Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury

Abstract

Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated alpha(M)beta(2) integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disease, the capture of erythrocytes by alpha(M)beta(2) microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produce vascular damage and lung injury. Inactivation of E-selectin or alpha(M)beta(2) prevents tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thromboinflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

Figures

Figure 1. Heterotypic interactions of RBC and platelets with leukocyte microdomains are induced during inflammation

(a) Heterotypic interactions between leukocytes and platelets in a TNF-α- stimulated C57BL/6 mouse. Fluorescently conjugated antibodies to L-selectin (blue) and CD41 (red) allow identification of the trailing edge of crawling leukocytes and platelets, respectively. The short arrow indicates an interacting RBC, and the asterisks show interacting platelets (green for those mediated by the trailing edge, white for the leading edge). The large arrow points to the direction of blood flow. Scale bar = 10 µm. (b) Frequency of interactions between RBCs and leukocytes in venules of mice where surgical trauma or trauma plus TNF-α administration preceded imaging. n = 5 mice. ***, p<0.001, Mann-Whitney test. (c) Frequency of interactions between leukocytes and platelets. n=5–6 mice. ***, p<0.001, Mann-Whitney test. (d) Contribution of the leading and trailing edges in RBC interactions after TNF-α treatment. n = 4 mice; ‡, p<0.005. (e) Contribution of leukocyte microdomains to platelet interactions in wild-type mice, treated or not with TNF-α. n = 5 mice per group; ‡, p<0.005 compared to Trailing group, paired t-test; NS, not significant.

Figure 2. RBC and platelet interactions depend on E-selectin and its ligand ESL-1

(a) Number of RBC captures per adherent leukocyte in wild–type, Selp−/− and Sele−/− mice. n = 32–40 venules from 5–7 mice per group. *, p<0.05 compared to WT, **, p<0.01 compared to the other groups; Kruskal-Wallis test with Dunn’s multigroup comparison. (b) Reduced number of platelet captures per adherent leukocyte in Sele−/− mice. n = 32–42 venules from at least 5 mice per group. ***, p<0.0001. (c) Contribution of leukocyte microdomains to platelet interactions in TNF-α-treated mice. n = 5 mice per group. **, p<0.01, Mann-Whitney test. ‡, p=0.003 and NS, not significant compared to Trailing groups, paired t-test. (d) RBC-leukocyte interactions in wild-type, Selplg−/− and Cd44−/− mice. n = 31–42 venules from 5–7 mice per group. *, p<0.05, Kruskal-Wallis test with Dunn’s multigroup comparison. (e) RBC captures by leukocytes transduced with control (Scrmbl) or shESL-1 lentiviral vectors. n = 4–6 mice per group. ***, p<0.0001 compared to GFP− cells from the same lentiviral group, paired t-test. (f) nRBC interactions in mice pretreated with inhibitors to Src kinases (PP2), p38 MAPK (SB203580) or Syk (Piceatannol), or vehicle control (DMSO). n = 33–44 venules from 6 mice per group. *, p<0.05 compared to all other groups, Kruskal-Wallis test with Dunn’s multigroup comparison.

Figure 3. Heterotypic interactions with RBC and platelets are mediated by the leukocyte integrin αMβ2

(a) Micrographs of adherent leukocytes obtained by MFIM analyses of TNF-α-treated C57BL/6 mice injected with labeled antibodies against L-selectin (red) and anti-αM (green). αMβ2 integrin expression is homogenously distributed, including where an RBC (arrowhead) is shown interacting at the leading edge of an adherent leukocyte. Scale bar = 10 µm. (b) RBC-WBC interactions in Itgam−/− mice compared to wild-type (WT) controls. n = 41–42 venules from 6–7 mice per group. **, p<0.001, Mann-Whitney test. (c) Platelet–WBC interactions in Itgam−/− mice compared to WT. n = 35–36 venules from 5–6 mice per group. **, p<0.0001, Mann-Whitney test. (d) Contribution of leukocyte microdomains to platelet captures in TNF-α-treated Itgam−/− mice. n = 5 mice. ‡, p=0.008, paired t-test compared to Trailing values.

