Pathogenic T cells in murine lupus exhibit spontaneous signaling activity through phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways

Objective. To determine the activation status of two cytoplasmic signaling pathways, phosphatidylinositol 3-kinase (PI 3-kinase) and the mitogen-activated protein kinase (MAPK) family. Methods. We studied the pathogenic CD4+ T cells that drive disease in the parent-into-F₁ mouse model of lupus-like chronic graft-versus-host disease (GVHD). We determined immunoprecipitated kinase activity for PI 3-kinase and MAPK members (Raf-1, extracellular signal-regulated kinase 1 [ERK-1], c-Jun N-terminal kinase 1 [JNK-1], and p38 MAPK) from either unfractionated splenocytes or purified donor CD4+ T cells. Uninjected normal mice served as negative controls, and acute GVHD mice served as positive controls. Results. Compared with negative controls, unfractionated splenocyte kinase activity from chronic GVHD mice was significantly increased for PI 3-kinase and JNK-1, but not for Raf-1, p38 MAPK, or ERK-1. Increased PI 3-kinase and JNK-1 activity was also seen in acute GVHD splenocytes, as was increased Raf-1 and p38 MAPK activity. The pattern of increased PI 3-kinase and JNK-1 activity seen in unfractionated chronic GVHD splenocytes was also seen in isolated donor, but not host, CD4+ T cells from chronic GVHD mice, indicating that donor CD4+ T cell signaling activity accounted for at least a portion of the activity observed in unfractionated splenocytes. Increased ERK-1 activity was not seen in either donor or host CD4+ T cells. This pattern of cytoplasmic signaling pathway in donor CD4+ T cells was associated with increased T cell receptor membrane signaling activation (Lck and Fyn phosphorylation) and increased transcription activation (phosphorylation of inhibitor of nuclear factor κB), confirming the biologic significance of these observations. Conclusion. The pathogenic T cells driving disease in this murine model exhibit activation in the form of spontaneous cytoplasmic signaling pathway activity that can be detected without in vitro restimulation and involves a T cell-specific (PI 3-kinase) and a nonspecific stress/cytokine pathway (JNK-1). These results raise the possibility that a full characterization of the signaling pathways active in pathogenic lupus T cells might lead to new therapeutic targets.