Ellinor Kenne

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.

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.

Contact system revisited: an interface between inflammation, coagulation, and innate immunity.

The contact system is a plasma protease cascade initiated by factor XII (FXII) that activates the proinflammatory kallikrein‐kinin system and the procoagulant intrinsic coagulation pathway. Anionic surfaces induce FXII zymogen activation to form proteolytically active FXIIa. Bacterial surfaces also have the ability to activate contact system proteins, indicating an important role for host defense using the cooperation of the inflammatory and coagulation pathways. Recent research has shown that inorganic polyphosphate found in platelets activates FXII in vivo and can induce coagulation in pathological thrombus formation. Experimental studies have shown that interference with FXII provides thromboprotection without a therapy‐associated increase in bleeding, renewing interest in the FXIIa‐driven intrinsic pathway of coagulation as a therapeutic target. This review summarizes how the contact system acts as the cross‐road of inflammation, coagulation, and innate immunity.

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.

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

BackgroundBrain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear.MethodsIn this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evans blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data.ResultsNeutrophil depletion did not significantly affect Evans blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury.ConclusionOur results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice.

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.

Leukotriene B4-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin)

In humans, heparin‐binding protein (HBP) and the potent chemotactic lipid leukotriene B4 (LTB4) are important mediators of innate immune reponses. Here we show that human neutrophils (PMNs) challenged with LTB4 (30 s to 5 min) release HBP as determined by Western blot analysis. This response peaks at 100 nM of agonist and is mediated by the BLT1 receptor. Proteinphosphatase‐1 (30 μM) and wortmannin (0.5 μM) block the LTB4‐mediated HBP release from PMNs, which suggests involvement of the 1‐phosphatidylinositol 3‐kinase intracellular pathway during degranulation. Furthermore, postsecretory supernatants from LTB4‐stimulated PMNs induce intracellular calcium mobilization in endothelial cells in vitro and increase in vascular permeability in vivo, as assessed in a mouse model of pleurisy. Selective removal of HBP from the supernatant significantly reduces these activities attributing a key role to HBP in the LTB4‐induced change in vascular permeability. This lipid‐protein axis could offer novel opportunities for pharmacological intervention in key steps of the vascular response to inflammation.—Di Gennaro, A., Kenne, E., Wan, M., Soehnlein, O., Lindbom, L., Haeggstrom, J.Z. Leukotriene B4‐induced changes in vascular permeability are mediated by neutrophil release of heparinbinding protein (HBP/CAP37/azurocidin). FASEB J. 23, 1750–1757 (2009)

Factor XII: a drug target for safe interference with thrombosis and inflammation

Data from experimental animal models revealed an essential role for factor XII (FXII) in thrombotic occlusive diseases. In contrast to other blood coagulation factors, deficiency in the protease is not associated with abnormal bleeding from injury sites (hemostasis) in patients or in animals. Cumulatively, these findings suggest that FXII could be targeted as a new method of anticoagulation that is devoid of bleeding risks. An FXIIa-neutralizing antibody, 3F7, has been developed that inhibited thrombosis in an extracorporeal membrane oxygenation (ECMO) system as efficiently as heparin. However, in sharp contrast to heparin, 3F7 treatment was not associated with an increase in therapy-associated hemorrhage. In this review, we summarize current knowledge of FXII physiology and pharmacology.

ApoE−/−/Lysozyme MEGFP/EGFP mice as a versatile model to study monocyte and neutrophil trafficking in atherosclerosis

OBJECTIVES Intravital microscopy is a useful tool for studying leukocyte trafficking in atherosclerosis. However, distinction between various subclasses of leukocytes using this technology is lacking. Therefore, we generated ApoE(-/-)/Lysozyme M(EGFP/EGFP) mice and investigated whether targeted cell types could be visualized by in vivo microscopy and whether absence of lysozyme M will influence atherosclerosis. METHODS We crossed male ApoE(-/-) mice with mice homozygous for a knock-in mutation of enhanced green fluorescent protein (EGFP) in the lysozyme M locus (Lys(EGFP/EGFP)) creating ApoE(-/-)/Lys(EGFP/EGFP) mice. Mice were sacrificed at the age of 26 weeks. Blood was collected for serum lipid analysis, differential white blood cell count and flow cytometry. Lesion area was determined on en face mounted aortas and sections from aortic roots were stained for immunohistochemistry. Atherosclerotic lesions were also studied by confocal- and intravital microscopy. RESULTS Basic parameters, such as white blood cell count, cholesterol profile, lesion area and plaque composition was unaltered in ApoE(-/-)/Lys(EGFP/EGFP) mice compared to ApoE(-/-) mice. Fluorescent neutrophils and monocytes were clearly visualized by intravital fluorescence and confocal microscopy. Fluorescent cells were distributed primarily in the periphery of atherosclerotic lesions indicating a preference for recruitment in these areas. CONCLUSIONS ApoE(-/-)/Lys(EGFP/EGFP) mice will serve as a useful model to study leukocyte trafficking in atherosclerosis and how different subsets of leukocytes influence atherogenesis.

Immune cell recruitment to inflammatory loci is impaired in mice deficient in basement membrane protein laminin α4

For leukocytes to penetrate the vessel wall, they need to interact sequentially with the endothelial lining and the perivascular BM. The matrix protein laminin‐411 is a major constituent of the vascular BM. The laminin α4 chain is a component of laminin‐411 and has structural and signaling functions. Here, we addressed the role of BM laminin α4 in leukocyte recruitment to inflammatory loci. We used several recruitment models in Lam4−/− and WT mice to determine whether lack of laminin‐411 in the perivascular BM influences extravasation of inflammatory cells. Recruitment of all major leukocyte subsets (neutrophils, monocytes, and lymphocytes) was reduced in Lam4−/− mice compared with WT. With the use of intravital microscopy, we concluded that this decrease was a result of impaired diapedesis through the vessel wall, as neither leukocyte adhesion to the endothelial lining nor migration in extravascular tissue was hampered in Lam4−/− mice. Collectively, our data suggest a reduced ability of immune cells to penetrate the vessel wall in mice deficient in laminin α4.