Masafumi Hasui

Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat‐killed Staphylococcus aureus

Neutrophils, short‐lived leucocytes that die by apoptosis, play an important role in the first stage of defense against bacterial infections. It has been reported that phagocytosis of intact bacteria or Candida albicans can accelerate neutrophil apoptosis. However, the mechanism of phagocytosis‐mediated neutrophil apoptosis is not well characterized. In this study, we evaluated whether ingestion of heat‐killed Staphylococcus aureus (S. aureus) enhances neutrophil apoptosis and whether this type of apoptosis is mediated by oxidative stress by using antioxidants and polymorphonuclear leucocytes (PMNs) from patients with chronic granulomatous disease (CGD). Co‐culture of PMNs with varying doses of S. aureus resulted in accelerated PMN death in a dose‐ and time‐dependent manner. Increased PMN apoptosis was observed by both Annexin V and PI staining. Similar results were observed in PMNs of CGD patients. Dimethyl sulphoxide (DMSO, an OH• scavenger) did not significantly inhibit either S. aureus‐ingested PMN apoptosis or spontaneous PMN apoptosis. On the other hand glutathione (GSH, an H2O2 scavenger) significantly inhibited both types of apoptosis.

Increased nitric oxide production by neutrophils from patients with chronic granulomatous disease on trimethoprim–sulfamethoxazole

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim-sulfamethoxazole (TMP-SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP-SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs (p<0.05). TMP-SMX with LPS significantly increased compared with LPS-stimulated samples at clinical (n=5, p<0.05) and 10-fold clinical concentrations (n=5, p<0.01). TMP-SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations (n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP-SMX has a role of bactericidal activity instead of O(2)(-) in host defense mechanism.

Dizygotic twin sisters with myelokathexis: Mechanism of its neutropenia

Dizygotic twin sisters were first found to have neutropenia at 1 year of age when evaluated for recurrent pulmonary infections. Since then they have remained neutropenic (0.05∼0.5 × 109/l). Despite of their neutropenia, myeloid hyperplasia was evident on a marrow smear examination, and a number of cells were hypersegmented with fine interlobular bridging with chromatin strands and cytoplasmic vacuolation. Electron microscopy showed apoptotic cells with condensed nuclei and apoptotic bodies in the cytoplasm. Although life span, hydrogen peroxide production, phagocytosis, spreading, and chemotaxis of peripheral neutrophils were normal, the survival of bone marrow neutrophils in both infants was markedly decreased when compared with that of normal bone marrow neutrophils. During the bone marrow culture apoptotic neutrophils were observed at an earlier stage in both patients than in normal controls, biochemically and morphologically. Morphology of bone marrow neutrophils in both patients resembled that of cultured control bone marrow neutrophils. Peripheral neutropenia and appearance of characteristic neutrophils in the bone marrow in myelokathexis are considered to be an expression of apoptosis of bone marrow neutrophils. Am. J. Hematol. 62:106–111, 1999.

Difference in changes of membrane fluidity of polymorphonuclear leukocytes stimulated with phorbol myristate acetate and formyl-methionyl-leucyl-phenylalanine: role of excited oxygen species.

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N‐formyl‐methionyl‐leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer‐forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time‐ and dose‐dependent. FMLP at 10‐5 M induced a decrease, while FMLP at 10‐7 M induced a rapid increase. On stimulation with 10‐7 M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10‐5 M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.

Abnormal Membrane Fluidity as a Cause of Impaired Functional Dynamics of Chemoattractant Receptors on Neonatal Polymorphonuclear Leukocytes: Lack of Modulation of the Receptors by a Membrane Fluidizer

