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.

Figure 1. mRNA expression of proinflammatory cytokines.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres (A) and in CaReS® matrixes (B). Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. mRNA expression was quantified by qRT-PCR. The expression of IL-1, TNF and IL-6 was downregulated in IL-4 expressing scaffolds as compared to non-transfected controls on stimulation.

Figure 2. mRNA expression of enzyme mediators.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres (A) and in CaReS® matrixes (B). Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. mRNA expression was quantified by qRT-PCR. The expression of iNOS and COX-2 was downregulated in IL-4 expressing scaffolds as compared to non-transfected controls on stimulation.

Figure 3. mRNA expression of regulatory mediators.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres (A) and in CaReS® matrixes (B). Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. mRNA expression was quantified by qRT-PCR. The expression of IGF-1, IL-1ra and IL-4 was up-regulated in IL-4 expressing scaffolds as compared to non-transfected controls on stimulation.

Figure 4. Detection of IL-4 protein.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres and in CaReS® matrixes. Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. Cell lysate and culture media were used to measure IL-4 production by sandwich ELISA. A lower IL-4 protein expression was found in cells conditioned with pCOX-2.cIL-4 as compared to those with pcDNA3.1.cIL-4 in lysates (A) and culture media (B). In both constructs, the expression was high as compared to non-transfected chondrocytes.

Figure 5. mRNA expression of collagens and aggrecan.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres (A) and in CaReS® matrixes (B). Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. mRNA expression was quantified by qRT-PCR. The expression of COL1a1 was downregulated whereas that of COL2a1 and aggrecan was up-regulated in IL-4 expressing scaffolds as compared to non-transfected controls on stimulation.

Figure 6. Release of total collagen and sGAG.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres and in CaReS® matrixes. Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. Supernatants were used to measure total collagen and sGAG release by respective assays. A diminished release of both collagen (A) and sGAG (B) was observed in the IL-4 expressing scaffolds as compared to non-transfected chondrocytes on stimulation.

Figure 7. Determination of NO production.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres and in CaReS® matrixes. Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. Culture media were used to measure NO levels by using Griess' reagent system. There was a reduced NO production in IL-4 expressing scaffolds (A). However, there was no reduction in the NO production when the neutralizing anti-cIL-4 antibody was added in the culture media from both scaffolds (B).

Figure 8. PGE₂ determination.

IL-4 transfected (pcDNA3.1.cIL-4 and pCOX-2.cIL-4) and non-transfected chondrocytes were seeded in alginate microspheres and in CaReS® matrixes. Both scaffolds were stimulated with rcIL-1β (100 ng/ml) and rcTNFα (50 ng/ml) for 96 h. Culture media were used to measure PGE₂ levels by using PGE₂ enzyme immunoassay kit. A diminished production of PGE₂ was observed in IL-4 expressing scaffolds as compared to non-transfected.