IL-17 expressing B cells.

<div><p>A) Percentage of CD19⁺ B cells within IL-17⁺CD3^- lymphocytes in RA patients (n = 8) and healthy controls (n = 8) in unstimulated PBMC or after stimulation with PMA/i for 20 h; B+C) freshly thawed PBMC or PBMC cultured for 20 h in RPMI-1640 medium with the addition of Brefeldin A for 16h (n = 5 and n = 8, respectively) in RA patients and healthy controls; B) Percentage of CD19⁺ B cells within IL-17⁺CD3^- lymphocytes; C) Percentage of IL-17⁺ cells within CD3^-CD19⁺ B cells; D) Percentage of IgD⁺CD27^- naïve B cells, IgD⁺CD27⁺ non-switched memory B cells and CD24⁺⁺CD38⁺⁺ regulatory B cells of IL-17⁺CD3^-CD19⁺ B cells of freshly thawed PBMC of healthy controls (n=3); E) Percentage of IL-17⁺ cells in EBV-transformed B cell lines without stimulation; F) Representative gel-electrophoresis of amplificates of IL-17A RT-PCR of unstimulated freshly thawed CD4⁺ and CD19⁺ MACS-sorted cells of a healthy donor: CD4⁺ T cells (lane 1), CD19⁺ B cells (lane 2), CD4^-CD19^- cells (lane 3); of a RA patient: CD4⁺ T cells (lane 4), CD19⁺ B cells (lane 5), CD4^-CD19^- cells (lane 6); and of the EBV-transformed B cell lines: Olga (lane 7), AMAI (lane 8); upper level: amplificates of IL-17A, lower level: corresponding control amplificates of β-actin, Marker: Puc 8 mix ladder; The data shown are representative results of RT-PCR of 3 healthy donors and 3 RA patients.</p> <p>*p < 0.05; **p < 0.01, ns = not significant; columns represent the mean and bars indicate 95% CI.</p></div

Influence of in vitro culture on IL-17⁺ cells.

<div><p>A-D) Comparison of freshly thawed cells and cells cultured for 20 h in RPMI-1640 medium with the addition of Brefeldin A for 16 h (n = 5 and n = 8, respectively) in RA patients and healthy controls; A) Percentage of CD3⁺ T cells within IL-17⁺ lymphocytes; B) Percentage of CD4⁺ cells within IL-17⁺CD3⁺ T cells; C) Percentage of CD11b⁺ cells within IL-17⁺CD3^- lymphocytes; D) Percentage of CD56⁺ NK cells within IL-17⁺CD3^- lymphocytes.</p> <p>*p < 0.05, **p < 0.01,***p < 0.001, ns = not significant, columns represent the mean and bars indicate 95% CI.</p></div

Gating strategy and IL-17⁺ T cells in RA patients and healthy controls.

<div><p>A) Exemplary gating strategy shown on representative pseudo-colour dot-plots of unstimulated PBMC of a RA patient. Dead cells were excluded using EMA-viability dye. Lymphocytes were gated (FSC vs. SSC), followed by the subgating of CD3⁺ and IL-17⁺ cells. IL-17⁺CD3⁺ T cells and IL-17⁺CD3^- non-T cells were further specified. Frequencies in each subgate are expressed as percentage of their parent population; B-D) Comparative analysis of CD3⁺ lymphocytes in PBMC stimulated with PMA/i, PHA or incubated in RPMI-1640 without stimulation for 20 h in RA patients and healthy controls; B) Percentage of CD3⁺ T cells within the IL-17⁺ lymphocytes; C) Percentage of CD4⁺ and CD8⁺ cells within the IL-17⁺CD3⁺ T cell population, D) Percentage of CD4⁺ cells within CD3⁺ T cells; E-F) Comparative analysis of IL-17 expression in different T cell populations stimulated with PMA/i, PHA, a pool of 23 peptides of MHC-class II-restricted T cell epitopes or incubated in RPMI-1640 without stimulation for 20 h; E) Percentage of IL-17⁺ cells within CD3⁺CD4⁺ Th cells; F) Percentage of IL-17⁺ cells within CD3⁺CD8⁺ Tc cells; G) Percentage of IL-17⁺ cells within CD3⁺CD4⁺CD8⁺ double positive T cells.</p> <p>Data from healthy controls (n = 20) and RA patients (n = 20). *p < 0.05; **p < 0.01, ***p < 0.001,ns = not significant, column represents the mean and bars indicate 95% CI.</p></div

IL-17⁺ non-T cells.

<div><p>A-F) Comparative analysis of PBMC stimulated with PMA/i for 20h or incubated in RPMI-1640 medium with the addition of Brefeldin A for 16 h in RA patients and healthy controls; A) Percentage of CD3^- non-T cells within IL-17⁺ lymphocytes (RA n = 20, healthy controls n = 20); B) Percentage of CD56⁺ NK cells within IL-17⁺CD3^- non-T cells; C) Percentage of IL-17⁺ cells within CD3^-CD56⁺ NK cells; D) Percentage of CD14⁺ monocytes in IL-17⁺CD3^- non-T cells within the lymphocyte gate and within the monocyte gate (FSC vs. SSC); E) Percentage of IL-17⁺ cells within CD3^-CD14⁺ monocytes within the monocyte gate; F) Percentage of CD11b⁺ cells within IL-17⁺CD3^- lymphocytes.</p> <p>Data B)-F) PMBC of healthy controls (n = 8) and RA patients (n = 8).</p> <p>*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant, column represents the mean and bars indicate 95% CI.</p></div

Expression of FGFR1, HE4 and α5β1 integrin on NCAM positive cells in incipient renal fibrosis.

<p>(<b>A</b>) Double immunofluorescent labeling of NCAM and FGFR1; merge of these two markers clearly shows that all NCAM+ cells coexpressed FGFR1 (white arrows); diffuse NCAM expression on interstitial cells; strong FGFR1 expression on bold vessels (white stars) and diffuse expression on interstitial cells; x200. (<b>B</b>) Double immunofluorescent labeling of NCAM and HE4; merge of NCAM and HE4 revealed single cells coexpressing both markers (white arrow); x400. (<b>C</b>) Double immunofluorescence labeling of NCAM and α5β1; merge of these two markers clearly shows co-expression of NCAM and α5β1 on renal interstitial cells in area of incipient fibrosis (white arrows); x600. <b>(D)</b> Double immunofluorescence labeling of NCAM and αSMA; merge of these two markers showed no overlapping of NCAM and αSMA on renal interstitial cells in area of incipient fibrosis, although areas of NCAM⁺ and SMA⁺ interstitial cells are close to each other; x100. Staining techniques are described in detail under Material and Methods.</p

Differences in relative mRNA expression levels of molecules relevant for renal fibrosis among NCAM positive cells from normal and renal interstitium with incipient fibrosis, obtained by laser capture microdissection.

<p><b>(A)</b> Relative mRNA expression levels of various molecules relevant for renal fibrosis; data are presented with mean values and standard error bars; *- indicate statistically significant difference, p<0.05; graph is made of mean values in order to unify variable presentations, although only αSMA, SLUG and ALK3 followed normal distribution; thus, some variables differed extremely among cases and consequently these data did not display normal distribution; due to influence of these extreme values on the mean value presented in the graph, bars are high but without statistical significance (such as BMP7); Student’s t test was used for variables with normal distribution both in control and kidneys with fibrosis: αSMA, SLUG and ALK3; due to high variability of other variables, exclusively in diseased kidneys, we applied nonparametric Mann Whitney U test to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis. <b>(B)</b> NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescene, clone EP257Y, x400. <b>(C)</b> MMP-9 positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunofluorescence, clone 6-6B, x400. <b>(D)</b> arrows indicate the overlapping of NCAM and MMP-9 in interstitial cells.</p

NCAM positive interstitial staining among various kidney diseases and their relationship to severity of renal interstitial fibrosis.

