Molecular Immunology 64 (2015) 18–25
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Activation of calcium-sensing receptor increases TRPC3/6 expression in T lymphocyte in sepsis Qiu-yue Wu a,1 , Ming-rui Sun b,1 , Chun-li Wu a , Yang Li c , Jing-jing Du a , Jing-ya Zeng a , Hai-liang Bi a , Yi-hua Sun a,∗ a
Department of Clinical Laboratory, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001, China c Department of Rheumatology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China b
a r t i c l e
i n f o
Article history: Received 19 September 2014 Accepted 23 October 2014 Available online 12 November 2014 Keywords: Calcium-sensing receptor Transient receptor potential channel Sepsis Lymphocytes Cytokine
a b s t r a c t Sepsis is a systemic inflammatory response syndrome induced by infection. T Lymphocytes play an important role in this disease. Transient receptor potential (TRP) channels and calcium-sensing receptors (CaSR) are expressed in lymphocytes to promote intracellular Ca2+ release. However, data about the link between CaSR and TRP channels in septic T lymphocytes are few. In this study, by Ca2+ imaging and Western blotting, we found that in septic rat peripheral blood T lymphocytes expressions of TRPC3 and TRPC6 proteins are higher. The SR/ER Ca2+ ATPase inhibitor thapsigargin (TG) and CaSR agonist NPS R-568 also increased expressions of TRPC3 and TRPC6 proteins, which were reversed by PLC-IP3 channel blocker U73122 and TRPC channels inhibitor SKF96365. By Ca2+ imaging, we found that the depletion of ER Ca2+ stores by TG elicited a transient rise in cytoplasmic Ca2+ , followed by sustained increase depending on extracellular Ca2+ . But, SKF96365, not Verapamil (L-type channels inhibitor) and NiCl2 (Na+ /Ca2+ exchanger inhibitor), inhibited the relatively high [Ca2+ ]i . NPS R-568 also resulted in the same effect, and the duration of [Ca2+ ]i increase was eliminated completely by U73122 and was reduced in the absence of [Ca2+ ]o . NPS R-568 and TG increased the apoptotic ratio of septic T lymphocytes, which can be suppressed by SKF96365 and U73122. These results suggested that CaSR activation promoted the expression of TRPC3 and TRPC6 and enhanced T lymphocytes apoptosis through PLC-IP3 signaling pathway in sepsis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sepsis is a systemic inflammatory syndrome due to extraordinary immune system response to the microorganism or its toxin in the bloodstream (Lever and Mackenzie, 2007; Levy et al., 2003). Despite the rapid progress in medical care has made over the past decades, sepsis remains a leading cause of mortality in intensive care units (ICU) (Wheeler and Bernard, 1999). Hence, research into the mechanism of sepsis is urgently needed. CaSR is a member of the superfamily of G-protein-coupled receptors (GPCRs) (Wang et al., 2006). CaSR maintains Ca2+ homeostasis in the body and plays an important role in cell differentiation, apoptosis, hormone secretion, etc. Wang et al. (2003) have demonstrated that extracellular calcium and CaSR activator increased the intracellular concentration of calcium through the PLC-inositol 1,
∗ Corresponding author. Tel.: +86 451 86616691; fax: +86 451 87503325. E-mail address: [email protected] (Y.-h. Sun). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2014.10.018 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
4, 5-triphosphate (PLC-IP3 ) pathway in adult rat heart. Sun et al. (Li et al., 2013) found CaSR existence in normal human peripheral blood T lymphocytes. Stimulation of cell surface G-protein- or tyrosine-kinasecoupled receptors often evoked an increase in the IP3 level to empty the internal stores and the subsequent Ca2+ influx from the extracellular environment via calcium channels located on the plasma membrane, which are called store operated channel (SOC). The TRP channels are a heterogeneous family of calcium-permeable cation channels, which are now subdivided into seven subfamilies (Cerny and Huber, 2011). Among them, the transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the SOC channels (Ambudkar et al., 2006). Several TRP channels are known to be expressed and functional in lymphocytes (Launay et al., 2004; Philipp et al., 2003). Among them, TRPC1 and TRPC6 channels are connected to cell proliferation, whereas TRPC3 and TRPC4 primarily have pro-apoptotic action (Shapovalov et al., 2011). Wenning et al. (2011) found that TRPC3 modulated Ca2+ -dependent proliferation of primary CD4+ T-cells. Many researchers have found that ther are a certain
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relationship between CaSR and TRP channels. Hiani et al. (El Hiani et al., 2006) demonstrated that the activation of the CaSR by an increase in [Ca2+ ]o or spermine promoted an elevation in [Ca2+ ]i through PLC cascade in MCF-7 cells and the cationic current was highly Ca2+ selective, which were inhibited by the TRPC inhibitors. We have also reported (Sun et al., 2010; Feng et al., 2011) that CaSR activation contributed to apoptosis and stimulated TRPC3 and TRPC6 channels in neonatal rat ventricular myocytes. But the relation between CaSR and TRP channels and the function of these channels in T lymphocytes in sepsis remain unknown, although both TRP channels and CaSR have been reported to play important roles in lymphocytes. It is increasingly being recognized that lymphocyte apoptosis is a vital process in the pathogenesis of sepsis (Chang et al., 2007; Lang and Matute-Bello, 2009). However, the triggers of lymphocyte apoptosis during sepsis are yet unclear. We have proved that CaSR activation can enhance cardiomyocyte and T lymphocyte apoptosis. How is it to modulate sepsis? Therefore, in this study we aim to investigate the role of CaSRs and TRPCs on septic rat T lymphocytes and their relation with the PLC-IP3 pathway.
