In-vitro validation of cytokine neutralizing antibodies by testing with ovine mononuclear splenocytes

Cytokines have gained increasing attention as therapeutic targets in inflammation-related disorders and inflammatory conditions have been investigated in sheep. Monoclonal antibodies (mAbs) specific for the ovine pro-inflammatory cytokines interleukin (IL)-1β and IL-6 could be used to study the effects of blocking pro-inflammatory cytokines in sheep. Ovine-specific IL-1β and IL-6 proteins and mAbs specific for these molecules were produced and the ability of the mAbs to neutralize the proteins was tested in cultures of ovine splenic mononuclear cells. Expression of nuclear factor (NF)-κβ and signal transducer and activator of transcription (STAT)-3 was evaluated by western blotting and densitometric quantification. Treatment with purified IL-1β and IL-6 proteins increased NF-κβ (P < 0.001) and STAT-3 (P < 0.01) expression, respectively, in cell culture. Treatment with these proteins that were pre-incubated with IL-1β and IL-6 mAbs attenuated (P < 0.01) these effects. These results confirm the bioactivity of ovine IL-1β and IL-6 proteins and the neutralizing capacity of anti-ovine-IL-1β and -IL-6 mAbs in vitro. These mAbs could be used to investigate anti-inflammatory strategies for attenuation of the effects of these pro-inflammatory cytokines in sheep.