Effect Of Lead On Intracellular Calcium, Calmodulin And Proliferation In Lymphocyte

Background and objective Lead is a highly toxic heavy metal,exposure to which can produce a wide range of adverse health effects. It has been shown to exert toxicity effects on various target organs including the central nervous system, digestive system, hematopoietic tissue, kidney and immune system. Immune system is an important target organ of lead. Although many studies have addressed the toxic effects of lead on immune system, the mechanisms of immunotoxicity induced by lead are still unclear.Lead is absorbed into bloodstream first, and then it equilibrates rapidly with extracellular fluid. Around 95% of the absorbed lead is stored in the bones, where it becomes insoluble state as lead phosphate and deposites. A samll amount is distributed to soft tissues in the form of phosphate salt. Pb²⁺ can enter cells by ion channels and distributes in nucleus, cytosol , mitochondria and lysosomes. It is well-known that Ca²⁺ plays a pivotal role in signal transduction and other cellular processes, including maintenance of lymphocyte normal function and antigen recognition. These Ca²⁺ respond to an array of physiological stimuli and extracellular chemical messengers that act either directly on the channels or via intracellular signals. Even minute changes in intracellular Ca²⁺ levels can have a major impact on cellular activities. Because ionic radius and ionic charge of Pb²⁺ are similar to those Ca²⁺, examining the effect of Pb²⁺on lymphocyte intracellular Ca²⁺ ([Ca²⁺]i) is the basis of understanding the immunotoxicity of Pb²⁺. Since most Ca²⁺ responsive proteins do not directly bindfree Ca²⁺, a cellular response to [Ca²⁺]i signal first requires binding to an intermediary protein, which then in turn transduces the signal into downstream effects. The most important Ca²⁺ transducer is calmodulin (CaM). CaM is one of the best understood of the Ca²⁺ binding regulatory proteins in eukaryotic cells. Calmodulin is activated by its binding to four Ca²⁺ ions. Activation of calmodulin has now been implicated in conferring calcium sensitivity to over 30 different target proteins. It may be the important calcium-calmodulin-activated pathway of signal transduction during lymphocyte activation. The result of lymphocyte signals transduction is a series of specific gene activation, which induces lymphocyte proliferation and activation. Practice has proved that gene activation cascades events play a key role in lymphocyte activation, which is crucial to IL-2 induction. We hypothesize that the effect of Pb²⁺ on IL-2 secretion by lymphocyte is a major outcome of lead immunotoxicity. Therefore, investigation of the [Ca²⁺]i, CaM, and IL-2 levels, as well as cell proliferation on the molecular level is the basis of understanding the immunotoxicity of lead.Part I The effect of lead on intracellular Ca²⁺ in lymphocytes Objective To investigate the effect of different lead concentration on quiescent and active lymphocytes [Ca²⁺]i.Methods (1) Lymphocytes were separated from mice spleen and exposed to various concentration of lead in vitro. Lymphocytes were divided into the following four groups: 10^-6 M PbCl₂ group, 10^-5M PbCl₂ group, 10^-4 M PbCl₂ group and control group; (2) Measurement of lymphocyte [Ca²⁺]i was labeled with Fura2-AM and fluorescence was monitored using a Thermo Labsystems Fluoroskan Ascent, USA . Results (1) Effects of different concentration of PbCl₂ on lymphocytes[Ca²⁺]i: The lymphocyte [Ca²⁺]i level of control group ranged from 80.23-91.84 nM. The levels oflymphocyte [Ca²⁺]i increased during the 2-20 min lead exposure period, and the increase depended on either the exposure time or the exposure concentration. The lymphocyte [Ca²⁺]i increased to the highest level after 10 min of lead exposure. The levels of lymphocyte [Ca²⁺]i increased significantly to 159.69±29.46 nM and 162.49±21.35 nM in the 10^-5 M PbCl₂ and 10^-4 M PbCl₂ groups, respectively, when compared with the control group (P<0.05). The difference between the 10^-5 M PbCl₂ and 10^-4 M PbCl₂ groups, however, was not significant (P>0.05). A slight decrease was observed for lymphocyte [Ca²⁺]i level after 20 min of lead exposure, and the levels of lymphocyte [Ca²⁺]i in either the 10^-5 MPbCl₂ or 10^-4 MPbCl₂ group had statistically difference from that in control group (P<0.05). After 1-24 hr of lead exposure, however, lymphocyte [Ca²⁺]i levels were not significantly different from that in control group (P >0.05); (2) The effect of lead on lymphocyte [Ca²⁺]i with incubation in the absence of Ca²⁺: The lymphocyte [Ca²⁺]i level of control group ranged from 86.86 to 96.40 nM. 10-20 min of lead exposure resulted in significance increase of lymphocyte [Ca²⁺]i in both the 10^-5 M PbCl₂ and 10^-4 M PbCl₂ groups (P<0.05). However,the difference between 10^-5 MPbCl₂ and 10^-4 MPbCl₂ groups was not statistically significant (P>0.05) ; (3) The effect of lead on lymphocyte [Ca²⁺]i with the use of CaM antagonist W-7: The lymphocyte [Ca²⁺]i level of control group ranged from 66.97 to 87.50 nM. When the lymphocytes were pretreated with W-7, lymphocyte [Ca²⁺]i