Platelets Guide Leukocytes to Their Sites of Extravasation

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells "find" their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation

Endothelial expression of adhesion and signaling molecules, composition of the vascular wall, and shear rates in the venular microvasculature.

<p>Representative confocal microscopy images of ICAM-1/CD54, VCAM-1/CD106, ICAM-2/CD102, PECAM-1/CD31, JAM-A, and CCL2 expression in venular endothelial cells of the cremaster muscle of WT mice before (open dots) and after (filled dots) stimulation with CCL2 (<b>A</b>; scale bar 50 μm). Panels show quantitative expression levels of these proteins in dependency of the venular diameter as well as the corresponding venular shear rates (mean ± SEM; <i>n</i> = 3–4 per group; #<i>p</i> < 0.05 versus PBS). Panels (<b>B</b>) show representative images and quantitative data for the number of perivascular macrophages, pericytes, and collagen IV LERs of venular microvessels in dependency of the venular diameter (scale bar 50 μm; mean ± SEM; <i>n</i> = 3 per group).</p

Interactions of platelets, neutrophils, and iMOs in the microvasculature.

<p>Interactions of neutrophils (red) and iMOs (green) with endothelial cells (blue) were analyzed in the microvasculature of the cremaster muscle of CX₃CR1^GFP/+ mice by multichannel in vivo microscopy (<b>A</b>; scale bar 100 μm). Panels show results for intravascularly rolling and firmly adherent as well as transmigrated neutrophils or iMOs in venules after 6 h of intrascrotal stimulation with PBS or CCL2 receiving a platelet-depleting anti-CD42b mAb (<b>B</b>), a neutrophil-depleting anti-Ly-6G mAb (<b>C</b>), or isotype control antibodies in dependency of the venular diameter (mean ± SEM for <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus PBS; *<i>p</i> < 0.05 versus isotype control).</p

Consequences of interactions of neutrophils with platelets for the extravasation of neutrophils.

<p>Binding of ICAM-1/CD54 or VCAM-1/CD106 to neutrophils isolated from the peripheral blood of WT mice was assessed upon exposure to recombinant murine CD40L/CD154, P-selectin/CD62P, L-selectin/CD62L, or PSGL-1/CD162 by flow cytometry. P-selectin-elicited binding of ICAM-1/CD54 to murine neutrophils was quantified upon blockade of PSGL-1/CD162 or inhibition of MAPK (<b>A</b>, upper panels; mean ± SEM; <i>n</i> = 4–6 per group; #<i>p</i> < 0.05 versus unstimulated; *<i>p</i> < 0.05 versus isotype control or vehicle). Conformational changes of β2 integrins in human neutrophils were analyzed by using conformation-specific mAbs (<b>A</b>, lower panels). Using multichannel in vivo microscopy on the CCL2-stimulated cremaster muscle of CX₃CR1^GFP/+ mice, intravascular rolling flux and firm adherence of neutrophils were analyzed (mean ± SEM; <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus unstimulated; *<i>p</i> < 0.05 versus isotype control or vehicle). Neutrophil extravasation was evaluated by using a peritonitis assay. Panels (<b>B</b>) show quantitative data for CCL2-challenged animals receiving blocking mAbs directed against Mac-1/CD11b, LFA-1/CD11a, VLA-4/CD49d, ICAM-1/CD54, ICAM-2/CD102, VCAM-1/CD106, PECAM-1/CD31, or JAM-A, or isotype control antibodies (mean ± SEM; <i>n</i> = 4–6 per group; *<i>p</i> < 0.05 versus isotype control).</p

Graphical synopsis.

<p>Schematic illustration of the sequential steps in the leukocyte recruitment cascade including platelet-directed guidance of leukocytes to their site of extravasation.</p

Consequences of interactions of iMOs with platelets and neutrophils for the extravasation of iMOs.

<p>Interactions of intravascularly adherent neutrophils and iMOs were analyzed by multichannel in vivo microscopy. Panel shows quantitative data for interactions (>30 s) between intravascularly adherent neutrophils and iMOs in animals receiving blocking mAbs directed against CD62L or PSGL-1/CD162, or istoype control antibodies (<b>A</b>; mean ± SEM; <i>n</i> = 4 per group; *<i>p</i> < 0.05 versus isotype control; scale bar 10 μm). Binding of ICAM-1/CD54 or VCAM-1/CD106 to iMOs isolated from the peripheral blood of WT mice was assessed upon exposure to recombinant murine CD40L/CD154, P-selectin/CD62P, L-selectin/CD62L, or PSGL-1/CD162 by flow cytometry (<b>B</b>; mean ± SEM; <i>n</i> = 4–6 per group; #<i>p</i> < 0.05 versus unstimulated; *<i>p</i> < 0.05 versus isotype control or vehicle). P-selectin-elicited binding of ICAM-1/CD54 to murine iMOs was quantified upon blockade of PSGL-1/CD162 or inhibition of MAPK (<b>B</b>, upper panels). Conformational changes of β2 integrins in human iMOs were analyzed by using conformation-specific mAbs (mean ± SEM; <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus unstimulated; <b>B,</b> lower panels). Using multichannel in vivo microscopy on the CCL2-stimulated cremaster muscle of CX₃CR1^GFP/+ mice, intravascular rolling flux and firm adherence of iMOs were analyzed. Extravasation of iMOs was evaluated by using a peritonitis assay. Panels (<b>C</b>) show quantitative data for CCL2-challenged animals receiving blocking mAbs directed against Mac-1/CD11b, LFA-1/CD11a, VLA-4/CD49d, ICAM-1/CD54, ICAM-2/CD102, VCAM-1/CD106, PECAM-1/CD31, or JAM-A, or isotype control antibodies (mean ± SEM; <i>n</i> = 4–6 per group; *<i>p</i> < 0.05 versus isotype control).</p