2. Materials and methods 2.1. Materials CaSR activator NPS R-568 was purchased from TOCTIS and CaSR antagonist NPS 2143 was from Santa Cruz Biotechnology. AntiTRPC1 Ab, anti-TRPC3 Ab and anti-TRPC 6 Ab were from Abcam. The PLC-IP3 pathway blocker U73122 and its inactive analog U73343, the TRPC inhibitor SKF96365, the SR/ER Ca2+ ATPase inhibitor thapsigargin (TG) and Fluo-3/AM were all obtained from Sigma.
2.2. Induction of sepsis model by CLP Wistar rats (10–12 weeks old), weighing 200–250 g each, were used in the experiments according to an approved protocol (Animal Research Institute of Harbin Medical University). CLP-induced polymicrobial sepsis was performed as previously described (Rittirsch et al., 2009). Briefly, rats were anesthetized with chloral hydrate and a midline abdominal incision was made. The cecum was mobilized, ligated below the ileocecal valve, and punctured twice with a 21-gauge needle to induce polymicrobial peritonitis. The abdominal wall was closed in two layers. Shamoperated mice, which were used as the control group, underwent the same procedure, including opening the peritoneum and exposing the bowel, but without ligation and needle perforation of the cecum. After surgery, the rats were injected with 300 l/100 g physiologic saline solution for fluid resuscitation. All rats had unlimited access to food and water both pre- and post-operatively. Peripheral blood T lymphocytes were harvested at 20 h after surgery. The successful CLP rat model should show the following signs: (1) The rectal temperature increases by 1 ◦ C compared with preoperation. (2) Leukocytosis (white-cell count, >12,000/mm3 ). (3) Food intake decreases, lethargy, and piloerection. (4) Pyuria, diarrhea, and eye secretions; Skin appears petechiae or ecchymosis. (5) Autopsy shows a peritoneal opacity exudate, inflatable jejunum, swelling cecum and adhesion, and puncture needle, which are obvious. (6) Blood cultures show bacteria growth, especially E. coli and enterococcus.
Fig. 1. The expressions of TRPC1/3/6 proteins in septic rat peripheral blood T lymphocytes. T Lymphocytes were cultured by the CaSR agonist NPS R-568 (10 mM) for 18 h, pretreated with the TRP channel inhibitor SKF96365 (5 mM) or the SR/ER Ca2+ ATPase inhibitor thapsigargin (TG, 1 M) or the PLC-IP3 pathway blocker U73122 (10 M) for 1 h. Western blot showed that in the CLP group, expressions of TRPC3 and TRPC6 proteins increased. NPS R-568 and TG increased expression of TRPC3 and TRPC6 proteins markedly, which were reversed by SKF96365 and U73122 (A, B), but they had no significant effect on expression of TRPC1 (C). Expression profiles of TRPC3 and TRPC6 proteins were quantified by densitometry (D). ∗ P < 0.05 vs. control group, # P < 0.05 vs. CLP group, P < 0.05 vs. CLP + NPS R-568 group, P < 0.05 vs. CLP + NPS R-568 group. Western blot results are representative of three experiments.
2.3. Blood samples EDTA anticoagulant peripheral blood (10 ml) was collected from the rats. First, we obtained the lymphocytes by inverted-culture mononuclear leucocytes, which were purified by lymphocyte separation medium (TBD biological manufacture CO., LTD, Tianjin, China). Then, the T lymphocytes were purified by negative selection using the RosetteSepTM human T cell enrichment cock-tail (15021, StemCell Technologies, Vancouver, BC) according to the manufacturer’s instructions. The cells were then incubated in the RPMI-1640 medium in at 37 ◦ C. After 12 h, the cells were treated with PHA (1 g/ml) for activation. 2.4. Western blotting analysis The T lymphocytes with different conditions (as described in the results) were harvested and lysed on ice with protein lysate containing Phenylmethylsulphonylfluoride (PMSF) for 30 min. The protein concentration was determined by using the Bradford protein assay with BSA as standard. Total proteins (20 g) were subjected to 10% SDS-PAGE and blotted onto nitrocellulose
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Fig. 2. [Ca2+ ]i detection in septic rat peripheral blood T lymphocytes. T Lymphocytes were grown in 96-well plates at 37 ◦ C for 3 days and were dyed by fluo-3/AM. In the absence of extracellular Ca2+ , thapsigargin (TG, 1 M) induced a transient increase in intracellular Ca2+ (A); the subsequent addition of extracellular calcium (2 mM) induced the sustained high [Ca2+ ]i , which can persist for several minutes (B). SKF96365 (1 M) inhibited the elevated [Ca2+ ]i (C). Verapamil (10 M) and NiCl2 (10 M) had no significant effect (D). The role of NPS R-568 (10 M) in increasing [Ca2+ ]i (E) was abolished by TG (F). Representative traces were shown in A–E with representative data from 5 to 8 cells.
membrane at 4 V for 20 min. After blocked in TBS-T containing 5% (w/v) skimmed milk at 37 ◦ C for 1 h, the membranes were then incubated overnight at 4 ◦ C with various antibody: anti-TRPC1 Ig (1:500) or anti-TRPC3 Ig (1:1000) or anti-TRPC6 Ig (1:500),
respectively and incubated with anti-IgG antibody conjugated with alkaline phosphates diluted 1:2000 in TBS-T for 1 h at room temperature. Antibody–antigen complexes were detected by using Western Blue® Stabilized Substrate for Alkaline Phosphatese.