<p><b>(A)</b> Frequency of interstitial NCAM positivity among various kidney diseases. <b>(B)</b> Number of detected NCAM⁺ cells per field of view on ×400 magnification in controls and in diseased kidneys with regard to severity of interstitial renal fibrosis (IRF); p values after applying Mann-Whiteny U test. <b>(C-D)</b> FSGS with slight interstitial fibrosis (IRF-1) without tubular atrophy exhibiting an increased diffuse NCAM interstitial positivity detected on slides from paraffin-embedded tissue. (C) PAS, x400. (D) Immunoperoxidase staining, NCAM clone 123C3.D5, x400. <b>(E-G)</b> Lupus nephritis with NCAM positive interstitial cells detected focally around tubuli in the area with slight IRF (IRF-1). (E) PAS, x400. (F) Massone trichrome staining, x400. (G) Immunoperoxidase staining, NCAM clone 123C3.D5, x400.</p

Double immunofluorescent labeling of NCAM with erythropoietin (EPO) and granzyme B.

<p><b>(A-C)</b> NCAM positive interstitial cells did not express EPO. Merge of NCAM (clone Eric-1) and EPO, cryostat sections, double immunofluorescent labeling, x400. <b>(D)</b> Diffuse NCAM (clone EP257Y) staining in peritubular incipient interstitial fibrosis of FSGS case, without any granzyme B positivity, cryostat section, double immunofluorescene, x400. <b>(E)</b> Overlapping of NCAM (clone EP257Y) and granzyme B in a single cell within the whole biopsy core of the case illustrated in previous picture, cryostat section, double immunofluorescence, x400. <b>(F)</b> Mononuclear interstitial inflammatory infiltrate of lupus nephritis, arrow indicates two NCAM+ cells without overlapping with granzyme B, cryostat section, double immunofluorescence, x400.</p

Presence of NCAM and its isoforms in normal and fibrotic kidneys.

<p>(<b>A</b>) RT-PCR: three NCAM isoforms in different renal samples, fibrosis was present in 3 cases, lanes IV, VIII and X. (<b>B</b>) RT-PCR: presence of all NCAM isoforms in FSGS. (<b>C</b>) RT-PCR: presence of all NCAM isoforms in MPGN. (<b>D</b>) Same case as Fig (B): increased NCAM expression in areas with slight fibrosis on cryostat section, immunoperoxidase, clone Eric-1, x200. (<b>E</b>) Same tissue as Fig (C): NCAM positivity in peritubular incipient interstitial fibrosis shown on cryostat section, immunoperoxidase, clone Eric-1, x400.</p

Isolation of NCAM positive renal interstitial cells by laser capture microdissection (LCM) and changes in relative mRNA NCAM isofroms expression levels in incipient renal fibrosis.

<p><b>(A)</b> Slide performed on cryostat section and stained by NCAM, clone Eric-1, with widespread NCAM expression, prior laser capture microdissection (arrow indicates the first selected NCAM positive cell for further LCM, while arrowhead shows second selected area). <b>(B)</b> Slide with rare NCAM cells within normal interstitium prior LCM. <b>(C), (D)</b> and <b>(E)</b> the same slides as Fig (A) and (B) after LCM procedure. <b>(F)</b> Relative expression levels of NCAM mRNAs isoforms, determined by quantitative real-time PCR (qRT-PCR), in NCAM⁺ cells captured by LCM from normal and from renal tissue with incipient IRF, data are presented with mean values and standard error bars; due to high variability of variables, exclusively in diseased kidneys, nonparametric Mann Whitney U test was applied to assess the difference in mRNA levels between controls and diseased kidneys; there were 6 samples (2 cases in triplicates) of control cases and 42 (14 cases in triplicates) samples of cases with incipient renal fibrosis.</p