ABSTRACT: Membrane properties associated with chemoattractant-mediated cellular responsiveness of neonatal polymorphonuclear leukocytes (PMN) were analyzed using n-formylmethionyl-leucyl-phenylalanine. Inasmuch as aliphatic alcohols as a membrane fluidizer can enhance the chemoattractant binding and affect subsequent cellular responsiveness in adult PMN, neonatal PMN were studied for such properties by their treatment with iso-propyl alcohol, an aliphatic alcohol. The alcohol (<2.5%) treatment enhanced the N-formylmethionyl-leucyl-phenylalanine binding to adult PMN, but there were no changes in the N-formylmethionyl-leucyl-phenylalanine binding to neonatal PMN. Although the N-formylmethionyl-leucyl-phenylalanine-induced subsequent responsiveness including migration, lysosomal enzyme release and superoxide anion production were modulated by the alcohol treatment in adult PMN, there was no such modulation in neonatal PMN. Because membrane fluidity is largely involved in the regulation of the receptor functions, the membrane fluidity of neonatal PMN was next measured by an excimer-forming lipid technique in flow cytometry. The membrane fluidity value (0.45 ± 0.037) of neonatal PMN was lower than that (0.74 ± 0.072) of adult PMN (p < 0.01). Although the aliphatic alcohol enhanced the membrane fluidity of adult PMN, it did not affect the membrane fluidity of neonatal PMN. We conclude that there is abnormal membrane fluidity as a cause of impaired functional dynamics of the chemoattractant receptors, which appears to underlie the defective modulation of cell functions by the membrane fluidizer in neonatal PMN.

Phagocytosis of heat-killed Staphylococcus aureus by eosinophils: comparison with neutrophils.

Eosinophils are characterized by several functional properties, such as chemotaxis, adhesion, superoxide anion production, and degranulation. In this article, we have studied the role of bacterial ingestion by eosinophils in comparison with that by neutrophils. Eosinophils and neutrophils were purified by using the Percoll gradient method followed by selection with CD16‐coated immunomagnetic beads and centrifugation through a Ficoll‐Hypaque gradient combined with dextran sedimentation, respectively. Both cells were preincubated with anti‐FcγRIIa mAb (CD32 mAb), anti‐FcγRIIIb mAb (CD16 mAb), anti‐CR3 (CD11b mAb), or anti‐CR1 (CD35 mAb) before being examined for phagocytosis of opsonized heat‐killed Staphylococcus aureus (S. aureus). Phagocytosis and production of hydrogen peroxide were simultaneously measured by flow cytometry using S. aureus labeled with propidium iodide and stained with 2′,7′‐dichlorofluorescein diacetate. Eosinophils showed significantly lower activity than neutrophils in both phagocytosis and hydrogen peroxide production. Phagocytosis by both cells was decreased by heat‐inactivated serum. Phagocytosis by neutrophils was significantly inhibited by CD16 mAb and CD32 mAb, whereas that by eosinophils was only inhibited by CD35 mAb. Whereas the mechanism of phagocytosis by neutrophils was mediated by CD16 and CD32, that of eosinophils was modulated by complement receptor 1 (CD35).

A case of Kawasaki Disease associated with syndrome of inappropriate secretion of antidiuretic hormone

Kawasaki disease (KD) is an acute vasculitis of unknown aetiology with varied clinical manifestations. Although coronary arteritis is common in the course of KD, central nervous system involvement is rare. We report a case of KD in an infant who developed convulsions and apnoea during his illness associated with syndrome of inappropriate secretion of antidiuretic hormone (SIADH).

Differential diagnosis and clinical course of autoimmune neutropenia in infancy: comparison with congenital neutropenia

Aim: Autoimmune neutropenia in infancy (AIN) is caused by granulocyte‐specific autoantibodies. Clinical presentation and diagnosis have not been well studied, resulting in cumbersome diagnostic investigations and unnecessary treatment such as granulocyte colony‐stimulating factor (G‐CSF) therapy. Methods: Clinical, laboratory and immunological data of 18 infants with AIN were evaluated. Granulocyte‐specific autoantibodies were detected by the direct granulocyte immuno‐fluorescence test (D‐GIFT), indirect granulocyte immunofluorescence test (I‐GIFT) and immuno‐blotting. Results: The average age of onset and resolution of neutropenia in AIN was 7.4 ± 3.4 mo (mean ± SD) and 20.4 ± 4.9 mo, respectively. Sixteen of the 18 patients presented with mild infectious symptoms; the other 2 patients were detected by chance and presented with no infectious symptoms. D‐GIFT was positive in all patients, and I‐GIFT was positive in 17 of these 18 patients. Most patients showed preferential binding to neutrophils from NA(1 + 2−)‐phenotyped donors by I‐GIFT and immunoblotting. An antibiotic (sulfamethoxazole‐trimethoprim) was given to 15 patients for prophylaxis. G‐CSF was given to only one infectious patient.