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Fig. 3. Involvement of the PLC-IP3 pathway in CaSR-activation-induced [Ca2+ ]i increases. T Lymphocytes were cultured in PBS without Ca2+ . And extracellular Ca2+ (2 mM) and NPS R-568 (10 M) induced [Ca2+ ]i increased in the presence of the inactive analog U73343 (10 M)(A, B), which were abolished in the presence of the PLC-IP3 pathway inhibitor U73122 (10 M)(C, D). Representative traces were shown in A–D with data from 5 to 8 cells.
Anti--actin IgG (Boster Biological Technology Ltd., China) was used (1:400) as the house-keeping internal control. Quantitative comparisons of various proteins under various experimental conditions were performed using a Personal DensitometerTM (Molecular Dynamics, Bio-Rad).
described in the results, and intracellular fluo-3 fluorescence measurements continued. The changes of [Ca2+ ]i were represented as fluorescence intensity (FI). During the experiment, FI of fluo-3 in lymphocytes was recorded by using a laser scanning confocal microscope (Insight Plus-IQ, Meridian Instruments Inc, MI, USA) with excitation at 488 nm from an argon ion laser.
2.5. Intracellular calcium measurement T Lymphocytes were grown in 96-well plates at 37 ◦ C for 3 days, and then switched to serum-free medium. After 24 h, the medium was removed; acetoxymethyl ester of fluo-3 (fluo-3/AM) in 1% PBS was added to each well and left for 30 min at 37 ◦ C. Then, cells were rinsed twice with PBS to remove the remaining dye and 200 l of medium was added. The changes of [Ca2+ ]i were represented as fluorescence intensity (FI). During the experiment, FI of fluo-3 in lymphocytes was recorded by using a laser scanning confocal microscope (Insight Plus-IQ, Meridian Instruments Inc, MI, USA) with excitation at 488 nm from an argon ion laser. After the baseline fluorescence of T lymphocytes loaded with fluo-3/AM was measured, NPS R-568, NPS 2143, TG, U73122, U73343, Verapamil or NiCl2 were added to the medium as was
2.6. Apoptosis detection The apoptotic ratio was measured by flow cytometry analysis. The PBS-washed T lymphocytes were incubated with 5 l Annexin V-Phycoerythrin (Annexin V-PE) and 5 l 7-Amino-Actinomycin (7-AAD) for 15 min at room temperature in the dark according to manufacturer’s guidelines. 2.7. Statistical analysis All values are expressed as means ± SE. Comparisons among the groups were performed using one way variance (ANOVA) followed by LSD test. Values of P < 0.05 were considered statistically significant.
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3. Results 3.1. The expressions of TRPC proteins in septic rat peripheral blood T lymphocytes Western blotting was used to observe the expressions of TRPC1, TRPC3 and TRPC6 proteins in septic rat peripheral blood T lymphocytes. The results showed that T lymphocytes from septic rats expressed higher levels of TRPC3 and TRPC6 than those from control rats. Further, the CaSR agonist NPS R-568 and the Ca2+ ATPase (SERCA) irreversible inhibitor TG increased the expressions of TRPC3 and TRPC6 proteins. The TRPC inhibitor SKF-96365 and the PLC-IP3 channel blocker U73122 decreased the overexpression of TRPC3 and TRPC6 proteins induced by NPS R-568. There was no significant alteration among the different conditions in terms of expression of the TRPC1 protein (Fig. 1). 3.2. TG activates TRPC channels In this experiment, we used the Ca2+ ATPase (SERCA) irreversible inhibitor TG to verify that TRPCs have been activated by
the depletion of ER Ca2+ store in T lymphocytes. The results showed that TG induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , and the addition of Ca2+ induced a sustained [Ca2+ ]i increase that remained for about 5 min. SKF96365 reversed the sustained increases. But Verapamil (L-type channels inhibitor) and NiCl2 (Na+ /Ca2+ exchanger inhibitor) had no significant effect on the elevated [Ca2+ ]i . NPS R-568 could not increase [Ca2+ ]i after TG had taken effect in the absence of extracellular calcium (Fig. 2). 3.3. Involvement of the PLC pathway in CaSR-agonist-induced intracellular calcium increase In order to validate the dependence of CaSR-agonist-evoked Ca2+ entry by the PLC-IP3 pathway, the PLC-IP3 blocker U73122 and its inactive analog U73343 were used. T Lymphocytes were cultured in PBS without Ca2+ but with U73122 (10 M) or U73343 (10 M). Then, extracellular Ca2+ (2 mM) and NPS R-568 (10 M) were added. The results showed that pretreatment of septic rat peripheral blood T lymphocytes with U73122 prevented the [Ca2+ ]i increase induced by extracellular calcium or NPS R-568, whereas U73343 had no such inhibitory effect (Fig. 3).
Fig. 4. TRPC inhibitor reduced the elevated [Ca2+ ]i induced by extracellular Ca2+ and NPS R-568. Sustained elevation of [Ca2+ ]i in response to [Ca2+ ]o (2 mM) and NPS R-568 (10 M) depended on extracellular calcium (A, B). SKF96365 (1 M) reversed Ca2+ - or NPS R-568-induced [Ca2+ ]i increase (C, D), whereas the effects of Verapamil and NiCl2 were insignificant (E, F). NPS R-568 without extracellular Ca2+ cannot sustain the high level intracellular Ca2+ concentration (G). Representative traces are shown in A–F with averaged data.
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Fig. 4. (Continued ).
