Titanium Ions Promote Exogenous Calcium-Dependent Calcium Influx in Activated Jurkat T Cells: A Possible Mechanism to Explain Its Immunostimulatory Properties.

Titanium and its alloys have been widely used in dental and orthopedic implants. Owing to the biotribocorrosion behavior of implants in simulated oral environment, Ti(IV) ions could be released into surrounding tissues. Current studies have found that Ti(IV) ions could affect the biological activities of immune cells in adjacent tissues and subsequently jeopardize the long-term performance of implant prostheses. However, the potential mechanism underlying its immunomodulatory properties remains unclear. Calcium signaling has been confirmed to be involved in regulation of lymphocyte immune function. Therefore, we hypothesize that Ti(IV) ions modulated T cell function through the change of intracellular calcium concentrations. This study is aimed at exploring the role of intracellular calcium responses in the modulatory effect of Ti(IV) ions on unactivated and phytohemagglutinin-activated Jurkat T cells. Here, we confirmed that Ti(IV) ions within a certain concentration range induced CD69 expression on both unactivated and activated T cells in our study. Additionally, the combined stimulation with Ti(IV) ions and PHA increased expression of IL-1β, TNF-α, and RANKL. Furthermore, we found that treatment with Ti(IV) induced a transitory increase in the levels of [Ca2+]i in activated Jurkat cells, dependent on the presence of exogenous calcium. Treatment with different doses of Ti(IV) for 24 h significantly increased the levels of [Ca2+]i in the activated Jurkat cells in a dose-dependent manner, but had little effect in the unactivated cells. Treatment with Ti(IV) did not significantly affect the PLCγ1 activation and inositol-1,4,5-trisphosphate (IP3) secretion in Jurkat cells. Taken together, these data indicated that Ti(IV) enhanced calcium influx during the T cell activation, independent of IP3-mediated intracellular calcium release. Our work provides insights into the mechanism involved in the regulation of lymphocyte behaviors under the effect of Ti(IV) ions, which may help to develop therapeutic strategies for dental implant failures.


Introduction
The titanium implant, as a primary component of the dental implant system, was designed to be durable with a high wear resistance, targeting improvement of masticatory function for patients suffering tooth loss. However, growing evidence suggests that such implants can release wear particles and metal ions into surrounding tissues due to a biotribocorrosion process [1][2][3]. Metal ions released from implants can affect biological activities such as immune functions and have been reported to lead to the premature failure of metallic implants [4,5].
Previous researches have shown that the titanium ions can induce the activation of monocytes/macrophages, neutrophils, and T lymphocytes and stimulate the production of proinflammatory and bone remodeling cytokines [6][7][8]. Treatment with Ti(IV) can enhance dendritic cell-mediated T cell responses, particularly for proinflammatory Th1 response [9]. More interestingly, previous in vivo studies have shown that T lymphocytes in the peri-implant microenvironment exhibit stronger activation and proliferation in patients with loosen implants [10,11]. Given that activated T cells can secrete inflammatory cytokines, which can result in inflammation-associated bone loss [12,13], further understanding the precise role of Ti(IV) in regulating T cell activation will be of significance.
During the T cell activation, engagement of T cell receptor (TCR) by antigen determinant initiates T cell activation by increasing the levels of intracellular calcium ([Ca 2+ ] i ) [14]. The increased levels of [Ca 2+ ] i can come from intracellular pool, which is positively controlled by inositol-1,4,5trisphosphate (IP 3 ). Furthermore, the increased levels of [Ca 2+ ] i can also stem from enhanced calcium influx [15]. The increased levels of [Ca 2+ ] i are crucial for subsequent function of activated T cells, such as functional differentiation and effector cytokine production [16,17]. However, there is no information on whether and how Ti(IV) can modulate the levels of [Ca 2+ ] i in T cells.
In this study, we address a potential effect, mediated by Ca 2+ signaling, of titanium ions on Jurkat T lymphocytes and investigate the expression levels of proinflammatory and bone remodeling cytokines.

Materials and Methods
2.1. Cell Culture. Human acute T lymphocyte leukemia Jurkat (Clone E6-1) cells were obtained from the Cell Resource Center of Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a 5% CO 2 atmosphere. The cells (1 × 10 6 cells/well) were stimulated with, or without, 5 μg/ml PHA (Sigma, St. Louis, USA) in 6-well plates for 24 h and treated with different reagents for subsequent experiments.

Flow Cytometric Detection of CD69 Expression.
For analysis of CD69 expression on the cell surface, Jurkat T cells were incubated in 6-well plates as described above and then treated with Ti(IV) chloride (Sigma, St. Louis, USA) at different concentrations (5, 25, 50, or 100 μM). The purity of Ti(IV) chloride was >99.995% as determined by trace metal analysis, and the Ti(IV) chloride solution did not contain detectable bacterial endotoxin lipopolysaccharide (LPS), determined using the ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript, NJ, USA), according to the manufacturer's instruction. After 24 h of incubation, cells were harvested and then labeled with 5 μl anti-CD69-PE antibody (eBioscience, CA, USA) for 30 min at room temperature. The percentage of T cell-expressing CD69 was determined using flow cytometer (Becton Dickinson, NJ, USA).

Flow Cytometric Analysis of Intracellular Ca 2+
Concentration. The unactivated and activated Jurkat cells (1 × 10 6 cells/well) were treated in triplicate with Ti(IV) ions (5, 25, 50, or 100 μM) in 6-well plates in culture medium containing Ca 2+ (2 mM) or calcium-free medium for 24 h. The cells were harvested and adjusted to 1 × 10 6 cells/ml. The cells were loaded with 5 μM fluo-3/AM for 45 min in the presence of 0.02% Pluronic F-127. After being washed, the cells were analyzed by flow cytometry in an influx spectrofluorometer (Becton Dickinson, USA) with excitation at 488 nm and emission at 526 nm. The maximal fluorescence (F max ) was determined over 5 min after lysing the cells and subcellular organelles with Triton X-100 (0.2% v/v, Sigma). The minimum fluorescence (F min ) was determined over a further 5 min following treatment with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N ′ ,N ′ -tetraacetic acid (EGTA). The fluorescent signal values were converted to [Ca 2+ ] i using the following equation: where K d (450 nM) is the dissociation constant of the fluo-3/Ca 2+ complex, F is the measured fluorescence intensity, F min is the minimum fluorescent signals at a very low Ca 2+ condition, and F max is the fluorescent signals measured at a high Ca 2+ condition.
2.6. Western Blot Analysis of PLCγ1 Phosphorylation. Jurkat T cells were stimulated as described above for 24 h, washed with cold PBS, and lysed in RIPA buffer containing 2 mM PMSF for 20 min on ice. After centrifugation and quantification of protein concentrations, the cell lysates (30 μg/lane) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% fat-free dry milk in TBST and were probed with rabbit anti-PLCγ1, anti-p-PLCγ1, or anti-β-actin. The bound antibodies were detected with horseradish peroxidase-(HRP-) conjugated secondary antibodies and visualized using the enhanced chemiluminescent reagents (Amersham, Little Chalfont, UK).

Measurement of IP 3 by ELISA.
The levels of IP 3 in the supernatants of cultured cells were measured by enzymelinked immunosorbent assay (ELISA). Briefly, the unactivated and activated Jurkat cells (1 × 10 6 cells/well) were treated in triplicate with, or without, Ti(IV) ions (5, 25, 50, or 100 μM) in 6-well plates for 24 h. Their supernatants were harvested and the levels of IP 3 in the supernatants of cultured cells were measured by ELISA using the specific kit (Cusabio Biotech, China), according to the manufacturer's protocol. The reactions were measured at 450 nm in the microplate reader (Molecular Devices). The concentrations of IP 3 were determined by a standard curve established using different concentrations of IP 3 provided.

Statistical
Analysis. All data were present as the mean ± standard deviation (SD). Statistically significant differences between group means were determined by analysis of variance (ANOVA) using the SPSS18.0 program. Post hoc testing was performed for intergroup comparisons using the least significance difference (LSD) test. A P value of <0.05 was considered statistically significant.

Ti(IV) Ions Promote CD69 Expression on Both
Unactivated and Activated T Cells. To evaluate the effect of Ti(IV) ions on T cell activation, we assessed the expression of its early marker CD69. Flow cytometry analysis showed that Ti(IV) ions dose-dependently enhanced CD69 expression on both unactivated and PHA-activated T cells ( Figure 1). Furthermore, the enhancement effect of Ti(IV) ions on CD69 expression was more significant in activated T cells than that in unactivated cells.

Ti(IV) Ions
Increase Expression of IL-1β, TNF-α, and RANKL in Activated T Cells. To examine whether the stimuli from Ti(IV) ions affect the expression of proinflammatory and bone remodeling cytokines, cells were treated with Ti(IV) ions in the presence or absence of PHA for 24 h and then the expression levels of IL-1β, TNF-α, and RANKL were measured. Compared with unactivated T cells, the expression of IL-1β, TNF-α, and RANKL in activated T cells was significantly increased by Ti(IV) ions ( Figure 2). Furthermore, the stimulatory activities of Ti(IV) ions on this cytokine production were concentration-dependent.

Effect of Ti(IV) Ions on PLCγ1
Phosphorylation. The phosphorylation of PLCγ1 leads to the activation of IP 3 , which is a critical regulator of intracellular calcium release from the calcium pool during the T cell activation [18]. We measured the changes in the levels of PLCγ1 phosphorylation in Jurkat cells after exposed to Ti(IV) for 24 h. As shown in Figure 6, treatment with either dose of Ti(IV) did not significantly change the levels of PLCγ1 phosphorylation in both unactivated and activated cells regardless of Ti(IV) dosages. Hence, Ti(IV) did not affect the activation of PLCγ1 in Jurkat T cells.

