SEARCH

SEARCH BY CITATION

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2451-sup-0001-FigureS1.pdf315K

Supplementary Figure 1. The migration inducing effect of Lcn2 on PMNs was not blocked by Calphostin or Wortmannin. (A, B) 1×106 freshly isolated human PMNs were allowed to migrate for 30 min in a Boyden chemotaxis chamber. PMNs were preincubated with calphostin [5nM] of wortmannin [50nM]. Graphs show lower quartile, median and upper quartile (boxes) and minimum/maximum ranges (whiskers).

Supplementary Figure 2. Enterobactin does not change chemotaxis properties of Lcn2. rmLcn2 [10nM] was mixed with enterobactin at a ratio of 1:1 10 min prior to usage in chemotaxis assay. 1×106 freshly isolated human PMNs were allowed to migrate for 30 min in a Boyden chemotaxis chamber. Graphs show lower quartile, median and upper quartile (boxes) and minimum/maximum ranges (whiskers).

Supplementary Figure 3. S. typhimurium detection in the skin was significantly increased in Lcn2-/- 48 hours after intradermale infection. 300 CFU S. typhimurium in 50μL NaCl [0.9%] were injected intradermally into Lcn2+/+ and Lcn2-/- mice. Intradermal NaCl [0.9%] administration was used as negative control. 48 hours later mice were sacrificed and the skin at the injection site was used for histological examination. Immunofluorescent staining of salmonella antigen CSA-1 was performed as described in Materials and Methods. Representative skin sections from three independent experiment (n = 6) are shown. Magnification x40; Zeiss (AxioCam MRc5). Graphs show lower quartile, median and upper quartile (boxes) and minimum/maximum ranges (whiskers). Quantification was performed as described in Materials and Methods.

Supplementary Figure 4. Leukocyte invasion at the sites of infection 48 hours after infection. 300 CFUs S. typhimurium in 50μL NaCl [0.9%] were injected intradermally into Lcn2+/+ and Lcn2-/- mice. Intradermal NaCl [0.9%] administration was used as negative control. 48 hours later mice were sacrificed and skin at the injection site was used for histological examination. Representative skin sections out of three independent experiment (n = 6) are shown indicating comparable numbers of inflammatory cells in the skin sections Lcn2+/+ and Lcn2-/- mice. Magnification x40; Zeiss (AxioCam MRc5).

Supplementary Figure 5. Isolation of PMNs as described in “Materials and Methods” shows a purity greater than 95%. Heparin-anticoagulated blood of 3–4 mice was pooled and PMNs were isolated as described in “Materials and Methods”. Isolated PMNs were stained with anti-mouse Ly6G FITC (1A8) for subsequent FACS analysis.

Supplementary Figure 6. The extracellular expression of CXCR2 of Lcn2-/- PMNs is significantly reduced compared to Lcn2+/+ mice. 200 μL of blood was drawn by retroorbital blood puncture of untreated Lcn2-/- and Lcn2+/+ mice at the age of 8 weeks. Whole blood was prepared for analysis of PMNs expression markers by means of FACS analysis as described in Materials and Methods. A granulocyte gate was set and Ly6G positive cells were analysed for CXCR2 surface expression. Data are shown as mean ± SEM of 4 mice. Student`s t-test was used for statistical analysis.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.