Chloroquine inhibits Ca²⁺ permeable ion channels-mediated Ca²⁺ signaling in primary B lymphocytes

Abstract

Background

Chloroquine, a bitter tastant, inhibits Ca²⁺ signaling, resulting in suppression of B cell activation; however, the inhibitory mechanism remains unclear.

Results

In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca²⁺ increases in mouse splenic primary B lymphocytes, which were markedly inhibited by chloroquine. Under Ca²⁺-free conditions, TG elicited transient Ca²⁺ increases, which additionally elevated upon the restoration of 2 mM Ca²⁺. The former were from release of intracellular Ca²⁺ store and the latter from Ca²⁺ influx. TG-induced release was inhibited by 2-APB (an inhibitor of inositol-3-phosphate receptors, IP₃Rs) and chloroquine, and TG-caused influx was inhibited by pyrazole (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3) and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca²⁺ increases induced by the engagement of B cell receptor (BCR) with anti-IgM.

Conclusions

These results indicate that chloroquine inhibits Ca²⁺ elevations in splenic B cells through inhibiting Ca²⁺ permeable IP₃R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.

Background

Chloroquine is a bitter tastant [1–4], which was used to treat malaria [5] and immune-related diseases such as rheumatic disease, systemic lupus erythematosus [6], early-stage AIDS [7] and chronic graft-versus-host disease [8]. Moreover, it inhibits Ia molecule biosynthesis [9] and CpG DNA-induced protection [10] in B cells. These results imply that chloroquine might be an immunosuppressant of B cell activation. Cytosolic Ca²⁺ increases play an important role in B cell development [9], survival [11], activation [12], and differentiation [13, 14], cytokine production [12] and cell death [15]. Ca²⁺ increases in B cells are induced by antigen or anti-B cell receptor (BCR) ligation. BCR antibodies bind to the BCR, resulting in the phosphorylation of tyrosine in phospholnositide-specific phospholipase C (PLC). The phosphorylated PLC catalyzes phosphatidylinositol-4, 5-bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃). IP₃ binds to IP₃Rs located on the surface of the endoplasmic reticulum (ER). The activated IP₃Rs then mediate Ca²⁺ release from the ER, leading to increases in intracellular Ca²⁺ [15, 16]. In addition, Ca²⁺ increases can also be induced by thapsigargin (TG) [17]. However, whether and how bitter tastant chloroquine inhibits Ca²⁺ increases in B cells remains unclear.

In this study, we found that chloroquine inhibited Ca²⁺ increases induced by TG and BCR engagement with anti-IgM through inhibiting Ca²⁺ permeable ion channels.

Results

Chloroquine decreases TG-induced increases of intracellular Ca²⁺

In this study, we sought to investigate the effect of chloroquine on intracellular Ca²⁺ in primary B lymphocytes from mouse spleens. As shown in Fig. 1a and b, the increases of Ca²⁺ were induced by TG, an inhibitor of the ER Ca²⁺ ATPase, which were inhibited by chloroquine. The dose-relationship of inhibition is shown in Fig. 1c. The IC₅₀ was 9.3 ± 0.7 mM (Fig. 1c). These results indicate that chloroquine inhibits TG-induced elevations of cytosolic Ca²⁺.

Fig. 1.

Chloroquine (chloro) blocks increases of Ca²⁺. a TG induced steady increases of Ca²⁺ in splenic primary B lymphocytes, which were blocked by chloro. The bold line represents the average values. b The average Ca²⁺ levels from 178 cells. c Dose-dependent inhibition of chloro on TG-induced Ca²⁺ increases. ***p < 0.001. These results indicate that chloro attenuates TG-induced elevations of Ca²⁺

Mechanism of chloroquine-caused inhibition on TG-induced Ca²⁺ increases

To study whether chloroquine inhibits extracellular Ca²⁺ entry, we performed the following experiments. Intracellular Ca²⁺ store was first depleted by TG under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA), which resulted in transient increases of Ca²⁺. Ca²⁺ (2 mM) was then restored in the extracellular solutions, which induced additional increases and were markedly blocked by chloroquine (Fig. 2a). The Ca²⁺ levels in 155 cells were summarized (Fig. 2b). These data indicate that chloroquine inhibits Ca²⁺ influx.

Fig. 2.

