The leukocyte response in inflammation is characterized by an initial recruitment of polymorphonuclear leukocytes (PMN) preceding a second wave of monocytes to the site of injury or infection. In the mouse, 2 populations of monocytes have been identified, Gr1 ؊ CCR2 ؊ CX3CR1 hi resident monocytes and Gr1 ؉ CCR2 ؉ CX3CR1 lo inflammatory monocytes. Here, intravital microscopy of the musculus cremaster and a subcutaneous air pouch model were used to investigate a possible link between PMN extravasation and the subsequent emigration of inflammatory monocytes in response to local stimulation with PAF. In mice that were made neutropenic by injection of a PMNdepleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell IntroductionPolymorphonuclear leukocytes (PMN) dominate the initial leukocyte influx to sites of acute infection and inflammation. 1 This first wave of PMN extravasation precedes a second wave of monocyte extravasation. Recruited PMN are thought to trigger this cellular switch by releasing soluble factors that initiate monocyte recruitment, 2-4 much of which may be mediated by ready-made PMN granule proteins deposited at the site of inflammation. 5,6 Indeed, supernatants of activated PMN from patients with specific granule deficiency lacking proteins in their primary, secondary, and tertiary granules show a reduced capacity to attract monocytes despite normal monocyte chemotaxis in vitro to other stimuli. 7 After this initial observation, several PMN-derived granule proteins with monocyte-chemotactic activity were identified, among them LL-37, cathepsin G, human neutrophil peptide 1-3 (HNP1-3, ␣-defensins), and heparin-binding protein (HBP, also known as CAP37 and azurocidin). [8][9][10][11] Their action was found to be pertussis toxin (PTx)-sensitive and several receptors were suggested to mediate the chemotactic effect. [11][12][13] Peripheral blood monocytes constitute a heterogeneous population of circulating leukocytes in both humans 14 and mice. 15 In the murine blood, 2 monocyte subsets can be distinguished based on their expression of CX3CR1, CCR2, and Gr1. Whereas resident monocytes (Gr1 Ϫ CCR2 Ϫ CX3CR1 hi ) home to noninflamed tissues, inflammatory monocytes (Gr1 ϩ CCR2 ϩ CX3CR1 lo ) are predominantly recruited to sites of inflammation by mechanisms involving CCR2. 15 These inflammatory monocytes were recently shown to be of critical importance in diverse inflammatory and infectious diseases. [16][17][18][19] In this study, we investigated the significance of the initial PMN efflux for the subsequent extravasation of monocytes. Our results demonstrate that PMN seed granule proteins in the tissue which contribute to mobilization specifically of inflammatory monocytes. Functionally, the PMN-dependent invasion of inflammatory monocytes results in a more vigorous immune response as shown by enhanced cytokine release and bacterial clearance at the site of inflammation. Methods AnimalsWild-type C57BL/...

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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 ∼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 μm in diameter), but not small (<45 μm), 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.

Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T-lymphocytes in lesions. However, recent data suggest that neutrophils also may be of importance in atherogenesis. Here, we use apolipoprotein E (ApoE)-deficient mice with fluorescent neutrophils and monocytes (ApoE ؊/؊ /Lys EGFP/EGFP mice) to specifically study neutrophil presence and recruitment in atherosclerotic lesions. We show by flow cytometry and confocal microscopy that neutrophils make up for 1.8% of CD45 ؉ leukocytes in the aortic wall of ApoE ؊/؊ /Lys EGFP/EGFP mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. However, neutrophils accumulate at sites of monocyte high density, preferentially in shoulder regions of lesions, and may even outnumber monocyte/macrophages in these areas. Furthermore, intravital microscopy established that a majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque. These data demonstrate neutrophilic granulocytes as a major cellular component of atherosclerotic lesions in ApoE ؊/؊ mice and call for further study on the roles of these cells in atherogenesis. Recruitment of immune cells to the arterial intima is central to atherogenesis. Current dogma emphasizes the role of macrophages and T-lymphocytes in promoting plaque development and destabilization.1,2 However, the most abundant white blood cell in the circulation, the neutrophilic granulocyte, has until recently rarely been associated with the development of atherosclerosis. Nonetheless, proteins typically secreted by neutrophils are abundant in lesions, [3][4][5][6][7] and systemic neutrophil counts appear to correlate closely with severity of atherosclerosis in humans.8 Similar observations were also recently made in the murine system in which increased peripheral neutrophil count was associated with enhanced plaque size, whereas the opposite was true when neutrophils were depleted from the circulation. There are also data that indicate presence of neutrophils in lesions of low-density lipoprotein (LDL) receptor-deficient mice.5 Despite these findings, data on potential roles of neutrophils in atherogenesis are rare in the literature.We recently crossed apolipoprotein E-deficient ApoE mice, which allow for sensitive detection of neutrophils in atherosclerotic plaques. 11 Here, we study the presence and spatial distribution of neutrophils in atherosclerotic arteries of these mice. We demonstrate that neutrophils are present in substantial numbers in aortic plaque. Moreover, their contribution is higher in shoulder regions of plaque, which are areas of high inflammatory activity. Intravital microscopy further revealed that neutrophils are the main cell population that interacts with atherosclerotic endothelium, suggesting an ongoing recruitment of neutrophils to lesions. These data demonstrate that neutrophils represen...

