Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

Abstract

Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1β and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca²⁺ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca²⁺ responses were assessed through the Ca²⁺ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca²⁺ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca²⁺ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

Keywords: Cell biology.

Fig. 1

Actin filament and immunostaining of COMP. Unstimulated chondrocytes and chondrocytes stimulated with IL-1β (5 ng/ml) or LPS (10 ng/ml) for 24 h were stained with Alexa™488-conjugated (red) phalloidin probe and for the chondrogenic matrix molecule COMP (green). The nuclei visualized with Hoescht33258 (blue). Untreated chondrocytes were dominated by F-actin organized in stress fibers and pronounced COMP-staining (A, D). Chondrocytes stimulated with IL-1β (B, E) or LPS (C, F) exhibited a more retracted organization and occasionally ring structures were demonstrated (white arrows). COMP-staining similar to unstimulated chondrocytes was observed (n = 3-4). Control sections with isotype staining were used (data not shown).

Fig. 2

Time-dependent evoked Ca²⁺ responses. All chondrocytes were stimulated in a Ca²⁺ imaging system over time with glutamate (10⁻³ M) (A), 5-HT (10⁻⁵ M) (B) or ATP (10⁻⁴ M) (C). The areas under the Ca²⁺ peaks (AUC) of Ca²⁺ transients were calculated. The cells were from 3–4 coverslips, and from 3 different seeding times. One-way ANOVA followed by Turkey’s post hoc test was used for statistical analysis (n = 43) *p < 0.05, **p < 0.01.

Fig. 3

Glutamate evoked Ca²⁺ responses in inflammatory chondrocytes. All cells were stimulated in a Ca²⁺ imaging system, and the areas under the Ca²⁺ peaks (AUC) of Ca²⁺ transients were calculated. Chondrocyte Ca²⁺ responses to glutamate (10⁻³ M) when stimulated with IL-1β (5 ng/ml) or LPS (10 ng/ml) for 24 h, unstimulated cells were used as control (A). The cells were from 3–4 coverslips, and from 3 different seeding times. The appearance of the Ca²⁺ transients is visualized; results are shown from a typical experiment (B). For statistical analysis, a paired student’s t-test was used to compare unstimulated cells and IL-1β or LPS stimulated cells (n = 30), *p < 0.05, **p < 0.01.

Fig. 4

5-HT evoked Ca²⁺ responses in inflammatory chondrocytes. All cells were stimulated in a Ca²⁺ imaging system, and the areas under the Ca²⁺ peaks (AUC) of Ca²⁺ transients were calculated. Chondrocyte Ca²⁺ responses to 5-HT (10⁻⁵ M) when stimulated with IL-1β (5 ng/ml) or LPS (10 ng/ml) for 24 h, unstimulated cells were used as control (A). The cells were from 3–4 coverslips, and from 3 different seeding times. The appearance of the Ca²⁺ transients is visualized; results are shown from a typical experiment (B). For statistical analysis, a paired student’s t-test was used to compare unstimulated cells and IL-1β or LPS stimulated cells (n = 30), *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

ATP evoked Ca²⁺ responses in inflammatory chondrocytes. All cells were stimulated in a Ca²⁺ imaging system, and the areas under the Ca²⁺ peaks (AUC) of Ca²⁺ transients were calculated. Chondrocyte Ca²⁺ responses to ATP (10⁻⁴ M) when stimulated with IL-1β (5 ng/ml) or LPS (10 ng/ml) for 24 h, unstimulated cells were used as control (A). The cells were from 3–4 coverslips, and from 3 different seeding times. The appearance of the Ca²⁺ transients is visualized; results are shown from a typical experiment (B). For statistical analysis, a paired student’s t-test was used to compare unstimulated cells and IL-1β or LPS stimulated cells (n = 30), *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

A-F. The effect of IL-1β and LPS treatment on TLR4 and some membrane transporter levels in chondrocytes. Chondrocytes were treated with IL-1β (5 ng/ml) or LPS (10 ng/ml) for 24 h, and unstimulated cells were used as control. A-E, The cells were analyzed by western blotting with antibodies against TLR4, connexin 43 (Cx 43), Na⁺/K⁺ ATPase, GLT-1, GLAST-1, and β-actin as indicated on the figure. The blots are representative of three different horses. F, Representative Ponceau staining to confirm protein loading, which correlated to β-actin levels indicating it to be an appropriate internal control for normalization of protein levels.; Full size image is shown in supplementary documents (Fig. 6) of (Fig. 6A-F).

Fig. 7

A-E. The relative intensities of protein bands (mean ± SE) are shown in bar graphs for TLR4, connexin 43 (Cx 43), Na⁺/K⁺ ATPase, GLT-1, GLAST-1. Intensity were quantified using the ImageLab software in the linear exposure range and normalized to β-actin from the same blot. The intensity of the control protein band was set to 1.