Chloro inhibits extracellular Ca²⁺ influx. a TG induced small transient increases of Ca²⁺ under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA). Following the restoration of 2 mM Ca²⁺, large sustained elevations occurred and were declined by chloro. b The average Ca²⁺ levels from 155 cells. ***p < 0.001. These results indicate that chloro inhibits extracellular Ca²⁺ influx

We next investigated whether chloroquine inhibits TG-induced intracellular Ca²⁺ release. Under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA), the incubation of chloroquine abolished TG-induced transient Ca²⁺ increases and the restoration of 2 mM Ca²⁺-caused additional elevations (Fig. 3a). This phenomenon was observed in 241 cells. These results indicate that chloroquine blocks Ca²⁺ release from the ER.

Fig. 3.

Chloro blocks TG-induced Ca²⁺ release by inhibiting IP₃Rs on the ER membrane. a After cells were incubated with chloro, neither TG nor the addition of 2 mM Ca²⁺ induced increases of Ca²⁺. These experiments were performed in 241 cells. These data suggest that chloro inhibits TG-induced Ca²⁺ release. b Following cells were incubated with 2-APB, an IP₃R blocker, TG failed to induce increases of Ca²⁺ under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA). These experiments were conducted in 129 cells. These data suggest that 2-APB inhibits TG-induced Ca²⁺ release from intracellular Ca²⁺ stores by inhibiting IP₃Rs on the ER membrane

We then studied which Ca²⁺ release pathway was inhibited by chloroquine. It has known that Ca²⁺ release is mainly mediated by IP₃Rs and RyRs. 2-APB is a blocker of IP₃R [20]. As shown in Fig. 3b, 2-APB abrogated TG-induced increases of Ca²⁺ under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA). These inhibitions were observed in 129 cells. These results indicate that chloroquine inhibits IP₃R-mediated Ca²⁺ release from the ER.

Next, we investigate the role of RyRs, since which mediate Ca²⁺ release from the ER [21]. Cells were stimulated with caffeine, a selective activator of RyRs, which failed to increase Ca²⁺ (Fig. 4). These results indicate that these cells have no functional RyRs, suggesting that RyRs do not contribute to TG-induced Ca²⁺ release.

Fig. 4.

Caffeine fails to trigger increases of Ca²⁺. Caffeine did not trigger increases of Ca²⁺ under 2 mM Ca²⁺ conditions. These were observed in 227 cells. These results suggest that these cells do not have functional RyRs

Chloroquine inhibits Ca²⁺ influx as shown in Fig. 2, we then studied the underlying mechanism. In previous studies, it has been found that chloroquine blocked TRPC3 and/or STIM/Orai channels, resulting in decreases of Ca²⁺ in airway smooth muscle cells [3, 18] and in murine CD4⁺ thymocytes [4]. As shown in Fig. 5, TG-induced increases were declined by Pyr3, an inhibitor of TRPC3 and/or STIM/Orai channels, suggesting that chloroquine inhibits TRPC3 and/or STIM/Orai channels-mediated Ca²⁺ influx.

Fig. 5.

Pyr3 attenuates TG-induced Ca²⁺ elevations. a TG-induced sustained increases of Ca²⁺ were reduced by Pyr3, a selective inhibitor of TRPC3 and STIM/Orai channels. b The average values from 145 cells. ***p < 0.001. These results suggest that TRPC3 and/or STIM/Orai channel mediate TG-induced Ca²⁺ increases

Chloroquine inhibits BCR engagement-induced Ca²⁺ elevations

We finally observed the effect of chloroquine on BCR engagement-induced Ca²⁺ increases. Our previous results indicate that the engagement of BCR with anti-IgM induces Ca²⁺ elevations in B cells [16]. As shown in Fig. 6, anti-IgM induced Ca²⁺ increases. However, such increases were potently inhibited by the incubation of chloroquine, although that the statistical results show that anti-IgM still induced a significant elevation. These results indicate that chloroquine inhibits BCR engagement-induced Ca²⁺ elevations.

Fig. 6.

Chloro inhibits anti-IgM-induced Ca²⁺ increases. a Anti-IgM induced Ca²⁺ increases. b Chloro failed to affect the level of Ca²⁺, however, inhibited anti-IgM-induced increases were inhibited by chloro. c The summary results. NS: p > 0.05; ***p < 0.001. These results indicate that chloro blocks BCR engagement-induced Ca²⁺ increases

Discussion

In the present study, our results indicate that chloroquine depresses Ca²⁺ increases induced by TG and anti-IgM via inhibiting the intracellular Ca²⁺ release mediated by IP₃R channels and inhibiting the extracellular Ca²⁺ influx mediated by TRPC3 and/or STIM/Orai channels.