3.4. TRPC inhibitor reduces the sustained increase of [Ca2+ ]i induced by extracellular calcium and NPS R-568 To further prove the involvement of TRPCs in intracellular calcium increase induced by CaSR activation, we tested the effects of various inhibitors on the persisting high [Ca2+ ]i induced by [Ca2+ ]o and NPS R-568. We found that NPS R-568 elicited an intracellular calcium increase that could persist up to more than 5 min in the presence of extracellular calcium, longer than that in the absence of extracellular calcium. However, SKF96365 quickly brought the increased [Ca2+ ]i to a lower level. Verapamil and NiCl2 had no obvious effects on the sustained time of high [Ca2+ ]i . This effect of NPS R-568 is similar to that of extracellular calcium. NPS R-568 without extracellular Ca2+ cannot sustain the high level intracellular Ca2+ concentration (Fig. 4). 3.5. Apoptosis of lymphocytes In septic rat peripheral blood T lymphocytes, NPS R-568 would initiate cell apoptosis theoretically. As shown in results, compared with the control group the amount of apoptotic cells in the CLP group greatly increased and NPS R-568 exacerbated the apoptosis degree in the CLP rats. In this study, we also found that both SKF96365 and U73122 decreased the septic rat T lymphocytes apoptosis induced by NPS R-568 (Fig. 5).
4. Discussion Calcium acts as a second messenger in many cell types, including lymphocytes. Resting lymphocytes maintain a low concentration of Ca2+ . However, engagement of antigen receptors induces calcium influx from the extracellular space by several routes. A chief mechanism of Ca2+ entry in lymphocytes is through TRP channels. Sustained elevation in intracellular Ca2+ concentration plays a central regulatory role in immune inflammatory response. In this study, we first confirmed that the NPS R-568-induced expression of TRPC3 and TRPC6 proteins was upregulated in septic rat peripheral blood T lymphocytes by the PLC-IP3 pathway. Further, we proved that U73122 prevented [Ca2+ ]i increase induced by both [Ca2+ ]o and NPS R-568, whereas U73343 had no such effect. Meanwhile, we found that the TRPC inhibitor SKF96365, more sensitive than L-type Ca2+ channels and Na+ /Ca2+ exchanger inhibitor, attenuated the sustained time of [Ca2+ ]i increase induced by [Ca2+ ]o and NPS R-568. These results proved that CaSR and TRPC channels co-regulated the intracellular Ca2+ concentration. T Lymphocytes apoptosis is a critical process in immune inflammatory response. An increase in cytosolic Ca2+ might be a key initiator of the intracellular signaling of apoptosis. From the results, we found that the apoptotic ratio of T lymphocytes in sepsis rats increased more than 3-fold than the sham control group and NPS R568 exacerbated the apoptosis degree. On the other hand, U73122 and SKF96365 significantly attenuated the cell apoptosis induced
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Fig. 5. Representative figures of flow cytometry results (A–F) and rate of apoptotic cells measured by flow cytometry (G). Results showed that the apoptotic ratio of septic T lymphocytes increased 2-fold more than the sham control group and the CaSR activator NPS R-568 exacerbated the apoptosis degree, whereas U73122 and SKF96365 significantly attenuated cell apoptosis induced by NPS R-568. * P < 0.05 vs. control group, ** P < 0.05 vs. CLP group, , P < 0.05 vs. NPS R-568 group.
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by NPS R-568. These results further confirmed that CaSR activation promoted the T lymphocytes apoptosis through PLC-IP3 pathway accompanied by TRPC channels. All the data showed that CaSR activation induced TRPC expression in septic rat peripheral blood T lymphocytes and sustained levels of increased calcium to promote the apoptosis of septic rat T lymphocytes. Sepsis, a systemic inflammatory response caused by infection or injury, can lead to the development of tissue damage, septic shock, multiple organ dysfunction syndrome (MODS) and even death (LaRosa and Opal, 2011). Many therapeutic attempts for sepsis targeting “early inflammatory mediators” (such as TNF-a and IL-6) came in spite of the narrow therapeutic window provided by these cytokines (Abraham et al., 1995; Pruitt et al., 1995). In recent years, growing evidence has demonstrated that “late-phase” mediator is more important and CaSR plays a critical role in the generation and development of sepsis by acting as a key late-phase mediator. In another article (Wu et al., 2014), we have shown that CaSR activation was accompanied with the production change of different cytokines and apoptosis degree in T lymphocytes in septic rats. And, apoptosis degree and intracellular calcium concentration has close relationship with the cytokine secretion. Inflammation is a complex process. One of the important steps of agonist induced inflammation has been shown to require increased cytoplasmic Ca2+ turnover for proper signal transduction (Pani et al., 2012; Kelly et al., 2010). Arun Chauhan team researched the mechanisms of Helminth induced suppression of macrophage activation. The results from that study demonstrated inflammatory responses in macrophages induced by lower levels of LPS strongly correlated with the reduction in TRPC1-SOCE. Moreover, parasite ligands treated cells also displayed reduction in LPS induced NFkB activation (Chauhan et al., 2014). This was supported by their earlier studies demonstrating dependence of NFkB activation on TRPC1-mediated SOCE (Pani et al., 2013). So, regulation of T-lymphocyte apoptosis is very important in modulating the production of cytokines and the subsequent influence on the sepsis. To reduce the T-lymphocyte apoptosis in sepsis, we would promote them to produce more pro-inflammatory cytokines that making the patient competitive to opportunistic infections, although, it may also damage the tissue. Therefore, according to our results, inhibiting CaSR or TRPC channels activity and/or applying PLC-IP3 pathway blocker may be more effective ways to help sepsis patients achieve better therapeutic outcomes. Acknowledgements This research is supported by projects of theNational Natural Science Foundation of China (81101315, 81371892). References Abraham, E., Wunderink, R., Silverman, H., Perl, T.M., Nasraway, S., Levy, H., Bone, R., Wenzel, R.P., R. alk, B., Allred, R., 1995. Efficacy and safety of monoclonal antibody to human tumor necrosis factor a in patients with sepsis syndrome. JAMA: J. Am. Med. Assoc. 273, 934–941.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Activation of calcium-sensing receptor increases TRPC3/6 expression in T lymphocyte in sepsis Qiu-yue Wu a,1 , Ming-rui Sun b,1 , Chun-li Wu a , Yang Li c , Jing-jing Du a , Jing-ya Zeng a , Hai-liang Bi a , Yi-hua Sun a,∗ a
