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Expression of Lectin-Like Transcript 1, the Ligand for CD161, in Rheumatoid Arthritis.

Chalan P, Bijzet J, Huitema MG, Kroesen BJ, Brouwer E, Boots AM - PLoS ONE (2015)

Bottom Line: FACS analysis of digested ST confirmed LLT1 expression by CD68+ cells.Elevated systemic sLLT1 was found in all patient groups.Serum levels of sLLT1 were increased in all patient groups (patients with early- and late-stage RA, seropositive arthralgia and spondyloarthropathy) when compared to healthy subjects.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT

Objectives: Precursor Th17 lineage cells expressing CD161 are implicated in Rheumatoid Arthritis (RA) pathogenesis. CD4+CD161+ T-cells accumulate in RA joints and may acquire a non classical Th1 phenotype. The endogenous ligand for CD161 is lectin-like transcript 1 (LLT1). CD161/LLT1 ligation may co-stimulate T-cell IFN-γ production. We investigated the presence and identity of LLT1-expressing cells in RA synovial fluid (SF) and synovial tissue (ST). We also assessed levels of soluble LLT1 (sLLT1) in different phases of RA development.

Methods: Paired samples of peripheral blood mononuclear cells (MC) and SFMC (n = 14), digested ST cells (n = 4) and ST paraffin sections (n = 6) from late-stage RA were analyzed for LLT1 expression by flow cytometry and immunohistochemistry. sLLT1 was measured using a sandwich ELISA. Sera and SF from late-stage RA (n = 26), recently diagnosed RA patients (n = 39), seropositive arthralgia patients (SAP, n = 31), spondyloarthropathy patients (SpA, n = 26) and healthy controls (HC, n = 31) were assayed.

Results: In RA SF, LLT1 was expressed by a small proportion of monocytes. In RA ST, LLT1-expressing cells were detected in the lining, sublining layer and in areas with infiltrates. The LLT1 staining pattern overlapped with the CD68 staining pattern. FACS analysis of digested ST confirmed LLT1 expression by CD68+ cells. Elevated systemic sLLT1 was found in all patient groups.

Conclusions: In RA joints, LLT1 is expressed by cells of the monocyte/macrophage lineage. Serum levels of sLLT1 were increased in all patient groups (patients with early- and late-stage RA, seropositive arthralgia and spondyloarthropathy) when compared to healthy subjects.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemical detection of LLT1 expression in RA ST cells.To study surface-expressed LLT1 at the site of inflammation, 6 synovial tissue biopsies obtained from hand, shoulder or knee joints from long-standing, treated RA patients who underwent joint replacement surgery or synovectomy were processed for immunohistochemistry. Representative pictures showing immunohistochemical staining of consecutive tissue slides from 2 late-stage RA patients stained with antibodies against LLT1, CD68, CD3 and CD20cy (A,B). Graphs in C depict the results of the semiquantitative scoring (mean + SD) performed by 3 independent researchers. Scoring of the staining of all the markers was performed according to a 4-point scale: 0 = no positive cells; 1 = <5% positive cells; 2 = 5–50% positive cells; 3 = >50% positive cells. Three to five different pictures of each slide section (n = 6 different sections per biopsy) were taken. In each picture the lining, sublining, lymphoid infiltrate area’s (defined based on CD3 and CD20cy staining) and blood vessels were scored separately for the expression of LLT1, CD68, CD3 and CD20cy. Mouse monoclonal anti-LLT1, clone 4C7 (Abnova) was used.
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pone.0132436.g002: Immunohistochemical detection of LLT1 expression in RA ST cells.To study surface-expressed LLT1 at the site of inflammation, 6 synovial tissue biopsies obtained from hand, shoulder or knee joints from long-standing, treated RA patients who underwent joint replacement surgery or synovectomy were processed for immunohistochemistry. Representative pictures showing immunohistochemical staining of consecutive tissue slides from 2 late-stage RA patients stained with antibodies against LLT1, CD68, CD3 and CD20cy (A,B). Graphs in C depict the results of the semiquantitative scoring (mean + SD) performed by 3 independent researchers. Scoring of the staining of all the markers was performed according to a 4-point scale: 0 = no positive cells; 1 = <5% positive cells; 2 = 5–50% positive cells; 3 = >50% positive cells. Three to five different pictures of each slide section (n = 6 different sections per biopsy) were taken. In each picture the lining, sublining, lymphoid infiltrate area’s (defined based on CD3 and CD20cy staining) and blood vessels were scored separately for the expression of LLT1, CD68, CD3 and CD20cy. Mouse monoclonal anti-LLT1, clone 4C7 (Abnova) was used.

