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Gottron's papules exhibit dermal accumulation of CD44 variant 7 (CD44v7) and its binding partner osteopontin: a unique molecular signature.

Kim JS, Bashir MM, Werth VP - J. Invest. Dermatol. (2012)

Bottom Line: Mechanically stretching cultured fibroblasts for 6 hours induced CD44v7 mRNA and protein, whereas IFN-γ treatment induced OPN mRNA and protein.OPN alone did not induce CD44v7, but stretching dermal fibroblasts in the presence of OPN increased human acute monocytic leukemia cell line (THP-1) monocyte binding, which is blunted by anti-CD44v7 blocking antibody.C4S, CD44v7, and OPN are three molecules uniquely present in Gottron's papules that contribute to inflammation individually and in association with one another.

View Article: PubMed Central - PubMed

Affiliation: New York University School of Medicine, New York, New York, USA.

ABSTRACT
The accumulated mucin in non-Gottron's dermatomyositis (DM) lesions is primarily chondroitin-4-sulfate (C4S), which is immunomodulatory in vitro. Gottron's papules are a particularly resistant manifestation of DM that often persist after other lesions have resolved with therapy. We examined non-Gottron's DM lesions and Gottron's papule skin biopsies for C4S, CD44 variant 7 (CD44v7), a chondroitin sulfate-binding isoform causally implicated in autoimmunity, and osteopontin (OPN), a CD44v7 ligand implicated in chronic inflammation. Gottron's papule dermis contained more C4S and CD44v7 than non-Gottron's lesions. Normal skin showed less CD44v7 over joints relative to Gottron's lesions. All DM dermis had increased OPN compared with healthy skin. Mechanically stretching cultured fibroblasts for 6 hours induced CD44v7 mRNA and protein, whereas IFN-γ treatment induced OPN mRNA and protein. OPN alone did not induce CD44v7, but stretching dermal fibroblasts in the presence of OPN increased human acute monocytic leukemia cell line (THP-1) monocyte binding, which is blunted by anti-CD44v7 blocking antibody. C4S, CD44v7, and OPN are three molecules uniquely present in Gottron's papules that contribute to inflammation individually and in association with one another. We propose that stretch-induced CD44v7 over joints, in concert with dysregulated OPN levels in the skin of DM patients, increases local inflammatory cell recruitment and contributes to the pathogenesis and resistance of Gottron's papules.

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Mechanical stretching of fibroblasts, particularly in combination with osteopontin treatment, augments the adhesion of THP-1 monocytes, in a process dependent on CD44v7Confluent monolayers of cultured human dermal fibroblasts grown on flexible membranes were incubated overnight without or with osteopontin (OPN), without or with stretch, as indicated. Unbound OPN was rinsed away, and then adherence of fluorescently labeled THP-1 monocytes onto these monolayers was assessed. Wells were incubated for one hour following treatment with anti-CD44v7 blocking antibody or isotype control Ig, and then washed just before addition of the labeled monocytes. Representative fluorescent micrographs of adherent monocytes (green dots) are shown. a. Control fibroblasts; b. Mechanically stretched fibroblasts; c. Mechanically stretched fibroblasts pretreated with recombinant OPN; d. Counts of adherent, fluorescent monocytes per high-power field (HPF; means±SEM, n=6), P<0.0001 by ANOVA; (***, P<0.001; **, P<0.01; *, P<0.05; n.s., not significant).
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Figure 6: Mechanical stretching of fibroblasts, particularly in combination with osteopontin treatment, augments the adhesion of THP-1 monocytes, in a process dependent on CD44v7Confluent monolayers of cultured human dermal fibroblasts grown on flexible membranes were incubated overnight without or with osteopontin (OPN), without or with stretch, as indicated. Unbound OPN was rinsed away, and then adherence of fluorescently labeled THP-1 monocytes onto these monolayers was assessed. Wells were incubated for one hour following treatment with anti-CD44v7 blocking antibody or isotype control Ig, and then washed just before addition of the labeled monocytes. Representative fluorescent micrographs of adherent monocytes (green dots) are shown. a. Control fibroblasts; b. Mechanically stretched fibroblasts; c. Mechanically stretched fibroblasts pretreated with recombinant OPN; d. Counts of adherent, fluorescent monocytes per high-power field (HPF; means±SEM, n=6), P<0.0001 by ANOVA; (***, P<0.001; **, P<0.01; *, P<0.05; n.s., not significant).

Mentions: Based on these findings, we hypothesized that the increased CD44v7 in association with its ligand osteopontin could play a crucial role in the localized inflammation of Gottron’s papules. To study the functional effect of increased CD44v7 ligation with osteopontin on immune cell recruitment, we incubated dermal fibroblast monolayers overnight, without or with stretching, without or with recombinant human osteopontin (rOPN), followed by rinsing. Fluorescently labeled THP-1 monocytes were then co-cultured with the prepared fibroblast monolayers, followed by rinsing to remove non-adherent monocytes, and the adherent monocytes were visualized with an inverted fluorescent microscope. We found that overnight incubation of fibroblast monolayers in medium supplemented with rOPN had no effect on THP-1 adhesion in the absence of stretch (Figure 6). Stretching the fibroblasts in the absence of rOPN supplementation resulted in an increase in the number of adherent THP-1 cells 1.8 times unstretched, no-rOPN controls (Figure 6, p<0.01). Stretching fibroblasts overnight in medium supplemented with rOPN resulted in an increase in THP-1 adhesion 2.2 times that of stretched fibroblasts without rOPN (p<0.001) and 2.3 times that of unstretched, no-OPN controls (p<0.001).


