Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors.

Chronic granulomatous diseases of childhood (CGD) are a group of disorders of phagocytic cell superoxide (O2.-) production (respiratory burst). Anion exchange chromatography separated from normal neutrophil cytosol a 47-kilodalton neutrophil cytosol factor, NCF-1, that restored activity to defective neutrophil cytosol from most patients with autosomally inherited CGD in a cell-free O2.--generating system. A 65-kilodalton factor, NCF-2, restored activity to defective neutrophil cytosol from one patient with autosomal CGD. NCF-1, NCF-2, and a third cytosol fraction, NCF-3, were inactive alone or in pairs, but together replaced unfractionated cytosol in cell-free O2.- generation. Neutrophils deficient in NCF-1, but not NCF-2, did not phosphorylate the 47-kilodalton protein. It is proposed that NCF-1, NCF-2, and NCF-3 are essential for generation of O2.- by phagocytic cells and that genetic abnormalities of these cytosol components can result in the CGD phenotype.

Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease.

A 47-kilodalton neutrophil cytosol factor (NCF-47k), required for activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase superoxide (O2-.) production, is absent in most patients with autosomal recessive chronic granulomatous disease (AR-CGD). NCF-47k cDNAs were cloned from an expression library. The largest clone predicted a 41.9-kD protein that contained an arginine and serine-rich COOH-terminal domain with potential protein kinase C phosphorylation sites. A 33-amino acid segment of NCF-47k shared 49% identity with ras p21 guanosine triphosphatase activating protein. Recombinant NCF-47k restored O2-. -producing activity to AR-CGD neutrophil cytosol in a cell-free assay. Production of active recombinant NCF-47k will enable functional regions of this molecule to be mapped.

Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src.

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.

Signaling through CD40 rescues IgE but not IgG or IgA secretion in X-linked immunodeficiency with hyper-IgM.

The ligand for CD40 (CD40L) is a membrane protein on activated T cells that induces B cell proliferation and differentiation. Several mutations of the CD40L gene were reported responsible for defective class switching of B cells in an X-linked immunodeficiency with hyper IgM (X-HIM). We studied four affected males from three families and found three independent mutations including new mutations of CD40L gene. In every X-HIM patient tested, however, anti-CD40 plus IL-10 did not induce class switching from IgM to IgG or IgA, even in the presence of Staphylococcus aureus Cowan I strain (SAC). CD4+ T cell clones, expressing CD40L on their surface, also did not rescue IgG or IgA induction by X-HIM peripheral blood B cells in vitro. But signaling through CD40 induced both B cell proliferation and IgE secretion when IL-4 was added to the culture. Taken together, these results show that in vitro signaling through CD40 rescues IgE but not IgG or IgA secretion by peripheral blood X-HIM B cells and suggest that in vivo CD40 and CD40L interaction might be necessary for IgG and IgA differentiation in X-HIM.

Translocation of guinea pig p40-phox during activation of NADPH oxidase.

The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-gamma-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox, and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-gamma-s-independent mechanism.

Characterization of the GTP-dependent activation of the superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We characterized the cell-free activating system of the superoxide (O2-)-producing NADPH oxidase of pig neutrophils. Activation of the oxidase required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resulted in separation of the cytosolic fraction into two fractions, the flow-through and bound fractions, which synergistically supported the O2- production with the membrane fraction in the absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas only the bound fraction besides the membrane fraction was required for the activation in the presence of GTP gamma S. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchange chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding native factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein responsible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-ADP-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of the ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding protein for the activation and the lost ability was recovered by the addition of GTP gamma S. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.

Modulation of degranulation and superoxide generation in human neutrophils by unsaturated fatty acids of odd carbon numbers.

Unsaturated fatty acids of odd carbons, 13:1(12), 17:1(10trans), 19:1(7) and 19:1(10) inhibited release of myeloperoxidase (MPO) from fMet-Leu-Phe-cytochalasin B-treated neutrophils. The inhibitory effect was smaller than that of aseanostatins which have been isolated as microbial-derived free fatty acids with a methyl blanch (i-14:0 and ai-15:0) (Journal of Antibiotics (1991) 44, 524-532). These unsaturated fatty acids also inhibited lactoferrin release by the same treatment. On the other hand, 13:1(12), 15:1(10) and 19:1(10) inhibited fMet-Leu-Phe-stimulated superoxide generation of neutrophils, and the fatty acids 15:1(10), 17:(10) and 19:2(10,13) induced superoxide generation in both unstimulated cells and the cell-free system. However, none of unsaturated fatty acids of odd carbons tested inhibited beta-glucuronidase release, whereas 15:1(10), 17:1(10), 19:1(10) and 19:2(10,13) rather enhanced an increase in beta-glucuronidase activity liberated from cells at high concentrations over 35 microM, indicating cellular damages by these fatty acids. These observations suggest that unsaturated free fatty acids having odd carbons such as 13, 15, 17 and 19 may act as modulators of neutrophil functions including degranulation and superoxide generation.

Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage.

HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.

Structural organization of the gene for CD40 ligand: molecular analysis for diagnosis of X-linked hyper-IgM syndrome.

CD40 ligand (CD40L) on activated T cells plays a crucial role for Ig heavy-chain class switching and the mutation of the gene for this ligand in the X-chromosome causes immunodeficiency with hyper-IgM (X-HIM). We isolated and characterized the human genomic clone for CD40 L to obtain information about the transcriptional regulatory regions of the gene and to develop a molecular diagnostic method for X-HIM patients. The genomic DNA isolated was over 12 kb long containing 5 exons that cover full sequence of mRNA for the ligand. DNA motif analysis based on transcription factor database revealed the presence of a GATA 1 box at around -265 bp. The typical TATA box, CAT site or GC rich region was not found in the 5' flanking region. However, two possible TATA like sequences were found at around -27 and -136 bp. Using the oligonucleotide primers corresponding to the introns, we performed a PCR-SSCP analysis of each exon from a patient with X-HIM syndrome and detected abnormality in exon 5. When sequenced, dinucleotide deletion in this exon was found in the patient and in one X allele of his mother as the only different sequence from that of the control gene. This procedure is simple and could be used for diagnosis of the X-HIM syndrome.

Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase.

NADPH oxidase, a superoxide-producing enzyme in phagocytic cells, consists of membrane-associated cytochrome b558 and cytosolic components (p47-phox, p67-phox, p40-phox, rac 1/2). Activation of NADPH oxidase is accompanied by the phosphorylation of cytosolic components (p47-phox and p67-phox). In this study, we have examined whether p40-phox, a newly identified cytosolic component, is phosphorylated during neutrophil activation, and the relationship between p40-phox phosphorylation and NADPH oxidase activation. When 32P-labeled guinea pig neutrophils were stimulated by phorbol 12-myristate 13-acetate, p40-phox was phosphorylated as p47-phox. It is interesting that phosphorylation of p40-phox was markedly inhibited by protein kinase C inhibitor, H-7, but not by casein kinase II inhibitor, A-3, and H-7 inhibited translocation of p40-phox and activation of NADPH oxidase. Furthermore, purified protein kinase C but not casein kinase II directly phosphorylated p40-phox of p40-phox/p47-phox/p67-phox complex. Together these observations suggest that p40-phox is phosphorylated by protein kinase C during neutrophil activation, and phosphorylation of p40-phox may be important for the activation of NADPH oxidase.

Association of the interleukin-4 receptor alpha chain with p47phox, an activator of the phagocyte NADPH oxidase in B cells.

Interleukin (IL)-4 plays an important role in IgE synthesis in B cells and in Th2 differentiation in T cells. IL-4 conducts its biological activities through binding to the IL-4 receptor (IL-4R) on the surface of target cells. IL-4R are thought to be composed of the IL-4R alpha chain (IL-4R alpha) and either the IL-2R gamma chain or the IL-13R alpha chain. We have previously shown that the membrane-proximal portion in the cytoplasmic domain of the human IL-4R alpha (hIL-4R alpha) is critical for proliferation, generation of germline epsilon transcript, and activation of STAT6, based on analyses of truncated hIL-4R alphas. In this study, we found that p47phox, an activator of the phagocyte NADPH oxidase, binds to this portion by the two-hybrid system. Furthermore, we observed the association of p47phox with the hIL-4R alpha in B cells derived from a normal donor. These results suggest that p47phox is involved in the signal transduction of IL-4 in B cells. However, activation of STAT6, CD23 expression, and IgE synthesis induced by IL-4 were not affected in p47phox-deficient patients, which raises the possibility that p47phox may be important in other signaling activities as well in B cells.

Suppression of superoxide anion production by interleukin-10 is accompanied by a downregulation of the genes for subunit proteins of NADPH oxidase.

Interleukin-10 (IL-10) inhibited the production of superoxide anion (02-) by both unactivated and interferon-gamma (IFN-gamma)-activated human monocytes. Simultaneous addition of IL-10 with IFN-gamma at the start of incubation was necessary for an optimal inhibitory effect. The degree of inhibition was substantially comparable to that of IL-4, and the combination of suboptimal concentrations of IL-10 and IL-4 produced an additive effect. A similar effect was also obtained when viral IL-10 (vIL-10) was used instead of IL-10. The inhibitory effect of IL-10 was accompanied by the reduced accumulation of transcripts for heavy chain subunit of cytochrome b558 (gp9l-phox) and 47-kD cytosolic factor (p47-phox), components of the O2--generating NADPH oxidase system. Reduction of the mRNAs was distinct within 24 hours. On the other hand, the induced O2- production by human monocytic leukemia cell lines (THP-1 and HL60) was not inhibited by IL-10. The amount of gp9l-phox and p47-phox mRNAs remained unchanged even in the presence of excess amount of IL-1O. Taken together, these results suggest that IL-10 inhibits 02- production by downregulation of the gp9l-phox and p47-phox genes in human monocytes.

Severe hypoplasia of lymphoid tissues in Mo1 deficiency.

A lymph node was examined from a 4-year-old female child with documented "Mo1" (CR3) deficiency. There was hypoplasia of the lymph node, with small, poorly delineated germinal centers and overall lymphopenia. Retrospective analysis of the clinical course of a decreased elder sister of the index patient indicated that the elder sister probably also had "Mo1" deficiency. Review of findings at autopsy revealed severe hypoplasia of all lymphoid organs with lymphopenia. Our observations suggest that lymphoid tissue hypoplasia may be a feature of CR3 deficiency and is likely a result of the defective lymphocyte adherence functions to endothelium associated with absent lymphocyte function-associated antigen (LFA-1).

Induction of CD40 in promyelocytic HL60 cells cultured with retinoic acid and/or various cytokines.

The antigenic protein CD40 on the surface of B lymphocytes plays an important role in their proliferation, immunoglobulin class switching, and rescue from apoptosis in the germinal center through interaction with T lymphocytes expressing CD40 ligand. The protein is also found on the cell surface of other antigen-presenting cells such as monocytes, dendritic cells, and thymic epithelium cells, but its presence in other myeloid cells has not been reported. We show here that CD40 protein is induced in promyelocytic HL60 cells, when cultured with retinoic acid, a vitamin that converts them to granulocyte-like cells. The cultured cells also expressed CD15, a marker for granulocytes, and cytochrome b(558), an essential component of the superoxide-generating system in phagocytes, on their surface. No detectable amount of mRNA for CD40 was found in naive HL60 cells, whereas a large amount of the message was induced in the cells cultured with the vitamin. Although CD40 expression was enhanced when the cells were further cultured with GM-CSF or IFN-gamma, expression of CD14, a marker for monocytes, was also enhanced. HL60 cells, therefore, express CD40 protein during differentiation not only toward monocytes but also toward granulocytes, at least transiently.

Characterization of the superoxide-generating system in human peripheral lymphocytes and lymphoid cell lines.

In B lymphocytes, but not T lymphocytes, isolated from human peripheral blood, we detected the four protein components essential for "the respiratory burst" by immunoblot analyses using peptide-directed antibodies. These are two membrane proteins, namely, 91- and 22-kDa subunits of cytochrome b558, and two cytosolic proteins with molecular masses of 47 and 65 kDa. Like in neutrophils, cytochrome b558 was expressed on the cell surface of peripheral B lymphocytes. Mean amounts (n = 8) of the 91-, 22-, 47-, and 65-kDa proteins, respectively, in peripheral B lymphocytes calculated from intensity of the blots were 0.011 +/- 0.003, 0.026 +/- 0.006, 0.179 +/- 0.022, and 0.039 +/- 0.013 relative to those in neutrophils on the basis of cell number. Epstein-Barr virus (EBV)-transformed cell lines derived from normal B lymphocytes and some B cell lines also possessed cytochrome b558 and two cytosolic proteins. Isolated human peripheral B lymphocytes generated the superoxide anion upon cross-linking of surface antigens such as IgM, IgD, IgG, HLA-DR, and CD19. EBV-transformants derived from normal peripheral B lymphocytes and B lymphoid cell lines also generated the superoxide anion when stimulated with various antibodies against surface antigens. These results indicate that peripheral B lymphocytes have substantial amounts of a superoxide-generating system identical to that in phagocytes and that the system is stimulated to generate the superoxide anion by the cross-linking of clonally expressed surface immunoglobulins or of certain surface antigens.

Neutrophil cytoskeletal disease.