levels displayed a dose-dependent increase, but [Ca²⁺]i levels did not reach the levels as that seen in the absence of W-7. After 5min of exposure, the levels of lymphocyte [Ca²⁺]i in either the 10^-5 MPbCl₂ or 10^-4 MPbCl₂ group were statistically different from that in control group (P<0.05). After 20 min exposure, lymphocyte [Ca²⁺]i remained at higher levels than that of the control group; (4) The effect of lead on lymphocyte [Ca²⁺]i under the voltage-dependent calcium channel blocker or Cl^- channel blocker: Pretreatment of lymphocyte with voltage-dependentcalcium channel blocker nifedipine (2.5 μmol/L) or Cl^-channel blocker, there was no significant change in [Ca²⁺]i levels when compared with those in the absence of blockers; (5) The effect of lead on activated lymphocyte [Ca²⁺]i: The levels of lymphocyte [Ca²⁺]i produced a large enhancement and showed a 4-5 folds increase with respect to quiescent cells. No significant differences were found in the lead exposure groups when compared with the control group.Conclusions (1) The effect of lead on lymphocyte[Ca²⁺]i was dose and time-dependent; (2) When the lymphocytes were exposed to PbCl₂ ranged 10^-4-10^-6M, the changes of lymphocyte [Ca²⁺]i levels appeared reversible; (3) CaM may play an important role in the process of the effect of lead on the lymphocyte [Ca²⁺]i; (4) Pretreatment of lymphocyte with voltage-dependent calcium channel blocker or Cl^- channel blocker, there was no significant change in [Ca²⁺]i levels when compared with those in the absence of blockers.Part II The effect of lead on the Calmodulin mRNA and Calmodulinin lymphocytesObjective To investigate the effect of lead on the lymphocyte CaM and analyze therelationship between calmodulin mRNA and calmdulin.Methods (1) Lymphocytes were separated from mice spleen and exposed to lead withvarious concentrations in vitro. (2) The CaM mRNA levels of lymphocytes weremeasured by RT-PCR. (3) The levels CaM protein of lymphocytes were measuredby Western blot.Results (1) The effect of lead on CaM mRNA in lymphocyte: After 1hr exposure, thelymphocyte CaM mRNA levels in both the 10^-5 M and 10^-6 M PbCl₂ groups wereincreased. After 6hr exposure, the CaM mRNA level in 10^-6M PbCl₂ group wassignificantly increased when compared with the control group. The expression of CaMmRNA in 10^-5MPbCl₂ group was slightly increased after 1hr of lead exposure, however, significantly decreased when compared with the control group after 6hr of lead exposure. The expression of CaM mRNA in 10^-4M PbCl₂ group was slight decreased after 1hr of lead exposure and significantly decreased after 6hr of lead exposure compared with the control group. After 12 hr or 24 hr of lead exposure, there were no significant differences in CaM mRNA expression among different groups; (2) Results of CaM immunohistochemistry and Western blot: CaM expression in lymphocyte was seen at the nuclear, cytoplasm and membrane. The result of Western blot displayed that the expression of CaM in 10^-4MPbCl₂ group was decreased significantly after 12 hr of exposure when compare with control group. There were no significant differences in CaM expression among different groups except 10^-4MPbCl₂ group; (3) The relationship between calmodulin mRNA and calmdulin: The results of Spearman correlated analysis showed there was no relationship between calmodulin mRNA expression and calmodulin.Conclusions:(1) When the lymphocytes were exposed to PbCl₂ in the range of 10^-4-10^-6M, lower concentration of lead exposure could activate the CaM mRNA expression, whereas higher concentrations inhibited such. (2) Higher concentration of PbCl₂(10^-4 M) inhibited the CaM expression of lymphocytePart III The effect of lead on proliferation andIL-2 level of lymphocyteObjective To investigate the effect of lead on proliferation and IL-2 level of lymphocytes.Methods (1) Lymphocytes were separated from mice spleen and exposed to lead with various concentrations in vitro; (2) MTT analysis measured the effect of lead on proliferation of lymphocytes; IL-2 levels in supernatant of cultured lymphocytes wasmeasured by ELISA.Results (1) The effect of lead on proliferation in lymphocyte: Compared to control group, there was no statistical difference of SI in the 10^-6M PbCl₂ group. After 24 hr of lead exposure, the SI of lymphocyte decreased markedly in both the 10^-5MPbCl₂ group and the 10^-5MPbCl₂ group; (2) The effect of lead on IL-2 level of lymphocyte: After 12hr of lead exposure, the levels of IL-2 in 10^-4M PbCl₂ group significantly decreased when compared with that the control group. After 24hr of lead exposure, the levels of IL-2 in both the 10^-4M PbCl₂ group and the 10^-5M PbCl₂ group decreased markedly. There was no statistical difference of IL-2 levels between 10^-6MPbCl₂ group and control group; (3) Correlation between lymphocyte SI and IL-2 levels: After 24hr of lead exposure, there was a positive correlation between SI and IL-2 level of lymphocyte(r=0.65, p=0.022).Conclusions:(1) PbCl₂ (10^-5M - 10^-4M)inhibited the proliferation and the IL-2 secretion of lymphocyte ; (3) There was a positive correlation between SI and IL-2 level of lymphocyte.