Fig 2: Soluble variants of Siglec-5.Panel A: Domain structures of sSiglec-5/C4BP and sSiglec-5/Fc which are used within this study. Panel B: Analysis of purified sSiglec-5/C4BP and sSiglec-5/Fc via SDS-Page and Coomassie-staining under non-reduced and reduced conditions.

Fig 3: In vitro perfusion of primary leukocytes.Panel A: Freshly isolated PBMCs were incubated with in the absence or presence of soluble Siglec-5/Fc (sSg-5/Fc; 10 µg/ml) or Siglec-7/Fc (sSg-7/Fc; 10 µg/ml) prior to perfusion over P-selectin-coated biochips. Perfusion was performed at 2 dyn/cm2. Panel B: Freshly isolated PMNs were incubated with in the absence or presence of soluble Siglec-5/Fc (sSg-5/Fc; 10 µg/ml) or sSiglec-5/C4BP (sSg-5/C4BP; 10 µg/ml) prior to perfusion over P-selectin-coated biochips. Perfusion was performed at 0.5 dyn/cm2. For both panels, the number of rolling cells/microscopic field were determined and arbitrarily set at 100% for the control in the absence of Siglecs. Data represent mean ± SD of 3–4 perfusions. For each perfusion, a minimum of 3 fields were analyzed. P-values were determined in a 1-way ANOVA followed by Dunnett’s test.

Fig 4: In vitro perfusion of THP1-cells.Panel A: THP1-cells were incubated with in the absence or presence of sialidase prior to perfusion over P-selectin-coated biochips. The number of rolling cells/microscopic field were determined and arbitrarily set at 100% for non-desialylated THP1-cells. P-value was determined in a two-tailed unpaired t-test. Panel B: THP1-cells were incubated in the absence or presence of soluble Siglec-5/Fc (sSg-5/Fc) or Siglec-7/Fc (sSg-7/Fc) prior to perfusion over VCAM1-coated biochips. Plotted is the number of adhering cells/microscopic field. Panels C & D: THP1-cells were incubated in the absence or presence of soluble Siglec-5/Fc (sSg-5/Fc; 10 µg/ml) or Siglec-7/Fc (sSg-7/Fc; 10 µg/ml) prior to perfusion over E-selectin or P-selectin-coated biochips. Plotted is the number of rolling cells/microscopic field. P-values were determined in a 1-way ANOVA followed by Dunnett’s test. Panel D: THP1-cells were incubated in the absence or presence of sSiglec-5/Fc or anti-PSGL1 antibodies prior to perfusion over P-selectin-coated biochips. Plotted is the number of rolling cells/microscopic field, with this number being set as 100% in the absence of sSiglec-5/Fc or anti-PSGL1 antibodies. P-value was calculated via multiple t-tests according to the Holm-Sidak method. Panels F & G: representative image-sequence over a 4-sec period of THP1-cells rolling over P-selectin in the absence (panel F) or presence (panel G) of sSiglec-5/Fc. For each image-sequence, six cells have been highlighted to visualize their movements over time. For panels A–E: data represent mean ± SD of 3–5 perfusions. For each perfusion, a minimum of 3 fields were analyzed. All perfusions were performed at 0.5 dyn/cm2.

Fig 5: Involvement of sSiglec-5/C4BP in the kinetics of leukocyte recruitment during a sterile inflammation in mice.Leukocytes were labeled with fluorescent dye Syto59. Panel A: Representative images depicting the course of leukocyte recruitment during a sterile inflammation over time in control mice or mice treated with sSiglec-5/C4BP (0.8 mg/kg via injection in the lateral tail vein). Panels B-C-D: The total Integral Fluorescent Intensity (IFI) over the 5-h observation period was quantified in control mice or in mice treated with Siglec-5. Data represent the IFI of the full microscopic field (panel B), the IFI of the vessels (panel C) or the IFI of the surrounding tissue (panel D). Plotted are mean ± SEM from 4 mice per group and 4 vessels per mouse (n = 16). P-values were determined using a two-tailed unpaired t-test. Panels E & F: the average rolling velocity (panel E) and path duration (panel F) were determined for each vessel segment of each mouse included in the study at 4 different time-points during the observation period. Each data point represents the mean of 3–4 cells.