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Published on: September 16, 2012
Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells
A L Rivas1, E S Kimball, F W Quimby
1Department of Pathology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14850, USA.
Insights
This study investigated canine cytokine bioassays for interleukin-2 (IL2) and interleukin-6 (IL6). Canine cells showed specific responses to IL2 and IL6, impacting proliferation and antibody synthesis.
Area of Science:
- Immunology
- Cell Biology
- Veterinary Science
Background:
- Cytokine bioassays are crucial for understanding immune responses.
- Inter-species cross-reactivity can affect assay accuracy.
- Canine cytokine responses require detailed investigation.
Purpose of the Study:
- To investigate the inter-species cross-reactivity of interleukin-2 (IL2) and interleukin-6 (IL6) bioassays in canines.
- To analyze the kinetics of canine peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM).
- To determine cytokine release, responsiveness, cell proliferation, antibody synthesis, and cell surface phenotype.
Main Methods:
- Canine PBMC were stimulated with PWM and supplemented with human recombinant IL2 (rIL2).
- Cell proliferation, de novo antibody synthesis, and cell surface phenotype (CD4+/CD8+ ratio) were measured.
- Cytokine-like activities (IL2 and IL6) in culture supernatants were assessed using mouse cell lines and inhibitory antibodies.
Main Results:
- Mitogen-stimulated canine PBMC proliferated and synthesized antibodies, with a shift towards CD8+ phenotype initially, then CD4+ after IL2 addition.
- IL2 supplementation reduced de novo antibody synthesis, suggesting a negative correlation with proliferation.
- Supernatants contained IL2-like and IL6-like activities, peaking at different time points (2 d.p.s. for IL2, 4-6 d.p.s. for IL6).
Conclusions:
- Canine PBMC exhibit distinct responses to IL2 and IL6 stimulation.
- The study provides insights into canine immune kinetics and cytokine bioassay validation.
- Findings are relevant for veterinary immunology research and diagnostic assay development.
Abstract:
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.). Mitogenically 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.).