The aim of this study was to investigate whether chloroquine inhibits increases of Ca²⁺ and the underlying mechanism. We found that chloroquine inhibited increases of Ca²⁺ induced by TG (Fig. 1). To define the inhibitory mechanism, we investigated the pathways that mediated TG-induced Ca²⁺ elevations. Intracellular Ca²⁺ increases will be mediated by Ca²⁺ permeation channels on the plasma and the ER membrane [22]. These have been demonstrated by the results that under Ca²⁺-free conditions, TG induced transient increases and followed by additional elevations upon the restoration of 2 mM Ca²⁺ (Fig. 2), since the former resulted from release and the latter from influx. Moreover, the release was mediated by IP₃R in the ER membrane (Fig. 3a, b), because that 2-APB blocked release-induced Ca²⁺ elevations. IP₃Rs have three isoforms (IP₃R1, IP₃R2, and IP₃R3), which can be inhibited by 2-APB [20]. While, the influx was mediated by TRPC3 and/or STIM/Orai channels in the plasma membrane. Because that Pyr3 (Fig. 5) blocked TG-induced sustained increases, which will be mediated by influx based on the results shown in Fig. 2. These data indicate that TG-induced intracellular Ca²⁺ elevations were mediated by above described ion channels, and which will be inhibited by chloroquine and then resulting in decreases. These results are consistent with previous findings that chloroquine blocks TRPC3 and/or STIM/Orai channels resulting in decreases in Ca²⁺ levels in smooth muscle cells [1, 3] and in murine CD4⁺ thymocytes [4]. In addition, BCR engagement frequently occurs in vivo, which then results in immunological responses. Therefore, we observed whether the engagement can induce Ca²⁺ increases and are blocked by chloroquine. The results show that chloroquine inhibited BCR engagement-induced Ca²⁺ increases (Fig. 6).

Ca²⁺ is a crucial second messenger that modulates many cellular processes in splenic B cells, such as cell differentiation and activation [14, 23]. Therefore, our data would indicate that chloroquine might be a potent inhibitor for B cells-mediated immunological responses and inflammations.

Conclusions

Chloroquine inhibits Ca²⁺-permeable ion channels in the plasma and the ER membranes, resulting in decreases of Ca²⁺. These findings suggest that chloroquine would be an immunosuppressant.

Methods

Animals

6- to 8-week-old BALB/c male mice were purchased from the Hubei Provincial Center for Disease Control and Prevention, Wuhan, China. The mice were housed under controlled temperature (21–23 °C) and light (lights on between 08:00 and 20:00) conditions and were provided adequate water and food. All housing and experiments were performed in according with the Guide for the Institutional Animal Care and Use Committee of the South-Central University for Nationalities.

Reagents

Fura-2 AM was purchased from Invitrogen (Eugene, OR, USA). Pyrazole-3 (Pyr3) and 2-Aminoethoxydiphenyl borate (2-APB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thapsigargin (TG) was purchased from Cayman (Tallinn, Estonia). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Rockville, MD, USA). Anti-B220-PE and anti-IgM antibodies were purchased from BD Pharmingen (San Diego, CA, USA). All of the other chemicals were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).

Isolation of B cells

B lymphocytes were isolated from mouse spleens as previously described [16]. Briefly, after the animals were killed, the spleens were removed and placed in RPMI 1640 medium containing 10% FBS and 1 mM l-glutamine. The spleens were then gently teased apart with two G27 syringe needles. The non-cellular tissues were removed by filtering this preparation through a 70-μM nylon mesh. The cells were maintained at room temperature.

Measurement of intracellular Ca²⁺

Intracellular Ca²⁺ was measured using fura-2 AM as previously described [18, 19]. The cells were loaded with 2.5 μM fura-2 AM. Paired 340/380 fluorescence images were acquired using a TILL imaging system (FEI Munich GmbH, Munich, Germany), and the fluorescence ratios represent the intracellular Ca²⁺ levels. The B cells were identified by anti-B220-PE.

Data analysis and statistics

All of the data are presented as the mean ± SEM. The n values represent the number of cells. Unpaired Student’s t tests were performed to identify significant differences between the means. Differences with p < 0.05 were considered statistically significant.

Abbreviations

TG

thapsigargin

IP₃Rs

inositol-3-phosphate receptors

TRPC3

transient receptor potential C3

STIM

stromal interaction molecule

BCR

B cell receptor

PLC

phospholnositide-specific phospholipase C

PIP₂

phosphatidylinositol-4, 5-bisphosphate

DAG

diacylglycerol

IP₃

inositol-1,4,5-trisphosphate

ER

endoplasmic reticulum

2-APB

2-aminoethoxydiphenyl borate

Pyr3

pyrazole-3

FBS

fetal bovine serum

RyRs

ryanodine receptors