data_sheet_1_Neutrophils and Activated Macrophages Control Mucosal Immunity by Proteolytic Cleavage of Antileukoproteinase.docx

<p>Antileukoproteinase or secretory leukocyte peptidase inhibitor is a small protein which protects the mucosal linings against excessive proteolysis, inflammation, and microbial infection. We discovered that gelatinase B or matrix metalloproteinase (MMP)-9, a secreted zinc-dependent endopeptidase typically found at sites of inflammation, destroys antileukoproteinase by cleavages within both of its two functional domains: the anti-microbial N-terminal and the anti-proteolytic C-terminal domains. Cleaved antileukoproteinase possessed a significantly lower ability to bind lipopolysaccharides (LPS) and a reduced capacity to inhibit neutrophil elastase (NE) activity. Whereas intact antileukoproteinase repressed proinflammatory transcript [prostaglandin-endoperoxide synthase 2 (PTGS2) and IL6] synthesis and protein secretion [e.g., of MMP-9] in human CD14⁺ blood monocytes stimulated with LPS, this effect was reduced or lost for cleaved antileukoproteinase. We demonstrated the in vivo presence of antileukoproteinase cleavage fragments in lower airway secretions of non-cystic fibrosis bronchiectasis patients with considerable levels of neutrophils and, hence, elastase and MMP-9 activity. As a comparison, other MMPs (MMP-2, MMP-7, and MMP-8) and serine proteases (NE, cathepsin G, and proteinase 3) were also able to cleave antileukoproteinase with similar or reduced efficiency. In conclusion, in specific mucosal pathologies, such as bronchiectasis, neutrophils, and macrophage subsets control local immune reactions by proteolytic regulation, here described as the balance between MMPs (in particular MMP-9), serine proteases and local tissue inhibitors.</p

Additional file 1: of End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis

Table S1. Detailed overview of the used primary and secondary antibodies, and chromogens. Superscript numbers (1–5) above the catalogue number indicate which primary antibody is combined with which secondary antibody and chromogen. Abbreviation: RTU = ready to use. Table S2. Inter- and intra-observer variability in the counting of the myeloid and lymphoid cells, expressed by means of the Spearman’s rank correlation coefficient. Significant correlations are indicated with *, with *** = p < 0.001. Table S3. Overview of the number of analysed follicles in each compartment for every included tissue block. CF11-20 represents the female CF patients. Table S4. Quantification of the myeloid cell types and lymphoid follicles according to localization (subdivided in the three compartments: airways, parenchyma and perivascular). The p-values in the right-hand column are the result of Kruskal-Wallis 1-way ANOVA testing. Significant differences with airways are indicated with *, with * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. These values are the results of Dunn’s post hoc testing. Figure S1. Histological section of formalin-fixed paraffin-embedded CF lung tissue. Section was stained for mast cells (tryptase). Representative image showing an airway surrounded by circular fibrosis suggestive of constrictive bronchiolitis. The bronchiole is accompanied by its blood vessel. Abbreviations: AW = airway, BV = blood vessel. Scale bar = 100 μm. Figure S2. Serial histological sections of formalin-fixed paraffin-embedded CF lung tissue. Both images show the same lymphoid follicles located in the proximity of an airway. Panel A shows a CD20 staining of all B cells lying organised in germinal centres. These are surrounded by a paracortex staining positively for CD3 T cells (panel B). High endothelial venules (green arrowhead) allowing extravasation of naïve B and T cells into the lymphoid follicle are located in the T cell area. AW = airway. Scale bar = 50 μm. (DOCX 19517 kb