Osamu Koshio

Localization of chemotactic activity and 64 kD protein phosphorylation for human polymorphonuclear leukocytes in N-terminus of the chemotactic protein LUCTIL-8☆

A synthetic peptide, AVLPRSAKEL (LU10), the N-terminal amino acid sequence of chemotactic protein (LUCT/IL-8), showed chemotactic activity to polymorphonuclear leukocytes (PMN) with an ED50 of 5 nM for comparable to that of LUCT. Native LUCT and LU10 specifically induced the phosphorylation of 64 kD protein of PMN, and serine residue in the 64 kD protein was major phosphorylated amino acid. Furthermore, native LUCT enhanced the release of myeloperoxidase and beta-glucuronidase from PMN in the presence of cytochalasin B and FMLP, but LU10 did not. These results strongly suggest that the active site for both chemotactic stimulation and 64 kD protein phosphorylation is localized on the sequence of N-terminal 10 amino acids of LUCT.

Analysis of membrane antigens on neutrophils from patients with sepsis.

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.

Effects of Grepafloxacin on the Function of Human Polymorphonuclear Leukocytes and the Phosphorylation of p38 Mitogen-Activated Protein Kinase

Background: Some new quinolones (NQs) modulate polymorphonuclear leukocyte (PMN) functions. We investigated these effects on PMN functions at concentrations <10 μg/ml. Methods: Chemotactic activity and the production of reactive oxygen species (ROS) were measured using a 48-well chemotaxis chamber and by luminol-dependent chemiluminescence (CL) activity, respectively. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was measured by Western blot using specific antibodies to its phosphorylation sites. Results: Grepafloxacin (GPFX) at concentrations >5 μg/ml increased the chemotactic activity and ROS production of PMNs after stimulation with N-formylmethionyl-leucyl-phenylalanine (fMLP), whereas prulifloxacin (PUFX) showed no effect. In contrast to PUFX, GPFX at concentrations >1 μg/ml stimulated the phosphorylation of p38 MAPK. Conclusions: GPFX enhanced the chemotactic activity and ROS production of PMNs after stimulation with fMLP at concentrations <10 μg/ml. These effects of GPFX on PMNs could be in part due to the enhancement of p38 MAPK phosphorylation.

Downregulation of immunomodulator gene expression in LPS-stimulated human polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole

Lansoprazole (LPZ) has anti-inflammatory activity and repairs cells damaged by phagocytic cells. In the present study, we evaluated the effects of LPZ on gene expression, especially that of immunomodulator genes, in human polymorphonuclear leukocytes (PMNs) activated by lipopolysaccharide (LPS). Several concentrations of LPZ (final concentrations, 0–10 µg/ml) were added to the PMNs (1 × 106 cells/ml), which were stimulated with LPS (100 ng/ml) and incubated at 37°C for 1 or 3 h. When LPS-stimulated PMNs were treated with LPZ at ≥5.0 µg/ml for 1 h, mRNA expression levels of CXCR1/2 and TNFα were suppressed in a dose-dependent manner. The gene expression level of CD14 was also downregulated by LPZ at ≥0.1 µg/ml, with expression suppressed to 50% by 10 µg/ml LPZ. However, LPZ at 0.01–5.0 µg/ml had no significant effect on the expression of TLR-4 or CD11b/CD18 mRNA. LPZ at 10 µg/ml downregulated the levels of these mRNAs to 80% and 50%, respectively. On the other hand, when the reaction period was extended to 3 h with the same conditions, all mRNA expression levels were downregulated by ≥0.01 µg/ml LPZ, in a dose-dependent manner. LPZ may suppress the biological functions of PMNs, such as chemotaxis and inflammatory chemokine production.

Formyl-Met-Leu-Phe-dependent serine kinase for a 64,000 molecular weight protein of polymorphonuclear leukocytes in a cell-lysate system

In the Triton X-100-treated polymorphonuclear leukocytes (PMN), which were stimulated with formyl-Met-Leu-Phe (FMLP) for 1 min, a 64,000 molecular weight protein (p64) was preferentially phosphorylated by the incubation with [gamma-32P]ATP in the presence of Mg2+, but not in the presence of Ca2+. Phosphoamino acid analysis of pp64 revealed that the p64-kinase was a serine-specific protein kinase. The p64 was maximally phosphorylated in the first minute, suggesting that the rapid phosphorylation was related to the initial reaction for activation of the FMLP-stimulated PMN functions. The FMLP-stimulated phosphorylation of p64 was slightly inhibited by the addition of cGMP in the reaction mixture. However, addition of cAMP, the cyclic nucleotide-dependent kinase inhibitor (H-8), protein kinase C-inhibitor (H-7) or Ca/calmodulin-dependent kinase inhibitor (W-7), showed no effect on the phosphorylation. These data suggest that phosphorylation of p64 seems to be a novel protein kinase specific to p64.

Production of anti-Candida antibodies in mice with gut colonization of Candida albicans.

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.

Role of F4/80+Mac-1high adherent non-parenchymal liver cells in concanavalin A-induced hepatic injury in mice

Aim:  Non‐parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80+Mac‐1high+ cells were involved in the regulative feedback‐modulated regulation of inflammatory responses during concanavalin A (Con A)‐induced hepatitis.

Glucose-stimulated phosphorylation of the 64-kDa protein of human polymorphonuclear leukocytes in a cell-free system

Glucose-stimulated phosphorylation of 64-kDa protein using a greater than 30 kDa fraction of human polymorphonuclear leukocytes in a dose-dependent fashion with 33 microM for maximum stimulation and 1.4 microM for ED50. None of the glucose derivatives and metabolites of glycolysis stimulated phosphorylation, but glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-diphosphate inhibited the glucose-stimulated phosphorylation, strongly suggesting phosphorylation by glucose-regulated protein kinase. Cyclic nucleotides and the protein kinase inhibitors H-7, H-8, W-7 and staurosporine did not affect phosphorylation.