Development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction

Abstract

Impairment of immune function is suggested to play a contributing role for the increasing incidence of infectious diseases in the harbor porpoise (Phocoena phocoena) of the North and Baltic Seas. Both, lymphocyte-transformation-assay of peripheral blood lymphocytes (PBMC) and detection of cytokine expression are important tools for the characterization of the cellular immune response. To evaluate optimal parameters for the lymphocyte-transformation-assay isolated blood lymphocytes from four healthy harbor porpoises were stimulated with different concentrations of concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Cell proliferation was measured photometrically after 72 h using 5-bromo-deoxyuridine-assay and stimulation indices were calculated. The expression of pro- and anti-inflammatory cytokines such as interleukin (IL)-2, IL-4, IL-6, IL-10, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha was investigated in control and mitogen-stimulated lymphocytes using reverse-transcription polymerase chain reaction (RT-PCR). Primers for IL-2, IL-4 and IL-6 were selected from published cDNA-sequences of other cetaceans. Established canine and human primers were taken for the detection of TNF-alpha, TGF-beta, IL-10 and the house keeping transcript glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Specificity of the amplicon was confirmed by DNA sequence analysis and comparison with nucleotide sequences of other marine and terrestrial mammals. Con A and PHA represented the most powerful mitogens for harbor porpoise lymphoid cells at concentrations of 5 and 2 microg/ml, respectively, while PWM induced a comparatively low maximum proliferation at a concentration of 2 microg/ml. GAPDH was amplified in non-stimulated and all mitogen-stimulated cells. With the exception of IL-10 none of the other cytokines were detected in non-stimulated cells. Transcription of IL-4, IL-6, IL-10, TNF-alpha and TGF-beta-mRNA was observed after incubation with all the three phytomitogens, whereas IL-2 was only detected in Con A and PWM treated cells. Lymphocyte-transformation-assay and RT-PCR for detection of cytokines will allow to investigate possible impaired immune function in the harbor porpoise in future studies.