Katsu Saionji

Evaluation of the expression of human CAP18 gene during neutrophil maturation in the bone marrow

To understand the gene expression of CAP18 (18‐kDa cationic antibacterial protein), a member of cathelicidins, we evaluated mRNA and protein expression of CAP18 using human bone marrow cells and mature neutrophils. Northern blot analysis revealed that CAP18 mRNA was expressed more abundantly in bone marrow cells than mature neutrophils, whereas Western blot analysis indicated that CAP18 protein was more abundant in mature neutrophils than bone marrow cells. Consistent with this, in situ hybridization using bone marrow cells demonstrated that the expression of CAP18 mRNA was neutrophil lineage‐specific and was observed primarily in myelocytes (>95%) with limited expression in more immature cells (promyelocytes) and mature cells (metamyelocytes, band cells, and segmented neutrophils). Furthermore, immunohistochemical study indicated that, coincident with the increase of CAP18 mRNA levels, CAP18‐positive cells increased markedly at myelocyte stage, and the increased levels remained almost constant (>95%) in metamyelocytes, band cells, and segmented neutrophils, although the mRNA levels were remarkably reduced in these cells. Together these observations indicate that CAP18 gene transcription likely occurs lineage‐ and stage‐specifically at the myelocyte stage of neutrophil maturation in the bone marrow and results in the synthesis and cytoplasmic accumulation of CAP18, which is present in the subsequent stages of neutrophil maturation. J. Leukoc. Biol. 64:845–852; 1998.

Expansion of CD14+CD16+ Blood Monocytes in Patients with Chronic Renal Failure Undergoing Dialysis: Possible Involvement of Macrophage Colony-Stimulating Factor

A novel subpopulation of blood monocytes coexpressing CD16 antigen and low levels of CD14 antigen (CD14+CD16+ monocytes) has recently been identified, and expansion of these CD14+CD16+ monocytes has been reported under some pathological conditions. In this study, we examined the immunophenotype of blood monocytes in patients with chronic renal failure (CRF) who were undergoing hemodialysis (HD, n = 52) or continuous ambulatory peritoneal dialysis (CAPD, n = 36) using two-color immunofluorescence flow cytometry. The percentage and absolute number of CD14+CD16+ monocytes were significantly higher (p < 0.001) in both HD and CAPD patients compared with those in healthy control subjects. We also determined the plasma concentrations of hematopoietic growth factors and cytokines using an enzyme-linked immunosorbent immunoassay. The plasma levels of macrophage colony-stimulating factor (M-CSF) were markedly increased in both HD and CAPD patients relative to the normal controls. The plasma M-CSF levels correlated significantly with the number of CD14+CD16+ monocytes in the whole group of subjects. These findings suggest that elevated endogenous M-CSF levels may participate in the expansion of CD14+CD16+ monocytes in CRF patients undergoing dialysis.

Altered surface expression of effector cell molecules on neutrophils in myelodysplastic syndromes

The surface expression of effector cell molecules on neutrophils was examined in 18 patients with myelodysplastic syndromes (MDS) and 20 healthy control subjects. The MDS patients were further classified as low clinical risk (L‐MDS, n = 7) and high clinical risk (H‐MDS, n = 11). The expression of Fc receptors for IgG (FcR), complement receptors (CR) and cellular adhesion molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. The effect of granulocyte colony‐stimulating factor (G‐CSF) and tumour necrosis factor‐alpha (TNF) on L‐selectin shedding and CR up‐regulation on neutrophils was also examined. The percentage of FcRI‐positive neutrophils and CD11b/CR3 expression on neutrophils were significantly increased in the H‐MDS patients when compared to the controls. In contrast, the expression of FcRII, FcRIII, L‐selectin, LFA‐1 and CD18 on neutrophils was significantly reduced in the H‐MDS patients compared with the controls. The L‐MDS neutrophils exhibited lower expressions of CR1, L‐selectin, LFA‐1 and CD18 than those of the controls. Neutrophils from some H‐MDS patients showed impaired L‐selectin shedding and CR up‐regulation after stimulation with G‐CSF or TNF, although these were not significantly different when assessed in the whole H‐MDS group. These findings suggest that an altered surface expression of effector cell molecules and an impaired modulation of cellular adhesion molecules on neutrophils may contribute to the increased susceptibility to bacterial infections in MDS patients.

In vivo Administration of Granulocyte Colony-Stimulating Factor Increases the Surface Expression of Sialyl-Lewisx on Neutrophils in Healthy Volunteers

We examined the in vivo effect of granulocyte colony-stimulating factor (G-CSF) on the surface expression of putative counterligands for endothelial selectins on neutrophils in healthy volunteers. G-CSF (50 µg/m2/day) was administered subcutaneously to 5 healthy volunteers for 4 days. The expression of surface antigens on neutrophils was determined by flow cytometry and monoclonal antibodies. G-CSF administration increased the number of leukocytes, mainly of neutrophils, which was associated with an increase in the expression of the high-affinity Fc receptor for IgG (FcRI, CD64) and CD14 on neutrophils. G-CSF administration decreased the surface expression of L-selectin on neutrophils, whereas it increased the expression of sialyl-Lewisx but not Lewisx on neutrophils. These findings suggest that G-CSF participates in the neutrophil-endothelial cell interactions in vivo by modulating the expression of adhesion molecules and ligands for endothelial selectins on neutrophils.

Epidemiologic Typing of Methicillin-Resistant Staphylococcus aureus by Macrorestriction Analysis of Genomic DNA Using Two Different Restriction Enzymes

Ninety-seven methicillin-resistantStaphylococcus aureus (MRSA) isolates from patients, staff and the environment in a teaching hospital in Japan were typed by pulsed-field gel electrophoresis (PFGE). Two different macrorestriction enzymes (SmaI andSstII) were used to digest the DNA. In this study, after identifying the dominant pattern, the isolates were classified into 4 categories including indistinguishable, closely related, possibly related, and unrelated. While closely related isolates were identified as a subtype, possibly related isolates showing differences of 4 to 6 fragments were difficult to identify either as a subtype or different type. Although there were similar trends in isolate identification by restriction patterns ofSmaI andSstII, some isolates showed a discrepancy. There were 9 isolates categorized as possibly related by eitherSmaI orSstII restriction pattern, and while 2 of the 9 displayed possibly related patterns by both enzymes, 7 showed discrepancies. Among the latter 7 isolates, 1 displayed an indistinguishable pattern by the other macrorestriction enzyme, 5 showed closely related patterns, and 1 showed an unrelated pattern. These discrepancies may help delineate a more precise differentiation of the isolates. Indices of the discriminatory ability of PFGE typing bySmaI andSstII were 0.768 and 0.742. The combination of these 2 enzymes led to an increase in discrimination (D=0.8045). The use of PFGE typing bySmaI andSstII in parallel may enhance discrimination.