Einar E. Eriksson

Neutrophil secretion products pave the way for inflammatory monocytes

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 PMN-depleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell count but was restored by local treatment with secretion of activated PMN. Components of the PMN secretion were found to directly activate inflammatory monocytes and further examination revealed PMN-derived LL-37 and heparin-binding protein (HBP/CAP37/azurocidin) as primary mediators of the recruitment of inflammatory monocytes via activation of formyl-peptide receptors. These data show that LL-37 and HBP specifically stimulate mobilization of inflammatory monocytes. This cellular cross-talk functionally results in enhanced cytokine levels and increased bacterial clearance, thus boosting the early immune response.

Neutrophil primary granule proteins HBP and HNP1–3 boost bacterial phagocytosis by human and murine macrophages

In acute inflammation, infiltrating polymorphonuclear leukocytes (also known as PMNs) release preformed granule proteins having multitudinous effects on the surrounding environment. Here we present what we believe to be a novel role for PMN-derived proteins in bacterial phagocytosis by both human and murine macrophages. Exposure of macrophages to PMN secretion markedly enhanced phagocytosis of IgG-opsonized Staphylococcus aureus both in vitro and in murine models in vivo. PMN secretion activated macrophages, resulting in upregulation of the Fcgamma receptors CD32 and CD64, which then mediated the enhanced phagocytosis of IgG-opsonized bacteria. The phagocytosis-stimulating activity within the PMN secretion was found to be due to proteins released from PMN primary granules; thorough investigation revealed heparin-binding protein (HBP) and human neutrophil peptides 1-3 (HNP1-3) as the mediators of the macrophage response to PMN secretion. The use of blocking antibodies and knockout mice revealed that HBP acts via beta2 integrins, but the receptor for HNP1-3 remained unclear. Mechanistically, HBP and HNP1-3 triggered macrophage release of TNF-alpha and IFN-gamma, which acted in an autocrine loop to enhance expression of CD32 and CD64 and thereby enhance phagocytosis. Thus, we attribute what may be a novel role for PMN granule proteins in regulating the immune response to bacterial infections.

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)

Distinct Infiltration of Neutrophils in Lesion Shoulders in ApoE−/− Mice

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.

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.

Intravital Microscopy on Atherosclerosis in Apolipoprotein E–Deficient Mice Establishes Microvessels as Major Entry Pathways for Leukocytes to Advanced Lesions

Background— There has been considerable speculation about the role of lesion microvessels in the accumulation of leukocytes in atherosclerosis. However, direct study of microvascular recruitment of leukocytes in lesions has not been performed, and the quantitative role for this route of entry is unclear. Methods and Results— Here, microvascular recruitment of leukocytes was studied in advanced lesions in 12- to 24-month-old apolipoprotein E–deficient (ApoE−/−) mice. Histology and transmission electron microscopy demonstrated the presence of mainly adventitial, but also intimal, microvessels. Interactions between leukocytes and endothelium occurred in lesion venules. Leukocyte rolling was largely P-selectin dependent; however, residual rolling was mediated by L-selectin and endothelial P-selectin glycoprotein ligand 1. Leukocyte adhesion was significant and was attenuated in mice treated with antibodies against P-selectin, CD18, or both before preparation for intravital microscopy, suggesting acute activation of these 2 molecules by surgical trauma. Nonetheless, the density of firmly arrested leukocytes was 100-fold higher in lesion venules compared with the arterial lumen even in mice pretreated with antibodies against P-selectin and CD18, indicating strong recruitment of cells from venules that is unrelated to experimental manipulation. Fluorescent myelomonocytic cells in ApoE−/− mice carrying a knock-in mutation for enhanced green fluorescent protein (EGFP) in the lysozyme M locus (ApoE−/−/lysMEGFP/EGFP mice) were distributed specifically around lesion venules, but not around arterioles or capillaries, further indicating ongoing extravasation from venules into plaque tissue. Conclusions— These findings provide strong data for microvascular recruitment of leukocytes in atherosclerosis and indicate roles for L-selectin and P-selectin glycoprotein ligand 1 in this process.

Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages

Macrophages represent a multi‐functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil‐derived granule proteins in the anti‐microbial activity of macrophages. Neutrophil secretion obtained by antibody cross‐linking of the integrin subunit CD18 (X‐link secretion) or by treatment with N‐Formyl‐Met‐Leu‐Phe (fMLP secretion) induced a several‐fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca2+. Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X‐link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti‐microbial therapy.

