Arthur J. Verhoeven

Leukocyte adhesion deficiency type 1 (LAD-1)/variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins.

Leukocyte adhesion deficiency (LAD) is characterized by the inability of leukocytes, in particular neutrophilic granulocytes, to emigrate from the bloodstream towards sites of inflammation. Infectious foci are nonpurulent and may eventually become necrotic because of abnormal wound healing. LAD-1 is characterized by the absence of the beta2 integrins (CD11/CD18) on leukocytes. When expression is completely absent, patients often die within the first year. However, low levels of beta2 expression may result in a milder clinical picture of recurrent infection, which offers a better prognosis. In this paper, we describe the in vivo and in vitro findings on a patient with clinical features of a mild LAD-1 disorder, i.e., suffering from bacterial infections without apparent pus formation in the presence of a striking granulocytosis, showing no delayed-type hypersensitivity reaction upon skin testing, no specific antibody generation, but normal in vitro T cell proliferation responses after immunization. Expression levels of CD11/CD18 proteins were completely normal, but leukocyte activation did not result in CD11/ CD18 activation and high-avidity ligand-binding. In vitro chemotaxis and endothelial transmigration of the neutrophils as well as leukocyte aggregation responses were almost absent. On the other hand, beta1 and beta3 integrin-mediated adhesion functions were completely normal. During follow-up, a bleeding tendency related to decreased beta3 activation became clinically apparent, different from previously described cellular adhesion molecule variants. Therefore, this is the first well-documented case of a clinical combined immunodeficiency syndrome that results from nonfunctional CD11/CD18 molecules, and thus designated LAD-1/ variant.

FUNCTIONAL ASSOCIATION BETWEEN THE HUMAN MYELOID IMMUNOGLOBULIN A FC RECEPTOR (CD89) AND FCR GAMMA CHAIN : MOLECULAR BASIS FOR CD89/FCR GAMMA CHAIN ASSOCIATION

FcR γ chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (FcεRI), and the class I and IIIA IgG receptors (FcγRI and FcγRIIIa). Here, we demonstrate that the Fc receptor γchain associates with FcαR in transfected IIA1.6 B lymphocytes. FcαR could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR γchain, a physical interaction with FcαR could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of FcαR promotes association with FcR γchain. We therefore constructed FcαR molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between FcαR and FcR γchain was observed only with a positively charged residue (Arg209 or His209) present within the FcαR transmembrane domain. These data show that transmembrane signal transduction by the FcαR is mediated via FcR γchain, and that FcαR requires a positively charged residue within the transmembrane domain to promote functional association.

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%‐500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%‐25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF‐B9 cells resulted in colocalization of both proteins in the canalicular membrane. Conclusion: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1‐related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential β‐subunit or chaperone for ATP8B1 in hepatocytes. (HEPATOLOGY 2007.)

LAD-1/variant syndrome is caused by mutations in FERMT3

Leukocyte adhesion deficiency-1/variant (LAD1v) syndrome presents early in life and manifests by infections without pus formation in the presence of a leukocytosis combined with a Glanzmann-type bleeding disorder, resulting from a hematopoietic defect in integrin activation. In 7 consanguineous families, we previously established that this defect was not the result of defective Rap1 activation, as proposed by other investigators. In search of the genetic defect, we carried out homozygosity mapping in 3 of these patients, and a 13-Mb region on chromosome 11 was identified. All 7 LAD1v families share the same haplotype, in which 3 disease-associated sequence variants were identified: a putative splice site mutation in CALDAGGEF1 (encoding an exchange factor for Rap1), an intronic 1.8-kb deletion in NRXN2, and a premature stop codon (p.Arg509X) in FERMT3. Two other LAD1v patients were found to carry different stop codons in FERMT3 (p.Arg573X and p.Trp229X) and lacked the CALDAGGEF1 and NRXN2 mutations, providing convincing evidence that FERMT3 is the gene responsible for LAD1v. FERMT3 encodes kindlin-3 in hematopoietic cells, a protein present together with integrins in focal adhesions. Kindlin-3 protein expression was undetectable in the leukocytes and platelets of all patients tested. These results indicate that the LAD1v syndrome is caused by truncating mutations in FERMT3.

An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37° followed by a one‐step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 ± 4% at 89 ± 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml.

Diversion of first blood volume results in a reduction of bacterial contamination for whole‐blood collections

Background and Objectives In a previous study we established a reliable setpoint for the prevalence of bacteria in whole blood. In the present study we investigated the possible preventive effect, of diversion of the first 10 ml of a blood donation, on the bacterial contamination rate.

The effect of the transfusion of stored RBCs on intestinal microvascular oxygenation in the rat

BACKGROUND: Although it is known that the transfusion of stored RBCs does not always improve tissue O2 consumption under conditions of limited tissue oxygenation, the efficiency of O2 delivery to the microcirculation by stored RBCs has never been determined.

Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for β-glucan-bearing particles

We investigated the role of the beta-glucan receptor, Dectin-1, in the response of human neutrophils to unopsonized Saccharomyces cerevisiae and its major beta-glucan-containing capsular constituent, zymosan. Although reported to be indispensable for yeast phagocytosis in murine phagocytes, human Dectin-1 was not involved in the phagocytosis of S. cerevisiae or zymosan by human neutrophils. Phagocytosis of yeast particles proved to be completely dependent on CD11b/CD18, also known as complement receptor 3 (CR3). The findings were supported by data with neutrophils from a patient suffering from Leukocyte-Adhesion Deficiency type-1 (LAD-1) syndrome lacking CD11b/CD18. In addition, neither the priming by zymosan of the fMLP-induced NADPH-oxidase activity in human neutrophils nor the secretion of IL-8 by human neutrophils in response to zymosan preparations was affected by blocking anti-Dectin-1 antibodies or laminarin as a monovalent inhibitor. As shown by neutrophils from an IRAK-4-deficient patient, the zymosan-induced IL-8 release was also independent of TLR2. In summary, our data show that Dectin-1, although indispensable for recognition of beta-glucan-bearing particles in mice, is not the major receptor for yeast particles in human neutrophils.

The influence of tetracyclines on T cell activation

Minocycline has been shown to have an anti‐inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell‐mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose‐dependent inhibition of T cell proliferation and reduction in production of lL‐2. interferon‐gamma (IFN‐γ) and tumour necrosis faetor‐alpha (TNF‐α). Besides an inhibition of IL‐2 production, mitiocycline exerted its effect on T cell proliferation by induction of a decreased IL‐2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3‐induced intracellular Ca2+ mobilization. Taken together. the results clearly indicate that minocycline has immunomodulating effects on human T cells.

Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

Background Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Δψm), which is usually generated by the respiratory chain complexes. We investigated the source of Δψm in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Δψm for the generation of ATP. Methods and Principal Findings Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Δψm was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Δψm. Complex V did not maintain Δψm by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils. Conclusions We show that neutrophils can maintain Δψm via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.