The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation

Abstract

Background: Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation.

Methods: Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+) chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated.

Results: In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163(+) chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+) chondrocytes with enhanced phagocytic activity were present in Col-II(+) chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+) chondrocytes were also found in isolated Col-II(+) chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163(+) cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+) chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05).

Conclusions: An increased number of CD163(+) chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.

Figure 1. Enhanced phagocytic activity and increased CD163 and TNF-α expression in degraded TMJ cartilage.

A: The gross surface morphology of rat temporomandibular joint (TMJ) condyles from control (4C) and experimental (4E, 8E, 12E) groups. Pit lesions are indicated by arrows. B: Comparison of the mRNA levels of MMP-3, MMP-9, CD163, TNF-α, IL-1, ACP-1, integrin-β1 and integrin-α4 in the condylar cartilage of control (C) and experimental (E) groups. C: Transmission electron micrographs of TMJ cartilage from control group (left top panel) and the regions with grossly damaged cartilage from experimental groups (the others panels). The apoptotic (outlined with the red dashed line) and necrotic chondrocytes are shown by arrow heads. Note that within the degraded TMJ cartilage some cells were phagocytizing neighboring apoptotic and necrotic cells. D: Serial sections of condylar cartilage from the 8-week old control (upper panels) and experimental (lower panels) groups, stained with H&E (HE), or co-stained with CD163 and TUNEL. F: fibrous layer; P: proliferative layer; H: hypertrophic layer. E: Comparison of the protein levels of CD163 and TNF-α in the condylar cartilage of control (C) and experimental (E) groups by Western blotting (left panel). Graph representing the quantification of the Western blotting results, normalized to the expression of β-actin. *P<0.05, **P<0.01. 4C: 4-week old control group; 4E: 4-week old experimental group; 8C: 8-week old control group; 8E: 8-week old experimental group; 12C: 12-week old control group; 12E: 12-week old experimental group.

Figure 2. Increase in CD163⁺ cells with enhanced phagocytic activity in experimentally-induced arthritic cartilage of rat TMJs.

A: Flow cytometry analysis and comparison of the percentage of total CD163⁺ cells and CD163⁺ cells with phagocytic activity within isolated type II collagen expressing (Col-II⁺) cells from TMJ cartilage from the 8-week experimental group and their age-matched controls. B: Confocal microscope images of the CD163⁺ cells and assessment of their phagocytic activity in primary cells isolated from TMJ cartilage. The images reveal an increase in CD163⁺ cells and enhanced co-localization with the FITC-labeled cell debris in 8-week experimental group compared with the age-matched controls. C: Serial confocal images (1–4) of the primary cells isolated from TMJ cartilage of 3-week old rats co-cultured with DiO-labeled cellular debris. Sections were stained with a CD163 antibody and a Cy3-conjugated secondary antibody. Note that the CD163⁺ cells showed membrane staining (red) and the cell debris (green) was located inside the cell membrane. D: Dynamic observation of the phagocytic process involving living CD163⁺ cells sorted from TMJ cartilage engulfing cellular debris. Note that the DiO-labeled debris is undergoing phagocytosis by the CD163⁺ cell indicated within the white box. E: Comparison of the nitric oxide (NO) concentration and amount of intracellular reactive oxygen species (ROS) in the primary cells isolated from TMJ cartilage from 8-week experimental group and their age-matched controls. *P<0.05.

Figure 3. CD163⁺ chondrocytes in normal joint cartilage of 3-week old rats.

A: Immunohistochemical staining of CD163 in cartilage from the TMJ and knee. The CD163⁺ cells located below the superior zone of the TMJ and knee cartilage, show intense membrane and cytoplasmic staining (arrows). Rat liver and muscle were selected as positive and negative controls, respectively, for the detection of CD163. Membrane staining of CD163⁺ cells was observed in liver (indicated by arrows), but no CD163⁺ cells were detected in muscle. As additional controls, TMJ and knee cartilage was also stained with an isotype control antibody. B: Flow cytometric analysis and graphical representation of the percentage of total CD163⁺ cells and CD163⁺ cells with phagocytic activity within the Col-II⁺ cells isolated from TMJ cartilage (n = 3; *P<0.05).

Figure 4. Exogenous TNF-α increased CD163 expression in primary chondrocytes from TMJ cartilage of 3-week old rats.

A: The primary cells isolated from TMJ cartilage of 3-week old rats were positive for type II collagen (Col-II) and aggrecan, as detected by immunofluorescence and toluidine blue, respectively (400× magnification). B: A time-course of induction of CD163 mRNA expression in primary cells isolated from TMJ cartilage and treated with 10 ng/ml of TNF-α. C–D: Flow cytometric analysis and graphical representation of the percentage of CD163⁺ cells within the primary cells isolated from TMJ cartilage and treated with 10 ng/ml of TNF-α.

Figure 5. TNF-α increased the phagocytic activity of CD163⁺ cells isolated from 3 week old rat TMJ cartilage.

A–B: Flow cytometry analysis (A) and graphical representation (B) of the percentage of total CD163⁺ cells and CD163⁺ cell with phagocytic activity within the primary cells isolated from TMJ cartilage and treated with vehicle, TNF-α alone, or TNF-α and a CD163 neutralizing antibody.

Figure 6. TNF-α increased the phagocytic and migratory activities of CD163⁺ cells isolated from rat TMJ cartilage.

A–B: Confocal microscope images (A) and graphical representation (B) of the numbers of CD163⁺ cells and their phagocytic activity within primary cells isolated from TMJ cartilage and treated with vehicle, TNF-α alone, or TNF-α and a CD163 neutralizing antibody. The co-localization of the CD163⁺ cell with DiO-labeled cell debris (arrows), indicates that the cell debris is undergoing phagocytosis by the CD163⁺ cells, as shown in the insets. Bar: 50 µm. C: Transwell assay combined with immunohistochemical staining of CD163 indicates the migratory potential of CD163⁺ cells in response to 10 ng/ml TNF-α, which is impaired in the presence of the TNF-α neutralizing antibody (AT, 1 µg/ml). Arrows indicate the migrating CD163⁺ cells. Five fields were selected at random (at 200× magnification), and the number of CD163⁺ cells and total cells in each image were counted. **P<0.01.

Figure 7. Increased expression of CD163 and TNF-α in knee cartilage from osteoarthritis patients.

A and B: Toluidine blue and immunohistochemical staining of CD163 and TNF-α. C: Quantification of CD163⁺ and TNF-α⁺ cells from immunohistochemistry samples comparing the knee cartilage from patients with osteoarthritis (OA) or amputees (control). D: Comparison of the mRNA levels of TNF-α and CD163 in Col-II⁺ cells isolated from knee cartilage from OA patients or amputees (control). *P<0.05.