Direct Observations In Vivo on the Role of Endothelial Selectins and α4 Integrin in Cytokine-Induced Leukocyte-Endothelium Interactions in the Mouse Aorta

The molecular mechanisms underlying leukocyte recruitment in large arteries have been extensively studied using histological techniques on fixed tissues. However, there are no reports that address the dynamics of leukocyte recruitment in large arteries in vivo. We developed an intravital microscopy technique for direct observation of leukocyte-endothelium interactions in the mouse aorta. Circulating leukocytes were labeled intravasally with rhodamine 6G and microscopically visualized within the aorta, allowing direct analysis of leukocyte rolling and adhesion. In untreated vessels, leukocyte-endothelium interactions were virtually absent. However, local pretreatment with cytokines interleukin-1beta and tumor necrosis factor-alpha induced clear-cut leukocyte rolling and adhesion, compatible with normal blood flow and wall shear rate. High shear decreased rolling leukocyte flux and increased leukocyte rolling velocity, thus decreasing the tendency for firm adhesion. Leukocyte rolling was almost abolished by an antibody blocking the function of P-selectin, whereas function-blocking antibodies against E-selectin and the alpha(4)-integrin subunit decreased rolling leukocyte flux to 51+/-34% (mean +/- SD) and 59+/-11% of the value before antibody treatment, respectively. In addition, inhibition of E-selectin function, but not of alpha(4) integrin, resulted in increased leukocyte rolling velocity (from 48+/-32 to 71+/-32 microm per second). Taken together, we introduce the first model for direct studies of leukocyte-endothelium interactions in a large artery in vivo and demonstrate cytokine-induced shear-sensitive leukocyte rolling that is critically dependent on P-selectin and modulated by E-selectin and alpha(4) integrin.

Engagement of β2 integrins induces surface expression of β1 integrin receptors in human neutrophils

Induction of β1 integrin (CD49/CD29) expression in polymorphonuclear leukocytes (PMN) has been shown to be associated with transendothelial migration recently. Yet, β1 integrin expression is relatively insensitive to cell activation with soluble agonists, such as N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP). We hypothesized that β2 integrins (CD11/CD18), critically involved in PMN adhesion and extravasation, may play a role in regulating β1 integrin expression in PMN. Antibody cross‐linking of CD18, mimicking adhesion‐dependent engagement of β2 integrins, resulted in rapid, tyrosine kinase‐dependent upregulation of β1 integrins. This response was potentiated by simultaneous chemoattractant (fMLP) stimulation of PMN. Moreover, upregulation of β1 integrins evoked by CD18 cross‐linking was found to support adhesion of fMLP‐stimulated PMN to matrix proteins and also was critical for the ability of PMN to migrate in collagen gels in response to a gradient of fMLP. Taken together, these data demonstrate that engagement of β2 integrins in human PMN induces β1 integrin expression in these cells of significance for their migration in the extravascular tissue. Thus, β2 integrins may serve the function to regulate PMN locomotion in extravascular tissue via receptor crosstalk with β1 integrins.

Powerful Inflammatory Properties of Large Vein Endothelium In Vivo

Objective—Inflammatory responses of large vein endothelium are of importance in pathological processes such as venous thrombosis, chronic venous congestion, and vein graft atherosclerosis. However, the inflammatory properties of large vein endothelium are unclear. Methods and Results—In this study, we used several microscopy techniques to investigate the inflammatory properties of large vein endothelium in vivo. We show that the endothelium in the mouse inferior vena cava (IVC) possesses powerful inflammatory properties that are distinct from the less inflammatory reactive aortic endothelium and virtually identical to endothelial responses in postcapillary venules. Inflammatory stimulation with tumor necrosis factor-&agr; induced strong expression of cell adhesion molecules (CAMs) in the IVC. These CAMs promoted recruitment of leukocytes, platelets, and erythrocytes to the vein wall. The inflammatory responses altered endothelial structure and increased endothelial permeability in the IVC. Accumulation of blood cells and endothelial damage were markedly reduced in mice deficient in the endothelial leukocyte recruitment molecules E-selectin and P-selectin, indicating a central role for these molecules in driving structural and functional changes of IVC endothelium. Conclusions—These findings provide the first comprehensive demonstration of the inflammatory capacity of large vein endothelium and emphasize the actions of endothelial cells as targets in large vein disease.