Leukocyte infiltration in atherosclerosis has been extensively investigated by using histological techniques on fixed tissues. In this study, intravital microscopic observations of leukocyte recruitment in the aorta of atherosclerotic mice were performed. Interactions between leukocytes and atherosclerotic endothelium were highly transient, thereby limiting the ability for rolling leukocytes to firmly adhere. Leukocyte rolling was abolished by function inhibition of P-selectin (P<0.001, n=8), whereas antibody blockage of E-selectin (n=10) decreased rolling leukocyte flux to 51 +/- 9.9% (mean+/-SE, P<0.01) and increased leukocyte rolling velocity to 162 +/- 18% (P<0.01) of pretreatment values. Notably, function inhibition of the integrin alpha(4) subunit (n=5) had no effect on rolling flux (107+/-25%, P=0.782) or rolling velocity (89+/-6.1%, P=0.147), despite endothelial expression of vascular cell adhesion molecule 1 (VCAM-1). Leukocytes interacting with atherosclerotic endothelium were predominantly neutrophils, because treatment with antineutrophil serum decreased rolling and neutrophil counts in peripheral blood to the same extent. In conclusion, we present the first direct observations of atherosclerosis in vivo. We show that transient dynamics of leukocyte-endothelium interactions are important regulators of arterial leukocyte recruitment and that leukocyte rolling in atherosclerosis is critically dependent on the endothelial selectins. This experimental technique and the data presented introduce a novel perspective for the study of pathophysiological events involved in large-vessel disease.

IMPORTANCETo our knowledge, it is unknown whether a prehospital stroke triage system combining symptom severity and teleconsultation could accurately select patients for primary stroke center bypass and hasten delivery of endovascular thrombectomy (EVT) without delaying intravenous thrombolysis (IVT). OBJECTIVE To evaluate the predictive performance of the newly implemented Stockholm Stroke Triage System (SSTS) for large-artery occlusion (LAO) stroke and EVT initiation. Secondary objectives included evaluating whether the Stockholm Stroke Triage System shortened onset-to-puncture time for EVT and onset-to-needle time (ONT) for IVT. DESIGN, SETTING, AND PARTICIPANTS This population-based prospective cohort study conducted from October 2017 to October 2018 across the Stockholm region (Sweden) included patients transported by first-priority ("code stroke") ambulance to the hospital for acute stroke suspected by an ambulance nurse and historical controls (October 2016-October 2017). Exclusion criteria were in-hospital stroke and helicopter or private transport. Of 2909 eligible patients, 4 (0.14%) declined participation. EXPOSURES Patients were assessed by ambulance nurses with positive the face-armspeech-time test or other stroke suspicion and were evaluated for moderate-to-severe hemiparesis (Ն2 National Institutes of Health stroke scale points each on the ipsilateral arm and leg [A2L2 test]). If present, the comprehensive stroke center (CSC) stroke physician was teleconsulted by phone for confirmation of stroke suspicion, assessment of EVT eligibility, and direction to CSC or the nearest primary stroke center. If absent, the nearest hospital was prenotified.MAIN OUTCOMES AND MEASURES Primary outcome: LAO stroke. Secondary outcomes: EVT initiation, onset-to-puncture time, and ONT. Predictive performance measures included sensitivity, specificity, positive and negative predictive values, the overall accuracy for LAO stroke, and EVT initiation. RESULTSWe recorded 2905 patients with code-stroke transports (1420 women [49%]), and of these, 323 (11%) had A2L2+ teleconsultation positive results and were triaged for direct transport to CSC (median age, 73 years [interquartile range (IQR), 64-82 years]; 55 women [48%]). Accuracy for LAO stroke was 87% (positive predictive value, 41%; negative predictive value, 93%) and 91% for EVT initiation (positive predictive value, 26%; negative predictive value, 99%). Endovascular thrombectomy was performed for 84 of 323 patients (26%) with triage-positive results and 35 of 2582 patients (1.4%) with triage-negative results. In EVT cases with a known onset time (77 [3%]), the median OPT was 137 minutes (IQR, previous year, 206 minutes [IQR,; n = 75) (P < .001). The regional median ONT (337 [12%]) was unchanged at 115 minutes (IQR, previous year, 115 minutes [IQR,; n = 360) (P = .79). The median CSC IVT door-to-needle time was 13 minutes (IQR,(10)(11)(12)(13)(14)(15)(16)(17)(18) 116 [4%]) (previous year, 31 minutes [IQR,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35...