D. Roos

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.

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

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.

Neutrophil activation in sickle cell disease.

Vascular occlusion is the main cause of the morbidity and mortality observed in patients with sickle cell disease (SCD). Increasing evidence indicates that (activated) neutrophils could play an important role in the initiation and propagation of vaso‐occlusive processes in SCD. In this study, the activation state of neutrophils in sickle cell patients was analyzed by determining the level of expression of neutrophil antigens such as CD62L, CD11b, CD66b, CD63, and Fcγ receptors. We also analyzed plasma levels of lactoferrin, elastase, soluble (s)CD16 (sFcγRIII), and serum levels of soluble (s)CD62L (sL‐selectin) as neutrophil activation markers in these patients. Significant differences were observed in the activation state of neutrophils in non‐symptomatic sickle cell patients compared to healthy HbAA controls as exemplified by significant decrease in L‐selectin expression, enhanced expression of CD64, and increased levels of soluble markers like sL‐selectin, elastase, and sCD16. During vaso‐occlusive crisis the differences were even more pronounced. These results show neutrophils to be activated in sickle cell patients, suggesting a role of importance in the pathophysiology of sickle cell disease. J. Leukoc. Biol. 66: 411–415; 1999.

A Point Mutation in gp91-phox of Cytochrome b558 of the Human NADPH Oxidase Leading to Defective Translocation of the Cytosolic Proteins p47-phox and p67-phox

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patients neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.

Phagocytosing human neutrophils inactivate their own granular enzymes.

During phagocytosis, neutrophils generate reactive oxygen metabolites and release lysosomal enzymes into the extracellular medium. We have investigated the possibility that these enzyme are inactivated by the oxygen compounds. Phagocytosing neutrophils from 12 patients with chronic granulomatous disease, which do not generate these oxygen metabolites, released two to three times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference proved to be due to a decrease of approximately 20% of the total activity of these enzymes in normal neutrophils, but not in neutrophils of patients with chronic granulomatous disease. This inactivation of enzymes took place during phagocytosis of opsonized zymosan particles as well as during stimulation of normal cells with phorbol myristate acetate. The inactivation was not due to formation of inhibitors. The lysosomal enzymes were not activated when the neutrophils were stimulated under anaerobic conditions. Addition of catalase, superoxide dismutase, or albumin gave no protection against the oxidative damage; reduced glutathione gave partial protection. The oxidative inactivation was more pronounced in the presence of azide. Measurement of the activity and the amount of protein of acid alpha-glucosidase in the cells showed that the specific activity of this enzyme decreased by approximately 50% during 30 min of phagocytosis. This indicates that the inactivation of the lysosomal enzymes takes place in the phagolysosomes, before the enzymes have leaked into the extracellular medium.

A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

Influence of structurally different lipid emulsions on human neutrophil oxygen radical production

The aim of this study was to evaluate immunomodulatory properties of lipid emulsions applied in parenteral nutrition by measuring neutrophil oxygen radical production (the ‘respiratory burst’) after lipid incubation.

Different proteolytic mechanisms involved in Fc gamma RIIIb shedding from human neutrophils.

The Fcγ receptor type IIIb (CD16) is highly expressed on human neutrophils and is found in a soluble form in plasma and in other body fluids. Upon activation of neutrophils in vitro, FcγRIIIb is shed from the cell surface by proteolytic cleavage. We have now investigated the effect of metalloproteinase inhibitors and a serine proteinase inhibitor on the shedding of FcγRIIIb induced by phorbol 12‐myristate 13‐acetate (PMA) or cytochalasin B (cyto B) + N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP). Metalloproteinase inhibitors blocked to a large extent PMA‐induced, but not cyto B + fMLP‐induced shedding of FcγRIIIb. Inhibition of members of the ADAM (a disintegrin and metalloproteinase) family appeared most efficient. In contrast, the serine protease inhibitor N‐methoxysuccinyl‐alanine‐alanine‐proline‐valine‐chloromethylketone (MeOsuc‐AAPV‐CMK) largely blocked cyto B + fMLP‐induced, but not PMA‐induced shedding of FcγRIIIb. Metalloproteinase inhibitors in combination with the serine proteinase inhibitor resulted in full inhibition of FcγRIIIb shedding induced by either PMA or cyto B + fMLP. The shedding of FcγRIIIb that accompanied apoptosis was inhibited by 60% in the presence of inhibitors of metalloproteinases but was insensitive to inhibition of serine proteinases. These results show that distinct types of proteolytic enzyme are involved in the stimulus‐induced shedding of FcγRIIIb from human neutrophils and suggest that these proteinases may become differentially activated under various physiological or pathological conditions.

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis

Anti‐neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegeners granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3‐ANCA do not always correspond to clinical disease activity.