Exuberant expression of chemokine genes by adult human articular chondrocytes in response to IL-1beta

Abstract

Objective: To provide a more complete picture of the effect of interleukin-1 beta (IL-1beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach.

Design: Chondrocytes from human knee cartilage were cultured in medium containing IL-1beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed.

Results: IL-1beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1beta. The phenotype induced by IL-1beta was partially reversed by TGF-beta1, but not by BMP-2. In the presence of IL-1beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1beta, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappaB).

Conclusion: IL-1beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta1 has the ability to antagonize some of the phenotype induced by IL-1beta.

Figure 1. RT-PCR validation of microarray

Chondrocytes from normal human knee cartilage were treated with 10 ng/ml of IL-1β for 24 h. RNA extracted from these cells was used for microarray analysis as well for RT-PCR validation. Control cells (C) received vehicle only. Total RNA samples from treated and untreated cells were analyzed by RT-PCR with the primers indicated in Table 1. GAPDH was used as reference for gel loading. Quantification of individual bands was done by NIH IMAGE J software analysis with PCR in the linear range of product (Table 2).

Figure 2. Dose response of chemokines and matrix molecules to IL-1β

(A) Chondrocytes from normal human knee cartilage were treated with 0.01, 0.1 and 1 ng/ml of IL-1β for 24 h. Control cells (C) received vehicle only. RT-PCR was done with indicated primers and GAPDH was used as reference. (B) Quantitation of RT-PCR analysis with gene expression normalized to GAPDH expression using NIH IMAGE J software. Pixel intensity is given in arbitrary units (AU).

Figure 3. Time course of response of chemokines and matrix molecules to IL-1β

Chondrocytes from preserved areas of cartilage from knees of OA patients were treated with 0.1 ng/ml of IL-1β for 0, 1, 4, 8, 12, and 24 h. Control cells (C) received vehicle only. RT-PCR was done with indicated primers with GAPDH used as reference.

Figure 4. Response of normal and OA chondrocytes to IL-1β

Chondrocytes from normal human knee cartilage and preserved areas of OA knee cartilage were exposed to 0.1 ng/ml of IL-1β for 24 h. Control cells (C) received vehicle alone. Genes which showed an increase in expression from OA chondrocytes without addition of IL-1β are denoted by *.

Figure 5. Reversal of IL-1β-induced phenotype by TGF-β1

Chondrocytes from normal knee cartilage and preserved areas of OA knee cartilage were exposed to 0.1 ng/ml of IL-1β for 24 h. Control cells (C) received vehicle alone. After 24 h, media containing IL-1β was removed and fresh media containing 10 ng/ml of TGF-β1 was added for 48 h. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading. Lanes denoted as (IL-1β) indicate that cells were treated with IL-1β for 24 h and were not treated with TGF-β1; lanes denoted as TGF-β1 (IL-1β) indicate cells received TGF-β1 after removal of IL-1β; ^ indicates that the bands were too low to quantify; ~ indicates no change.

Figure 6. Effect of FGF-18 on IL-1β-induced phenotype

Normal and OA chondrocytes were treated with 0.01 ng/ml of IL-1β for 24 h. At 24 h, 200 ng/ml of FGF-18 was added and cultured for another 24 h. Control cells (C) received vehicle alone. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading.

Figure 7. Effect of IL-1β on cartilage explants in culture

Cartilage explants from the preserved areas of knees of OA patients were treated with 10 ng/ml of IL-1β for 24 h. Control tissue (C) received vehicle alone. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading.