Reversal of the Abnormal Development of T Cell Subpopulations in the Thymus of Autoimmune MRL-lpr/lpr Mice by a Polyamine Biosynthesis Inhibitor

Abstract

Polyamines—putrescine, spermidine, and spermine—are a group of positively charged organic molecules that are present in all living cells. They are important regulators of cell growth and differentiation, but the precise mechanism of their action is not known. Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. Recent studies demonstrated that down-regulation of polyamine biosynthesis by irreversible inhibition of ODC with difluoromethylornithine (DFMO) is a novel therapeutic approach for the treatment of murine lupus in autoimmune MRL-lpr/lpr mice. Since murine lupus in this strain is associated with a major alteration in thymic T cell subopulations, we questioned whether abnormal polyamine biosynthesis contributes to aberrant T cell maturation in the thymus of MRL-lpr/lpr mice. Thymocytes were analyzed for cell surface markers, CD4 and CDS by 2-color flow cytometry using their respective monoclonal antibodies. The proportion of thymocyte subsets in disease-free mice (8–10 week of age) was ∼72% double positive (DP; CD4+CD8+) cells, 5–7% double negative (DN; CD4-CD8-) cells, 11–16% CD4+ cells and 7–8% CD8+ cells. At 14 weeks of age, a stage of clinical disease expression, thymocytes were marked by the presence of ∼40% DN cells and ∼25% DP cells. The percentage of T cell subsets in untreated (-) and DFMO-treated (+) mice at 14 weeks were as follows: CD4-CD8-, 40113 (-) versus 19±4* (+); CD4+CD8+, 25+14 (-) versus 47±10* (+); CD4+, 24±3 versus 17±5 (+); CD8+, 8.7±2.1 (-) versus 14+3.5* (+) (*P < 0.05; n= 4.6). High levels of immunoreactive ODC protein was detected at this age using flow cytometry with anti- ODC antibody. HPLC analysis showed a 2-fold increase in intracellular putrescine and spermidine levels in thymocytes of 14-week old MRL-lpr/lpr mice compared to those of 10-week old mice. ODC activity and polyamine levels were significantly lower in DFMO-treated mice than that of untreated controls. HPLC analysis further showed that DFMO downregulated polyamine biosynthesis. These results demonstrate a major role for polyamines in the development of CD4 and CD8 expression and the functional maturation of T cells. Polyamine-induced alterations in T cell development appear to contribute to autoimmune disease in MRL-lpr/lpr mice since polyamine depletion with DFMO-treatment prolonged their life-span and prevented renal injury.

Key Words:

• Polyamines
• autoimmune
• MRL-lpr/lpr mice
• T cell development
• ornithine decarboxylase
• difluoromethylornithine