title: Evidence for regulated interleukin-4 expression in chondrocyte-scaffolds under in vitro inflammatory conditions.
abstract: OBJECTIVE
  To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions.
  
  
  METHODS
  Mature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4.
  
  
  RESULTS
  cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2) in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent.
  
  
  CONCLUSIONS
  Regulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.
authors: Institute of Immunology and Molecular Biology, Department of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.
authors: Muhammad Farooq
authors: MF
authors: Rai
authors: Thomas
authors: T
authors: Graeve
authors: Sven
authors: S
authors: Twardziok
authors: Michael F G
authors: MF
authors: Schmidt
mesh_terms: D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology*; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis; D004285:Dogs; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D006025:Glycosaminoglycans / Q000378:metabolism; D007249:Inflammation / Q000235:genetics* / Q000473:pathology*; D018836:Inflammation Mediators / Q000378:metabolism; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D009500:Neutralization Tests; D009569:Nitric Oxide / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D053674:Aggrecans; D057134:Antibodies, Neutralizing; D006025:Glycosaminoglycans; D018836:Inflammation Mediators; D012333:RNA, Messenger; D015847:Interleukin-4; D009569:Nitric Oxide; D003094:Collagen; D015232:Dinoprostone