Figure 4. E-selectin and ESL-1 modulate regional αMβ2 activity on adherent leukocytes in vivo

(a) Frequency of fluosphere (beads) binding by F4/80− and F4/80+ adherent leukocytes. Data obtained from analyses of 84 fluosphere captures from 4 mice. ***, p<0.001, t-test. (b) Time-lapse micrographs of in vivo fluosphere capture by two representative adherent leukocytes (asterisks). Leading edges are outlined with dotted lines and L-selectin clusters are in red. Sequence of capture of fluospheres at the times indicated. A movie depicting capturing events is shown in Supplementary Video 3. The histogram (right panel) shows the quantitation of the fluospheres captured by the leading and trailing edges from 98 events identified in 7 mice; ‡, p=0.0005. Scale bar = 10 µm. (c) Binding of albumin-coated fluospheres to leukocytes in wild-type and Itgam−/− mice. Each dot represents the average number of fluospheres bound per leukocyte in individual venules. n = 40 venules from 4–5 mice per group. **, p<0.001, Mann-Whitney test. (d) Binding of albumin-coated fluospheres to leukocytes in wild-type, Selp−/− and Sele−/− mice. n = 33–43 venules from 4–7 mice per group. *, p<0.05 compared to the other groups; Kruskal-Wallis test. (e) Binding of fluospheres in wild-type, Selplg−/− and Cd44−/− mice. n = 30–47 venules from 4–6 mice per group. (f) Fluosphere capture in leukocytes transduced with shESL-1 or scrambled sequence lentiviral vectors. n = 5 mice per group. ***, p<0.0001 compared to GFP− cells from the same lentiviral group, paired t-test.

Figure 5. Antibody-induced lung injury requires platelet-leukocyte interactions and is blocked by antibodies to E-selectin and αMβ2

(a) Protein accumulation in BAL fluids after anti-H2b or H2d antibody administration. αPlt, platelet-depleted mice; n = 7–9 mice. **, p<0.01. (b) Platelet counts from mice in (a). n = 9–10 mice; ***, p<0.001. (c) Left panels are representative micrographs of platelet-leukocyte interactions before (pre-H2d) and after (post-H2d) anti-H2d administration (L-selectin, blue; CD41, red). Arrow, direction of flow. Scale bar = 10 µm. Right panel, frequency of platelet-leukocyte interactions in control or anti-E-selectin-treated (9A9) mice. §, p<0.01 and NS, not significant, paired t-test. #, p<0.05, unpaired t-test. (d) Contribution of microdomains to interactions in the rIgG group. n = 5 mice; *, p=0.01. (e) Representative micrographs depicting vascular permeability after anti-H2d or anti-H2b treatment (FITC-dextran, blue). Scale bar = 100 µm. (f) FITC-dextran extravasation in mice treated with rat IgG, anti-E-selectin (9A9) or anti-αMβ2 (M1/70) antibodies. n = 20–32 venules. **, p<0.01; ***, p<0.001. (g) FITC-dextran extravasation in platelet-depleted (αPlt) or control mice after anti-H2d administration. n = 16–20 venules. §, p = 0.002. (h) Effect of P-selectin (RB40.34), E-selectin (9A9) and αMβ2 (M1/70) inhibition in lung injury. n = 7–13 mice. *, p<0.05; **, p<0.001. (i) Percentage of ROS-positive adherent leukocytes in control (Ctrl), platelet-depleted (αPlt) or anti-E-selectin antibody-treated (9A9) mice; n = 30–53 venules. *, p<0.05; ***, p<0.001 compared to all other groups. (j) Protein content in BAL fluids of mice treated with saline (Ctrl) or n-acetyl-cysteine (N-AcC). n = 5–14 mice. *, p < 0.05, unpaired t-test.

Figure 6. Vaso-occlusion in sickle cell disease requires E-selectin-mediated activation of αMβ2

(a) Individual contributions of P- and E-selectins in sRBC capture by adherent leukocytes using genetically deficient mice or function-blocking antibodies. n = 9–17 mice. §, p< 0.01 compared to time 0–90 in the same group; *, p<0.05; **, p<0.01; ***, p<0.01 compared to the SCD group at the same time point; ¶, p<0.05 compared to the SCD Selp−/− group at the same time point; Kruskal-Wallis test. (b) Analysis of blood flow rates from the experiments shown in (a), at the 181–270 time point. SS, sickle cell; n = 9–16 mice; *, p < 0.01, one-way ANOVA. (c) Representative micrographs showing albumin-coated fluospheres bound to adherent leukocytes in hemizygous (SA) or homozygous (SS) SCD mice. Scale bar = 10 µm. (d) Binding of albumin-coated fluospheres to leukocytes in SA and SS chimeras with wild-type endothelium or Sele−/− endothelium. Each circle represents the average number of fluospheres bound per leukocyte for an individual venule, and bars represent mean values. n = 24–34 venules from 3–5 mice per group. **, p<0.001 compared to the two other groups; Kruskal-Wallis test. (e) Experimental scheme to assess the role of αMβ2 in vaso-occlusion. (f) αMβ2 blockade reduced the number of sRBC interactions with leukocytes. n = 6 mice per group; *, p = 0.001. (g) Kaplan-Meier survival curves after TNF-α treatment in control and M1/70 treated groups. n = 6 mice per group. Log-rank test, p< 0.002.