Quantitative measurement of the bactericidal capability of neutrophils from patients and carriers of chronic granulomatous disease.

Previous studies examined the ability of polymorphonuclear leukocytes (PMN) from patients and carriers of chronic granulomatous disease (CGD) to kill small numbers of bacteria in vitro. The purpose of the present study was to accurately measure the number of microorganisms killed by CGD PMN when the initial number of bacteria per neutrophil was increased. Bactericidal capacities of PMN from seven patients and five proven carriers of CGD were examined with five test ratios ranging from 1.25 to 100 bacteria/neutrophil. CGD PMN killed a limited number of catalase-positive S. aureus but the number of bacteria killed increased progressively as the initial test ratio was advanced. At the highest ratio (100:1) patient PMN killed a mean of nine S. aureus per cell (normal 48 +/- 13.2 SD). PMN from patients with CGD killed increasing numbers of catalase-negative S. faecalis with normal efficiency, reaching a mean of 14 S. faecalis killed per cell at 100:1. Individual variation in the ability of CGD PMN to kill S. Aureus was apparent but the degree of PMN bactericidal effectiveness did not correlate with the patient's clinical course. PMN from asymptomatic carriers of proven sex-linked CGD also had a wide spectrum of staphylocidal activity which ranged from normal to levels comparable to CGD patients.     

Receptor-mediated regulation of superoxide production in human neutrophils stimulated by phorbol myristate acetate.

Human neutrophils contain receptors for phorbol myristate acetate (PMA), a complex lipid that induces them to generate superoxide (O (2)). Binding of PMA to these receptors displays specificity, reversibility, and high affinity. The receptor's apparent KD was approximately 0.29 nM and multiple copies (approximately 2.1 +/- 0.6 x 10(5)) were present per neutrophil. We found that the timing and magnitude of the neutrophil's respiratory burst were set independently. The onset of O (2) production occurred after a lag that was inversely proportional to the initial concentration of added PMA. The extent (rate) of O (2) production was directly proportional to the fractional occupancy of the receptor by PMA. Dual regulatory controls, such as those we noted when neutrophils were stimulated by PMA, could afford metabolic stability in the face of transient or low intensity stimuli without compromising quick and powerful responses to larger disturbances.     

Leukotriene B4 metabolism in neutrophils of patients with chronic granulomatous disease: phorbol myristate acetate decreases endogenous leukotriene B4via NADPH oxidase-dependent mechanism

We studied the effect of phorbol myristate acetate (PMA) on endogenous leukotriene B4 (LTB4) metabolism of calcium ionophore A23187-stimulated human neutrophils. Preincubation of normal neutrophils with PMA significantly suppressed the recovery of endogenous LTB4 induced by A23187. PMA did not suppress the recovery of LTB4 produced by neutrophils from patients with chronic granulomatous disease (CGD), which is known to be defective in NADPH oxidase activation to produce reactive oxygen species (ROS). PMA inhibited the formation of omega-oxidation products of LTB4, but enhanced arachidonic acid release in normal and CGD neutrophils. Furthermore, 5-lipoxygenase activity of 10,000 x g supernatants from normal neutrophils pretreated with PMA was equivalent to that of the controls. Decrease in LTB4 recovery was not attributed to the suppression of the intracellular Ca2+ increase. Thus, it is suggested that reactive oxygen species (ROS) produced by PMA may directly affect endogenous LTB4 and convert it into metabolite(s) distinct from omega-oxidation products.     

NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.

Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.     

Phorbol myristate acetate stimulates ATP-dependent calcium transport by the plasma membrane of neutrophils.

We studied the effect of phorbol myristate acetate (PMA) on the plasma membrane ATP-dependent calcium pump in neutrophils. Plasma membrane-enriched fractions ("podosomes") from PMA-stimulated guinea pig neutrophils exhibited a twofold stimulation of ATP-dependent calcium transport when compared with control podosomes. The stimulatory effect was rapid (beginning less than 2 min after exposure to PMA) and reached maximal values within 5 min. PMA increased the maximum velocity but not the affinity of the calcium pump for Ca++. Pump activation was not preceded by a rise in cytosolic free calcium concentration [Ca++]i, as assessed by the intracellularly trapped fluorescent calcium indicator Quin 2, but instead slightly lowered [Ca++]i and prevented the rise in [Ca++]i normally induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. These results suggest that the calcium pump in the plasma membrane of neutrophils may be stimulated by calcium-independent pathways, and that this activation could be one of the earliest events mediating some of the effects of phorbol esters.     

Cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Correlation to changes in the cytosolic free Ca2+ level and action of phorbol myristate acetate.

We have studied how cytosolic free Ca2+ ([Ca2+]i) changes and phorbol myristate acetate (PMA) exposure affects ligand-independent cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Mere incubation primed neutrophils to double their binding of fMet-Leu-Phe. This spontaneous increase of peptide binding was unaffected by changes in the extracellular calcium concentration. However, depression of the [Ca2+]i totally abolished the increased binding of fMet-Leu-Phe. Scatchard-Plot analysis revealed that the observed increase of peptide binding was due to an increased number of receptors. Normalization of the [Ca2+]i in cells where it was initially depressed resulted in a slow but progressive increase in fMet-Leu-Phe binding. The rate of receptor recruitment could be enhanced by rapidly increasing the [Ca2+]i by addition of ionomycin. Addition of PMA to cells with near maximal receptor expression led to a marked reduction of fMet-Leu-Phe binding without affecting [Ca2+]i. These observations suggest the existence of a dual regulatory mechanism for up- and down-regulation of fMet-Leu-Phe receptors on the cell surface of human neutrophils.     

Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease.

The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.     

Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (myeloperoxidase [MPO], mannose binding lectin [MBL], Fcgamma receptors IIa, IIIa, IIIb, TNF-alpha, and IL-1 receptor antagonist), each containing a physiologically relevant polymorphism predicted to influence the host inflammatory response, were selected for analysis. Genotypes of MPO (P = 0.003) and FcgammaRIIIb (P = 0.007) were strongly associated with an increased risk for GI complications, while an FcgammaRIIa (P = 0.05) genotype was suggestive for an association. Patients with all three associated genotypes had the highest risk for GI complications (P < 0.0001). The risk of AIDs was strongly associated with variant alleles of MBL (P = 0.01) and weakly associated with an FcgammaRIIa genotype (P = 0.04). Patients with variant forms of both MBL and FcgammaRIIa had the highest risk of developing an AID (P = 0.003).     

Leukotriene biosynthesis by polymorphonuclear leukocytes from two patients with chronic granulomatous disease.

Polymorphonuclear leukocytes (PMNL) isolated from two patients with chronic granulomatous disease (CGD) were tested for their ability to metabolize arachidonic acid to lipoxygenase products including 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). Analyses of incubations of these PMNL with arachidonic acid and the calcium ionophore A23187 did not differ from simultaneous controls in the production of LTB4, other 5,12-dihydroxy-eicosatetraenoic acids, or monohydroxy-eicosatetraenoic acids. The clinical diagnosis of CGD was confirmed in both cases by determination of PMNL chemiluminescence. Leukocytes from both patients failed to generate active oxygen species in response to either LTB4 or formyl-methionyl-leucyl-phenylalanine. The observation of arachidonic acid oxidation in the absence of superoxide anion precludes a role for the active oxygen species in this metabolic process. These studies clearly dissociate the ionophore-induced leukocyte respiratory burst from the oxidation of arachidonate to the leukotrienes. In addition, the defect of CGD appears to be unrelated to the ability of PMNL to carry out arachidonate oxygenation.     

Skewing of X-chromosome inactivation in three generations of carriers with X-linked chronic granulomatous disease within one family

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by impairment of intracellular microbicidal activity of phagocytes. Mutations in one of the four known NADPH-oxidase components preclude generation of superoxide and related antimicrobial oxidants, leading to the phenotype of CGD. Defects in gp91-phox, encoded by CYBB, lead to X-linked CGD, responsible for approximately 70% of all CGD cases. The aim of the study was to evaluate the hypothesis that age-related skewing of X-chromosome inactivation, as described in several CGD families, is caused by preferential survival of bone marrow clones with an inactive NADPH oxidase. MATERIALS AND METHODS: We studied the neutrophils from three patients and four carriers in three generations of a Turkish family with X-linked CGD. Carrier detection was carried out by the dihydrorhodamine (DHR)-1,2,3 assay, which measures on a per-cell basis the NADPH oxidase-dependent oxidation of DHR by phagocytes. The X-chromosome inactivation pattern was determined with the HUMARA assay in DNA from leucocytes as well as in DNA from a buccal smear of the four carriers. RESULTS: The three patients were identified by a negative DHR test, and the mutation in their CYBB gene was characterized by DNA sequencing. Moreover, we found an age-related degree of skewing of X-chromosome inactivation in the leucocytes of the four X-CGD carriers, both at the protein level (NADPH oxidase activity) and at the DNA level (HUMARA assay). However, similar skewing of X-chromosome inactivation was found in the buccal DNA from these women. CONCLUSIONS: These novel findings indicate that the age-related degree of skewing was probably a chance finding, not related to preferential survival of NADPH oxidase-deficient precursor cells, because this enzyme is not expressed in (buccal) epithelial cells.     

Studies in normal and chronic granulomatous disease neutrophils indicate a correlation of tubulin tyrosinolation with the cellular redox state

A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.     

T cell stimulation via the erythrocyte receptor. Synergism between monoclonal antibodies and phorbol myristate acetate without changes of free cytoplasmic Ca++ levels

We observed that certain E-receptor antibodies (CD2 antibodies) can induce proliferation of resting human T cells in the presence of PMA, while other CD2 antibodies fail to have such an effect. The same CD2 antibodies that were mitogenic in the presence of PMA (9.6, X11, VIT13), but not the nonreactive ones, were also able to induce T cell proliferation via the so-called alternative pathway of T cell activation, i.e., when added pairwise in certain combinations to T cells in the absence of PMA. While the simultaneous addition of two comitogenic CD2 antibodies (9.6 or X11 plus VIT13) or the addition of a single nonmitogenic CD3 antibody (VIT3) led to a clearcut elevation of intracellular Ca++ levels, no such effect could be observed after the addition of one CD2 antibody alone. Even in the presence of PMA, one comitogenic CD2 antibody alone was unable to trigger a significant Ca++ response, although this combination induced a proliferative response. These data indicate that, distinguishable by their influence on free cytoplasmic Ca++, there are two different mechanisms of T cell activation via CD2. While simultaneous triggering with two antibodies leads to cell proliferation preceded by an increase of Ca++ levels, stimulation with one antibody plus PMA results in proliferation without a measurable early Ca++ response. We conclude that T cells treated by certain CD2 antibodies alone already recognize an activation signal probably unrelated to Ca++ homeostasis, a signal that can further be developed by PMA to result in a completely developed proliferative response.     

Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions

Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.     

Cytochrome b deficiency in an autosomal form of chronic granulomatous disease. A third form of chronic granulomatous disease recognized by monocyte hybridization

Three patients (two sisters and a brother) in one family are described with chronic granulomatous disease. The granulocytes of these patients did not respond with a metabolic burst to various stimuli and failed to kill catalase-positive microorganisms. The magnitude of the cytochrome b signal in the optical spectrum of the patients' granulocytes was less than 4% of the normal value, whereas the amount of noncovalently bound flavin in these cells was normal. The mode of inheritance of the genetic defect in this family is autosomal because the granulocytes of both parents (first cousins) and a nonaffected sister of the patients expressed 70-80% of the normal cytochrome b signal, showed low-normal or subnormal oxidative reactions during stimulation, and did not display mosaicism in the stimulated nitroblue-tetrazolium slide test. Somatic cell hybridization was performed between the monocytes from the affected boy in this family with monocytes from either a cytochrome b-negative male patient with X-linked chronic granulomatous disease or a cytochrome b-positive male patient with the classic autosomal form of this disease. In both combinations, monocyte hybrids were observed with nitroblue tetrazolium reductase activity after stimulation with phorbol myristate acetate. This complementation of the oxidase activity required protein synthesis. Our results prove that the defect in this family is genetically distinct from that in the other two forms of chronic granulomatous disease. Moreover, our results also indicate that the expression of cytochrome b in human phagocytes is coded by at least two loci, one on the X chromosome and one on an autosome.     

Graves病对骨矿物质代谢的影响

目的通过骨密度测定以及骨代谢指标检测技术了解,甲状腺功能亢进性骨病的特征,探讨Graves病对骨矿物质代谢的影响。方法测定96例Graves病患者和35例正常对照者的血清FT3、FT4、TSH浓度、血清骨钙素(BGP)、血钙、血磷、尿羟脯胺酸(HOP)浓度和骨密度(BMD)。结果Graves病患者组血清FT3、FT4浓度高于正常对照组(P<0.01);血清BGP、尿HOP浓度与血清FT3浓度呈正相关(r=0.35,P<0.05;r=0.34,P<0.05),明显高于缓解组和正常对照组(P<0.01);血钙、血磷浓度均高于正常对照组(P<0.01);患者组L2～4、股骨颈、Ward's三角和大转子的骨密度均低于正常对照组(P<0.01);患者骨量丢失发生率为41%(39/96)。结论Graves病可导致骨矿物质代谢紊乱,并发骨量丢失和骨质疏松,应早发现及时治疗。     

