![Figure 3. Adhesion mediated by immobilized MK in vitro was CD18 dependent. (A-D) To measure adhesion of PMNs, cells were left untreated for control (w/o) or stimulated for 10 minutes at 37°C. Adhesion is expressed as a percentage of all cells added to poly-L-lysine (100%). (A) Adhesion of human or murine WT PMNs on immobilized FG left untreated (w/o) for control, or stimulated with soluble MK (10 ng/ml, 30 ng/ml, or 100 ng/ml), or TNF-α (100 ng/ml) (n = 18 [human]; n = 3 [murine]). (B) Adhesion of human or murine WT PMNs on immobilized FG or immobilized MK either left untreated (w/o) or stimulated with TNF-α (100 ng/ml) (n = 4 [human]; n = 4 [murine]). (C) Adhesion of murine WT PMNs on immobilized ICAM1 or MK. Cells were left untreated or preincubated with a CD18 function-blocking antibody (anti-CD18) for 20 minutes at RT. Subsequently, cells were left unstimulated (w/o) for control or stimulated with TNF-α (100 ng/ml) (n = 6 [ICAM1]; n = 9 [MK]). (D) Isolated CD18+/+ or CD18−/− murine PMNs on immobilized ICAM1 or MK. Diagrams show percentage of adhesion without stimulation (w/o) or after stimulation with TNF-α (100 ng/ml) (n = 3). (E) Induction of adhesion of murine WT PMNs under flow conditions (1 dyne/cm2). Microflow chambers were coated with P-selectin (10 µg/ml) and ICAM1 (12.5 µg/ml) alone (P-selectin + ICAM1), or combined with MK (P-selectin + ICAM1 + MK: 10 µg/ml). Data show the total number of adherent PMNs at indicated times (n = 5). (F) Adhesion of isolated MK+/+ and MK−/− PMNs under flow conditions. Microflow chambers were coated with P-selectin (10 µg/ml), ICAM1 (12.5 µg/mL), and CXCL1 (5 µg/ml) (n = 4). (G) Adhesion strengthening of adherent murine WT PMNs under gradually increasing shear stress (0.2-8.0 dyne/cm2). WT PMNs were seeded into microflow chambers coated with ICAM1 (ICAM1: 12.5 µg/ml) alone, or in combination with MK (ICAM1 + MK: 10 µg/ml), or CXCL1 (ICAM1 + CXCL1: 5 µg/ml) for 10 minutes before flow was applied as indicated. Adhesion strengthening was measured as the number of adherent PMNs in percent of initially adherent cells at 0.2 dyne/cm2 (100%) (n = 5). (A-E) Show mean ± SEM. *P < .05; ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/12/10.1182_blood-2013-06-510875/4/1887f3.jpeg?Expires=1724165936&Signature=hhby4kyqpPzgZ-g9waKSzZtg2zRlVUDbA8kGqcdx4dfxFyAi-duZdvVHIxpPt~e-HRfCoQA~1OTSDa4YtgTJwZLp5t757v8I2gRbnJr1O5AEi8IItzuu7ZlX53u-rnyJcWwnsEA1NiUNkZ67erJg8co6CyKU2PHWZVjenjmLw4E0XBdEgHVBAQe1abPQ9tJXWK5-4IqmBJvmlLq5kRCh33kT4BAI5w~Qi1zQ11dTx5MU8Rq7p8GRK8RW0HR0vEA3Lj9tv7wrW1210Ln76NnGPHDnVkkfJV1eUCgHxouc-p77Z4R4L~wYPjBa4-IDBtzlM8T5~eWz~p-uA6HqJKx2pQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Adhesion mediated by immobilized MK in vitro was CD18 dependent. (A-D) To measure adhesion of PMNs, cells were left untreated for control (w/o) or stimulated for 10 minutes at 37°C. Adhesion is expressed as a percentage of all cells added to poly-L-lysine (100%). (A) Adhesion of human or murine WT PMNs on immobilized FG left untreated (w/o) for control, or stimulated with soluble MK (10 ng/ml, 30 ng/ml, or 100 ng/ml), or TNF-α (100 ng/ml) (n = 18 [human]; n = 3 [murine]). (B) Adhesion of human or murine WT PMNs on immobilized FG or immobilized MK either left untreated (w/o) or stimulated with TNF-α (100 ng/ml) (n = 4 [human]; n = 4 [murine]). (C) Adhesion of murine WT PMNs on immobilized ICAM1 or MK. Cells were left untreated or preincubated with a CD18 function-blocking antibody (anti-CD18) for 20 minutes at RT. Subsequently, cells were left unstimulated (w/o) for control or stimulated with TNF-α (100 ng/ml) (n = 6 [ICAM1]; n = 9 [MK]). (D) Isolated CD18+/+ or CD18−/− murine PMNs on immobilized ICAM1 or MK. Diagrams show percentage of adhesion without stimulation (w/o) or after stimulation with TNF-α (100 ng/ml) (n = 3). (E) Induction of adhesion of murine WT PMNs under flow conditions (1 dyne/cm2). Microflow chambers were coated with P-selectin (10 µg/ml) and ICAM1 (12.5 µg/ml) alone (P-selectin + ICAM1), or combined with MK (P-selectin + ICAM1 + MK: 10 µg/ml). Data show the total number of adherent PMNs at indicated times (n = 5). (F) Adhesion of isolated MK+/+ and MK−/− PMNs under flow conditions. Microflow chambers were coated with P-selectin (10 µg/ml), ICAM1 (12.5 µg/mL), and CXCL1 (5 µg/ml) (n = 4). (G) Adhesion strengthening of adherent murine WT PMNs under gradually increasing shear stress (0.2-8.0 dyne/cm2). WT PMNs were seeded into microflow chambers coated with ICAM1 (ICAM1: 12.5 µg/ml) alone, or in combination with MK (ICAM1 + MK: 10 µg/ml), or CXCL1 (ICAM1 + CXCL1: 5 µg/ml) for 10 minutes before flow was applied as indicated. Adhesion strengthening was measured as the number of adherent PMNs in percent of initially adherent cells at 0.2 dyne/cm2 (100%) (n = 5). (A-E) Show mean ± SEM. *P < .05; ***P < .001.
