Abnormalities of Stromal Cells in Autoimmune Systemic Lupus Murine Model.

Mice of the BXSB strain develop autoimmune systemic lupus-like syndrome characterized by thrombocytopenic purpura, coronary vascular disease with myocardial infarction, glomerulonephritis, and autoantibodies. Defects in hematopoietic cells and lymphocytes have been reported in these strains. We have analyzed mesenchymal stem cells (MSCs) purified from autoimmune prone BXSB to determine whether stromal, and not only hematopoietic cells contribute to the development of autoimmune pathology in these mice. MSCs are progenitors within the bone marrow stroma that spontaneously differentiate into adipose, bone, cartilage, and stromal tissue. Stromal progeny of MSCs support engraftment of hematopoietic stem cells (HSCs) and promote hematopoiesis. We have previously demonstrated the capacity of bone marrow transplanted from healthy mice into autoimmune prone mice to prevent and treat the autoimmune pathology and reverse associated abnormalities. MSCs were purified from autoimmune BXSB bone marrow, and from normal C57Bl/6 mouse strains. Initially, adherent stromal cell populations from both murine cultures proliferated at similar rate, however fewer colony forming unit-fibroblasts (CFU-Fs) were obtained in cultures from BXSB stroma. After few passages, C57/Bl 6 stroma continued to reach 70% confluency within 2 weeks of splitting, while BXSB stromal culture continued to survive but stagnated and did not reach confluency. Both MSCs harvested from BXSB and C57/Bl6 lacked hematopoietic progenitors and did not develop CFU-E, CFU-GM, or CFU-GEMM when cultured in methyl cellulose medium supplemented with the appropriate hematopoietic growth factors. To examine interaction between diseased stroma and healthy marrow cells, stroma from autoimmune BXSB and healthy C57Bl/6 mice were separately placed in the bottom chambers of transwell plates. The top chambers contained marrow cells from healthy GFP-transgenic B6 mice and were separated by nylon mesh membrane (5μ pores) from the bottom chambers. More marrow cells migrated to the autoimmune MSCs well as shown by flow cytometry. This suggests that the autoimmune affected MSCs produce cyokines/mediators, or have cellular structure defects that attract healthy marrow cells. To investigate the latter possibility, we looked for expression of gap junction protein Connexin 43 which is important for MSC-HSC interaction and possibly subsequent expansion and proliferation. While healthy MSCs from C57/Bl6 mice expressed connexin 43, this expression was minimal in MSCs purified from autoimmune mice as shown by both immunostaining and Western blot analysis. These results indicate that marrow-derived MSCs have defective structure and function when compared with MSCs from healthy mouse strains. These data support the hypothesis that co-transplantation of MSCs along with HSCs for the treatment of advanced SLE and other autoimmune disease is imperative to restore healthy stromal cells in the diseased marrow, and to support hematopoiesis and promote tissue repair.