Importance of primary capture and L-selectin-dependent secondary capture in leukocyte accumulation in inflammation and atherosclerosis in vivo

Abstract

In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by approximately 20-50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 microm in diameter), but not small (<45 microm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.

Figure 2

Primary and secondary capture in the microcirculation. (A) Intravital microscopy images of leukocyte rolling, primary capture, and secondary capture in a cytokine-treated mouse cremaster muscle arteriole. Vessel diameter is indicated with D. Rolling leukocytes are indicated with white circles and arrows. P and S indicate primary and secondary capture, respectively. Large white arrow represents direction of flow. Bar, 50 μm. Images were taken at times 0, 0.72, 0.96, and 1.56 s. (B) The respective contribution of primary and secondary capture in cytokine-induced leukocyte rolling in arterioles and venules, and in trauma-induced rolling in venules. Mean ± SEM of the percentage of total capture that was made up of secondary capture is indicated.

Figure 2

Primary and secondary capture in the microcirculation. (A) Intravital microscopy images of leukocyte rolling, primary capture, and secondary capture in a cytokine-treated mouse cremaster muscle arteriole. Vessel diameter is indicated with D. Rolling leukocytes are indicated with white circles and arrows. P and S indicate primary and secondary capture, respectively. Large white arrow represents direction of flow. Bar, 50 μm. Images were taken at times 0, 0.72, 0.96, and 1.56 s. (B) The respective contribution of primary and secondary capture in cytokine-induced leukocyte rolling in arterioles and venules, and in trauma-induced rolling in venules. Mean ± SEM of the percentage of total capture that was made up of secondary capture is indicated.

Figure 1

The concept of capture and the influences of vessel type, pretreatment, vessel diameter, and WSR on capture in vivo. (A) Capture in vitro occurs through either of two distinct mechanisms. Leukocytes may attach directly to the endothelium and subsequently initiate rolling interactions, an event called primary capture. Alternatively, a freely flowing leukocyte can transiently interact with a previously rolling leukocyte, and subsequently initiate a rolling interaction with the endothelium immediately downstream of the previously rolling cell. This event is known as secondary capture. (B) RLF, capture, and detachment in arterioles and venules of the mouse cremaster muscle microcirculation after cytokine stimulation of the tissue, and in response to preparation trauma only. Capture represents the number of leukocytes that initiated rolling within the central part of the field of vision without previously having been in contact with the vessel wall. (C) Capture of leukocytes in various situations in the microcirculation plotted against leukocyte detachment from the endothelium (r = 0.843, P < 0.001). Data for venules and arterioles are indicated by dots or circles, respectively. (D) Capture of leukocytes from the free flow was plotted against vessel diameter and WSR. In arterioles, capture of leukocytes to the endothelium occurs in all vessels where leukocyte rolling is observed. In venules, capture is low at low WSR and in vessels of small diameters whereas in larger venules and at higher WSR, capture is prominent.

Figure 1

The concept of capture and the influences of vessel type, pretreatment, vessel diameter, and WSR on capture in vivo. (A) Capture in vitro occurs through either of two distinct mechanisms. Leukocytes may attach directly to the endothelium and subsequently initiate rolling interactions, an event called primary capture. Alternatively, a freely flowing leukocyte can transiently interact with a previously rolling leukocyte, and subsequently initiate a rolling interaction with the endothelium immediately downstream of the previously rolling cell. This event is known as secondary capture. (B) RLF, capture, and detachment in arterioles and venules of the mouse cremaster muscle microcirculation after cytokine stimulation of the tissue, and in response to preparation trauma only. Capture represents the number of leukocytes that initiated rolling within the central part of the field of vision without previously having been in contact with the vessel wall. (C) Capture of leukocytes in various situations in the microcirculation plotted against leukocyte detachment from the endothelium (r = 0.843, P < 0.001). Data for venules and arterioles are indicated by dots or circles, respectively. (D) Capture of leukocytes from the free flow was plotted against vessel diameter and WSR. In arterioles, capture of leukocytes to the endothelium occurs in all vessels where leukocyte rolling is observed. In venules, capture is low at low WSR and in vessels of small diameters whereas in larger venules and at higher WSR, capture is prominent.

