Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus

Mohammed Talha Shekhani, Toni S Forde, Altynai Adilbayeva, Mohamed Ramez, Askhat Myngbay, Yergali Bexeitov, Volkhard Lindner, Vyacheslav A Adarichev, Mohammed Talha Shekhani, Toni S Forde, Altynai Adilbayeva, Mohamed Ramez, Askhat Myngbay, Yergali Bexeitov, Volkhard Lindner, Vyacheslav A Adarichev

Abstract

Background: The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation.

Methods: Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1.

Results: Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01).

Conclusion: CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.

Keywords: Animal model; Cell motility; Rrheumatoid arthritis; Synoviocytes.

Figures

Fig. 1
Fig. 1
Collagen triple helix repeat containing 1 (CTHRC1) expression is inducible in arthritis. Histology of hind limb (a, b) and knee joint (c–f) sections of naïve and arthritic BALB/c mice. Ti tibia, Ta talus, N navicular bone of hind paw. In naïve mice, synovial membrane is normal (a, c, green arrowheads). In mice with experimentally induced arthritis at the seventh day post injection of anti-collagen antibodies, massive multicellular pannus (yellow arrowhead) and influx of granulocytes into synovial cavity (blue arrowhead) were prominent (b, d). Immunohistochemical staining for CTHRC1 was negative in the normal synovial membrane (e) in contrast to strong positive staining of the arthritic hyperplastic synovium (f, brown 3,3′-diaminobenzidine precipitate). The border between bone (Bn) and cartilage (Ct) is shown with a dashed line (f). Sections were counterstained with hematoxylin (e, f) or stained with hematoxylin-eosin (a–d). Scale bars are 100 μm (a, b) and 50 μm (c–f). The data and images are representative of the two independent experiments using BALB/c wildtype inbred mice and mice from (FVB/J × BALB/c)F1 hybrid genetic backgrounds
Fig. 2
Fig. 2
Collagen triple helix repeat containing 1 (CTHRC1) expression is inducible both in mouse and in human arthritis. Naïve and arthritic mouse paws were analyzed for Cthrc1 RNA message using RT-PCR of the total RNA isolated from paws of naïve mice and mice with collagen antibody-induced arthritis (CAIA) (a). Protein content in synovium was measured using ImageJ densitometry of immunohistochemical-stained histological sections (b). Student’s t test was used to estimate the difference between arthritic and naive groups. In patients with rheumatoid arthritis (RA) with an average 28-joint-count disease activity score of 3.61 (e), blood plasma contained a significantly higher absolute concentration of CTHRC1 (c) and C-reactive protein (CRP) (d) according to the Mann–Whitney U test. Horizontal lines show median with range. Gapdh glyceraldehyde 3-phosphate dehydrogenase
Fig. 3
Fig. 3
Immunohistochemical localization of collagen triple helix repeat containing 1 (CTHRC1) expressing cells in arthritic synovium. Immunohistochemical staining (IHC) for CTHRC1 was positive for hypercellular pannus of the synovial joints of the hind paws (a) and of the knee (b–f) of BALB/c mice with experimentally induced arthritis. Specificity of the IHC staining was confirmed using the complete staining protocol on Cthrc1 gene-deficient mouse skeletal tissues (g). An additional control is shown using Cthrc1-wildtype tissue sections that were incubated with secondary donkey anti-rabbit antibody alone, which produced no signal (h). CTHRC1+ cells were found in the arthritic pannus intimal lining (a, e, f) and at the bone-pannus and cartilage-pannus boundaries (a–d). Dotted line shows the border between cartilage (Ct) and bone (Bn) (b). Fct fibrocartilage, Syn synovial cavity. Sections were counterstained with alcian blue (c, d, g) or with hematoxylin (a, b, e, f, h). Scale bars are 100 μm (a), 50 μm (b, c, e, g, h), and 20 μm (d, f)
Fig. 4
Fig. 4