Department of Clinical Laboratory, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001, China c Department of Rheumatology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China b
a r t i c l e
i n f o
Article history: Received 19 September 2014 Accepted 23 October 2014 Available online 12 November 2014 Keywords: Calcium-sensing receptor Transient receptor potential channel Sepsis Lymphocytes Cytokine
a b s t r a c t Sepsis is a systemic inflammatory response syndrome induced by infection. T Lymphocytes play an important role in this disease. Transient receptor potential (TRP) channels and calcium-sensing receptors (CaSR) are expressed in lymphocytes to promote intracellular Ca2+ release. However, data about the link between CaSR and TRP channels in septic T lymphocytes are few. In this study, by Ca2+ imaging and Western blotting, we found that in septic rat peripheral blood T lymphocytes expressions of TRPC3 and TRPC6 proteins are higher. The SR/ER Ca2+ ATPase inhibitor thapsigargin (TG) and CaSR agonist NPS R-568 also increased expressions of TRPC3 and TRPC6 proteins, which were reversed by PLC-IP3 channel blocker U73122 and TRPC channels inhibitor SKF96365. By Ca2+ imaging, we found that the depletion of ER Ca2+ stores by TG elicited a transient rise in cytoplasmic Ca2+ , followed by sustained increase depending on extracellular Ca2+ . But, SKF96365, not Verapamil (L-type channels inhibitor) and NiCl2 (Na+ /Ca2+ exchanger inhibitor), inhibited the relatively high [Ca2+ ]i . NPS R-568 also resulted in the same effect, and the duration of [Ca2+ ]i increase was eliminated completely by U73122 and was reduced in the absence of [Ca2+ ]o . NPS R-568 and TG increased the apoptotic ratio of septic T lymphocytes, which can be suppressed by SKF96365 and U73122. These results suggested that CaSR activation promoted the expression of TRPC3 and TRPC6 and enhanced T lymphocytes apoptosis through PLC-IP3 signaling pathway in sepsis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Sepsis is a systemic inflammatory syndrome due to extraordinary immune system response to the microorganism or its toxin in the bloodstream (Lever and Mackenzie, 2007; Levy et al., 2003). Despite the rapid progress in medical care has made over the past decades, sepsis remains a leading cause of mortality in intensive care units (ICU) (Wheeler and Bernard, 1999). Hence, research into the mechanism of sepsis is urgently needed. CaSR is a member of the superfamily of G-protein-coupled receptors (GPCRs) (Wang et al., 2006). CaSR maintains Ca2+ homeostasis in the body and plays an important role in cell differentiation, apoptosis, hormone secretion, etc. Wang et al. (2003) have demonstrated that extracellular calcium and CaSR activator increased the intracellular concentration of calcium through the PLC-inositol 1,
∗ Corresponding author. Tel.: +86 451 86616691; fax: +86 451 87503325. E-mail address: [email protected] (Y.-h. Sun). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2014.10.018 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
4, 5-triphosphate (PLC-IP3 ) pathway in adult rat heart. Sun et al. (Li et al., 2013) found CaSR existence in normal human peripheral blood T lymphocytes. Stimulation of cell surface G-protein- or tyrosine-kinasecoupled receptors often evoked an increase in the IP3 level to empty the internal stores and the subsequent Ca2+ influx from the extracellular environment via calcium channels located on the plasma membrane, which are called store operated channel (SOC). The TRP channels are a heterogeneous family of calcium-permeable cation channels, which are now subdivided into seven subfamilies (Cerny and Huber, 2011). Among them, the transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the SOC channels (Ambudkar et al., 2006). Several TRP channels are known to be expressed and functional in lymphocytes (Launay et al., 2004; Philipp et al., 2003). Among them, TRPC1 and TRPC6 channels are connected to cell proliferation, whereas TRPC3 and TRPC4 primarily have pro-apoptotic action (Shapovalov et al., 2011). Wenning et al. (2011) found that TRPC3 modulated Ca2+ -dependent proliferation of primary CD4+ T-cells. Many researchers have found that ther are a certain
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relationship between CaSR and TRP channels. Hiani et al. (El Hiani et al., 2006) demonstrated that the activation of the CaSR by an increase in [Ca2+ ]o or spermine promoted an elevation in [Ca2+ ]i through PLC cascade in MCF-7 cells and the cationic current was highly Ca2+ selective, which were inhibited by the TRPC inhibitors. We have also reported (Sun et al., 2010; Feng et al., 2011) that CaSR activation contributed to apoptosis and stimulated TRPC3 and TRPC6 channels in neonatal rat ventricular myocytes. But the relation between CaSR and TRP channels and the function of these channels in T lymphocytes in sepsis remain unknown, although both TRP channels and CaSR have been reported to play important roles in lymphocytes. It is increasingly being recognized that lymphocyte apoptosis is a vital process in the pathogenesis of sepsis (Chang et al., 2007; Lang and Matute-Bello, 2009). However, the triggers of lymphocyte apoptosis during sepsis are yet unclear. We have proved that CaSR activation can enhance cardiomyocyte and T lymphocyte apoptosis. How is it to modulate sepsis? Therefore, in this study we aim to investigate the role of CaSRs and TRPCs on septic rat T lymphocytes and their relation with the PLC-IP3 pathway.
