Abstract
Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations– 1 μM (10−3 mol/m3), 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance–expansion of Treg cells and lymphocyte apoptosis–was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.
Highlights
Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil (MMF), an immunosuppressive drug which was recently introduced to veterinary medicine
The strategy for gating canine lymphocytes is presented in S1 Fig. The percentage of ConAstimulated lymphocytes in early apoptosis after culture with MPA at the highest concentration (100 10−3 mol/m3 (μM)) was lower in comparison with the control and MPA at concentrations of 1 μM and 10 μM (p
The results of this study indicated that the effect of MPA on lymphocyte apoptosis was dependent on the mechanism of cell activation and MPA concentration
Summary
Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil (MMF), an immunosuppressive drug which was recently introduced to veterinary medicine. MPA blocks production of guanine nucleotides by non-competitive and reversible inhibition of inosine 5'monophosphate dehydrogenase activity (IMPDH) [1, 2]. Literature data suggest that its effect goes beyond lymphocyte division arrest. In the veterinary literature there are suggestions of its application in myasthenia [12,13,14], immune-mediated hemolytic anemia (IMHA) [15,16,17], anaplastic anemia [18], immune-mediated thrombocytopenia [19, 20], pemphigus vulgaris, pemphigus foliaceus [21,22,23], inflammatory bowel disease (IBD) [24], glomerular diseases including glomerulonephritis [25, 26], immune-mediated arthritis [27] and necrotizing meningoencephalitis [28]
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