Figure 3

Secondary capture is L-selectin dependent and contributes to cytokine-induced leukocyte rolling in arterioles and trauma-induced rolling in venules in vivo. (A) RLFFs in the microcirculation after antibody blockage of P- or L-selectin. RLFF was determined as rolling flux divided by the total number of leukocytes traveling in the vessel estimated from flow and WBC. (B and C) Impact of inhibition of L-selectin on RLFF and capture efficiencies in cytokine-treated arterioles and in trauma-induced rolling in venules at early time-points. Results were obtained in arterioles and venules of WT and L^−/− mice (left panels), and in WT mice before and after antibody blockage of L-selectin (right panels). Figures are based on data presented in Table . Capture efficiency/mm² represents the ratio between leukocytes that were captured within the field of vision and the total number of leukocytes traveling in the vessel adjusted for differences in luminal vessel area. (D) The percentage of RLFF after function inhibition of L-selectin in venules compared with RLFF before antibody treatment plotted against vessel diameter. Antibody blockage of L-selectin function (which abolished secondary capture) decreased RLFF in large venules (>45 μm in diameter) whereas in venules of diameters less than 45 μm, RLFF remained unchanged.

Figure 4

Secondary capture increases leukocyte recruitment in large arteries and in atherosclerosis in vivo. (A) Characteristics of capture and rolling in the cytokine-treated mouse femoral artery. Data for rolling flux and capture are indicated. Leukocyte–endothelial interactions were abolished by treatment with a function-blocking antibody against P-selectin (not shown). (B) Leukocyte capture and rolling on atherosclerotic lesions in the aorta of ApoE⁰ and ApoE⁰/LDLR⁰ mice. Data for capture and rolling flux are shown. (C) Secondary capture on atherosclerotic lesions in the mouse aorta plotted against RLF. RLF/mm represents the flux of rolling leukocytes adjusted for differences in the width of the vessel section observed. Secondary capture/mm² represents the number of leukocytes that initiated contact with atherosclerotic endothelium through secondary capture adjusted for differences in luminal vessel area.

Figure 4

Secondary capture increases leukocyte recruitment in large arteries and in atherosclerosis in vivo. (A) Characteristics of capture and rolling in the cytokine-treated mouse femoral artery. Data for rolling flux and capture are indicated. Leukocyte–endothelial interactions were abolished by treatment with a function-blocking antibody against P-selectin (not shown). (B) Leukocyte capture and rolling on atherosclerotic lesions in the aorta of ApoE⁰ and ApoE⁰/LDLR⁰ mice. Data for capture and rolling flux are shown. (C) Secondary capture on atherosclerotic lesions in the mouse aorta plotted against RLF. RLF/mm represents the flux of rolling leukocytes adjusted for differences in the width of the vessel section observed. Secondary capture/mm² represents the number of leukocytes that initiated contact with atherosclerotic endothelium through secondary capture adjusted for differences in luminal vessel area.

Figure 4

Secondary capture increases leukocyte recruitment in large arteries and in atherosclerosis in vivo. (A) Characteristics of capture and rolling in the cytokine-treated mouse femoral artery. Data for rolling flux and capture are indicated. Leukocyte–endothelial interactions were abolished by treatment with a function-blocking antibody against P-selectin (not shown). (B) Leukocyte capture and rolling on atherosclerotic lesions in the aorta of ApoE⁰ and ApoE⁰/LDLR⁰ mice. Data for capture and rolling flux are shown. (C) Secondary capture on atherosclerotic lesions in the mouse aorta plotted against RLF. RLF/mm represents the flux of rolling leukocytes adjusted for differences in the width of the vessel section observed. Secondary capture/mm² represents the number of leukocytes that initiated contact with atherosclerotic endothelium through secondary capture adjusted for differences in luminal vessel area.

Figure 5

Secondary capture in arterial vessels induces formation of rolling clusters and rolling strings of leukocytes. (A) Demonstration of rolling clusters in arterial vessels. In A1, the number of leukocytes passing a reference line during 20 consecutive 3-s periods in a WT (top) and an L^−/− (bottom) arteriole are shown. The respective parameters of capture and rolling in the depicted experiments were: Rolling flux, 95 (WT) and 75 (L^−/−); primary capture, 16 (WT) and 22 (L^−/−); and secondary capture, 20 (WT) and 0 (L^−/−). In A2–A6, the buildup of a rolling cluster in a cytokine-stimulated femoral artery is demonstrated. D indicates vessel diameter. P and S indicate primary and secondary capture, respectively. Large arrow indicates direction of flow. Bar, 100 μm. Images were taken at times 0, 0.64, 1.24, 1.52, and 2.52 s. (B) Sequential video frames showing the formation of a rolling string on an atherosclerotic lesion in the right iliac artery of an ApoE⁰/LDLR⁰ mouse in vivo. The orientation of the microscopic images is shown in B1. In B2–B6, the lesion is visible in the bottom left as a bright area. Large arrow indicates direction of flow. Bar, 100 μm. Images were taken at times 0, 0.54, 2.36, 3.20, and 3.96 s.