Abnormally Differentiating Keratinocytes in the Epidermis of Systemic Sclerosis Patients Show Enhanced Secretion of CCN2 and S100A9  Joanna Nikitorowicz-Buniak, Xu Shiwen, Christopher P. Denton, David Abraham, Richard Stratton  Journal of Investigative Dermatology  Volume 134, Issue 11, Pages 2693-2702 (November 2014) DOI: 10.1038/jid.2014.253 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Abnormal terminal differentiation in the epidermis of systemic sclerosis (SSc) patients. (a) Control and SSc skin sections were stained with hematoxylin. Parakeratosis in the SSc stratum corneum (SC) is marked with arrows. The expression of differentiation markers in the epidermis of SSc patients and controls was detected by immunohistochemical staining using (c) anti-involucrin, (e) anti-loricrin, and (g) anti-filaggrin antibodies. Sections were counterstained with hematoxylin and representative images chosen to analyze (b) the thickness of the SSc epidermis from the basal to the cornified layer (healthy control (HC) n=6, SSc n=6) and expression bands of (d) involucrin (HC n=5, SSc n=7), (f) loricrin (HC n=6, SSc n=9), and (h) the thickness of filaggrin (HC n=5, SSc n=7). The mean thickness for each band was calculated from 10 measurements taken from each section; bar=20 mm. The results are presented as mean±SEM. Statistical significance was determined using the Mann–Whitney U test; *P⩽0.05, **P⩽0.0005. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Activated keratinocytes in the systemic sclerosis (SSc) epidermis. (a) Number of keratinocytes and an average area of the cells in (b) the basal and (c) spinous epidermal layers were measured in both SSc and control epidermis (healthy control (HC) n=5, SSc n=6). (d) The expression of proliferating cells in the epidermis of SSc patients and controls was detected by immunohistochemical staining using anti-ki-67 antibody (HC n=5, SSc n=6); bar=20 mm. (e) The mean percentage of proliferating cells was calculated from an average of six measurements of ki-67-positive cells taken from each section, divided by the total number of cells in the basal layer (HC n=5, SSc n=6). The results are presented as mean±SEM. Statistical significance was determined using the Mann–Whitney U test; *P⩽0.05, **P⩽0.005. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Increased concentration of CCN2, S100A9, and hepatocyte growth factor (HGF) in systemic sclerosis (SSc) epidermis–conditioned media. Forearm biopsies were taken from SSc patients and matched controls. The epidermis and dermis were separated at the basal membrane using trypsin/EDTA and the explants were incubated in serum-free media overnight. The conditioned media were then collected and used for analysis of selected soluble factors by ELISA and Luminex platform. An increased concentration in SSc epidermis–conditioned media compared with control (Ctrl) was detected with (a) CCN2 (healthy control (HC) n=8, SSc n=8), (b) HGF (HC n=12, SSc n=12), and (c) S100A9 (HC n=6, SSc n=6). No significant increase was detected in the levels of (d) platelet-derived growth factor (PDGF)-AA (HC n=6, SSc n=6), (e) vascular endothelial growth factor (VEGF)-A (HC n=12, SSc n=12), (f) G-CSF (HC n=12, SSc n=12), (g) fibroblast growth factor-2 (HC n=12, SSc n=12), (h) CCL20 (HC n=12, SSc n=12), (i) monocyte chemotactic protein-1 (MCP-1) (HC n=12, SSc n=12), (j) IL-1α (HC n=12, SSc n=12), (k) IL-1β (HC n=12, SSc n=12), (l) IL-1ra (HC n=12, SSc n=12), (m) IL-6 (HC n=6, SSc n=6), (n) IL-8 (HC n=6, SSc n=6), and (o) lactate dehydrogenase (LDH). The results are presented as mean±SEM. Statistical significance was determined using the Mann–Whitney U-test *P⩽0.05. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increased expression of CCN2 in the systemic sclerosis (SSc) epidermis and at the epidermal–dermal junction in early SSc. The expression of (a) CCN2 in the epidermis of early and established SSc patients and controls (Ctrl) was detected by immunohistochemical staining using anti-CCN2 antibody. Panels show a magnified view of (i) the epidermal–dermal junction and (ii) blood vessels. Anti-hepatocyte growth factor (HGF) antibody was used for the detection of (b) HGF in the skin. Magnified view of (i) the epidermis–dermis junction and (ii) the dermis. Sections were counterstained with hematoxylin and representative images chosen. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increased mRNA levels of CCN2 and S100A9 in systemic sclerosis (SSc) epidermal blister sheets. (a) Double immunofluorescent staining was performed to detect S100A9 (green) and S100A8 (red) in SSc and control skin sections. 4',6-Diamidino-2-phenylindole (DAPI) (blue) was used to stain nuclei. RNA was extracted from epidermal blister sheets taken from healthy controls (HC n=9), dcSSc (diffuse cutaneous SSc) (n=24), and lcSSc (limited SSc) (n=8). Relative expression of (b) CCN2 mRNA, (c) S100A8 mRNA, and (d) S100A9 mRNA was measured with quantitative reverse transcriptase in real time. The values were normalized relative to TUBB mRNA expression using the ΔΔCT method and the results presented as mean±SEM. The Mann–Whitney U-test was used to calculate statistical significance; *P⩽0.05. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The effects of S100A9 on dermal fibroblasts. Fibroblasts from SSc patients (n=5) and controls (n=6) were incubated with different concentrations of rhS100A9. After 48 hours, (a) proliferation and (b) migration were assessed and normalized against untreated samples. Fibroblasts were incubated with rhS100A9 to assess its effects on the expression of CCN2. (c) Relative expression of CCN2 mRNA after 24-hour treatment. (d) CCN2 levels in cell lysates after 48-hour treatment (samples were originally run in different order and have been juxtaposed in the figure). (e) Densitometry of the western blot was measured and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cells were pretreated with Toll-like receptor 4 (TLR4) inhibitor TAK-242 (1 μM) before treatment with rhS100A9 (1.5 μg ml−1). (f) CCN2 levels in cell lysates after 48-hour treatment. (g) Densitometry of the western blot was measured and normalized against GAPDH. (h) Relative expression of CCN2 mRNA after 24-hour treatment. The values were normalized relative to TBP (TATA-binding protein) mRNA expression using the ΔΔCT method. Each panel represents the results of two independent experiments. Data were presented as mean±SD. Statistical significance was assessed by Student’s paired t-test; *P⩽0.05. HC, healthy controls. Journal of Investigative Dermatology 2014 134, 2693-2702DOI: (10.1038/jid.2014.253) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions