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.

Ubiquinone-5 is reduced by superoxide in the aerobic state by NADPH oxidase of guinea pig macrophages.

The reduction of ubiquinone-5 (Q1) by the phagocytosis-specific NADPH oxidase of guinea pig macrophages was not inhibited by superoxide dismutase (SOD) at concentrations usually used for O2- assay but was inhibited at about 100-times higher concentrations. Titration of the reaction with SOD and a comparison with that of xanthine oxidase showed that the inhibition was not due to the semiquinone oxidation accelerated by a removal of O2- but due to the accelerated dismutation of O2- which otherwise reduces the quinone. Molecular oxygens are therefore preferential electron acceptors in the NADPH oxidase even in the presence of Q1.

Levels of serum granulocyte colony-stimulating factor in patients with cerebrovascular diseases.

The role of leukocytes in the pathogenesis of cerebrovascular disease, in particular, cerebral ischemic disease has recently become a focus of research. Several studies have reported that a positive correlation between increased functional activities of neutrophils and the risk of cerebral ischemic disease. Granulocyte colony-stimulating factor (G-CSF) is known to be not only a granulocyte proliferating factor but also a potent activator of mature neutrophils. In this study, we measured the serum G-CSF levels in 143 patients with cerebrovascular diseases and in 100 patients with other diseases, using our established enzyme-linked immunosorbent assay (ELISA) for G-CSF The minimal detection level was 20 pg/ml G-CSF. In patients with cerebral infarction, G-CSF could be detected in 18.3% and in patients with cerebral hemorrhage, it could be detected in 9.8% of analyzed samples. On the other hand, 6% of the patients with other diseases had measurable levels of G-CSF. The differences among these three groups were statistically significant according to the chi 2 test (p < 0.01). Our findings that there was a significantly high frequency of elevated levels of G-CSF among patients with cerebrovascular diseases, may indicate that the action of G-CSF as a potent activator of neutrophils plays some role in the occurrence of cerebrovascular disease, in particular, cerebral infarction.

[Clinical study on the effect of natural alpha-interferon (HLBI) in the treatment of adult T-cell leukemia].

The phase II trial of natural interferon-alpha (HLBI) in treatment of adult T-cell leukemia was carried out as a cooperative study. Of the 24 cases which could be evaluated, 3 cases in crisis type and 5 cases in chronic type with lymphadenopathy and/or skin infiltration achieved PR, giving a response rate of 33.3%. The anti-tumor effect of HLBI for skin lesion could be assessed in 16 cases with skin infiltration, giving a response rate of 50.0% (5 CR and 3 PR) and demonstrating a high efficacy. Of the 31 eligible patients, side effects were recognised in 27 (87.1%). Major subjective and objective symptoms were fever (38.7%), fatigue (25.8%), anorexia (12.9%) and nausea (12.9%), and leukopenia (22.6%), granulocytopenia (38.7%), thrombocytopenia (38.7), elevation of GPT (12.9%) and GOT (12.9%) were observed.

NADPH binding component of neutrophil superoxide-generating oxidase.

The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.

NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease.

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.

Effects of varying the injection routes of PSK or OK-432 on development of immune responses against a superimposed antigen.

The present study examined functional and morphological modifications of development of immune responses to an unrelated antigenic stimulus by an ongoing response to a biological response modifier such as PSK or OK-432. Development of the immune responses was found to be strictly dependent upon the site where the ongoing response was occurring. In some cases, injection into the intraperitoneal site slightly augmented the development of a humoral immune response against a superimposed antigen, sheep erythrocytes, when the antigen had been injected into the site within 3 h after an injection of PSK. Thereafter, however, inhibition of the response was observed. Subcutaneous injection into the foot pad, but not into the upper chest, augmented the response. This augmentation occurred only when a superimposed antigen was injected into the site "prepared" with a previous injection of PSK, and showed a bell-shaped dependence on the dose of PSK. The foot pad site was also associated with an augmented development of a delayed type hypersensitivity against sheep erythrocytes when the antigen was injected into the "prepared" site. Morphological examination shows accumulation of inflammatory cells at the sites 2 to 7 days after an injection of PSK. The macrophages in the foot pad retained their "activated" state for at least 7 days after the injection whereas those in the peritoneal cavity were not sufficiently "activated" even after 2 days.

Activation of neutrophils NADPH oxidase by PMA: cytosol activity is translocated in phorbol-primed neutrophils.

1. Translocation of cytosol activity in phorbol-primed neutrophils was studied. 2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA. 3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane. 4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane. 5. These observations suggested that cytosol activity was translocated in PMA-primed cells.

Identification of the NADPH-binding subunit of the respiratory burst oxidase.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.

Response of ligated rabbit ileal loop to Clostridium perfringens type C strains and their toxic filtrates.

The vegetative cells and toxic filtrates of Clostridium perfringens type C strains were injected into ligated rabbit ileal loops and the responses were observed. Out of 12 strains examined, 2 strains showed positive reaction in this test, when the vegetative cells were injected. One of these 2 strains was an enterotoxigenic and beta-toxigenic and the other was beta- and delta-toxigenic but not enterotoxigenic. Culture filtrates containing beta or delta toxin also showed fluid accumulation in the rabbit ileal loop. Histological findings of loops injected with culture filtrates containing beta toxin showed separated and effaced villi, hemorrhage in the mucosa, engorged vessels, inflammatory cell infiltration, and hyperplasia of the lymphoid tissue.