Neutrophils and other phagocytes migrate to the site of infection, ingest pathogens, and destroy them after releasing granule contents and active oxygen. These activities of the cells are closely associated with a rapid reorganization of the cytoskeleton, in which actin polymerizes, cross-links, anchors to the membrane and depolymerizes under the control of various actin-associated proteins. Defect in actin or its associated proteins results in neutrophil cytoskeletal disease where abnormality primarily appears as motility or chemotactic defect of the cells. Although their molecular mechanisms have not been elucidated, neutrophil actin dysfunction and neutrophil actin dysfunction with abnormal 47- and 89-kd proteins have been reported. Recently, abnormal-beta-actin disease and disease with Rac 2 mutation, both of which accompany neutrophil chemotactic dysfunction, were analyzed at the molecular level. These diseases are systemic, but neutrophil dysfunction of the patients is remarkable. Here we review the literature on diseases due to cytoskeletal abnormality. Many other diseases with actin or actin-associated protein dysfunction may be reported in the near future.

Unusual case of diffuse connective tissue disease with nodule formations in muscle, lung, kidney and brain.

A 9-year-old boy had nodular masses in the left gastrocnemius muscle and lung, right kidney and right frontal area of the brain. Laboratory examinations, including positive anti-nuclear antibody, anti-RNP antibody, RA and CRP, hyper-gamma-globulinemia and accelerated ESR, showed characteristic of diffuse connective tissue disease (DCTD). Biopsy specimens were obtained from the thymus and from masses in the left lung and gastrocnemius muscle. The thymus showed hyperplasia, and a mass in the lung showed nonspecific inflammatory reactions such as connective tissue proliferation and cellular infiltration. Biopsied muscle also showed severe connective tissue proliferation, cellular infiltration, variation in fiber size and thickened blood vessel walls. In addition, abnormalities, including thickening of the basement membrane and an almost occluded narrow lumen of capillaries, were found on electron microscopy examination. Steroid administration led to remarkable improvement of the symptoms. These results suggested that DCTD is responsible for these symptoms.

[Gene therapy for inherited diseases using heamatopoietic stem cells--gene therapy for patients with chronic granulomatous disease].

The possibility of gene therapy for inherited diseases with a single gene mutation in Figure 1 had been verified by the successful treatment with bone marrow transplantation. As the gene therapy method and theory has been progressing rapidly, it is expected that gene therapy will overcome the complications of bone marrow transplantation. Of these inherited diseases, chronic granulomatous disease (CGD) is the one of the most expected disease for gene therapy. CGD is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp91 phox and p22 phox, and four cytosolic components, p47 phox, p67 phox, rac p21 and p40 phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10%/o vs. 23%) and that of gp91 phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91 phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp 91 phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91 phox to CD34+ hematopoietic stem cell from the patients with gp91 phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.

[Molecular bases of chronic granulomatous disease--analysis of the involvement of cytosol factors for NADPH oxidase].

During the past 5 years, the discovery of cell-free superoxide generation system (Bromberg Y, Pick E: Cell Immunol 88:213-221, 1984) has been revolutionized our understanding of phagocyte superoxide generation. Using cell-free system, it was clarified that NADPH oxidase for superoxide generation was comprised of components present in both the plasma membrane as well as in the cytosol. This oxidase could be kept inactive by keeping its components separated from each other within the cell and then quickly bringing them together in the plasma membrane upon activation. We analyzed cytosol components with column method, and clarified that 3 neutrophil cytosol factors (NCF-1/-2/-3) was necessary for reconstitute the cytosol activity which was missing in an autosomal recessive type of Chronic Granulomatous Disease (CGD) patients (Nunoi H, et al:Science 242:1298-1301, 1988). NCF-1/-2 were analyzed with B1 antibody and found their molecular weight as 47 and 67 kilodalton respectively. One of autosomal CGD patients was missing NCF-67k and the others were missing NCF-47k. NCF-47k and -67k were cloned with this antibody and sequenced and expressed as recombinant NCF-47k/-67k using baculovirus/insect cell system. Using these recombinants, we are trying to purify NCF-3 which is reported as small G protein in these days. Using monoclonal antibodies against these recombinants, we analyzed tissues with immunohistochemical methods and are trying to classify the type of CGD patients in Japan. In summary, at least five oxidase components now have been identified (alpha and beta chain of cytochrome b558 (gp91-phox, p22-phox) and NCF-47k/-67k/-3 (p47-phox/p67-phox/delta-1 or SOCI)).