Mechanisms underlying interactions of platelets with endothelial cells, neutrophils, and iMOs.

<p>Representative confocal microscopy images of vWF expression in venular endothelial cells of the cremaster muscle before and after stimulation with CCL2 (<b>A;</b> scale bar: 50 μm). Panel shows quantitative expression levels of this protein in dependency of the venular diameter (mean ± SEM; <i>n</i> = 3 per group; #<i>p</i> < 0.05 versus PBS). Panels (<b>B</b>) show quantitative data for intravascular firm adherence of platelets to endothelial cells in CCL2-stimulated cremasteric venules of animals receiving blocking antibodies or inhibitors directed against vWF, GPIbα, GPIIbIIIa, CD40L/CD154, CD40, P-selectin/CD62P, PSGL-1/CD162, PECAM-1/CD31, or ICAM-2/CD102, or control antibodies or vehicle as assessed by multichannel in vivo microscopy on the cremaster muscle of CX₃CR1^GFP/+ mice (mean ± SEM; <i>n</i> = 4 per group; *<i>p</i> < 0.05 versus control antibody or isotype control or vehicle). Panels (<b>C, D</b>) show quantitative data for interactions (>30 s) between intravascularly adherent platelets and adherent neutrophils or iMOs in animals receiving blocking antibodies directed against CD40L/CD154, CD40, P-selectin/CD62P, PSGL-1/CD162, PECAM-1/CD31, or ICAM-2/CD102, or isotype control antibodies (mean ± SEM; <i>n</i> = 4 per group; *<i>p</i> < 0.05 versus isotype control). Representative multichannel in vivo microscopy images of Mac-1/CD11b and L-selectin/CD62L expression on the surface of intravascularly adherent neutrophils in CCL2-stimulated cremasteric venular microvessels (scale bar: 10 μm; <b>E</b>). Panels show quantitative data for the expression of Mac-1/CD11b or L-selectin/CD62L with respect to the relative localization of intravascularly adherent neutrophils to adherent platelets or in platelet-depleted animals (mean ± SEM; <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus platelet-depleted; *<i>p</i> < 0.05 versus no colocalization with platelets).</p

Endothelial cell interactions of platelets in the microvasculature.

<p>Interactions of platelets (white) and endothelial cells (blue) were analyzed in the microvasculature of the inflamed cremaster muscle of CX₃CR1^GFP/+ mice by multichannel in vivo microscopy (<b>A</b>; scale bar 100 μm). Panels show results for intravascularly rolling and firmly adherent platelets in venules after 6 h of intrascrotal stimulation with phosphate-buffered saline (PBS) or CCL2 in dependency of the venular diameter (mean ± standard error of the mean [SEM] for <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus PBS). Panel (<b>B;</b> scale bar: 10 μm) shows the relative localization of platelets (white), neutrophils (red), and iMOs (green) to PECAM-1/CD31-immunoreactive endothelial junctions (blue; mean ± SEM for <i>n</i> = 3 per group). Panel (<b>C</b>) shows the adhesion dynamics of platelets, neutrophils, and iMOs during the course of the acute inflammatory response (mean ± SEM for <i>n</i> = 3–4 per group).</p

Role of platelets for the transmigration efficacy of leukocytes.

<p>Representative multichannel in vivo microscopy serial images of intravascularly adherent platelets (white) capturing an intraluminally crawling neutrophil (red) or iMO (green; <b>A</b>; arrows indicate direction of blood flow; scale bar 20 μm). Representative multichannel in vivo microscopy images as well as quantitative analysis of neutrophils and iMOs during their successive extravasation from sites of intravascularly adherent platelets (white; <b>B</b>; mean ± SEM; <i>n</i> = 4 per group; scale bar 20 μm). Panels (<b>C</b>) show quantitative data on the transmigration efficacy of total leukocytes, neutrophils, and iMOs in CCL2-stimulated cremasteric venules of animals receiving platelet-depleting mAbs or isotype control antibodies (mean ± SEM; <i>n</i> = 4 per group; *<i>p</i> < 0.05 versus isotype control).</p