Interleukin-1 inhibits osmotically induced calcium signaling and volume regulation in articular chondrocytes

Abstract

Objective: Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca(2+)](i)). The goal of this study was to examine the hypothesis that interleukin-1 (IL-1), a pro-inflammatory cytokine associated with osteoarthritis, influences osmotically induced Ca(2+) signaling.

Methods: Fluorescence ratio imaging was used to measure [Ca(2+)](i) and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10-ng/ml IL-1alpha. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-1Ra), Ca(2+) mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca(2+)](i), F-actin remodeling, volume adaptation and active volume recovery.

Results: In response to osmotic stress, chondrocytes exhibited transient increases in [Ca(2+)](i), generally followed by decaying oscillations. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease (RVD) following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca(2+)](i) in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca(2+)](i) in those cells that did respond. Co-treatment with IL-1Ra, thapsigargin, or toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization.

Conclusions: IL-1 alters the normal volumetric and Ca(2+) signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases. These findings provide further insights into the mechanisms by which IL-1 may interfere with normal physiologic processes in the chondrocyte, such as the adaptation or regulatory responses to mechanical or osmotic loading.

Figure 1. Representative trace of [Ca²⁺]_i response of chondrocytes to hypo-osmotic stress

Articular chondrocytes exposed to hypo- or hyper-osmotic stress medium responded by mobilizing [Ca²⁺]_i, followed by decaying oscillations. The y-axis on this plot represents the fluorescence ratio (Fluo-3 AM excitation: Fura Red AM excitation) normalized to the ratio before exposure to osmotic stress.

Figure 2

(a) Magnitude of peak [Ca²⁺]_i and time to peak in chondrocytes after exposure to hypo-osmotic stress Control chondrocytes responded to hypo-osmotic stress with a 2.5-fold increase in [Ca²⁺]_i within 83 seconds of exposure. Pre-treatment of cells with IL-1 had no effect on the fold increase of [Ca²⁺]_i or the time to peak. Data is expressed as mean ± standard deviation. * p<0.05 vs. control, n=14–20 cells, ANOVA with Fisher’s PLSD post hoc test. (b) Magnitude of peak [Ca²⁺]_i and time to peak in chondrocytes after exposure to hyper-osmotic stress. Control chondrocytes responded to hyper-osmotic stress with a 3-fold increase in [Ca²⁺]_i within 234 seconds of exposure. Pre-treatment of cells with IL-1 had no effect on the fold increase of [Ca²⁺]_i but significantly increased the time to peak to 412 seconds. Data is expressed as mean ± standard deviation. * p<0.05 vs. control, n=14–20 cells, ANOVA with Fisher’s PLSD post hoc test.

Figure 3

(a) Characteristic volume response of chondrocytes to hypo-osmotic stress Cell volume change after acute osmotic stress was measured using a custom image analysis algorithm. This trace shows cell volume over time for an untreated chondrocyte and an IL-1 treated chondrocyte exposed to 240 mOsm medium at time t=0. Cells respond to hypo-osmotic stress by rapidly swelling and attempting to regulate their volume. IL-1 treatment inhibited volume regulation in chondrocytes. (b) Characteristic volume response of chondrocytes to hyper-osmotic stress. Cell volume change after acute osmotic stress was measured using a custom image analysis algorithm. This trace shows cell volume over time for a single untreated chondrocyte exposed to 440 mOsm medium at time t=0. Cells respond to hyper-osmotic stress by rapidly shrinking with no appreciable volume regulation.

Figure 4. Cell swelling and regulatory volume decrease in chondrocytes exposed to hypo-osmotic stress

Control chondrocytes responded to osmotic stress with a rapid increase in cell volume to approximately 150% of initial volume followed by RVD to approximately 125% of initial volume. Pre-treatment of cells with IL-1 had no effect on cell swelling, but reduced the capacity for RVD. Inhibition of the IL-1 effect by IL-1Ra and thapsigargin successfully restored normal RVD. Cells pre-treated with toxin B before IL-1 exposure swelled normally, and showed significant volume regulation although final volume was slightly higher than in the control cells. * p<0.05 vs. untreated control, n=31–43 cells, ANOVA with Fisher’s PLSD post hoc test.

Figure 5. Extent of cell shrinking in chondrocytes exposed to hyper-osmotic stress

Control chondrocytes responded with a rapid decrease in cell volume to 80% of initial volume. Pre-treatment of cells with IL-1 reduced the extent of cell shrinking to 86% of initial volume. Inhibition of the IL-1 effect by IL-1Ra, thapsigargin, or toxin B restored volume adaptation to levels similar to control. * p<0.05 vs. untreated control, n=32–40 cells, ANOVA with Fisher’s PLSD post hoc test.

Figure 6. Cellular F-actin content in isolated chondrocytes after exposure to hypo-osmotic stress with and without IL-1 treatment

Hypo-osmotic stress led to a decrease in cellular F-actin within 2 minutes in both control and IL-1 treated chondrocytes. This disruption was followed by a gradual recovery to levels indistinguishable from the initial condition within 10 minutes. Conversely, after IL-1 treatment, chondrocytes did not significantly recover their cellular F-actin content by 10 minutes. Data is expressed as mean ± standard deviation. * p<0.05 vs. control group at 1 hour, n= 14–20, ANOVA with Fisher’s PLSD post hoc test.

Figure 7. Time constant of cell shrinking in chondrocytes exposed to hyper-osmotic stress

Control chondrocytes responded to hyper-osmotic stress with a rapid decrease in cell volume (τ=17.9 seconds). Pre-treatment of cells with IL-1 reduced the rate of cell shrinking as reflected by the greater time constant (τ=32.4 seconds). Inhibition of the IL-1 effect by IL-1Ra ((τ=19.8 seconds), thapsigargin (τ=16.6 seconds), and toxin B (τ=20.2 seconds) restored the rate of cell volume change to levels similar to control. * p<0.05 vs. untreated control, n=32–40 cells, ANOVA with Fisher’s PLSD post hoc test.