Co-localization of cells expressing synovial markers. Sagittal plane histological sections of the medial aspect of inflamed knee joints were stained with hematoxylin-eosin (a), macrophage-specific calcium-binding protein IBA1 (b, j), alpha smooth muscle actin (α-SMA) (c, d, l), collagen triple helix repeat containing 1 (CTHRC1) (e, f, k), cadherin CDH11 (g–i). Sections were counterstained with hematoxylin or alcian blue. Meniscus (m) was used to align images; femur (f), tibia (t). Area of the pannus-meniscus junction labeled with yellow arrowheads (c, e, g) are zoomed in at panels d, f, and h. Representative immunohistochemical (IHC) images at panels i–l show the area of pannus-meniscus junction at higher × 40 objective magnification. At this structure, cells positive for CDH11, CTHRC1 and α-SMA IHC staining demonstrated a similar gradient distribution with maximal signal at the meniscus fibrocartilage-pannus border, while macrophage-specific IBA1 showed dispersed localization. Scale bars are 100 μm (a–h) and 25 μm (i–l)
Fig. 5
Fig. 5
Collagen triple helix repeat containing 1 (CTHRC1) effects transwell cell migration. (a) Western immunoblotting detection of CTHRC1 in rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS), NIH 3T3, C3H/10 T1/2. RA-FLS express CTHRC1, but murine fibroblasts are negative for CTHRC1. (b) RA-FLS also express cadherin CDH11, alpha smooth muscle actin (α-SMA), and α-tubulin. Staining membrane with secondary antibodies alone was negative. (c) CTHRC1 is secreted into conditioned media upon transfection and overexpression in Chinese hamster ovary (CHO) cells. Lanes: (1) positive control 10 ng rhCTHRC1; (2) day 0; (3) day 1; (4) day 3; (5) day 5. (d) RA-FLS were treated directly in transwell chambers with rhCTHRC1 protein, while FBS concentration was varied from 0 %, 1–6 %. Cells that migrated through the membrane were stained with toluidine blue and counted under the microscope. Solid black columns represent cells treated with rhCTHRC1, gray columns are untreated cells. (e) murine C3H/10T1/2 fibroblasts were treated with rhCTHRC1 similarly to RA-FLS. (f) Serum from mice with acute CAIA (5 % serum mixed with 5 % FBS in complete media) showed the strongest promigratory effect. (g) Toluidine blue staining and microscopy of cells migrated through the membrane and spread on its opposite side after the completion of the experiment. Numerous dots are membrane pores of 8 μm in size. Data are representative of three similar but independent experiments. Statistical significance was calculated using Student’s t test: *p < 0.05; **p < 0.01 for treatment versus no treatment comparison
Fig. 6
Fig. 6
Time-lapse microscopy analysis of fibroblast migration under collagen triple helix repeat containing 1 (CTHRC1) treatment. NIH 3T3 fibroblasts seeded onto μ-slides were treated with rhCTHRC1 at 1000 ng/ml and observed using time-lapse microscopy. (a) Cell trajectories derived from stacks of images taken every 15 minutes during migration were presented as Rose plots to compare cell motility parameters. (b) Each cell trajectory was analyzed for accumulated distance (Ad), Euclidean distance (Ed), directness (Ed/Ad), and cell velocity (Ad/time). (c) Average values for 20–40 cells for each condition were compared. Solid black columns represent cells treated with rhCTHRC1, gray columns represent untreated cells. (d) Cell length was defined as a Euclidean distance between the most distant points, usually between the front of lamellipodia and the end of cell tail. Cell length is shown by a white bar. Phase contrast images of cells in the beginning and after 12 hours CTHRC1 treatment are presented. Scale bar is 20 μm. (e) Cell length was measured at the beginning and the end of observation period. Data represent four similar but independent experiments. Statistical significance was calculated using Student’s t test: *p < 0.05; **p < 0.01 for comparison of treatment versus no treatment

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