1. The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via β2 integrins (CD11/CD18)‏
by Ludwig T. Weckbach, Anita Gola, Michael Winkelmann, Sascha M. Jakob, Leopold Groesser, Julia Borgolte, Frank Pogoda, Robert Pick, Monika Pruenster, Josef Müller-Höcker, Elisabeth Deindl, Markus Sperandio, and Barbara Walzog
Blood
Volume 123(12):
March 20, 2014
©2014 by American Society of Hematology

2. MK was critical for leukocyte adhesion and extravasation in inflamed cremaster muscle venules.
MK was critical for leukocyte adhesion and extravasation in inflamed cremaster muscle venules. (A-C) Intravital microscopy of cremaster muscle postcapillary venules in MK+/+ and MK−/− mice 2 hours after intrascrotal administration of TNF-α (500 ng). (A) Number of rolling leukocytes; n = 21 venules from 5 MK+/+ mice, n = 24 venules from 5 MK−/− mice. (B) Leukocyte rolling velocity was analyzed offline; accumulated frequency of rolling velocity includes 224 cells from 5 MK+/+ mice and 221 cells from 5 MK−/− mice. (C) Number of adherent leukocytes as analyzed by intravital microscopy; n = 21 venules from 5 MK+/+ mice, n = 24 venules from 5 MK−/− mice. (D) Differential cell counts of perivascular leukocytes as assessed in cremaster muscle whole mounts were determined morphologically. (E) Analysis of leukocyte adhesion in cremaster muscle venules of MK+/+ or MK−/− mice during trauma-induced inflammation before (no treatment) and 10 minutes after systemic application of rMK using intravital microscopy. For MK+/+ mice, no treatment: n = 24 venules from 6 mice; rMK: n = 9 venules from 3 mice; for MK−/− mice, no treatment: n = 21 venules from 6 mice; rMK: n = 10 venules from 3 mice. (F) Flow cytometric analysis of cell surface expression of Gr-1, as well as expression of CD11a, CD11b, or CD18 in unstimulated (w/o) or TNF-α (100 ng/ml) treated PMNs from MK+/+ or MK−/− mice following 20 minutes incubation at 37°C. Histograms are representative of 3 independent experiments. (A-E) Show mean ± SEM. *P < .05; ***P < Eos, eosinophils; Others, lymphocytes and basophils; n.s., not significant.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology

3. Leukocyte extravasation was compromised in MK−/− mice during hypoxia-mediated inflammation.
Leukocyte extravasation was compromised in MK−/−mice during hypoxia-mediated inflammation. (A) Representative histological sections of Mm. gastrocnemii from MK+/+ or MK−/− mice 4 days after ligation of the femoral artery in the right leg (occ) and sham operation of the left leg (sham). Immunohistochemical staining of CD45+ cells (red) indicated by arrows. Bar = 100 μm. (B) Quantitative analysis of leukocyte extravasation measured as CD45+ cells per visual field; n = 3 independent experiments. Diagram shows mean ± SEM. ***P < .001.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology

4. Adhesion mediated by immobilized MK in vitro was CD18 dependent.
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 ( 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.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology

5. The high affinity conformation of β2 integrins was promoted by immobilized MK. (A-C) Flow cytometric analysis of the high affinity conformation of β2 integrins of human PMNs using the mAb24 antibody.
The high affinity conformation of β2integrins was promoted by immobilized MK. (A-C) Flow cytometric analysis of the high affinity conformation of β2 integrins of human PMNs using the mAb24 antibody. (A) Quantitative analysis of median fluorescence intensity of isolated PMNs left unstimulated (w/o) or stimulated with soluble MK at various concentrations (100 ng/ml or 30 μg/ml), or TNF-α (100 ng/ml) for 15 minutes at 37°C (n = 4). (B) Analysis of the high affinity conformation of β2 integrins of human PMNs using the mAb24 antibody or control antibody following incubation with microparticles coated with ICAM1 (ICAM1), ICAM1 and MK (ICAM1-MK), or MK (MK), and left untreated (w/o) or after stimulation with soluble MK (100 ng/ml), or TNF-α (100 ng/ml) for 15 minutes at 37°C. Histograms are representative of 6 independent experiments. (C) Quantitative analysis of median fluorescence intensity of mAb24 epitope expression of PMNs binding or nonbinding to coated microparticles (n = 6). (A, C) Show median ± SEM. ***P < .001.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology

6. MK binding to PMNs did not require CD18 or CD29.
MK binding to PMNs did not require CD18 or CD29. (A-C) Analysis of binding of Alexa Fluor 488-labeled MK to murine PMNs using flow cytometry. For control, PMNs were left unstained. (A-B) Cells were left untreated for control (w/o) or stimulated with TNF-α (100 ng/ml), CXCL1 (100 ng/ml), or MnCl2 (3 mM) for 20 minutes at 37°C. (A) Binding of MK to isolated PMNs from CD18+/+ or CD18−/− mice. (B) Binding of MK to isolated PMNs from CD29+/+ or CD29−/− mice. (C) Binding of MK to PMNs from WT mice. The α4 subunit was blocked with the anti-CD49d antibody or left unblocked (untreated), and the rat IgG2b antibody was used as control. Blocking was performed for 20 minutes at RT and cells were subsequently left unstimulated (w/o) or stimulated with TNF-α (100 ng/ml), or CXCL1 (100 ng/ml) for 20 minutes at 37°C. Histograms (A-C) are representative of 3 independent experiments.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology

7. Blocking of LRP1-impaired MK binding to PMNs
Blocking of LRP1-impaired MK binding to PMNs. (A) Binding of Alexa Fluor 488-labeled MK to WT PMNs using flow cytometry.
Blocking of LRP1-impaired MK binding to PMNs. (A) Binding of Alexa Fluor 488-labeled MK to WT PMNs using flow cytometry. For control, PMNs were left unstained. PMNs were treated with LRPAP (3 µM, blocked) for 20 minutes at RT or left untreated, and cells were subsequently left unstimulated (w/o) or stimulated with TNF-α (100 ng/ml), or CXCL1 (100 ng/ml) for 20 minutes at 37°C. Histograms are representative of 3 independent experiments. (B) Analysis of the high affinity conformation of β2 integrins of human PMNs using the mAb24 antibody following incubation with ICAM1 and MK-coated microparticles for 15 minutes at 37°C. Prior to exposure to microparticles, PMNs were treated with LRPAP (3, 6, 9 µM) or left untreated for control (w/o). Data represents percentage of median fluorescence intensity of PMNs interacting with beads after LRP1 blockade in comparison with unblocked samples (n = 5). (B) Shows median ± SEM. *P < .05.
Ludwig T. Weckbach et al. Blood 2014;123:
©2014 by American Society of Hematology