Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun 16;22(12):6461.
doi: 10.3390/ijms22126461.

Immunofluorescence Analysis of NF-kB and iNOS Expression in Different Cell Populations during Early and Advanced Knee Osteoarthritis

Marko Ostojic  1   2 , Ante Zevrnja  3 , Katarina Vukojevic  4   5 , Violeta Soljic  5
Affiliations

Immunofluorescence Analysis of NF-kB and iNOS Expression in Different Cell Populations during Early and Advanced Knee Osteoarthritis

Marko Ostojic et al. Int J Mol Sci. .

Abstract

Synovitis of the knee synovium is proven to be a precursor of knee osteoarthritis (OA), leading to a radiologically advanced stage of the disease. This study was conducted to elucidate the expression pattern of different inflammatory factors-NF-kB, iNOS, and MMP-9 in a subpopulation of synovial cells. Thirty synovial membrane intra-operative biopsies of patients (ten controls, ten with early OA, and ten with advanced OA, according to the Kellgren-Lawrence radiological score) were immunohistochemically stained for NF-kB, iNOS, and MMP9, and for different cell markers for macrophages, fibroblasts, leukocytes, lymphocytes, blood vessel endothelial cells, and blood vessel smooth muscle cells. The total number of CD68+/NF-kB+ cells/mm2 in the intima of early OA patients (median = 2359) was significantly higher compared to the total number of vimentin+/Nf-kB+ cells/mm2 (median = 1321) and LCA+/NF-kB+ cells/mm2 (median = 64) (p < 0.001 and p < 0.0001, respectively). The total number of LCA+/NF-kB+ cells/mm2 in the subintima of advanced OA patients (median = 2123) was significantly higher compared to the total number of vimentin+/NF-kB+ cells/mm2 (median = 14) and CD68+/NF-kB+ cells/mm2 (median = 29) (p < 0.0001). The total number of CD68+/iNOS+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/iNOS+ cells/mm2 and LCA+/iNOS+ cells/mm2 (p < 0.0001 and p < 0.001, respectively). The total number of CD68+/MMP-9+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/MMP-9+ cells/mm2 and CD5+/MMP-9+ cells/mm2 (p < 0.0001). Macrophages may have a leading role in OA progression through the NF-kB production of inflammatory factors (iNOS and MMP-9) in the intima, except in advanced OA, where leukocytes could have a dominant role through NF-kB production in subintima. The blocking of macrophageal and leukocyte NF-kB expression is a possible therapeutic target as a disease modifying drug.