Mentions: Following the detection of surface LLT1 by a proportion of SF monocytes, we next investigated the presence of LLT1-bearing cells in synovial tissue. To that end, synovial tissue specimens with low- to high-grade synovitis (median Krenn score of 5 [range 2–7]) [18] were obtained from six RA patients. Consecutive sections of synovial tissue were stained with antibodies against LLT1, CD68, CD3 and CD20cy (Fig 2). LLT1 staining was detected in all six biopsies and was predominant in the lining layer which showed pathological enlargement in all 6 biopsies analyzed (≥1 point according to Krenn et al [18]). LLT1 staining was also found in the sublining and in transitional areas consisting of different cell types [19]. Small numbers of LLT1+ cells were observed in scattered lymphoid infiltrates and more dense perivascular infiltrates. These synovial membrane areas were defined based on CD3 (T-cells) and CD20cy (B-cells) staining. Analysis of the results of the semi-quantitative scoring showed that 3/6 tissue biopsies had low (median score 1, indicating <5% positive cells) and 2/6 biopsies had moderate (median score 2, indicating 5–50% positive cells) expression of LLT1 in the lining layer. LLT1 expression in the sublining layer was found to be low (median score 1) in 4/6 and moderate (median score 2) in 2/6 biopsies while the infiltrate showed low and moderate LLT1 expression in 3/6 and 2/6 biopsies, respectively. In addition, some LLT1 staining of blood vessels was observed. The LLT1 staining pattern showed a marked overlap with CD68 staining. In all biopsies analyzed, CD68 expression was predominant in the lining and sublining layer and to a lesser extent in the lymphoid infiltrate areas.


Expression of Lectin-Like Transcript 1, the Ligand for CD161, in Rheumatoid Arthritis.

Chalan P, Bijzet J, Huitema MG, Kroesen BJ, Brouwer E, Boots AM - PLoS ONE (2015)

Immunohistochemical detection of LLT1 expression in RA ST cells.To study surface-expressed LLT1 at the site of inflammation, 6 synovial tissue biopsies obtained from hand, shoulder or knee joints from long-standing, treated RA patients who underwent joint replacement surgery or synovectomy were processed for immunohistochemistry. Representative pictures showing immunohistochemical staining of consecutive tissue slides from 2 late-stage RA patients stained with antibodies against LLT1, CD68, CD3 and CD20cy (A,B). Graphs in C depict the results of the semiquantitative scoring (mean + SD) performed by 3 independent researchers. Scoring of the staining of all the markers was performed according to a 4-point scale: 0 = no positive cells; 1 = <5% positive cells; 2 = 5–50% positive cells; 3 = >50% positive cells. Three to five different pictures of each slide section (n = 6 different sections per biopsy) were taken. In each picture the lining, sublining, lymphoid infiltrate area’s (defined based on CD3 and CD20cy staining) and blood vessels were scored separately for the expression of LLT1, CD68, CD3 and CD20cy. Mouse monoclonal anti-LLT1, clone 4C7 (Abnova) was used.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492745&req=5

