Background Human T lymphocytes promote osteolysis through the production of the osteoclastogenic cytokine RANKL. We have previously demonstrated that RANKL secretion is mediated by the simultaneous engagement of the lymphocyte function-associated antigen 2 (CD2) and T-cell receptor (TCR/CD3). However, the cell signaling events involved in its expression and release from T-cells are poorly understood.
Objectives By using a variety of signaling mutants of Jurkat as well as chemical inhibitors of cell signaling factors, we sought to elucidate the role of the TCR signaling complex and that of ion channel-mediated signaling cascades in the induction of RANKL by CD2.
Methods The human T-cell line Jurkat and several signaling mutant derivatives were exposed to various combinations of bead bound anti-CD3, CD2 and CD28 antibodies. Total soluble RANKL was determined by osteoprotegrin capture sandwich ELISA. A Jurkat clone with a stably integrated NFAT-luciferase reporter (NFAT-Luc) was used to assess the signaling events leading to NFAT activation, a known regulator of RANKL expression, in response to cross-linking of CD3, CD2 and CD28 for 4 hrs. Prior to being exposed to the bead-bound antibodies, these cells were treated with a panel of chemical inhibitors to a variety of signaling factors including kinases (PI3K, CaMKII, AKT and PKC), phosphatase (calcineurin), calmodulin (CaM), calcium (TRPC3) and potassium ion channels (Kv1.1, 1.2 and 1.5) at broad concentration ranges. Cytotoxicity was evaluated by CellTiterFluor.
Results Similar to primary human T lymphocytes, Jurkat cells secreted RANKL only in response to cross-linking of both CD2 and CD3. This process was dependent on ZAP70 signaling but was independent of the following components associated with TCR signaling: beta-chain of the TCR, CD45, and PLCgamma. Cross-linking of CD3 and CD28 failed to induce RANKL secretion in wild-type cells even though high levels of IL-2 were generated. A 2-fold higher level of NFAT-Luc activation was observed with CD2/CD3 co-ligation as compared to CD28/CD3 and from the panel of inhibitors, chlorpromazine HCl, an inhibitor of both calmodulin and K+-channel signaling, was more effective at blocking NFAT-Luc activation by CD2/CD3 co-ligation than that of CD28/CD3, suggesting that these signaling cascades contribute to RANKL expression regulated by NFAT in response to CD2. Additional inhibitors to either calcium or potassium signaling cascades (FK 506, a calcineurin inhibitor, and 4-aminopyridine, a Kv1.1 & 1.2 inhibitor, respectively) also blocked CD2/CD3-induced activation of the NFAT reporter without cytotoxicity; however, inhibition was not restricted to CD2 since CD28/CD3-induced signaling was also affected.
Conclusions Our results demonstrate that T-cells secrete RANKL in a ZAP70-dependent manner and only require the co-ligation of CD3 and CD2 in the absence of the co-stimulatory receptor CD28. Furthermore, calcineurin and voltage-gated K+-channel signaling cascades were shown to induce the activation of NFAT in response to CD2/CD3 co-ligation, suggesting that these pathways may contribute to the expression RANKL in the inflamed RA synovium by T-cells.
Acknowledgements Authors would like to thank Drs. Jochen Salfeld, Anna Yarilina and Eric Goedken for their discussion and critical review of the data.
Disclosure of Interest B. Harvey Shareholder of: AbbVie Inc., Employee of: AbbVie Inc., Z. Kaymakcalan Shareholder of: AbbVie Inc., Employee of: AbbVie Inc.