Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow cytometry.

Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [3H]-thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [3H]-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4(+)CD8(+), CD4(-)CD8alpha(hi), CD4(-)CD8alpha(lo) and gammadelta TCR(+) cells. PWM stimulation induced responses by CD4(+)CD8(+), CD4CD8alpha(hi) but not by CD4(-)CD8alpha(lo) or gammadelta TCR(+) cells. Con A stimulation resulted in a sustained proliferation of CD8alpha(hi) cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM(+) population (e.g. B cells). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs.

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin.

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.