pone.0132436.g002: Immunohistochemical detection of LLT1 expression in RA ST cells.To study surface-expressed LLT1 at the site of inflammation, 6 synovial tissue biopsies obtained from hand, shoulder or knee joints from long-standing, treated RA patients who underwent joint replacement surgery or synovectomy were processed for immunohistochemistry. Representative pictures showing immunohistochemical staining of consecutive tissue slides from 2 late-stage RA patients stained with antibodies against LLT1, CD68, CD3 and CD20cy (A,B). Graphs in C depict the results of the semiquantitative scoring (mean + SD) performed by 3 independent researchers. Scoring of the staining of all the markers was performed according to a 4-point scale: 0 = no positive cells; 1 = <5% positive cells; 2 = 5–50% positive cells; 3 = >50% positive cells. Three to five different pictures of each slide section (n = 6 different sections per biopsy) were taken. In each picture the lining, sublining, lymphoid infiltrate area’s (defined based on CD3 and CD20cy staining) and blood vessels were scored separately for the expression of LLT1, CD68, CD3 and CD20cy. Mouse monoclonal anti-LLT1, clone 4C7 (Abnova) was used.
Mentions: Following the detection of surface LLT1 by a proportion of SF monocytes, we next investigated the presence of LLT1-bearing cells in synovial tissue. To that end, synovial tissue specimens with low- to high-grade synovitis (median Krenn score of 5 [range 2–7]) [18] were obtained from six RA patients. Consecutive sections of synovial tissue were stained with antibodies against LLT1, CD68, CD3 and CD20cy (Fig 2). LLT1 staining was detected in all six biopsies and was predominant in the lining layer which showed pathological enlargement in all 6 biopsies analyzed (≥1 point according to Krenn et al [18]). LLT1 staining was also found in the sublining and in transitional areas consisting of different cell types [19]. Small numbers of LLT1+ cells were observed in scattered lymphoid infiltrates and more dense perivascular infiltrates. These synovial membrane areas were defined based on CD3 (T-cells) and CD20cy (B-cells) staining. Analysis of the results of the semi-quantitative scoring showed that 3/6 tissue biopsies had low (median score 1, indicating <5% positive cells) and 2/6 biopsies had moderate (median score 2, indicating 5–50% positive cells) expression of LLT1 in the lining layer. LLT1 expression in the sublining layer was found to be low (median score 1) in 4/6 and moderate (median score 2) in 2/6 biopsies while the infiltrate showed low and moderate LLT1 expression in 3/6 and 2/6 biopsies, respectively. In addition, some LLT1 staining of blood vessels was observed. The LLT1 staining pattern showed a marked overlap with CD68 staining. In all biopsies analyzed, CD68 expression was predominant in the lining and sublining layer and to a lesser extent in the lymphoid infiltrate areas.

Bottom Line: FACS analysis of digested ST confirmed LLT1 expression by CD68+ cells.Elevated systemic sLLT1 was found in all patient groups.Serum levels of sLLT1 were increased in all patient groups (patients with early- and late-stage RA, seropositive arthralgia and spondyloarthropathy) when compared to healthy subjects.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT

Objectives: Precursor Th17 lineage cells expressing CD161 are implicated in Rheumatoid Arthritis (RA) pathogenesis. CD4+CD161+ T-cells accumulate in RA joints and may acquire a non classical Th1 phenotype. The endogenous ligand for CD161 is lectin-like transcript 1 (LLT1). CD161/LLT1 ligation may co-stimulate T-cell IFN-γ production. We investigated the presence and identity of LLT1-expressing cells in RA synovial fluid (SF) and synovial tissue (ST). We also assessed levels of soluble LLT1 (sLLT1) in different phases of RA development.

Methods: Paired samples of peripheral blood mononuclear cells (MC) and SFMC (n = 14), digested ST cells (n = 4) and ST paraffin sections (n = 6) from late-stage RA were analyzed for LLT1 expression by flow cytometry and immunohistochemistry. sLLT1 was measured using a sandwich ELISA. Sera and SF from late-stage RA (n = 26), recently diagnosed RA patients (n = 39), seropositive arthralgia patients (SAP, n = 31), spondyloarthropathy patients (SpA, n = 26) and healthy controls (HC, n = 31) were assayed.

Results: In RA SF, LLT1 was expressed by a small proportion of monocytes. In RA ST, LLT1-expressing cells were detected in the lining, sublining layer and in areas with infiltrates. The LLT1 staining pattern overlapped with the CD68 staining pattern. FACS analysis of digested ST confirmed LLT1 expression by CD68+ cells. Elevated systemic sLLT1 was found in all patient groups.

Conclusions: In RA joints, LLT1 is expressed by cells of the monocyte/macrophage lineage. Serum levels of sLLT1 were increased in all patient groups (patients with early- and late-stage RA, seropositive arthralgia and spondyloarthropathy) when compared to healthy subjects.

No MeSH data available.


Related in: MedlinePlus