2. Materials and methods 2.1. Materials CaSR activator NPS R-568 was purchased from TOCTIS and CaSR antagonist NPS 2143 was from Santa Cruz Biotechnology. AntiTRPC1 Ab, anti-TRPC3 Ab and anti-TRPC 6 Ab were from Abcam. The PLC-IP3 pathway blocker U73122 and its inactive analog U73343, the TRPC inhibitor SKF96365, the SR/ER Ca2+ ATPase inhibitor thapsigargin (TG) and Fluo-3/AM were all obtained from Sigma.
2.2. Induction of sepsis model by CLP Wistar rats (10–12 weeks old), weighing 200–250 g each, were used in the experiments according to an approved protocol (Animal Research Institute of Harbin Medical University). CLP-induced polymicrobial sepsis was performed as previously described (Rittirsch et al., 2009). Briefly, rats were anesthetized with chloral hydrate and a midline abdominal incision was made. The cecum was mobilized, ligated below the ileocecal valve, and punctured twice with a 21-gauge needle to induce polymicrobial peritonitis. The abdominal wall was closed in two layers. Shamoperated mice, which were used as the control group, underwent the same procedure, including opening the peritoneum and exposing the bowel, but without ligation and needle perforation of the cecum. After surgery, the rats were injected with 300 l/100 g physiologic saline solution for fluid resuscitation. All rats had unlimited access to food and water both pre- and post-operatively. Peripheral blood T lymphocytes were harvested at 20 h after surgery. The successful CLP rat model should show the following signs: (1) The rectal temperature increases by 1 ◦ C compared with preoperation. (2) Leukocytosis (white-cell count, >12,000/mm3 ). (3) Food intake decreases, lethargy, and piloerection. (4) Pyuria, diarrhea, and eye secretions; Skin appears petechiae or ecchymosis. (5) Autopsy shows a peritoneal opacity exudate, inflatable jejunum, swelling cecum and adhesion, and puncture needle, which are obvious. (6) Blood cultures show bacteria growth, especially E. coli and enterococcus.
Fig. 1. The expressions of TRPC1/3/6 proteins in septic rat peripheral blood T lymphocytes. T Lymphocytes were cultured by the CaSR agonist NPS R-568 (10 mM) for 18 h, pretreated with the TRP channel inhibitor SKF96365 (5 mM) or the SR/ER Ca2+ ATPase inhibitor thapsigargin (TG, 1 M) or the PLC-IP3 pathway blocker U73122 (10 M) for 1 h. Western blot showed that in the CLP group, expressions of TRPC3 and TRPC6 proteins increased. NPS R-568 and TG increased expression of TRPC3 and TRPC6 proteins markedly, which were reversed by SKF96365 and U73122 (A, B), but they had no significant effect on expression of TRPC1 (C). Expression profiles of TRPC3 and TRPC6 proteins were quantified by densitometry (D). ∗ P < 0.05 vs. control group, # P < 0.05 vs. CLP group, P < 0.05 vs. CLP + NPS R-568 group, P < 0.05 vs. CLP + NPS R-568 group. Western blot results are representative of three experiments.
2.3. Blood samples EDTA anticoagulant peripheral blood (10 ml) was collected from the rats. First, we obtained the lymphocytes by inverted-culture mononuclear leucocytes, which were purified by lymphocyte separation medium (TBD biological manufacture CO., LTD, Tianjin, China). Then, the T lymphocytes were purified by negative selection using the RosetteSepTM human T cell enrichment cock-tail (15021, StemCell Technologies, Vancouver, BC) according to the manufacturer’s instructions. The cells were then incubated in the RPMI-1640 medium in at 37 ◦ C. After 12 h, the cells were treated with PHA (1 g/ml) for activation. 2.4. Western blotting analysis The T lymphocytes with different conditions (as described in the results) were harvested and lysed on ice with protein lysate containing Phenylmethylsulphonylfluoride (PMSF) for 30 min. The protein concentration was determined by using the Bradford protein assay with BSA as standard. Total proteins (20 g) were subjected to 10% SDS-PAGE and blotted onto nitrocellulose
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Fig. 2. [Ca2+ ]i detection in septic rat peripheral blood T lymphocytes. T Lymphocytes were grown in 96-well plates at 37 ◦ C for 3 days and were dyed by fluo-3/AM. In the absence of extracellular Ca2+ , thapsigargin (TG, 1 M) induced a transient increase in intracellular Ca2+ (A); the subsequent addition of extracellular calcium (2 mM) induced the sustained high [Ca2+ ]i , which can persist for several minutes (B). SKF96365 (1 M) inhibited the elevated [Ca2+ ]i (C). Verapamil (10 M) and NiCl2 (10 M) had no significant effect (D). The role of NPS R-568 (10 M) in increasing [Ca2+ ]i (E) was abolished by TG (F). Representative traces were shown in A–E with representative data from 5 to 8 cells.
membrane at 4 V for 20 min. After blocked in TBS-T containing 5% (w/v) skimmed milk at 37 ◦ C for 1 h, the membranes were then incubated overnight at 4 ◦ C with various antibody: anti-TRPC1 Ig (1:500) or anti-TRPC3 Ig (1:1000) or anti-TRPC6 Ig (1:500),
respectively and incubated with anti-IgG antibody conjugated with alkaline phosphates diluted 1:2000 in TBS-T for 1 h at room temperature. Antibody–antigen complexes were detected by using Western Blue® Stabilized Substrate for Alkaline Phosphatese.
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Fig. 3. Involvement of the PLC-IP3 pathway in CaSR-activation-induced [Ca2+ ]i increases. T Lymphocytes were cultured in PBS without Ca2+ . And extracellular Ca2+ (2 mM) and NPS R-568 (10 M) induced [Ca2+ ]i increased in the presence of the inactive analog U73343 (10 M)(A, B), which were abolished in the presence of the PLC-IP3 pathway inhibitor U73122 (10 M)(C, D). Representative traces were shown in A–D with data from 5 to 8 cells.
