Ministry of Education, Science, and Culture, and from Immunodiagnostic Laboratory.
REFERENCES 1. Paul, W. E., and Ohara, J., Annu. Rev. Immunol. 5,429, 1987. 2. Mizuguchi, J., Beaven, M. A., Ohara, J., and Paul, W. E., J. Immunol. 137,2215, 1986.
23%
SHORT COMMUNICATION
3. Justement, L., Chen, Z., Harris, L., Ransom, J., Sandoval, V., Smith, C., Rennick, D., Roehm, N., and Cambier, J., J. fmmunol. 137,3664, 1986. 4. Dennis, G. J., Mizuguchi, J., McMillan, V., Finkelman, F. D., Ohara, J., and Mond, J. J., J. Immunol. 138,4307, 1987. 5. Cambier, J. C., and Ransom, J. T., Annu. Rev. Immunol. 5, 175, 1987. 6. Yakura, H., Kawabata, I., Ashida, T., Shen, F.-W., and Katagiri, M., J. Immunol. 137, 1475, 1986. 7. Chiou, C. Y., and Malagodi, H., Brit. J. Pharmacol. 53,279, 1975. 8. Malagodi, M. H., and Chiou, C. Y., Eur. J. Pharmacol. 27,25, 1974. 9. Rittenhouse-Simmons, S., and Deykin, D., Biochim. Biophys. Acta 543,409, 1978. 10. Smith, R. J., and Iden, S. S., Biochem. Biophys. Res. Comm. 91,263, 1979. 11. Owen, N. E., and Villereal, M. L., Biochem. Biophys. Res. Comm. 109,762, 1982. 12. Jenden, D. L., and Fairhurst, A. S., Pharmacol. Rev. 21, 1, 1969. 13. Sutko, J. L., and Kenyon, J. L., J. Gen. Phqsiol. 82,385, 1983. 14. Sutko, J. L., Ito, K., and Kenyon, J. L., Fed. Proc. 44,2984, 1985. 15. Yakura, H., Kawabata, I., Ashida, T., and Katagiri, M., J. Immunol. 141,875, 1988. 16. Rabin, E. M., Mond, J. J., Ohara, J., and Paul, W. E., J. Immunol. 137, 1573, 1986. 17. Mond, J. J., Carman, J., Sarma, C., Ohara, J., and Finkelman, F. D., J. Immunol. 137,3534, 1986. 18. RosolT, P. M., Stein, L. F., and Cantley, L. C., J. Biol. Chem. 259,7056, 1984. 19. Rosoff, P. M., and Cantley, L. C., Proc. Nad. Acad. Sci. USA 80,7547, 1983.
IMMUNOLOGY
126,233-238
(1990)
SHORT COMMUNICATION Signal Transduction Mechanisms of la Induction in 6 Cells by lnterleukin 4 and lmmunoglobulin Receptors TOMOFXJMI
ASHIDA,
KOH-ICHI
KUBO,
ISAO KAWABATA,
MAKOTO
KATAGIRI,
MAMI OGIMOTO,* AND HIDETAKA YAKURA*T’ Department of Pathology, Asahikawa Medical College, Asahikawa 078, Japan; and *Tokyo Metropolitan Institutefor Neurosciences, Fuchu, Tokyo 183, Japan Received October 10, 1989; accepted November 7, 1989 Molecular mechanisms of signal transduction through receptors for interleukin 4 (IL-4) are still largely unknown. To elucidate the second messenger(s) of IL-4 action in mature B cells, we performed blocking experiments with inhibitors of various aspects of cellular responses, using Ia-inducing activity of IL-4 as a readout system. In the event, only agents that are shown to inhibit calcium ion (Ca*‘) release from intracellular stores, such as 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) and ryanodine, could block the IL-4 action in a dosedependent fashion. These results suggest that the process leading to the final expression of IL-4 action may be mediated, at some point, by the release of Ca*+ from intracellular stores. In the parallel experiments with antiimmunoglobulin (Ig) antibody, we found that amiloride, an inhibitor of Na+/H+ pump, blocks the Ia induction by anti-IgM antibody. Thus the Na+/H+ exchange system activated by anti-Ig antibody may be present in mature B cells. o 1990 Academic PRESS,I~C.
INTRODUCTION IL-4* has a wide array of biological activities on a variety of cell lineages. For example, it acts on resting B cells to increase cell size and Ia expression or synergistically with anti-Ig antibody to induce B cell proliferation, or acts on lipopolysaccharide (LPS)-activated B cells to induce IgG 1 and IgE antibody production. Now its effects extend to activation and differentiation of T cells, macrophages, mast cell lines, or precursors of erythroid, megakaryocyte and granulocyte series cells (reviewed in Ref. (1)). Various attempts to elucidate the molecular mechanisms of the signaling events through IL-4 receptor have been made (2-5). However, the nature of the second messengers generated upon the binding of IL-4 to its receptor is largely unknown ’ To whom correspondence should be addressed at TMIN, 2-6 Musashidai, Fuchu, Tokyo 183, Japan. * Abbreviations used: Ca*‘, calcium ion; IL-4, interleukin 4; mAb, monoclonal antibody(ies); MFI, mean fluorescence intensity; PK-C, protein kinase C TMB-8,3,4,Strimethoxybenzoic acid 8-(diethylamino)octyl ester.
233 0008-8749190
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
SHORT COMMUNICATION
except for the results that neither activation of protein kinase C (PK-C) nor mobilization of Ca*+ is implicated in the process (2-5). In this study, we tried to identify the second messenger of IL-4 action by blocking experiments with a battery of inhibitors of cellular responses. We selected the induction of Ia antigen expression as a readout system because this is a response of short duration and is accomplished solely by IL-4. The results showed that only agents known to inhibit Cazf release from intracellular stores could inhibit the induction of Ia expression by IL-4. In accord with previous reports, Ca*+ channel blockers or PKC inhibitors did not have any effects. It is therefore likely that although the binding of IL-4 to its receptor does not mobilize Ca*+ from the extracellular milieu, it may induce the release of Ca*+, albeit in a minute amount, from intracellular stores. From a parallel set of experiments in which Ia induction by anti-Ig antibody was examined, it was suggested that there exists Na+/H+ exchange activity induced by anti-Ig antibody in mature B cells. MATERIALS
AND METHODS
Mice. C57BL/6 CrSlc (B6) and DBA/2 CrSlc mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All mice were used at 6- 14 weeks of age. Antibodies and reagents. Goat anti-mouse IgM antibody was purchased from TAGO, Inc. (Burlingame, CA). Anti-Iab and -Iad monoclonal antibodies (mAbs) were purchased from Meiji Institute of Health Science (Tokyo, Japan) and Becton-Dickinson (Mountain View, CA), respectively. Biotinylation of anti-Iab antibody was performed as previously reported (6). Inhibitors and their sources were as follows: W- 13 (Seikagaku Kogyo Co., Ltd., Tokyo, Japan), omeprazol (Yoshitomi Pharmaceutical Industries, Ltd., Osaka, Japan), amiloride HCl dihydrate (Merck Sharp & Dohme Research Lab., Rahway, NJ), ryanodine (Calbiochem Corporation, La Jolla, CA), palmitoylcarnitine, nifedipine, verapamil, and TMB-8 (all from Sigma Chemical Co., St. Louis, MO). IL-4. Purified recombinant murine IL-4 was generously provided by Dr. Steven Gillis (Immunex Corporation, Seattle, WA). One unit defined by B cell costimulatory assays with anti-IgM antibody was equivalent to about 10 pg of the purified material. Cellpreparation. B cells were prepared by eliminating T cells from spleen cells with the two cycles of complement-dependent cytotoxicity, first with mAb Thy-l.2 and then with mAb Ly- 1.2 and Ly-2.2. Immunofluorescence analysis showed that the treated population was 95% surface Ig+, 95% Lyb-2+ and 3% Thy-l+. Cell cycle analysis performed as previously described (6) revealed that 94.8% of the population was at Go phase, 1.1% at G, phase, and 2.1% at S + G2/M phase. Cell culture. B cells (106/ml) from B6 or DBA/2 mice were cultured in RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), 2-mercaptoethanol(5 X 10-j M) and 2% penicillin-streptomycin in the presence of purified recombinant IL-4 (200-400 pg/ ml) or goat anti-mouse IgM ( 10 pg/ml) for 16- I8 hr. Various blockers were included in the culture at the concentrations which had been demonstrated to cause no harmful effects on cell viability. la induction assay. FACS analysis was performed as previously described (6). Cells cultured as above were harvested and washed twice. B6 B cells were first incubated
SHORT COMMUNICATION
Fluorescence
235
Intensity
FIG. 1. Effects of inhibitors on la induction by IL-4. B cells (106/ml) from B6 mice were cultured for 18 hr without any agents (0) or with purified recombinant IL-4 (200 pg/ml) in the absence (C) or presence (*) of 25 @TMB-8 (A), 10 pM verapamil (B), 10 &fpalmitoylcamitine (C), or 10 PLLMW-13 (D). Cells were harvested, washed, and incubated first with biotinylated anti-Iab mAb and then with FITC-avidin. The treated cells were analyzed with a Shomedic CS-20.