Focal segmental glomerulosclerosis in a boy with Dent-2 disease

Sirs, Proximal tubular dysfunction is, in general, more common when the nephrotic syndrome is associated with focal segmental glomerulosclerosis (FSGS) than with minimal-change nephrotic syndrome. Although it has been considered that the nonselective proteinuria in FSGS causes proximal tubular cell injury with the resultant tubulointerstitial damage and scarring, Copelovitch et al. have recently postulated that the tubular dysfunction or damage is the primary renal insult and that FSGS may be a secondary epiphenomenon in some patients with congenital tubulopathies [1]. We encountered a patient who fits into this concept. The patient was a 4-year-old Japanese boy referred to us for further evaluation of significant proteinuria detected by a mass screening program for renal disease in Japan at 3 years of age. He was first referred to a local hospital where renal biopsy was performed because of persistent proteinuria for several months. Pathological diagnosis of FSGS was made (Fig. 1). His past medical history and family history were unremarkable. Physical examination on admission showed that the boy’s height was 95.2 cm (−2.4 S.D.), his weight was 15.1 kg (−1 S.D.), and his blood pressure was 102/ 68 mmHg. There were no dysmorphic features. Initial serum concentrations were all normal: sodium 139 mEq/L, potassium 3.8 mEq/L, chloride 103 mEq/L, carbon dioxide 25 mEq/L, calcium 9.9 mg/dl, phosphorous 3.8 mg/dl, albumin 4.5 g/dl, and creatinine 0.27 mg/dl. Twenty-fourhour protein excretion varied from 892 mg/day to 1,242 mg/day. Repeated urinalysis did not detect glucosuria or hematuria. From these findings, the presumptive diagnosis of idiopathic FSGS was made and the treatment consisting of steroid and cyclosporine A had been initiated without a significant reduction in the amount of proteinuria. During the treatment over the following 11 months, quantitative analysis of the low-molecular weight protein in urine, such as beta-2-microglobulin (BMG), was repeatedly performed and showed extraordinarily high values (BMG: 84,800 μg/l–185,000 μg/l, normal T, p.P509S). This same mutation has been neither previously described nor observed in 200 control chromosomes from healthy individuals. No mutations were detected in CLCN5. Our patient was diagnosed as having Dent-2 and was weaned off steroids and cyclosporine A over the following month. He is now 6 years old and otherwise healthy without any medication, although a significant amount of proteinuria is still present. K. Kaneko (*) :M. Hasui Department of Pediatrics, Kansai Medical University, 2-3-1 Shin-machi, Hirakata-shi, Osaka 573-1191, Japan e-mail: kanekok@hirakata.kmu.ac.jp

Two cases of autoimmune neutropenia possibly induced by β-lactam antibiotics in infants

This report describes two infants who had neutropenia develop after treatment with &bgr;-lactam antibiotics. The first patient, a 9-month-old girl, was administered flomoxef (FMOX); the second patient, a 14-month-old boy, was administered cefotiam (CTM). Both infants were found to have neutropenia 4 to 5 days after they were placed on respective antibiotics, and neutropenia had persisted despite antibiotics withdrawal for 1 to 3 months. Drug-induced lymphocyte stimulation test (DLST), granulocyte-bound antibodies, serum granulocyte antibodies, and immunoblotting analysis indicated that &bgr;-lactam antibiotics possibly triggered production of granulocyte autoantibody with resultant autoimmune neutropenia.