Gottron's papules exhibit dermal accumulation of CD44 variant 7 (CD44v7) and its binding partner osteopontin: a unique molecular signature.

Kim JS, Bashir MM, Werth VP - J. Invest. Dermatol. (2012)

Mechanical stretching of fibroblasts, particularly in combination with osteopontin treatment, augments the adhesion of THP-1 monocytes, in a process dependent on CD44v7Confluent monolayers of cultured human dermal fibroblasts grown on flexible membranes were incubated overnight without or with osteopontin (OPN), without or with stretch, as indicated. Unbound OPN was rinsed away, and then adherence of fluorescently labeled THP-1 monocytes onto these monolayers was assessed. Wells were incubated for one hour following treatment with anti-CD44v7 blocking antibody or isotype control Ig, and then washed just before addition of the labeled monocytes. Representative fluorescent micrographs of adherent monocytes (green dots) are shown. a. Control fibroblasts; b. Mechanically stretched fibroblasts; c. Mechanically stretched fibroblasts pretreated with recombinant OPN; d. Counts of adherent, fluorescent monocytes per high-power field (HPF; means±SEM, n=6), P<0.0001 by ANOVA; (***, P<0.001; **, P<0.01; *, P<0.05; n.s., not significant).
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Related In: Results  -  Collection

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Figure 6: Mechanical stretching of fibroblasts, particularly in combination with osteopontin treatment, augments the adhesion of THP-1 monocytes, in a process dependent on CD44v7Confluent monolayers of cultured human dermal fibroblasts grown on flexible membranes were incubated overnight without or with osteopontin (OPN), without or with stretch, as indicated. Unbound OPN was rinsed away, and then adherence of fluorescently labeled THP-1 monocytes onto these monolayers was assessed. Wells were incubated for one hour following treatment with anti-CD44v7 blocking antibody or isotype control Ig, and then washed just before addition of the labeled monocytes. Representative fluorescent micrographs of adherent monocytes (green dots) are shown. a. Control fibroblasts; b. Mechanically stretched fibroblasts; c. Mechanically stretched fibroblasts pretreated with recombinant OPN; d. Counts of adherent, fluorescent monocytes per high-power field (HPF; means±SEM, n=6), P<0.0001 by ANOVA; (***, P<0.001; **, P<0.01; *, P<0.05; n.s., not significant).
Mentions: Based on these findings, we hypothesized that the increased CD44v7 in association with its ligand osteopontin could play a crucial role in the localized inflammation of Gottron’s papules. To study the functional effect of increased CD44v7 ligation with osteopontin on immune cell recruitment, we incubated dermal fibroblast monolayers overnight, without or with stretching, without or with recombinant human osteopontin (rOPN), followed by rinsing. Fluorescently labeled THP-1 monocytes were then co-cultured with the prepared fibroblast monolayers, followed by rinsing to remove non-adherent monocytes, and the adherent monocytes were visualized with an inverted fluorescent microscope. We found that overnight incubation of fibroblast monolayers in medium supplemented with rOPN had no effect on THP-1 adhesion in the absence of stretch (Figure 6). Stretching the fibroblasts in the absence of rOPN supplementation resulted in an increase in the number of adherent THP-1 cells 1.8 times unstretched, no-rOPN controls (Figure 6, p<0.01). Stretching fibroblasts overnight in medium supplemented with rOPN resulted in an increase in THP-1 adhesion 2.2 times that of stretched fibroblasts without rOPN (p<0.001) and 2.3 times that of unstretched, no-OPN controls (p<0.001).

Bottom Line: Mechanically stretching cultured fibroblasts for 6 hours induced CD44v7 mRNA and protein, whereas IFN-γ treatment induced OPN mRNA and protein.OPN alone did not induce CD44v7, but stretching dermal fibroblasts in the presence of OPN increased human acute monocytic leukemia cell line (THP-1) monocyte binding, which is blunted by anti-CD44v7 blocking antibody.C4S, CD44v7, and OPN are three molecules uniquely present in Gottron's papules that contribute to inflammation individually and in association with one another.

View Article: PubMed Central - PubMed

Affiliation: New York University School of Medicine, New York, New York, USA.

ABSTRACT
The accumulated mucin in non-Gottron's dermatomyositis (DM) lesions is primarily chondroitin-4-sulfate (C4S), which is immunomodulatory in vitro. Gottron's papules are a particularly resistant manifestation of DM that often persist after other lesions have resolved with therapy. We examined non-Gottron's DM lesions and Gottron's papule skin biopsies for C4S, CD44 variant 7 (CD44v7), a chondroitin sulfate-binding isoform causally implicated in autoimmunity, and osteopontin (OPN), a CD44v7 ligand implicated in chronic inflammation. Gottron's papule dermis contained more C4S and CD44v7 than non-Gottron's lesions. Normal skin showed less CD44v7 over joints relative to Gottron's lesions. All DM dermis had increased OPN compared with healthy skin. Mechanically stretching cultured fibroblasts for 6 hours induced CD44v7 mRNA and protein, whereas IFN-γ treatment induced OPN mRNA and protein. OPN alone did not induce CD44v7, but stretching dermal fibroblasts in the presence of OPN increased human acute monocytic leukemia cell line (THP-1) monocyte binding, which is blunted by anti-CD44v7 blocking antibody. C4S, CD44v7, and OPN are three molecules uniquely present in Gottron's papules that contribute to inflammation individually and in association with one another. We propose that stretch-induced CD44v7 over joints, in concert with dysregulated OPN levels in the skin of DM patients, increases local inflammatory cell recruitment and contributes to the pathogenesis and resistance of Gottron's papules.

Show MeSH
Related in: MedlinePlus