Martin K. Wild

The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter

Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.

Complete Identification of E-Selectin Ligands on Neutrophils Reveals Distinct Functions of PSGL-1, ESL-1, and CD44

The selectins and their ligands are required for leukocyte extravasation during inflammation. Several glycoproteins have been suggested to bind to E-selectin in vitro, but the complete identification of its physiological ligands has remained elusive. Here, we showed that E-selectin ligand-1 (ESL-1), P-selectin glycoprotein ligand-1 (PSGL-1), and CD44 encompassed all endothelial-selectin ligand activity on neutrophils by using gene- and RNA-targeted loss of function. PSGL-1 played a major role in the initial leukocyte capture, whereas ESL-1 was critical for converting initial tethers into steady slow rolling. CD44 controlled rolling velocity and mediated E-selectin-dependent redistribution of PSGL-1 and L-selectin to a major pole on slowly rolling leukocytes through p38 signaling. These results suggest distinct and dynamic contributions of these three glycoproteins in selectin-mediated neutrophil adhesion and signaling.

Ultraviolet Radiation-Induced Regulatory T Cells Not Only Inhibit the Induction but Can Suppress the Effector Phase of Contact Hypersensitivity

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.

CD2 and the nature of protein interactions mediating cell-cell recognition

Summary: Rapid progress has recently been made in characterising the structures of leukocyte cell‐surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co‐receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell‐cell recognition. As an example of biological recognition, the defining characteristic of cell‐cell contact is that it involves che simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell‐cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand‐binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface‐shape complementarity, is the dominant feature of specific, low‐affinity protein recognition at die cell surface by CD2. The crystallographic analysis of the CD2‐binding domain of CD58 strongly supports this concept.

von Willebrand factor promotes leukocyte extravasation

von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollate-induced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier.

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that α4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.

Increased expression of syndecan-1 protects against cardiac dilatation and dysfunction after myocardial infarction

Background— The cell-associated proteoglycan syndecan-1 (Synd1) closely regulates inflammation and cell-matrix interactions during wound healing and tumorigenesis. The present study investigated whether Synd1 may also regulate cardiac inflammation, matrix remodeling, and function after myocardial infarction (MI). Methods and Results— First, we showed increased protein and mRNA expression of Synd1 from 24 hours on, reaching its maximum at 7 days after MI and declining thereafter. Targeted deletion of Synd1 resulted in increased inflammation and accelerated, yet functionally adverse, infarct healing after MI. In concordance, adenoviral gene expression of Synd1 protected against exaggerated inflammation after MI, mainly by reducing transendothelial adhesion and migration of leukocytes, as shown in vitro. Increased inflammation in the absence of Synd1 resulted in increased monocyte chemoattractant protein-1 expression, increased activity of matrix metalloproteinase-2 and -9, and decreased activity of tissue transglutaminase, associated with increased collagen fragmentation and disorganization. Exaggerated inflammation and adverse matrix remodeling in the absence of Synd1 increased cardiac dilatation and impaired systolic function, whereas gene overexpression of Synd1 reduced inflammation and protected against cardiac dilatation and failure. Conclusions— Increased expression of Synd1 in the infarct protects against exaggerated inflammation and adverse infarct healing, thereby reducing cardiac dilatation and dysfunction after MI in mice.

A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis.

The C-type lectin dendritic cell−specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.

P-Selectin Glycoprotein Ligand 1 Is Not Required for the Development of Experimental Autoimmune Encephalomyelitis in SJL and C57BL/6 Mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.

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.