Dirk Roos

Chronic Granulomatous Disease: The European Experience

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a “respiratory burst”, essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (∼1∶250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91phox deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with γ-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients.

Monitoring human basophil activation via CD63 monoclonal antibody 435

On activation of human basophilic granulocytes with anti-IgE or with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, the expression of the CD63 antigen on the cell surface, detected by monoclonal antibody (MAb) 435, increased up to 100-fold. The kinetics of CD63 up regulation and histamine release were identical, and a strong correlation was found between percentage of MAb 435-binding basophils and extent of histamine release. Immunoelectronmicroscopy demonstrated that the epitope for MAb 435 in resting basophils is located on the basophilic granule membrane. After basophil activation, MAb 435 bound to the exterior of the plasma membrane. Experiments with various doses of anti-IgE demonstrated that the binding of MAb 435 to basophilic granulocytes follows an all-or-nothing-like response per cell. Basophils either do not bind the MAb at all, or they bind a maximal amount of the MAb. We also measured the up regulation of the CD11/CD18 leukocyte adhesion complex. Here, too, we noted an increase in cell-surface exposure of all subunits after activation. This increase was not as strong as increase found with MAb 435. Thus, MAb 435 is an interesting new tool for investigating the activation of human basophils, in addition to the measurement of mediator release. This MAb may be useful for the detection of basophil activation in vivo.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration–dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of α1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.

Characterization and quantification of the peroxidase in human monocytes

1. 1. The properties of the peroxidase in human moncytes have been compared with those of myeloperoxidase in human granulocytes and with those of isolated human mycloperoxidase. 2. 2. The optical difference spectra of the reduced minus the oxidized states of monocyte and granulocyte homogenates show peaks at 472 and 635 nm that are identical with those observed in the spectrum of reduced isolate myeloperoxidase. 3. 3. Electron paramagnetic resonance spectra of monocytes, granulocytes and isolated myeloperoxidase show a rhombic high-spin haem iron signal with gx = 6.90 and gy = 5.07. 4. 4. Monocyte and granulocyte lystates show a peroxidative reaction with ortho-dianisidine and require the same optimal substrate concentrations. 5. 5. Monocyte and granulocyte homogenates, as well as isolated myeloperoxidase, catalyse the oxidation of iodide by H2O2. This reaction has an acid pH optimum of 4.5–5.5. 6. 6. Ingested zymosan particles are iodinated by monocytes as well as by granulocytes. 7. 7. On the basis of these observations we conclude that human monocytes contain myeloperoxidase. 8. 8. The concentration of myelperoxidase in monocytes is about 1.1 · 10−17 mol per cell. Granulocytes contain three times this amount of myeloperoxidase per cell.

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

[8] Purification and cryopreservation of phagocytes from human blood

Publisher Summary Three types of phagocytic cell are found in human blood: monocytes, neutrophilic granulocytes, and eosinophilic granulocytes. For their separation, both biological and physical properties of the cells can be used. The positive selection of phagocytes can be achieved by utilizing the adhesive or phagocytic properties of the cells. However, most methods utilize differences in specific gravity or size of the cells. Thus, either isopycnic centrifugation or velocity sedimentation can be used. Differences in electrophoretic mobility of blood cells have also been employed for their separation. Mostly, blood cells are first separated by isopycnic centrifugation into a light fraction containing mononuclear leukocytes (monocytes + lymphocytes), basophils and a few early precursor cells, and a heavy fraction containing erythrocytes and granulocytes. Separation of monocytes from lymphocytes and basophils and of neutrophils and eosinophils from each other can also be performed by isopycnic centrifugation, owing to the differences in specific gravity among the various types of blood cell. As neutrophils and eosinophils (together called “granulocytes”) have a higher specific gravity than other leukocytes, the common method of neutrophil purification is separation over a medium of 1.077 g/cm 3 and removal of the centrifugation has also been reported.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet‐rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22°C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation‐dependent antigens (GMP 140 from the alpha granules and a 53‐kDa glycoprotein from the lysosomal granules) was measured. β‐thromboglobulin (β‐TG) release was also determined. Disc‐to‐sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p < 0.01), had a higher β‐TG release (p < 0.01), bound more MoAbs against GP IIb/IIIa (p < 0.01), and expressed more GMP 140 and 53‐kDa glycoprotein (p < 0.01) than did BC‐ derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP‐derived platelets are more activated than BC‐derived platelets. This is most likely a result of the pelleting that follows the second high‐speed centrifugation of the PRP.

Hematologically important mutations: The autosomal recessive forms of chronic granulomatous disease (second update)

Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22-phox, NCF2, encoding p67-phox, NCF1, encoding p47-phox, and NCF4, encoding p40-phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.