Insulin-Like Growth Factor Binding Protein 6 in Rheumatoid Arthritis: A Possible Novel Chemotactic Factor?

Abstract

Objectives: Immune cell migration from the bloodstream to target tissues is a hallmark of rheumatoid arthritis (RA) pathogenesis. The role of chemoattractants, mainly chemokines, and their possible targeting for therapeutic purposes have been under intense investigation over the last few years but the results were not as satisfactory as expected. The insulin-like growth factor binding protein 6 (IGFBP6), a direct inhibitor of insulin-like growth factor (IGF)-II, also exerts IGF-independent effects including tumor cell migration in vitro. We aimed to assess the expression of this protein in serum, synovial fluid, and synovial tissue (ST) of RA patients and to identify its possible chemotactic role in this disorder.

Methods: IGFBP6 was measured in RA patients and healthy donors (HD) sera by Luminex xMAP^® technology and in ST of RA patients and osteoarthritis (OA) controls by immunofluorescence. The identification of circulating IGFBP6⁺ cells was evaluated by flow cytometry and an in vitro migration assay was arranged.

Results: We demonstrated that IGFBP6 is able to induce greater in vitro migration of RA as compared to HD and OA T lymphocytes and is overexpressed in serum and ST of RA patients. This in vitro chemotactic activity can be partially inhibited by dexamethasone.

Conclusion: Our findings suggest a pathogenic role of IGFBP6 in RA and support its possible targeting for therapeutic purposes.

Keywords: IGFBP6; cell migration; rheumatoid arthritis; synovial membrane.

Figure 1

Insulin-like growth factor binding protein 6 (IGFBP6) expression in serum (A) of rheumatoid arthritis (RA) patients and healthy donors (HD) and in synovial fluid (SF) of RA patients and osteoarthritis (OA) controls (C). IGFBP6 concentration is significantly higher in RA serum compared to HD and is significantly lower in RA SF compared to OA SF. Correlation between serum IGFBP6 and patients’ age (B), SF IGFBP6 and SF white blood cell (WBC) count (D), and SF platelet (PLT) count (E). SF IGFBP6 concentration is inversely correlated to both SF WBC and PLT counts. p values shown in panels (A,C) were calculated with Mann–Whitney U test.

Figure 2

Intracellular expression of insulin-like growth factor binding protein 6 (IGFBP6) in peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients and healthy donors (HD) (A) and representative flow cytometry dot plots (B,C) gated on total PBMCs of IGFBP6 expression in CD3⁺ cells and CD16⁺ cells in a patient with RA. In RA PBMCs CD16⁺IGFBP6⁺ cell percentage is higher compared to HD while the percentage of CD3⁺IGFBP6⁺ cells is lower compared to HD. In addition, in HD the largest proportion of IGFBP6⁺ T cells coexpresses CD45RA. All p values were calculated with Mann–Whitney U test.

Figure 3

Insulin-like growth factor binding protein 6 (IGFBP6) expression in synovial tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Representative microphotographs of synovial membrane sections from patients with RA (A,C,E) and patients with OA (B,D,F) are shown. (A,B) Hematoxylin and eosin (H&E) staining. (C,D) Double immunofluorescence staining for CD3 (red) and CD20 (green) with 4′,6-diamidino-2-phenylindole (DAPI, blue) counterstain for nuclei. RA synovial membrane displays either diffuse immune cell infiltrates or ectopic lymphoid structures composed of T and B cells (A,C). Few scattered T and B cells are present in OA synovium (B,D). (E,F) Immunofluorescence staining for IGFBP6 (red) with DAPI (blue) counterstain. IGFBP6 is strongly expressed in different cells of the lining and sublining layers of RA synovium (E). Faint expression of IGFBP6 is detected in OA synovium (F). Arrowheads indicate synovial lining layer. Arrows indicate microvessels in synovial sublining layer. Original magnification: ×10 (A,B), ×20 (C,D), and ×40 (E,F). Scale bar: 200 µm (A,B), 100 µm (C,D), and 50 µm (E,F). (G) Densitometric analysis of IGFBP6 immunofluorescent staining intensity in lining and sublining layers of RA (n = 7) and OA (n = 6) synovium expressed as optical density in arbitrary units (a.u.). Data are mean ± SD. Student’s t-test was used for statistical analysis.

Figure 4

Insulin-like growth factor binding protein 6 (IGFBP6) expression in different synovial resident cell types. Representative microphotographs of synovial sections from rheumatoid arthritis (RA) patients (A,C,E) and osteoarthritis (OA) controls (B,D,F) subjected to double immunofluorescence staining for IGFBP6 (red) and the pan-endothelial cell marker CD31 (green) (A,B), the fibroblast marker VIM (green) (C,D), or the macrophage marker CD68 (green) (E,F). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). In RA synovium, IGFBP6 is strongly expressed in CD31⁺ vascular endothelial cells (A), VIM⁺ fibroblast-like synoviocytes (FLS) (C), and CD68⁺ macrophages (E). Weak IGFBP6 immunopositivity is detectable in the same cell types of OA synovium (B,D,F). Insets: higher magnifications of double-immunopositive cells from the respective panels. Note the intense immunopositivity for IGFBP6 localized either in the cytoplasm or in the nucleus of RA synovial endothelial cells and FLS. Original magnification: ×40 (A–F). Scale bar: 50 µm (A–F).

Figure 5

Insulin-like growth factor binding protein 6 (IGFBP6) expression in synovial infiltrating immune cells. Representative microphotographs of synovial sections from rheumatoid arthritis (RA) patients (A,C) and osteoarthritis (OA) controls (B,D) double immunostained for IGFBP6 (red) and the T-cell marker CD3 (green) (A,B) or the monocyte marker CD14 (green) (C,D). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Numerous CD3⁺ T cells displaying strong immunopositivity for IGFBP6 (yellow cytoplasmic staining) are present in RA synovial membrane (A). Only a few CD14⁺ monocytes coexpressing IGFBP6 can be observed in RA synovium (C). Original magnification: ×40 (A–D). Scale bar: 50 µm (A–D).

Figure 6

In vitro migration assay. Insulin-like growth factor binding protein 6 (IGFBP6) is able to induce the migration of rheumatoid arthritis (RA) but not healthy donors (HD) and osteoarthritis (OA) peripheral blood mononuclear cells (A). The addition of dexamethasone (dex) in the upper chamber of the transwell partly inhibited such RA cell migration (B). The majority of RA-migrated cells are CD3⁺ T lymphocytes (C) and the addition of dex does not affect the proportion of migrated cells (D). All p values are calculated with Mann–Whitney U test.