Anti--actin IgG (Boster Biological Technology Ltd., China) was used (1:400) as the house-keeping internal control. Quantitative comparisons of various proteins under various experimental conditions were performed using a Personal DensitometerTM (Molecular Dynamics, Bio-Rad).
described in the results, and intracellular fluo-3 fluorescence measurements continued. The changes of [Ca2+ ]i were represented as fluorescence intensity (FI). During the experiment, FI of fluo-3 in lymphocytes was recorded by using a laser scanning confocal microscope (Insight Plus-IQ, Meridian Instruments Inc, MI, USA) with excitation at 488 nm from an argon ion laser.
2.5. Intracellular calcium measurement T Lymphocytes were grown in 96-well plates at 37 ◦ C for 3 days, and then switched to serum-free medium. After 24 h, the medium was removed; acetoxymethyl ester of fluo-3 (fluo-3/AM) in 1% PBS was added to each well and left for 30 min at 37 ◦ C. Then, cells were rinsed twice with PBS to remove the remaining dye and 200 l of medium was added. The changes of [Ca2+ ]i were represented as fluorescence intensity (FI). During the experiment, FI of fluo-3 in lymphocytes was recorded by using a laser scanning confocal microscope (Insight Plus-IQ, Meridian Instruments Inc, MI, USA) with excitation at 488 nm from an argon ion laser. After the baseline fluorescence of T lymphocytes loaded with fluo-3/AM was measured, NPS R-568, NPS 2143, TG, U73122, U73343, Verapamil or NiCl2 were added to the medium as was
2.6. Apoptosis detection The apoptotic ratio was measured by flow cytometry analysis. The PBS-washed T lymphocytes were incubated with 5 l Annexin V-Phycoerythrin (Annexin V-PE) and 5 l 7-Amino-Actinomycin (7-AAD) for 15 min at room temperature in the dark according to manufacturer’s guidelines. 2.7. Statistical analysis All values are expressed as means ± SE. Comparisons among the groups were performed using one way variance (ANOVA) followed by LSD test. Values of P < 0.05 were considered statistically significant.
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3. Results 3.1. The expressions of TRPC proteins in septic rat peripheral blood T lymphocytes Western blotting was used to observe the expressions of TRPC1, TRPC3 and TRPC6 proteins in septic rat peripheral blood T lymphocytes. The results showed that T lymphocytes from septic rats expressed higher levels of TRPC3 and TRPC6 than those from control rats. Further, the CaSR agonist NPS R-568 and the Ca2+ ATPase (SERCA) irreversible inhibitor TG increased the expressions of TRPC3 and TRPC6 proteins. The TRPC inhibitor SKF-96365 and the PLC-IP3 channel blocker U73122 decreased the overexpression of TRPC3 and TRPC6 proteins induced by NPS R-568. There was no significant alteration among the different conditions in terms of expression of the TRPC1 protein (Fig. 1). 3.2. TG activates TRPC channels In this experiment, we used the Ca2+ ATPase (SERCA) irreversible inhibitor TG to verify that TRPCs have been activated by
the depletion of ER Ca2+ store in T lymphocytes. The results showed that TG induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , and the addition of Ca2+ induced a sustained [Ca2+ ]i increase that remained for about 5 min. SKF96365 reversed the sustained increases. But Verapamil (L-type channels inhibitor) and NiCl2 (Na+ /Ca2+ exchanger inhibitor) had no significant effect on the elevated [Ca2+ ]i . NPS R-568 could not increase [Ca2+ ]i after TG had taken effect in the absence of extracellular calcium (Fig. 2). 3.3. Involvement of the PLC pathway in CaSR-agonist-induced intracellular calcium increase In order to validate the dependence of CaSR-agonist-evoked Ca2+ entry by the PLC-IP3 pathway, the PLC-IP3 blocker U73122 and its inactive analog U73343 were used. T Lymphocytes were cultured in PBS without Ca2+ but with U73122 (10 M) or U73343 (10 M). Then, extracellular Ca2+ (2 mM) and NPS R-568 (10 M) were added. The results showed that pretreatment of septic rat peripheral blood T lymphocytes with U73122 prevented the [Ca2+ ]i increase induced by extracellular calcium or NPS R-568, whereas U73343 had no such inhibitory effect (Fig. 3).
Fig. 4. TRPC inhibitor reduced the elevated [Ca2+ ]i induced by extracellular Ca2+ and NPS R-568. Sustained elevation of [Ca2+ ]i in response to [Ca2+ ]o (2 mM) and NPS R-568 (10 M) depended on extracellular calcium (A, B). SKF96365 (1 M) reversed Ca2+ - or NPS R-568-induced [Ca2+ ]i increase (C, D), whereas the effects of Verapamil and NiCl2 were insignificant (E, F). NPS R-568 without extracellular Ca2+ cannot sustain the high level intracellular Ca2+ concentration (G). Representative traces are shown in A–F with averaged data.
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Fig. 4. (Continued ).
