In-vitro Validation of Cytokine Neutralizing Antibodies by Testing with Ovine Mononuclear Splenocytes

Abstract

Summary 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 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.

Neutralizing anti-interleukin-1β antibodies modulate fetal blood–brain barrier function after ischemia

“…We have recently generated and purified pharmacological quantities of a specific mouse anti-ovine IL-1β mAb (anti-IL-1β mAb) using previously reported protocols and reagents (Rothel et al, 1997). We also confirmed that this neutralizing anti-IL-1β mAb is sensitive and specific for the ovine IL-1β protein and that it effectively neutralizes the biological effects of the ovine IL-1β in vitro (Chen et al, 2013). …”

“…Purification of anti-IL-1β mAb was performed as we previously described (Chen et al, 2013) with additional modifications as follows. The immunoglobulin G (IgG) from the anti-IL-1β mAb was purified from cell culture supernatants by affinity chromatography on Protein G Sepharose (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).…”

Interleukin-1β Transfer across the Blood–Brain Barrier in the Ovine Fetus

“…[13][14][15]37,38 Moreover, we have already shown that the highly specific ovine IL-6 protein that we have generated can cross the BBB of the adult mouse. 16,18 However, comparative quantitative aspects of transfer of other cytokines await future investigation. We would very much like to examine the ability of radiolabeled ovine IL-6 to cross the fetal BBB in future studies.…”

“…In this regard, we have recently shown that systemic infusions of anti-IL-1β monoclonal antibodies result in penetration of the monoclonal antibodies into the fetal brain, and that the monoclonal antibodies have biologic effects that include reducing the ischemia-related endogenous upregulation of IL-1β protein and the ischemia-related increases in BBB permeability measured with a nonspecific inert molecule (AIB) in the fetal brain. 18 …”

Interferon-γ: Promising therapeutic target in atherosclerosis

“…Additionally, there was also a decrease in the amount of lipid accumulation and number of macrophages in the necrotic core, which further improved plaque stability and reduced the risk of rupture. Furthermore, neutralizing antibodies have been used for other cytokines such as IL-1β and show great therapeutic promise [51,52] , therefore similar strategies could potentially be developed to use antibodies to achieve IFN-γ neutralization. Although targeting IFN-γ in atherosclerosis development may result in reduced lesion size and improved plaque stability, there are potential drawbacks that need to be assessed before IFN-γ targeting can be recommended therapeutically.…”