The p47phox mouse knock-out model of chronic granulomatous disease

Chronic granulomatous disease (CGD) is caused by a congenital defect in phagocyte reduced nicotinamide dinucleotide phosphate (NADPH) oxidase production of superoxide and related species. It is characterized by recurrent life-threatening bacterial and fungal infections and tissue granuloma formation. We have created a mouse model of CGD by targeted disruption of p47phox, one of the genes in which mutations cause human CGD. Identical to the case in human CGD, leukocytes from p47phox-/- mice produced no superoxide and killed staphylococci ineffectively. p47phox-/- mice developed lethal infections and granulomatous inflammation similar to those encountered in human CGD patients. This model mirrors human CGD and confirms a critical role for the phagocyte NADPH oxidase in mammalian host defense.     

The acid-base status does not influence the rate of acetate metabolism in hemodialyzed patients

The present study was undertaken to evaluate whether the acid-base status influences the rate of acetate metabolism in patients chronically hemodialyzed against acetate. Ten patients (5 "intolerant" and 5 "tolerant" to acetate) received in a randomized order and for three consecutive dialyses each of the six following infusions in the venous line of the dialyzer: NaHCO3 (22, 44 or 88 mEq/h), NaCl (22 or 44 mEq/h) or Dextrose 5% in water (30 mmol/h). Plasma acetate was measured at the end of the dialysis. Bicarbonate infusions increased significantly blood pH and plasma bicarbonate but did not change the plasma acetate concentration at the end of dialysis. We conclude that the rate of acetate metabolism is not modified by changes in the acid-base status within the range usually observed in hemodialyzed patients. A significant hypoxemia per dialysis was noted only in AT patients with lower plasma acetate and rapid acetate metabolism. We conclude that acetate metabolism (and not plasma acetate concentration) plays a significant role in dialysis-induced hypoxemia.     

Functional defect in neutrophil cytosols from two patients with autosomal recessive cytochrome-positive chronic granulomatous disease.

The kinetics of activation of the respiratory burst oxidase in the cell-free oxidase-activating system have been explained by a three-stage mechanism in which the membrane-associated oxidase components M: (a) take up a cytosolic factor S to form a complex M.S that is (b) slowly converted in the second stage to a precatalytic species [M.S]*, which finally (c) takes up two more (possibly identical) cytosolic components, C alpha and C beta, to successively generate [M.S]*C alpha, a low-activity (i.e., high Km) oxidase, and finally [M.S]*C alpha C beta, the ordinary (i.e., low Km) oxidase (Babior, B.M., R. Kuver, and J.T. Curnutte. 1988. J. Biol. Chem. 263:1713-1718). Studies with the cell-free oxidase-activating system from normal neutrophils and from neutrophils obtained from two patients with type II (autosomal recessive cytochrome-positive) chronic granulomatous disease (CGD) have suggested that (a) the defective element in the cytosol from patient neutrophils is S; (b) in normal neutrophil cytosol, S is limiting with respect to M; and (c) C alpha and C beta interact cooperatively with the activated precursor complex [M.S]*. It was further speculated that S might be identical to the nonphosphorylated progenitor of the phosphorylated 48-kD proteins that are missing in certain forms of CGD, and that other forms of type II CGD besides the one described in this report remain to be discovered.     

Expansion of genetically corrected neutrophils in chronic granulomatous disease mice by cotransferring a therapeutic gene and a selective amplifier gene

Hematopoietic stem cell gene therapy has not provided clinical success in disorders such as chronic granulomatous disease (CGD), where genetically corrected cells do not show a selective advantage in vivo. To facilitate selective expansion of transduced cells, we have developed a fusion receptor system that confers drug-induced proliferation. Here, a 'selective amplifier gene (SAG)' encodes a chimeric receptor (GcRER) that generates a mitotic signal in response to estrogen. We evaluated the in vivo efficacy of SAG-mediated cell expansion in a mouse disease model of X-linked CGD (X-CGD) that is deficient in the NADPH oxidase gp91phox subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91phox, and transplanted to lethally irradiated X-CGD recipients. Estrogen was administered to a cohort of the transplants, and neutrophil superoxide production was monitored. A significant increase in oxidase-positive cells was observed in the estrogen-treated mice, and repeated estrogen administration maintained the elevation of transduced cells for 20 weeks. In addition, oxidase-positive neutrophils were increased in the X-CGD transplants given the first estrogen even at 9 months post-transplantation. These results showed that the SAG system would enhance the therapeutic effects by boosting genetically modified, functionally corrected cells in vivo.