Expression of canine interleukin-4 in canine chondrocytes inhibits inflammatory cascade through STAT6.

Interleukin-4 (IL-4) is a pleiotropic cytokine with broad spectrum of biological effects on target cells. This study deals with the mammalian expression of canine IL-4 (cIL-4) in canine articular chondrocytes (CAC) and its ability to down-regulate pro-inflammatory cytokines, enzyme mediators and their catabolites. We transfected cIL-4 in CAC which were then stimulated with canine recombinant IL-1beta and TNFalpha or left as untreated control CAC. The cIL-4 protein was detected by Western blot analysis and quantified by sandwich ELISA utilizing monoclonal and polyclonal antibodies raised against recombinant cIL-4. Pro-inflammatory cytokines and enzyme mediators were quantified by quantitative real-time PCR, and nitrite production was measured by a calorimetric assay. Our results show that cIL-4 is expressed in CAC as a 17kD protein, which inhibits various cytokines and inflammatory mediators when stimulated by IL-1beta and TNFalpha. Thus, cIL-4 expressed in CAC is biologically active and suppresses inflammatory mediators in vitro. Since STAT6 (signal transducer and activator of transcription 6) was solely expressed in transfected CAC and not at all detectable in control cells, it is likely that IL-4 exerts its anti-inflammatory effects through STAT6 signaling. These studies allowed us to identify the canine form of STAT6 which we have partially sequenced.