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Objective: To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration., Methods: Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system., Results: An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration., Conclusion: Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs., (Copyright © 2013 by the American College of Rheumatology.)

Objective: To investigate the pattern of cytokine gene expression in human articular chondrocytes in culture in response to interleukin-1 beta (IL-1 beta). The effect of serum and variations in culture conditions was also studied., Methods: Messenger RNA was extracted from cells, reverse-transcribed to complementary DNA, and amplified by the polymerase chain reaction (PCR), using specific oligonucleotide primers. The PCR products were validated by restriction analysis with specific enzymes and by Southern blot analysis., Results: In cultured articular chondrocytes, IL-1 beta, IL-1 alpha, granulocyte colony-stimulating factor (CSF), and granulocyte-macrophage CSF cytokine genes were expressed only after induction by IL-1 beta. However, IL-6, IL-8, and macrophage CSF genes were expressed constitutively. The expression of IL-1 beta was dose and time dependent., Conclusion: Using PCR, it was possible to demonstrate gene expression for several cytokines in human articular chondrocytes in culture. It was evident that some cytokine genes were expressed constitutively and some were inducible by IL-1 beta.

The production of collagenase by human articular chondrocytes in response to interleukin-1 beta is inhibited in a dose-dependent manner by interferon-gamma (1-1,000 units/ml). The analysis of culture medium samples by Western blotting and the measurement of levels of tissue inhibitor of metalloproteinases suggest that the decrease in measurable collagenase activity is primarily due to the inhibition of procollagenase production. These results provide evidence of a role for interferon-gamma in limiting connective tissue degradation.

In cultured human articular chondrocytes, addition of tumor necrosis factor alpha (TNF alpha) stimulated caseinase activity over the range of 10(-11) M to 10(-7) M and stimulated prostaglandin E (PGE) production over the range of 10(-10) M to 10(-7) M. Maximal stimulation was observed at 10(-8)M TNF alpha for both activities. Gamma-interferon (gamma-IFN) had a variable effect on PGE production and no significant effect on caseinase activity in articular chondrocyte cultures over a concentration range of 0.1-1,000 units/ml. Co-incubation of TNF alpha with gamma-IFN enhanced PGE production and decreased caseinase activity. Concentrations as low as 1 unit/ml of gamma-IFN had significant effects on TNF-stimulated production of PGE and on caseinase activity. Resorption of human articular cartilage was stimulated by TNF alpha (10(-7) M) and was inhibited by gamma-IFN (1,000 units/ml). It is possible that cartilage breakdown in vivo may be modulated by such interactions between cytokines.