with biotinylated anti-Iab mAb for 20 min at 4°C and then with fluorescein isothiocyanate (FITC)-conjugated avidin (TAGO, Inc.) for 20 min at 4°C. For DBA/2 B cells, incubation was performed with anti-Iad mAb followed by FITC-conjugated protein A (Pharmacia Fine Chemicals, Uppsala, Sweden). After washing, cells were analyzed with a Shomedic cell sorter CS-20 (Showa Denko, K.K., Tokyo, Japan) or a JASCO FCS- 1 cell sorter (Japan Spectroscopic Co., Ltd., Tokyo, Japan). Percentage of reduction of Ia induction was calculated from mean fluorescence intensity (MFI) as follows: 1 _ (MFI with IL-4 plus inhibitor) - (MFI with medium) x ,oo (MFI with IL-4) - (MFI with medium) I RESULTS
AND DISCUSSION
The present study was conducted to elucidate the signal transduction mechanism through IL-4 receptor on mature B cells using Ia-inducing activity of IL-4 as an indicator. Inhibitors used for the experiments included a PK-C inhibitor (palmitoylcarnitine), Ca*+ channel blockers (verapamil and nifedipine), a calmodulin antagonist (W- 13) an Na+/H+ pump inhibitor (amiloride), intracellular Ca*’ inhibitors (TMB8 and ryanodine), and a K+/H+ ATPase inhibitor (omeprazol). Figure 1 shows the representative results of blocking experiments of Ia induction in B6 B cells by IL-4. As shown in Fig. 1A, TMB-8, an inhibitor of Ca*+ release from intracellular stores, strongly inhibited Ia expression induced by IL-4, whereas all the other inhibitors, such as verapamil (Fig. 1B), palmitoylcarnitine (Fig. 1C), and W- 13
236
SHORT COMMUNICATION TABLE 1 Dose-Response of Inhibitory Effect of TMB-8 and Ryanodine on Ia Induction by IL-4O Inhibitor” TMB-8
Ryanodine
Dose (PM)
Percentage of reductionb
25.00 12.50 6.25 3.12 50.00 25.00 12.50 6.25
82+0 59k2 35k 1 19k 1 84+0 44k2 2621 9+0
a DBA/2 B cells ( 106/ml) were cultured with or without purified recombinant IL-4 (400 pg/ml) for 16 hr in the presence or absence of graded doses of TMB-8 and ryanodine. Cells, washed twice, were first incubated with anti-Iad mAb and then with FITC-protein A. The treated cells were analyzed with a JASCO FCS- 1 cell sorter. b The results are expressed as mean percentage of reduction in MFI f SEM from three separate experiments.
(Fig. 1D), did not have any significant effects. In the experiments shown in Table 1, the inhibitory effect of TMB-8 was further examined on DBA/2 B cells, together with ryanodine, another inhibitor of Ca2+ release from intracellular stores. The inhibition caused by TMB-8 was dose-dependent, with maximal 82% reduction in MFI at 25 &4. More importantly, ryanodine also inhibited Ia induction by IL-4 in a dose-dependent fashion (Table 1). These results are mostly consistent with a prevailing view except for the involvement of Ca*+ in the process. Accumulating evidence suggests that the binding of IL4 to its receptor does not induce an increase in inositol phospholipid metabolism, PK-C translocation, or Ca*+ mobilization in resting B cells (2-5). It was shown in Fig. 1B and by others (4) that the influence of the Ca*’ channel antagonists (nifedipine and verapamil) on IL-Cinduced Ia expression is negligible. Moreover, it has been reported that there is no increase in the concentration of intracellular free Ca2+ upon stimulation by IL-4, as measured with fluorometric reagents (2,3). It was however found that two inhibitors of intracellular Ca2+ mobilization, TMB-8 and ryanodine, inhibit the IL-4 action (Fig. 1A and Table 1). TMB-8 has been postulated to reduce Ca*+ availability by stabilizing Ca2+ binding to intracellular stores and thereby inhibit the release of Ca2+ by external stimuli in platelets, neutrophils, muscles cells, and fibroblasts (7-l 1). Ryanodine (12) is also known to inhibit the release of Ca2+ from the sarcoplasmic reticulum in muscle cells ( 13, 14). Thus, one possibility would be that the full expression of IL-4 action might be controlled, at some point, by the release of Ca*’ from intracellular stores such as endoplasmic reticulum or possibly mitochondria, the amount of which is too low to be detected by the assays used in the previous reports. It is also formally possible, albeit less likely, that these inhibitors act at different aspects of cellular responses in lymphoid cell type. In either case, TMB-8 and ryanodine, together with Lyb-2 mAb (6, 15) and interferon-y ( 16, 17), should serve as important agents which help dissect the action of IL-4 in mature B cells.
231
SHORT COMMUNICATION TABLE 2 Effects of Various Inhibitors on la Induction by Anti-IgM Antibody” Inhibitor”
Dose (FM)
Percentage of reductionb
Palmitoylcarnitine TMB-8 Ryanodine w-13 Verapamil Omeprazol
IO 25 50 10 10 25
99 96 96 38 0 0
Amiloride
50 25 6
90 85 35
’ B6 or DBA/2 B cells (106/ml) were cultured with or without goat anti-mouse IgM antibody (10 pg/ml) for 16 hr in the absence or presence of highest nontoxic or graded doses of inhibitors. la expression was analyzed as described in Fig. I and Table 1. * Mean percentage of reduction in MFI from one of three experiments.
In the experiments performed as a control (Table 2), we found that Ia induction in mature B cells mediated by anti-IgM antibody was inhibited almost completely by palmitoylcamitine, TMB-8 and ryanodine, and partially by W- 13, but not by verapamil and omeprazol. Interestingly enough, amiloride, an inhibitor of the Na+/H+ exchange system, had a strong inhibitory effect on Ia induction by anti-IgM antibody. This was also dependent on the dose of amiloride, with highest 90% reduction attained at 50 PELM(Table 2). These results suggest that the activation pathway through Ig receptor leading to the induction of Ia antigen is modified by intracytoplasmic pH and at least a part of it may be mediated by the Na+/H+ exchange system. There have been several reports indicating that Na+ fluxes play an essential role in the differentiation of a pre-B cell line, 702/3, induced by LPS or phorbol esters ( 18, 19), but none for mature B cells. This is thus the first result suggesting that the Na+/ H+ exchange system is present in mature resting B cells activated through Ig receptor. In summary, the results presented herein suggest that although IL-4 does not utilize voltage-activated calcium gates in the plasma membrane, IL-4 action leading to Ia induction in mature B cells may be mediated in part by the release of Ca*’ from intracellular stores. In addition, it was suggested that there may exist Na+/H+ exchange activity in mature resting B cells activated by anti-Ig antibody. ACKNOWLEDGMENTS We thank Dr. Steven Gillis for generously providing recombinant IL-4. We are also grateful to Dr. Kazuo Ichihara, Department of Pharmacology, Asahikawa Medical College, for his helpful discussion. This work was supported in part by grants from the Ministry of Education, Science, and Culture, and from Immunodiagnostic Laboratory.
REFERENCES 1. Paul, W. E., and Ohara, J., Annu. Rev. Immunol. 5,429, 1987. 2. Mizuguchi, J., Beaven, M. A., Ohara, J., and Paul, W. E., J. Immunol. 137,2215, 1986.
23%
SHORT COMMUNICATION
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