Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells

The inter-species cross-reactivity of cytokine bioassays for interleukin-2 (IL2) and interleukin-6 (IL6) was investigated in the canine species. The kinetics of normal canine peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM), were analyzed in terms of cytokine release and responsiveness to cytokine stimulation, in conjunction with determination of cell proliferation, de novo antibody synthesis and cell surface phenotype. PBMC were stimulated with PWM at the beginning of the culture and human recombinant IL2 (rIL2) was added 3–4 days post stimulation (d.p.s.). nically stimulated cells proliferated and synthesized antibody in a linear fashion up to 6 d.p.s. Resting PBMC had a mean CD4+CD8+ ratio of 1.7:1; whereas cells stimulated with PWM were predominantly of CD8 phenotype at 7 d.p.s.. Three days after addition of IL2, stimulated cells were predominantly of the Thy+, sIg− CD4+, CD8− phenotype, with an increase in the CD4+CD8+ ratio. The magnitude of de novo antibody synthesis was lower in rIL2-supplemented cultures than in cultures stimulated only with PWM, and suggested a negative relationship between de novo antibody synthesis and proliferative responses of the same cultures. Supernatants from mitogen-stimulated cultures induced proliferation of mouse IL2- and IL6-dependent cell lines. Antibodies reactive with human IL2 or IL6 inhibited these responses. IL2-like activity in PWM-stimulated culture peaked by 2 d.p.s. and decreased thereafter. IL6-like activity peaked later (4–6 d.p.s.). eport demonstrates that two murine cytokine bioassays detect canine cytokines. Since cytokines may be used to define immune cells functionally, it is suggested that the combined assessment of the parameters described here may improve the evaluation of canine immune responses.