3.4. TRPC inhibitor reduces the sustained increase of [Ca2+ ]i induced by extracellular calcium and NPS R-568 To further prove the involvement of TRPCs in intracellular calcium increase induced by CaSR activation, we tested the effects of various inhibitors on the persisting high [Ca2+ ]i induced by [Ca2+ ]o and NPS R-568. We found that NPS R-568 elicited an intracellular calcium increase that could persist up to more than 5 min in the presence of extracellular calcium, longer than that in the absence of extracellular calcium. However, SKF96365 quickly brought the increased [Ca2+ ]i to a lower level. Verapamil and NiCl2 had no obvious effects on the sustained time of high [Ca2+ ]i . This effect of NPS R-568 is similar to that of extracellular calcium. NPS R-568 without extracellular Ca2+ cannot sustain the high level intracellular Ca2+ concentration (Fig. 4). 3.5. Apoptosis of lymphocytes In septic rat peripheral blood T lymphocytes, NPS R-568 would initiate cell apoptosis theoretically. As shown in results, compared with the control group the amount of apoptotic cells in the CLP group greatly increased and NPS R-568 exacerbated the apoptosis degree in the CLP rats. In this study, we also found that both SKF96365 and U73122 decreased the septic rat T lymphocytes apoptosis induced by NPS R-568 (Fig. 5).
4. Discussion Calcium acts as a second messenger in many cell types, including lymphocytes. Resting lymphocytes maintain a low concentration of Ca2+ . However, engagement of antigen receptors induces calcium influx from the extracellular space by several routes. A chief mechanism of Ca2+ entry in lymphocytes is through TRP channels. Sustained elevation in intracellular Ca2+ concentration plays a central regulatory role in immune inflammatory response. In this study, we first confirmed that the NPS R-568-induced expression of TRPC3 and TRPC6 proteins was upregulated in septic rat peripheral blood T lymphocytes by the PLC-IP3 pathway. Further, we proved that U73122 prevented [Ca2+ ]i increase induced by both [Ca2+ ]o and NPS R-568, whereas U73343 had no such effect. Meanwhile, we found that the TRPC inhibitor SKF96365, more sensitive than L-type Ca2+ channels and Na+ /Ca2+ exchanger inhibitor, attenuated the sustained time of [Ca2+ ]i increase induced by [Ca2+ ]o and NPS R-568. These results proved that CaSR and TRPC channels co-regulated the intracellular Ca2+ concentration. T Lymphocytes apoptosis is a critical process in immune inflammatory response. An increase in cytosolic Ca2+ might be a key initiator of the intracellular signaling of apoptosis. From the results, we found that the apoptotic ratio of T lymphocytes in sepsis rats increased more than 3-fold than the sham control group and NPS R568 exacerbated the apoptosis degree. On the other hand, U73122 and SKF96365 significantly attenuated the cell apoptosis induced
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Fig. 5. Representative figures of flow cytometry results (A–F) and rate of apoptotic cells measured by flow cytometry (G). Results showed that the apoptotic ratio of septic T lymphocytes increased 2-fold more than the sham control group and the CaSR activator NPS R-568 exacerbated the apoptosis degree, whereas U73122 and SKF96365 significantly attenuated cell apoptosis induced by NPS R-568. * P < 0.05 vs. control group, ** P < 0.05 vs. CLP group, , P < 0.05 vs. NPS R-568 group.
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by NPS R-568. These results further confirmed that CaSR activation promoted the T lymphocytes apoptosis through PLC-IP3 pathway accompanied by TRPC channels. All the data showed that CaSR activation induced TRPC expression in septic rat peripheral blood T lymphocytes and sustained levels of increased calcium to promote the apoptosis of septic rat T lymphocytes. Sepsis, a systemic inflammatory response caused by infection or injury, can lead to the development of tissue damage, septic shock, multiple organ dysfunction syndrome (MODS) and even death (LaRosa and Opal, 2011). Many therapeutic attempts for sepsis targeting “early inflammatory mediators” (such as TNF-a and IL-6) came in spite of the narrow therapeutic window provided by these cytokines (Abraham et al., 1995; Pruitt et al., 1995). In recent years, growing evidence has demonstrated that “late-phase” mediator is more important and CaSR plays a critical role in the generation and development of sepsis by acting as a key late-phase mediator. In another article (Wu et al., 2014), we have shown that CaSR activation was accompanied with the production change of different cytokines and apoptosis degree in T lymphocytes in septic rats. And, apoptosis degree and intracellular calcium concentration has close relationship with the cytokine secretion. Inflammation is a complex process. One of the important steps of agonist induced inflammation has been shown to require increased cytoplasmic Ca2+ turnover for proper signal transduction (Pani et al., 2012; Kelly et al., 2010). Arun Chauhan team researched the mechanisms of Helminth induced suppression of macrophage activation. The results from that study demonstrated inflammatory responses in macrophages induced by lower levels of LPS strongly correlated with the reduction in TRPC1-SOCE. Moreover, parasite ligands treated cells also displayed reduction in LPS induced NFkB activation (Chauhan et al., 2014). This was supported by their earlier studies demonstrating dependence of NFkB activation on TRPC1-mediated SOCE (Pani et al., 2013). So, regulation of T-lymphocyte apoptosis is very important in modulating the production of cytokines and the subsequent influence on the sepsis. To reduce the T-lymphocyte apoptosis in sepsis, we would promote them to produce more pro-inflammatory cytokines that making the patient competitive to opportunistic infections, although, it may also damage the tissue. Therefore, according to our results, inhibiting CaSR or TRPC channels activity and/or applying PLC-IP3 pathway blocker may be more effective ways to help sepsis patients achieve better therapeutic outcomes. Acknowledgements This research is supported by projects of theNational Natural Science Foundation of China (81101315, 81371892). References Abraham, E., Wunderink, R., Silverman, H., Perl, T.M., Nasraway, S., Levy, H., Bone, R., Wenzel, R.P., R. alk, B., Allred, R., 1995. Efficacy and safety of monoclonal antibody to human tumor necrosis factor a in patients with sepsis syndrome. JAMA: J. Am. Med. Assoc. 273, 934–941.
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