Fig. 8
CXCL4–DNA complex detection in vivo. a SSc plasma that was either positive (SSc pos, N = 12) or negative (SSc neg, N = 8) for CXCL4 content and HD plasma (that were all negative for CXCL4 content, N = 12) were plated on 96-well plates coated with a mouse anti-CXCL4 antibody. After washing, levels of CXCL4–DNA complexes were measured by using an anti-dsDNA antibody (see Methods, 1:300 dilution). Results are expressed as optical density (OD). Horizontal bars are the mean, vertical bars are s.e.m., P-values by Mann–Whitney test. b CXCL4–DNA immune complexes of CXCL4-positive patients (measured as in panel a) were plotted against IFN-α plasma levels to assess correlation between the presence of circulating CXCL4–DNA complexes and IFN-α in SSc patients (N = 12). Spearman’s correlation coefficient “r”, significance “P”, and sample size “N”, were indicated. c CXCL4 (upper panel) was immune-precipitated using an anti-CXCL4 antibody from plasma of SSc patients that were negative (SSc neg, N = 1) or positive (SSc pos, N = 4) for CXCL4 content, and from HD plasma (N = 3). The immune-precipitated material was run on a gel (8% acrylamide) and DNA was stained by ethidium bromide. In the middle panel, ethidium bromide staining of the same SSc and HD plasma immune-precipitated for IgG–immune complexes (IP IgG, see Methods); in the lower panel, ethidium bromide staining of plasma immune-precipitated for CXCL4 (IP CXCL4). Results are representative of one experiment of two performed, with different SSc and HD plasma. d LSM images relative to the presence of extracellular traps in skin biopsies from two different SSc patients out of four that show DNA filaments (blue, DAPI), CXCL4 in red (upper panels, patients SSc 04), or in green (lower panels, patient SSc 08), elastase in gray (lower panel) (magnification ×63; bar, 5 µm). CXCL4–DNA complexes are indicated by white arrowheads; high-power images of some of the indicated complexes are provided as insets for both patients. Results from two biopsies of eight analyzed
