Interleukin 1 beta-induced chloride currents are important in osteoarthritis onset: an in vitro study

Abstract

Persistent hypotonic and inflammatory conditions in the joint cavity can lead to the loss of cartilage matrix and cell death, which are the important mechanisms of osteoarthritis (OA) onset. Previous studies have confirmed that the existence of a hypotonic environment is a red flag for inflammation, as hypotonic environment induces the opening of the chloride channel of the cell and promotes chloride ion efflux, which prompts the cell volume to increase. Chloride channels play an important role in the regulation of mineralization and chondrocyte death. Here, we reported that OA chondrocytes showed a significant increase of cell death rate and the imbalance of cartilage matrix catabolism. We found that the distribution of skeleton protein F-actin was disordered. In addition, the volume-sensitive chloride current of OA chondrocytes decreased significantly with the increase of the expression levels of inflammation-related proteins caspase-1, caspase-3, and NLRP3. Moreover, interleukin-1β (IL-1β) showed a potential to activate the chloride current of normal chondrocytes. These results indicate that IL-1β-induced chloride channel opening in chondrocytes may be closely related to the occurrence of OA. This chloride channel opening process may therefore be a potential target for the treatment of OA.

Keywords: IL-1β; chloride channel; hypotonic; inflammation; osteoarthritis.

Figure 1.

Cell volume and mortality rate were increased in OA chondrocytes (A,B) Typical images of normal chondrocytes in a bright field and stained with toluidine blue, respectively. (C,D) Hematoxylin and eosin staining results of human normal or OA chondrocytes, respectively. The nuclei of chondrocytes are blue and the cytoplasm is transparent. (E) The integrity staining of freshly isolated chondrocyte membrane (the dye used was PI). Cells with incomplete membranes are in dark red fluorescent color. (F) The rate of cell death in normal and OA chondrocytes, respectively (n=126–155 cells in four views, **P<0.01 vs the normal group).

Figure 2.

Expression levels of COL I, COL II, and F-actin (A) The expression of COL I (red) identified by immunofluorescence in normal and OA chondrocytes. (B) The average fluorescence intensity of COL I (the mean±SE, n=3) (**P<0.01). (C) The expression of COL II (green). (D) The average fluorescence intensity of COL II (the mean±SE, n=3) (**P<0.01). (E) The difference in F-actin protein expression distribution between the normal group and OA group chondrocytes.

Figure 3.

Whole-cell patch-clamp recording of volume-sensitive chloride currents in normal and OA chondrocytes (A) The chloride ion fluorescence probe was used to detect the difference of chloride ion in normal and OA cartilage cells. The cultured P1 generation cells were seeded on a confocal dish and cultured overnight. Then isotonic (300 mOsmol/l) and hypotonic (160 mOsmol/l) extracellular fluid containing MQAE (10 mM) were added and incubated for 1 h. Green fluorescence is chloride ion fluorescent probe MQAE, excitation wavelength is 408 nm, false color: green. Red is cell membrane pigment, excitation wavelength is 543 nm, false color: red. (B) The pattern of whole-cell patch-clamp recording the chloride current. (C,D) Typical graphs of transient current under the hypotonic condition within 200 ms in normal or OA chondrocytes. (E,F) The typical time course of the Cl⁻ current activated by hypotonic bath solution in normal or OA chondrocytes. (G) The current–voltage (I–V) relationship recorded in normal or OA chondrocytes.

Figure 4.

Expression levels of caspase-1 and caspase-3 detected by immunofluorescence and immunohistochemical staining in normal and OA chondrocytes (A) The detection of caspase-1 and caspase-3 expressions by immunofluorescence. The digested chondrocytes are directly centrifuged on the slide for staining. The nuclei were stained with DAPI (blue), caspase-1 was labeled by Alexa Fluor 488 (green), and caspase-3 was labeled by Cy3 (red). Fluorescent images were obtained by confocal microscopy. (B) The detection of caspase-1 and caspase-3 expression levels by immunohistochemistry. Typical images were obtained with a pathological microscope. (C,D) The rate of positive cells for caspase-1 and caspase-3 (n=339–411 cells in five views, **P<0.01 vs the normal group; n=223–269 cells in five views, **P<0.01 vs the normal group).

Figure 5.

Expression level of NLRP3 detected by immunofluorescence and immunohistochemical staining in normal and OA chondrocytes (A) The detection of NLRP3 expression by immunofluorescence. The digested chondrocytes are directly centrifuged on the slide for staining. The nuclei were stained with DAPI (blue), and NLRP3 was labeled by the secondary antibody conjugated with FITC (green). (B) The detection of NLRP3 expression by immunohistochemistry. Typical images were obtained with a pathological microscope. (C) The rate of positive cells for NLRP3 (n=198–211 cells in eight views, **P<0.01 vs the normal group).

Figure 6.

IL-1β induces chloride currents in chondrocytes (A,B) The typical graphs of transient current under the conditions of an isotonic solution and isotonic solution containing 10 ng/ml of IL-1β within 200 ms in normal chondrocytes. (C) The typical time course of the Cl⁻ current activated by an isotonic solution containing 10 ng/ml of IL-1β in normal chondrocytes. (D) The current–voltage (I–V) relationship recorded in the context of an isotonic solution and isotonic solution containing IL-1β.

Figure 7.

The potential relationship between IL-1β-induced chondrocyte chloride current and OA The persistent hypotonic environment in joint fluid induces chondrocyte volume increase and Cl⁻ ion outflow. At the same time, it activates the NLRP3 inflammasome and then activates caspase-1, resulting in the release of IL-1β. IL-1β then activates the chloride channels of chondrocytes and continuously induces their opening, which leads to a pathological decrease in the chloride current. This may have a potential relationship with the occurrence of OA.