Shock waves co-stimulate T-cell proliferation and interleukin-2 expression through ATP release, P2 receptor and p38 mitogen activated protein kinase activation

Tiecheng YU; Yi ZHAO; Weilun CHEN; An JIN; Jianguo LIU

ABSTRACT

BACKGROUND:

The previous researches indicate that, shock waves can enhance the proliferation of T-cells and the expression of interleukin (IL)-2 through a mechanism that involves p38 mitogen activated protein kinase (MAPK)activation.

OBJECTIVE:

To investigate if adenosine triphosphate (ATP) release is an underlying mechanism through which low-density shock waves (LDSWs) augment T-cell function.

DESIGN:

Controlled repetitive measurement by groups, taking cells as subject.

SETTING:

Department of Orthopedics, the First Hospital of Jilin University.MATERIALS KDE-2001 Extracorporeal Shockwave Lithotripter (Beijing Zhongke Jian An Meditechs Co., Beijing, China).p38 MAPK inhibitor SB203580 1 mg (BioSource Inc., Camarillo, CA); p38 MAPK kit for detecting phosphorylation (Cell Signaling Technology, Inc. U.S.A.); P2 receptor inhibitor suramin 50 mg (BIOMOL Research Laboratories Inc., PA) was prepared into 0.02 mol/L solution by 1.749 2 mL IMDM. ATP enzyme apyrase 200 U (Sigma, U.S.A.); P2X7 receptor antagonist KN-62 (BioSource Inc., Camarillo, CA); ATP Bioluminescence Assay Kit CLS Ⅱ (Roche Diagnostics GmbH,Mannheim, Germany).

METHODS:

The experiment was carried out in the Orthopedic Laboratory of the First Clinical Hospital in Jilin University from January 2005 to December 2006. ①An Extracorporeal Shockwave Lithotripter (at 7 kV generator voltage, 0.3 μF capacitance, 23 MPa positive pressure, 0.18 mJ/mm2 energy flux density) was applied for LDSWs treatment ranging from 50 to 400 impulses. ②ATP release into the culture supernatant from Jurkat T-cells or human peripheral blood mononuclear cells (PBMCs) was determined with a specific ATP Bioluminescence Assay Kit. ③Negative control group excluded antagonist or inhibitor. Human PBMCs were used to determine the effect of LDSWs on activated T-lymphocyte proliferation. Human Jurkat cells were used to study the effects of LDSWs on IL-2 expression. Expression and phosphorylation of p38 MAPK in Jurkat T-cell were measured by Western Immunoblotting with anti-p38 MAPK antibodies and anti-p38 MAPK phospho-specific antibodies that recognized the phosphorylation (on Thr180/Tyr182).MAIN OUTCOME

MEASURES:

extra-cellular ATP release, IL-2 expression in cell suspension, cellular proliferation and the phosphorylation of p38 MAPK.

RESULTS:

①ATP release under the condition without LDSWs was obviously lower than that with LDSWs of 100, 150,200, 250, 300, 360 and 400 impulses (P ＜ 0.01), and ATP release increased with the LDSWs impulse.②Compared with negative control group, the additions of apyrase, KN-62 or suramin attenuated the 3H-TdR incorporation of the phytohemagglutinin-stimulated PBMCs or CD3/CD28-stimulated Jurkat T-cells, which were effected with LDSWs of 100,150, 200, 250, 300, 330 impulses at 0.18 mJ/mm2 (P＜ 0.01). IL-2 expression in the cellular supernatant was also significant increased (P ＜ 0.01). ATPase, KN-62 or suramin all decreased the effect of LDSWs on p38 MAPK of Jurkat T-cells.

CONCLUSION:

①LDSWs deform cellular membranes but have no effect on organelle, which results in ATP release from Jurkat cells. Exogenous ATP release activates P2X7 receptor and p38 MAPK, and increases IL-2 expression. LDSWs enhance T-cell proliferation and IL-2 expression through a mechanism that involves ATP release, P2X7 receptor and phosphorylized p38 MAPK activation. ②The release of ATP plays a key role in the mechanism through which LDSWs regulate the function of T cells.