Discussion
Depending on the sort of metals, concentration, exposure time, and the environment of affected immune cells, metal ions may either suppress or stimulate immune cell activity [19][20][21]. For example, Ti(IV) ions inhibit T and B cell activation in vitro [22], but stimulate monocytes/splenocytes to secrete higher concentrations of IL-1β, TNF-α, and IFN-γ in the presence of other stimulus [23,24]. Therefore, it is important to use resting or mitogen-activated T cells when exploring the mechanism underlying the immunomodulatory effect of Ti(IV) ions.
Jurkat cells, a human T cell leukemia cell line, were used to explore the effect and underlying mechanism of Ti(IV) ions on the biological behavior of T cells. In this study, we observed that Ti(IV) ions promote T cell activation and subsequent expression of proinflammatory cytokines, whereas the stimulatory effect was more pronounced in activated T cells. These results are compatible with data reported by Cadosch et al. [8] and the action mechanisms may be through interfering with signaling pathways involved in T cell activation and cytokine production.
Given that increased levels of intracellular calcium are necessary for activation-related downstream signaling and T cell function [16], it is possible that the effects of Ti(IV) ions may target the calcium signaling pathways in mitogenactivated T cells. In this work, we examined the effect of treatment with different doses of Ti(IV) on the dynamic changes in the levels of intracellular calcium in unactivated and PHA-activated Jurkat T cells. Our results indicate that  The elevated intracellular calcium can be the consequence of Ca 2+ release from intracellular calcium pools or/ and increased Ca 2+ influx from extracellular environment [25]. We found that treatment with any of the doses of Ti(IV) in calcium-contained medium significantly increased the levels of intracellular calcium in activated Jurkat cells in a dose-dependent manner. However, treatment with Ti(IV) in the presence of EGTA did not significantly increase the levels of intracellular calcium in both unactivated and activated Jurkat cells. Such data indicated that increased levels of intracellular calcium induced by Ti(IV) may depend on exogenous calcium. It is well known that activation-related signals can activate PLC, which hydrolyses PIP 2 to release IP 3 and release calcium from intracellular pool in activated T cells [26]. Inositol (1,4,5)-trisphosphate receptors (IP 3 Rs) respond to this elevation of IP 3 by releasing Ca 2+ , which can lead to the initiation of calcium signaling [27]. We found that treatment with Ti(IV) ions did not change the PLCγ1 phosphorylation and IP 3 levels in the supernatants of cultured unactivated and activated Jurkat T cells. These results further indicated that treatment with Ti(IV) ions has little effect on early IP 3 -mediated calcium release and the elevated levels of intracellular calcium by Ti(IV) depended on calcium influx in activated Jurkat T cells. Unlike other many types of cells, Ca 2+ release in activated T cells makes a relatively smaller contribution to the levels of [Ca 2+ ] i and downstream signaling than the exogenous calcium-dependent Ca 2+ influx [14,15]. Our data revealed that Ti(IV) ions promoted calcium influx in activated Jurkat cells in a dose-dependent manner. Thus, it appears that Ca 2+ influx, rather than the release of Ca 2+ from IP 3 -sensitive pool, may be crucial for the modulatory effects of Ti(IV) ions in activated T cells via the calcium signaling pathway. Calcium influx in T cells depends on a complex interplay among intracellular Ca 2+ stores, plasma membrane calcium channels, and mitochondrion, which determine the levels of [Ca 2+ ] i [28,29]. Prolonged Ca 2+ influx through calcium channels is crucial in activating the nuclear factor of activated T cells (NFAT), a Ca 2+ -sensitive transcription factor responsible for regulation of T cell activation and cytokine expression [30,31]. It was notable that even though Ti(IV) ions alone induced transitory calcium influx, the levels of [Ca 2+ ] i at 24 h posttreatment were not significant, which might not be enough to maintain NFAT in the nucleus. Evidence suggests that transient activation of NFAT was not sufficient to induce the expression of several cytokines [32]. These findings may explain why treatment with Ti(IV) ions increases the production of receptor activator of nuclear factor κB ligand (RANKL) in PHA-stimulated T cells, but the effect was not significant in unactivated T cells.
There are hints of evidence supporting the idea that the species Fe(C)Ti(N)-TF might provide a route for Ti(IV) entry into cells via the transferrin receptors after the release of metal ions from its implants [33,34]. Additionally, Ti(IV) has a strong affinity for phosphorous-containing molecules [35]. It is possible that Ti(IV) may bind to phosphorylated intracellular proteins, which often correspond to active functional states of enzymes or signaling proteins and interfere with signaling pathways. Further studies are necessary to explore the molecular mechanisms underlying the action of Ti(IV) in promoting Ca 2+ influx in activated T cells.
Taken together, the present study suggests that small amounts of titanium ions released from metal implants may enhance inflammatory T cell responses by increasing calcium  influx in activated T cells. These findings from this study are significant in understanding the immunostimulatory effect of Ti(IV) ions on T lymphocytes and its underlying mechanism, especially in the presence of other inflammatory mediators, and hence provide new insights into the process and therapeutic strategies of inflammatory osteolysis.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.