Keywords: inducible nitric oxide synthase; macrophage; nuclear factor kappa B; osteoarthritis; synovitis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Hystological staining of healthy (ctrl) (A), early osteoarthritic synovium (eOA) (B), and advanced osteoarthritic synovium (aOA) (C). The synovial membrane of patients in the control group shows no resident cells in the subintima and the intima (arrows). In patients with early OA, the synovium contains highly visible cell infiltration (frame) in the intima (arrow). In patients with advanced OA, lymphoid nodules (frame) are shown. Haematoxylin-eosin staining. Magnification ×20, scale bar 50 µm.
Figure 2
Figure 2
The distribution of vimentin+/NF-kB+, CD68+/NF-kB+, and LCA+/NF-kB+ (A), vimentin+/iNOS+, CD68+/iNOS+, and LCA+/iNOS+ (B), and vimentin+/MMP-9+, CD68+/MMP-9+, and CD5+/MMP-9 (C) positive cells per mm2 in the early osteoarthritis (OA), in the advanced osteoarthritis (OA) and control groups. Data were shown as mean ± SD. Significant differences (Kruskal–Wallis) are indicated by * p < 0.05, ** p < 0.001, *** p < 0.0001.
Figure 3
Figure 3
The synovial membrane of patients with early OA (eOA) (A–C). Vimentin positive (red) fibroblasts in the intima and subintima (frame), NF-kB positive cells (green) in the synovial intima and subintima (B). The merge of DAPI (blue) nuclear stain+vimentin+NF-kB (magnification of the frame in panel (A)) show numerous fibroblasts in the intima co-localized with NF-kB and vimentin (arrow), while the subintima displays vimentin positive fibroblasts (arrowhead) (C). The synovial membrane of patients with early OA (eOA) (D–F). CD68 positive (red) macrophages in the intima and subintima (frame). NF-kB positive cells (green) in the synovial intima and subintima (E). The merge of DAPI (blue)+CD68+NF-kB (magnification of the frame in panel (D)) show numerous macrophages in the intima co-localized with NF-kB and CD68 (arrow), while the subintima displayed CD68 positive macrophages(arrowhead) (F). The synovial membrane of patients with advanced OA (aOA) (G–I). LCA positive (red) lymphocytes in the intima, subintima, and lymph nodule (frame). NF-kB positive cells (green) in the synovial intima and subintima (H). The merge of DAPI (blue)+LCA+NF-kB (magnification of the frame in panel (G)) show numerous lymphocytes in the lymph nodule co-localized with NF-kB and LCA (arrow), while some lymphocytes displayed only LCA (arrowhead) (I). Magnification ×40 (first two columns), scale bar 25 µm and ×100 (last column), scale bar 10 µm.
Figure 4
Figure 4
The synovial membrane of patients with early OA (eOA) (A–C). Vimentin positive (red) fibroblasts in the subintima (frame). iNOS positive cells (green) in the synovial intima and subintima (B). The merge of DAPI (blue) nuclear stain+vimentin+iNOS (magnification of frame in panel A) show occasional fibroblasts in the subintima co-localized with iNOS and vimentin (arrow) (C). The synovial membrane of patients with early OA (eOA) (D–F). CD68 positive (red) macrophages in the intima and subintima (frame). iNOS positive cells (green) in the synovial intima and subintima (E). The merge of DAPI (blue)+CD68+iNOS (magnification of the frame in panel (D)) show numerous macrophages in the intima co-localized with iNOS and CD68 (arrow), while the subintima displayed CD68 positive macrophages(arrowhead) (F). The synovial membrane of patients with advanced OA (aOA) (G–I). LCA positive (red) lymphocytes in the intima, subintima, and lymph nodule (frame). iNOS positive cells (green) in the synovial intima and subintima (H). The merge of DAPI (blue)+LCA+iNOS (magnification of the frame in panel (G)) show no co-localization of iNOS and LCA, while a numerous number of lymphocytes display only LCA (arrowhead) (I). Magnification ×40 (first two columns), scale bar 25 µm and ×100 (last column), scale bar 10 µm.
Figure 5
Figure 5
The synovial membrane of patients with early OA (eOA) (A–C). Vimentin positive (red) fibroblasts in the intima and subintima (frame). MMP-9 positive cells (green) in the synovial intima and subintima (B). The merge of DAPI (blue) nuclear stain+vimentin+MMP-9 (magnification of the frame in panel (A)) show numerous fibroblasts in the intima co-localized with MMP-9 and vimentin (arrow), while the subintima displayed vimentin positive fibroblasts (arrowhead) (C). The synovial membrane of patients with early OA (eOA) (D–F). CD68 positive (red) macrophages in the intima and subintima (frame). MMP-9 positive cells (green) in the synovial intima and subintima (E). The merge of DAPI. (blue)+CD68+MMP-9(magnification of the frame in panel (D)) show numerous macrophages in the intima co-localized with MMP-9 and CD68 (arrow), while the subintima displayed CD68 positive macrophages(arrowhead) (F). The synovial membrane of patients with advanced OA (aOA) (G–I). CD5 positive (red) lymphocytes in the subintima (frame). MMP-9 positive cells (green) in the synovial intima and subintima (H). The merge of DAPI (blue)+CD5+iNOS (magnification of the frame in panel (G)) show the co-localization of MMP-9 and CD5 (arrow), while lymphocytes occasionally display only CD5 (arrowhead) (I). Magnification ×40 (first two columns), scale bar 25 µm and ×100 (last column), scale bar 10 µm.
Figure 6
Figure 6
The synovial membrane of patients. CD31 positive cells (red) in the blood vessels (frame) (A). CD31+NF-kB+ (merge) in the endothelial cells (B). The merge of CD31+NF-kB+DAPI (blue) nuclear; stain (C). CD31 positive cells (red) in the blood vessels (frame) (D). CD31+iNOS+ (merge) in the endothelial cells (E). The merge of CD31+iNOS+DAPI (blue) nuclear stain (F). Actin positive cells (red) in the blood vessels (frame) (G). Actin+NF-kB+(merge) in the smooth muscle cells (H). The merge of Actin+NF-kB+DAPI (blue) nuclear stain (I). Actin positive cells (red) in the blood vessels (frame) (J). Actin+iNOS+(merge) in the smooth muscle cells (K). The merge of Actin+iNOS+DAPI (blue) nuclear stain (L). eOA—early OA; aOA—advanced OA. Magnification ×40 (first two columns), scale bar 50 µm and ×100 (last column), scale bar 20 µm.
See this image and copyright information in PMC

References

    1. Loeser R.F., Goldring S.R., Scanzello C.R., Goldring M.B. Osteoarthritis: A disease of the joint as an organ. Arthritis Rheum. 2012;64:1697–1707. doi: 10.1002/art.34453. - DOI - PMC - PubMed
    1. Sokolove J., Lepus C.M. Role of inflammation in the pathogenesis of osteoarthritis: Latest findings and interpretations. Ther. Adv. Musculoskelet. Dis. 2013;5:77–94. doi: 10.1177/1759720X12467868. - DOI - PMC - PubMed
    1. Geurts J., Nasi S., Distel P., Müller-Gerbl M., Prolla T.A., Kujoth G.C., Walker U.A., Hügle T. Prematurely aging mitochondrial DNA mutator mice display subchondral osteopenia and chondrocyte hypertrophy without further osteoarthritis features. Sci. Rep. 2020;10:1296–1297. doi: 10.1038/s41598-020-58385-w. - DOI - PMC - PubMed
    1. Walsh D.A., Stocks J. New Therapeutic Targets for Osteoarthritis Pain. SLAS Discov. Adv. Life Sci. R&D. 2017;22:931–949. doi: 10.1177/2472555217716912. - DOI - PubMed
    1. Robinson W.H., Lepus C.M., Wang Q., Raghu H., Mao R., Lindstrom T.M., Sokolove J. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 2016;12:580–592. doi: 10.1038/nrrheum.2016.136. - DOI - PMC - PubMed