Xun Xie

Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins

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 α4 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.—Eriksson, E. E., Xie, X., Werr, J., Thoren, P., Lindbom, L. Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins. FASEB J. 15, 1149‐1157 (2001)

Characteristics of histamine‐induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery

The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte‐endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator‐induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. We first examined the relative importance of histamine and 5‐hydroxytryptamine (5‐HT) in acute mast cell‐dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)‐ and histamine2 (H2)‐receptor blockade. In contrast, the 5‐HT‐receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5‐HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose‐dependent; 5‐HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine‐induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). To examine the role of histamine‐receptors in the histamine‐induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1‐receptor antagonist), cimetidine, or ranitidine (H2‐receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H2‐receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine‐induced PMNL rolling by 82%. Furthermore, in contrast to an H3‐receptor agonist, challenge with either the H1‐receptor agonist 2‐thiazolylethylamine or two different H2‐receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. Treatment with the nitric oxide‐synthase inhibitor L‐NAME did not affect the histamine‐induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P‐selectin to rat isolated PMNLs. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1‐ and H2‐receptors, and lasted for 2–3 h. Moreover, the histamine‐induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P‐selectin ligand(s).

Neutrophil-Derived Heparin-Binding Protein (HBP/CAP37) Deposited on Endothelium Enhances Monocyte Arrest under Flow Conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca2+ that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.

Inhibitory effect of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion in rat mesenteric venules in vivo

1 Anti‐inflammatory actions of heparin and related glycosaminoglycans have been described in the literature. Here, we used intravital microscopy of the rat mesentery microcirculation to examine effects of locally administered heparin on leukocyte rolling and chemoattractant‐induced firm adhesion. 2 It was found that topical application of heparin reduced N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced leukocyte adhesion. Notably, the inhibitory action of heparin was not dose‐dependent, but rather a biphasic dose‐response was found, i.e. low (2 and 20 iu ml−1) and high (1000 iu ml−1) concentrations of heparin significantly reduced adhesion, whereas an intermediate dose (200 iu ml−1) was less effective. 3 Heparin, 2 and 20 iu ml−1, decreased rolling leukocyte flux, while having no effect on blood flow or total leukocyte flux. By contrast, heparin, 200 and 1000 iu ml−1, increased total leukocyte flux in parallel with a rise in volume blood flow resulting in recovery of the rolling leukocyte flux at these doses. Thus, the biphasic inhibitory action of heparin on fMLP‐induced firm adhesion could in part be attributed to changes in leukocyte delivery (i.e. blood flow) and rolling leukocyte flux induced by heparin. 4 When compensating for the influence of different rolling levels on fMLP‐evoked adhesion, a dose‐related inhibitory effect of heparin on the firm adhesive response per se was revealed. Similar results were obtained in a static adhesion assay in vitro where heparin 200 and 1000 iu ml−1 (but not 2 and 20 iu ml−1) significantly inhibited fMLP‐induced leukocyte adhesion in the absence of any modulatory influence on changes in rolling. 5 Our data show that locally administered heparin inhibits leukocyte rolling as well as chemoattractant‐induced firm adhesion in vivo which thus may contribute to the postulated anti‐inflammatory activity of this compound. However, because of interference with several microvascular functions, strict dose‐dependent responses to heparin treatment were not found, which illustrates the complex interplay between local blood flow, leukocyte rolling and chemoattractant‐induced adhesion as determinants of leukocyte recruitment to tissues in inflammation.

Microvascular mechanisms of histamine-induced potentiation of leukocyte adhesion evoked by chemoattractants

1 Intravital microscopy of the rat mesentery was used to examine interactions between histamine and the chemoattractant leukotriene B4 (LTB4) with regard to leukocyte adhesion in postcapillary venules. 2 Topical administration of histamine caused a four fold potentiation of LTB4‐induced leukocyte adhesion. 3 Histamine significantly increased the rolling leukocyte flux by 25%, and this effect of histamine on rolling was strictly blood flow‐dependent, i.e. we found significant positive correlations between both blood flow and total leukocyte flux and between total and rolling leukocyte flux, while no changes in leukocyte rolling fraction or rolling velocity were observed. Furthermore, histamine caused a clear‐cut increase in venular plasma protein leakage. 4 The platelet‐activating factor (PAF) receptor antagonist WEB 2086, which effectively inhibited adhesion of leukocytes evoked by exogenous PAF, did not reduce the potentiating effect of histamine on LTB4‐induced leukocyte adhesion. 5 The vasodilator acetylcholine (ACh) caused a moderate enhancement of LTB4‐induced leukocyte adhesion in proportion to its blood flow‐dependent 40% increase in rolling leukocyte flux. In contrast to histamine, ACh did not provoke vascular leakage of plasma proteins. 6 Taken together, our findings suggest that histamine plays an important pro‐inflammatory role in tissues where leukocyte rolling is already present, by potentiating chemoattractant‐induced firm leukocyte adhesion through a combination of microcirculatory changes such as increased rolling leukocyte flux and vascular permeability.

Human Cytomegalovirus Induces Inhibition of Macrophage Differentiation by Binding to Human Aminopeptidase N/CD13

Human CMV (HCMV) infection in immunocompromised patients is frequently associated with impaired immunological functions. We have recently found that HCMV inhibits cytokine-induced differentiation of monocytes into macrophages. In this study, we demonstrate that HCMV-induced inhibition of macrophage differentiation was dependent on binding of virus particles to the cell surface molecule CD13/aminopeptidase N, which involved Ca2+-dependent intracellular signaling pathways. We found that treatment of cells with the CD13-specific mAbs My7 and WM15 inhibited macrophage differentiation, and that My7 and WM15 induced a rise in intracellular Ca2+ in similar ways as HCMV. In contrast, binding of the CD13-specific Ab clone SJ1D1 blocked the ability of HCMV to inhibit macrophage differentiation, and blocked the HCMV-induced intracellular Ca2+ response. In addition, the Ca2+ modulator thapsigargin partially blocked the ability of HCMV to inhibit cellular differentiation. Furthermore, we demonstrate that recombinant viral glycoprotein gB was able to inhibit macrophage differentiation in similar ways as the virus. Thus, these results suggest that binding of HCMV to monocytes induces an intracellular rise of Ca2+, of which one result is a block in the ability of the cells to differentiate into macrophages. These observations suggest an efficient viral strategy to interfere with cellular differentiation pathways, and may also in part explain the generalized immunosuppression that is often observed in HCMV-infected patients.