Secretion of IFN-gamma by bovine peripheral blood mononuclear cells stimulated with Mycobacterium bovis protein fractions obtained by isoelectric-focusing

Due to the complexity and variety of biological effects found in Mycobacter ium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) ba sis, we approached the purification of M. bovis proteins through their isoe lectric point (pI). Twenty M. bovis culture filtrate protein extract (CFPE) isoelectric focused (IEF) protein fractions, confined between pi 3 and 10, were isolated. The MW of the major proteins isolated in the various fracti ons correlated with protein already reported 14-, 18-, 20-, 25-, 31-, 38-, 45-, 64-, 67- and 70 kDa by SDS-PAGE. Since several different pi fractions showed proteins of the same MW we tested the ability of all IEF fractions t o stimulate interferon-gamma (IFN-gamma) production by peripheral blood mon onuclear cells (PBMC) isolated from cattle with well defined M. bovis tuber culosis (TB) infection. In animals with few lesions IFN-gamma inductive IEF fractions were in the acid range. As the number of lesions increased, neut ral fractions were also inductive. Some fractions with relatively few prote ins induced as much IFN-gamma production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-gamma production by anergic cells . We conclude that IFN-gamma production in diseased animals is induced main ly by acidic mycobacterial proteins and that the response towards these pro teins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-gamma production in anergic status was severe ly affected. We found that this cytokine production is spontaneous and anti gen-independent. (C) 1999 Elsevier Science B.V. All rights reserved.