Fig 2: Interaction between ectodomains of Siglec-5 and PSGL1.Panel A: sSiglec-5/C4BP (closed circles) and sSiglec-5/Fc (open circles) were incubated with immobilized sPSGL1/Fc. Plotted is the response (OD450nm) versus sSiglec-5 concentration. Data represent mean ± SD of three independent experiments. Panel B: sSiglec-5/C4BP (10 µg/ml), sSiglec-5/Fc (10 µg/ml) or P-selectin (10 µg/ml) were pre-incubated with EDTA or polyclonal anti-Siglec-5 antibodies prior to the addition of sPSGL1/Fc-coated wells. Plotted is the response (OD450) for each of the preparations. P-values were determined in a 1-way ANOVA followed by a Tukey’s multiple comparison test. Panel C: Interaction between sPSGL1 and sSiglec-5/C4BP was examined using BLI-analysis. sPSGL1/Fc was immobilized onto amine-reactive biosensors and incubated with various concentrations of sSiglec-5/C4BP. A representative sensorgram of three independent experiments is plotted, showing the response (nm shift) versus time. Panel D: sPSGL1/Fc immobilized onto amine-reactive biosensors was incubated in the absence (grey bars) or presence (black bars) of sialidase (0.1 U/ml) before exposure to sSiglec-5/C4BP or P-selectin (both 10 µg/ml). Plotted is the residual response (expressed as percentage of response in the absence of sialidase, which was arbitrarily set at 100%) for both sSiglec-5/C4BP and P-selectin. Panels E & F: human or murine sPSGL1/Fc (hPSGL1 and mPSGL1) and BSA were immobilized onto amine-reactive biosensors and incubated with sSiglec-5/C4BP or sSiglec-5/Fc (7.5 µg/ml). Representative sensorgrams of two independent experiments is plotted, showing the response (nm shift) versus time.

Fig 3: 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.

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: 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.