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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

Flow-cytometric detection of LLT1 expression in RA ST cells.A) Percentages of CD3+ T-cells, CD19+ B-cells and CD68+ macrophages detected within the live cells gate of digested ST cells with flow cytometry. Briefly, necrotic cells were gated out based on the staining with the Fixable Viability Stain dye. Within the live cells gate lymphocytes and macrophages were gated based on FSC/SSC characteristics and CD68 expression, respectively. Within the lymphocyte gate T-cells and B-cells were gated based on CD3 and CD19 expression, respectively. Representative histogram overlays showing frequencies of B) LLT1+ and C) CD161+ cells within the populations of CD3+, CD19+ or CD68+ cells when compared to isotype control. D) Graphs show the percentages of LLT1 and CD161+ cells. Data from 4 independent donors were pooled. Bars represent the median value ± interquartile range. Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.
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pone.0132436.g003: Flow-cytometric detection of LLT1 expression in RA ST cells.A) Percentages of CD3+ T-cells, CD19+ B-cells and CD68+ macrophages detected within the live cells gate of digested ST cells with flow cytometry. Briefly, necrotic cells were gated out based on the staining with the Fixable Viability Stain dye. Within the live cells gate lymphocytes and macrophages were gated based on FSC/SSC characteristics and CD68 expression, respectively. Within the lymphocyte gate T-cells and B-cells were gated based on CD3 and CD19 expression, respectively. Representative histogram overlays showing frequencies of B) LLT1+ and C) CD161+ cells within the populations of CD3+, CD19+ or CD68+ cells when compared to isotype control. D) Graphs show the percentages of LLT1 and CD161+ cells. Data from 4 independent donors were pooled. Bars represent the median value ± interquartile range. Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.

Mentions: To further confirm LLT1 expression by synovial macrophages, we performed a flow cytometric analysis of LLT1 expression on cells derived from digested RA synovial tissue biopsies (n = 4). The digested ST cellular composition constituted mainly CD68+ cells, T-cells and few B-cells (Fig 3A). LLT1 expression was upregulated by ST CD68+ macrophages as evidenced by a shift in LLT1 mean fluorescence intensity (MFI, Fig 3B). In contrast, synovial tissue derived B- and T-cells were found to be LLT1 negative (Fig 3B and 3D). As before, a high percentage of CD161+ T-cells were detected [6]. CD161 expression was not detected on B-cells or on CD68+ macrophages (Fig 3C and 3D). Thus, we conclude that in the ST, LLT1 and CD161 are expressed in trans by CD68+ macrophages and CD4+ T-cells, respectively (Fig 3D). This would allow potential crosstalk of LLT1-bearing APC with CD161+ T-cells.


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)

Flow-cytometric detection of LLT1 expression in RA ST cells.A) Percentages of CD3+ T-cells, CD19+ B-cells and CD68+ macrophages detected within the live cells gate of digested ST cells with flow cytometry. Briefly, necrotic cells were gated out based on the staining with the Fixable Viability Stain dye. Within the live cells gate lymphocytes and macrophages were gated based on FSC/SSC characteristics and CD68 expression, respectively. Within the lymphocyte gate T-cells and B-cells were gated based on CD3 and CD19 expression, respectively. Representative histogram overlays showing frequencies of B) LLT1+ and C) CD161+ cells within the populations of CD3+, CD19+ or CD68+ cells when compared to isotype control. D) Graphs show the percentages of LLT1 and CD161+ cells. Data from 4 independent donors were pooled. Bars represent the median value ± interquartile range. Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.
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Related In: Results  -  Collection

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pone.0132436.g003: Flow-cytometric detection of LLT1 expression in RA ST cells.A) Percentages of CD3+ T-cells, CD19+ B-cells and CD68+ macrophages detected within the live cells gate of digested ST cells with flow cytometry. Briefly, necrotic cells were gated out based on the staining with the Fixable Viability Stain dye. Within the live cells gate lymphocytes and macrophages were gated based on FSC/SSC characteristics and CD68 expression, respectively. Within the lymphocyte gate T-cells and B-cells were gated based on CD3 and CD19 expression, respectively. Representative histogram overlays showing frequencies of B) LLT1+ and C) CD161+ cells within the populations of CD3+, CD19+ or CD68+ cells when compared to isotype control. D) Graphs show the percentages of LLT1 and CD161+ cells. Data from 4 independent donors were pooled. Bars represent the median value ± interquartile range. Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.
Mentions: To further confirm LLT1 expression by synovial macrophages, we performed a flow cytometric analysis of LLT1 expression on cells derived from digested RA synovial tissue biopsies (n = 4). The digested ST cellular composition constituted mainly CD68+ cells, T-cells and few B-cells (Fig 3A). LLT1 expression was upregulated by ST CD68+ macrophages as evidenced by a shift in LLT1 mean fluorescence intensity (MFI, Fig 3B). In contrast, synovial tissue derived B- and T-cells were found to be LLT1 negative (Fig 3B and 3D). As before, a high percentage of CD161+ T-cells were detected [6]. CD161 expression was not detected on B-cells or on CD68+ macrophages (Fig 3C and 3D). Thus, we conclude that in the ST, LLT1 and CD161 are expressed in trans by CD68+ macrophages and CD4+ T-cells, respectively (Fig 3D). This would allow potential crosstalk of LLT1-bearing APC with CD161+ T-cells.

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