Defective priming of the phagocyte oxidative burst in a child with recurrent intracellular infections.

Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms. Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation. This process is known as priming. We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms (Mycobacterium bovis, BCG, and Salmonella typhimurium). No T- or B-cell quantitative or qualitative defects were found. Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease. Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H(2)O(2) production after pretreatment with LPS (5 microg/mL for 30 min), TNFalpha (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10(-6) M for 5 min). In these conditions, H(2)O(2) production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H(2)O(2). Moreover, monocytes from the patient showed an impaired capacity to kill S. typhimurium in vitro. Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst. This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens.

Enzimopatías, inmunodeficiencia y trastornos de la función fagocítica / Enzymopathies, immune deficiency, and disorders of phagocytic function

Se presenta una revisión sobre las enzimopatías hereditarias asociadas con distintos trastornos inmunológicos. La deficiencia de adenosin deaminasa (ADA) se observa en una parte de los pacientes con inmunodeficiencia combinada grave, mientras que la disminución de la actividad de la nucleosido fosforilasa está presente en individuos con disfunción de los linfocitos T. El defecto de NADPH2-oxidasa y G6PD leucocitaria puede estar asociado con una enfermedad granulomatosa crónica y disminución de la actividad bactericida de los leucocitos respectivamente

Myeloperoxidase secretion during phagocytosis: a case of a patient with impaired bactericidal activity.

We describe a case of a 5-year-old male patient with prolonged and extensive osteomyelitis of the left femur. Staphylococcus aureus was grown from blood cultures taken upon admission and also from pus drained from an incised hip joint. A defect in immune function was suspected and neutrophil function was assessed. Chemotaxis and phagocytosis were normal, but phagocytosed S. aureus were not killed as efficiently as in control neutrophils. No inherent defect in the ability of these neutrophils to generate reactive oxidants was observed, but an unusual luminol-dependent chemiluminescence response was obtained during phagocytosis of latex beads or opsonized S. aureus: This was characterized by an initial rapid, but transient increase occurring within 1 min of addition of phagocytic stimulus. Whereas during phagocytosis of latex beads by control neutrophils less than 1% of the total myeloperoxidase activity was detected extracellularly, up to 15% was released from the patient's neutrophils. We propose that release of myeloperoxidase from the patient's neutrophils during phagocytosis reduces the intraphagosomal concentration of this enzyme and thus impairs the efficiency of intracellular killing of S. aureus.

Interferon induction and granulocyte function in patients with viral meningitis.

In 22 consecutive patients with acute viral meningitis we studied the ability of neutrophilic granulocytes to phagocytize yeast particles, the interferon (IFN) levels in serum and cerebrospinal fluid as well as the activity of the enzyme 2'-5' oligoadenylate synthetase in peripheral lymphoid cells. During the acute stage of illness a decreased phagocytosis of the neutrophils was demonstrated, while the adherence of yeast particles to the cells was increased. This was accompanied by the presence of IFN in the cerebrospinal fluid and an increase in the 2'-5' oligoadenylate synthetase activity of peripheral lymphoid cells. About two months after onset of symptoms the synthetase activity and the phagocytic activity of granulocytes were restored to normal levels. The results of this study may indicate that the impaired neutrophil function in viral disease is influenced by increases in IFN levels in vivo.

Generalised glucosephosphate isomerase (GPI) deficiency causing haemolytic anaemia, neuromuscular symptoms and impairment of granulocytic function: a new syndrome due to a new stable GPI variant with diminished specific activity (GPI Homburg).

A new glucosephosphate isomerase (GPI) variant is described which is characterised by very low specific activity in erythrocytes, granulocytes and muscle tissue, nearly normal stability, normal kinetic properties and a decreased electrophoretic mobility. The propositus suffers from a complex syndrome involving erythrocytes (congenital haemolytic anaemia), granulocytes (decreased production of superoxide anion and reduced bactericidal activity in vitro) and the neuromuscular system (myopathy, mental retardation). It is suggested that the clinical syndrome results from generalised GPI deficiency due to a decreased specific activity of the variant enzyme, which cannot be compensated by an increase of de-novo synthesis of GPI protein even in cells exhibiting active protein synthesis such as granulocytes and muscle cells.

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Deficiency of NADPH oxidase activity in chronic granulomatous disease.

NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.

Characterisation of the enzyme defect in chronic granulomatous disease.

Strikingly reduced activity of an enzyme, normally located in the plasma membrane of human neutrophils, has been demonstrated in a male patient with chronic granulomatous disease (C.G.D.). The subcellular distribution of N.A.D.H.-dependent reduction of nitroblue tetrazolium (N.B.T.) was determined in neutrophils because reduction of this dye by these patients is grossly impaired. Assayed at high concentrations of N.A.D.H. (1 mmol/1), N.B.T. is reduced by enzymes in the cytosol and mitochondria in addition to the plasma membrane by both normal and C.G.D. cells--properties which previously obscured the identity and location of this enzyme. At a more physiological concentration of N.A.D.H. (25 mumol/1), reduction of the dye by the plasma membrane, the principal site of N.B.T. reduction by normal neutrophils, was absent in the patient with C.G.D. It is suggested that absence or imperfect function of this reductase enzyme is the primary lesion in this disease.

Leukocyte glutathione peroxidase deficiency in a male patient with chronic granulomatous disease.

A male child with chronic granulomatous disease is described in whom glutathione peroxidase deficiency of leukocytes was identified. Stability and activity of G-6-PD and activity of NADPH oxidase were normal. The leukocytes of the parents showed intermediate activities of glutathione peroxidase, suggesting the possibility of autosomal recessive inheritance.

NADPH oxidase deficiency in X-linked chronic granulomatous disease.

We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity.