Evaluation of proliferation and cytokines production by mitogen-stimulated bovine peripheral blood mononuclear cells

Abstract

This in vitro study was conducted to evaluate lymphocyte blastogenic and cytokine production by bovine peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) mitogens, by using tetrazolium salt and ELISA tests, respectively. The results presented that Interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-17 and IFN-γ production in response to PWM mitogens was the highest and Con A the lowest amount and the median values of three mitogens were in the following order: PWM > PHA > Con A > cell control. In the case of IL-6, the production of this cytokine was the same amount for PWM and Con A and a lower amount for PHA stimulation. The results of this study not only showed a normal range for the production of these cytokines from PBMCs that were affected by mitogens, but it demonstrated that the bovine immune system at 2.5 to 3 months was post-natally matured enough to mount an effective immune response to mitogens as well as specific antigens.

Keywords: Bovine; Concanavalin A; Cytokine; Phytohemagglutinin; Pokeweed mitogen.

Fig. 1

The effect of culture period on blastogenic responses using the MTT assay. PBMCs were cultured at 5 × 10⁵ cells per well with mitogens (Con A at 5.0 μg mL^-1, PHA at 2.5 μg mL^-1, PWM at 2.5 μg mL^-1

Fig. 2

The effect of cell concentrations on blastogenic responses using the MTT assay concentration of PHA (a), PWM (b) and Con A (c). Each point represents the mean of triplicate cultures from fifteen calves.

Fig. 3

Cytokine production by PBMCs, from healthy calves after four days stimulation with PMW, PHA and Con A mitogens, as determined by ELISA. (A) for IL-2, (B) for IL-4, (C) for IL-5, (D) for IL-6, (E) for IL-10, (F) for IFN-γ, (G) for IL-17.