David Frommhold

RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease “congenital disorder of glycosylation IIc,” also termed “leukocyte adhesion deficiency II.” Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1-/- mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism.

Hypercoagulability Inhibits Monocyte Transendothelial Migration Through Protease-Activated Receptor-1-, Phospholipase-Cβ-, Phosphoinositide 3-Kinase-, and Nitric Oxide-Dependent Signaling in Monocytes and Promotes Plaque Stability

Background— Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. Methods and Results— De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TMPro/ProApoE−/− and FVLQ/QApoE−/− mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. Conclusions— Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.

Critical role of phospholipase Cγ2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis

1. 1. Jakus, 2. et al. J. Exp. Med. 2009 doi: 10.1084/jem.20081859 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20081859%26rft_id%253Dinfo%253Apmid%252F19273622%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%

α2,3‐Sialyltransferase‐IV is essential for L‐selectin ligand function in inflammation

L‐selectin belongs to the C‐type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L‐selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L‐selectin‐dependent rolling. To investigate the role of the α2,3‐sialyltransferase (ST3Gal)‐IV on L‐selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyers patch HEV of ST3Gal‐IV‐deficient mice and littermate control mice. In cremaster muscle venules with or without TNF‐α treatment, L‐selectin‐dependent rolling was almost completely abolished in ST3Gal‐IV–/– mice. In both models, L‐selectin interacts with P‐selectin glycoprotein ligand‐1 (PSGL‐1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L‐selectin ligands. In contrast, L‐selectin‐dependent rolling in Peyers patch HEV, which is mediated by unknown endothelial L‐selectin ligands, was not impaired in the absence of ST3Gal‐IV. Our in vivo data show that PSGL‐1, the molecule responsible for L‐selectin‐mediated leukocyte interactions in inflammation, is dependent on ST3Gal‐IV, while α2,3‐sialylation by ST3Gal‐IV is not necessary for L‐selectin ligand activity on high endothelial cells of Peyers patch HEV.

Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

BackgroundDuring inflammation, β2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.ResultsWe investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised β2-integrin-mediated outside-in signaling.ConclusionWe provide substantial evidence for an important role of Syk in mediating β2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.

A fundamental role of mAbp1 in neutrophils: impact on beta(2) integrin-mediated phagocytosis and adhesion in vivo.

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.

Leukocyte trafficking in a mouse model for leukocyte adhesion deficiency II/congenital disorder of glycosylation IIc.

Leukocyte adhesion deficiency II (LAD II), also known as congenital disorder of glycosylation IIc (CDG-IIc), is a human disease in which a defective GDP-fucose transporter (SLC35C1) causes developmental defects and an immunodeficiency that is based on the lack of fucosylated selectin ligands. Since the study of in vivo leukocyte trafficking in patients with LAD II is experimentally limited, we analyzed this process in mice deficient for Slc35c1. We found that E-, L-, and P-selectin-dependent leukocyte rolling in cremaster muscle venules was virtually absent. This was accompanied by a strong but not complete decrease in firm leukocyte adhesion. Moreover, neutrophil migration to the inflamed peritoneum was strongly reduced by 89%. Previous reports showed surprisingly normal lymphocyte functions in LAD II, which indicated sufficient lymphocyte trafficking to secondary lymphoid organs. We now found that while lymphocyte homing to lymph nodes was reduced to 1% to 2% in Slc35c1(-/-) mice, trafficking to the spleen was completely normal. In accordance with this, we found a defect in the humoral response to a T cell-dependent antigen in lymph nodes but not in the spleen. Taken together, Slc35c1(-/-) mice show strongly defective leukocyte trafficking but normal lymphocyte homing to the spleen, which may explain normal lymphocyte functions in LAD II.

Neutrophil and endothelial adhesive function during human fetal ontogeny

Attenuation of the immune response contributes to the high rate of neonatal infections, particularly in premature infants. Whereas our knowledge of innate immune functions in mature neonates is growing, little is known about the ontogeny of neutrophil recruitment. We investigated neutrophils and ECs in the course of gestation with respect to rolling and adhesive functions. With the use of microflow chambers, we demonstrate that the neutrophilˈs ability to roll and adhere directly correlates with gestational age. These adhesion‐related abilities are very rare in extremely premature infants (<30 weeks of gestation), which may correlate with our observation of markedly reduced expression of PSGL‐1 and Mac‐1 on neutrophils in preterm infants. In parallel, the capacity of HUVECs to mediate neutrophil adhesion under flow increases with gestational age. In addition, HUVECs from extremely premature infants exerting the lowest ability to recruit adult neutrophils show a diminished up‐regulation of E‐selectin and ICAM‐1. Finally, by following neutrophil function postnatally, we show that maturation of PMN recruitment proceeds equivalently during extra‐ and intrauterine development. Thus, PMN recruitment and EC adhesion‐related functions are ontogenetically regulated in the fetus, which might contribute significantly to the high risk of life‐threatening infections in premature infants.