Secretion of IFN-γ 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 Mycobacterium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) basis, we approached the purification of M. bovis proteins through their isoelectric 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 fractions 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 to stimulate interferon-γ (IFN-γ) production by peripheral blood mononuclear cells (PBMC) isolated from cattle with well defined M. bovis tuberculosis (TB) infection. In animals with few lesions IFN-γ inductive IEF fractions were in the acid range. As the number of lesions increased, neutral fractions were also inductive. Some fractions with relatively few proteins induced as much IFN-γ production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-γ production by anergic cells. We conclude that IFN-γ production in diseased animals is induced mainly by acidic mycobacterial proteins and that the response towards these proteins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-γ production in anergic status was severely affected. We found that this cytokine production is spontaneous and antigen-independent.