Hiroshi Fukumasu

Activation of Human Neutrophil by Cytokine-Activated Endothelial Cells

Abstract— Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokine-activated ECs potentiated the activity of neutrophil functions. The magnitude of O2− release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O2− on coculture with activated ECs. Anti–granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O2− release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O2− release by neutrophils. This stimulatory effect of activated EC supernatants on O2− release by neutrophils was abolished by anti–GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1–activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen–activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.

Plasma Granulocyte Colony Stimulating Factor Concentrations in Pregnant Women

We measured the plasma granulocyte colony stimulating factor (G-CSF) concentrations in pregnant women to evaluate the association between G-CSF and increased neutrophil counts that has been observed during pregnancy. We examined 96 pregnant and 10 nonpregnant women. The G-CSF concentrations were assayed using a sandwich enzyme immunoassay. The G-CSF concentrations in pregnant women were significantly higher at all times than those of nonpregnant women. The G-CSF concentrations were also significantly higher during than before labor. The plasma G-CSF concentrations were positively correlated with the neutrophil counts during the 3rd trimester only. In conclusion, increased G-CSF concentrations may be related to the increases in neutrophil counts in pregnant women, especially during the 3rd trimester.

Elevation in Plasma Alkaline Phosphatase Level during rhG-CSF Administration

Neutropenic patients suffering from gynecologic cancers treated with cancer chemotherapy were evaluated to investigate changes in levels of plasma alkaline phosphatase (ALP) during recombinant human granulocyte colony stimulating factor (rhG-CSF) administration, and to discern its causes. Plasma ALP, ALP isozymes, neutrophil alkaline phosphatase (NAP) activity, and morphological maturation of neutrophils were measured prior to, during, and after rhG-CSF administration. Plasma ALP values were significantly elevated (p < 0.05) during administration of rhG-CSF. ALP-3, the dominant alkaline phosphatase fraction of NAP, was the dominant isozyme that showed an increase in the plasma. Moreover, the elevation of plasma ALP-3 significantly correlated with the elevation of NAP activity (r = 0.939, p < 0.0001), and neutrophils in which NAP activity was induced were not limited to morphologically mature neutrophils. These results showed that rhG-CSF acts to stimulate neutrophils at all stages of maturation and causes an elevation of plasma ALP(ALP-3) concentrations in plasma.

Stimulation of fetal granulopoiesis by intrauterine injection of recombinant human granulocyte colony- stimulating factor into rat fetuses.

The small neutrophil reserve and exaggerated release of stored neutrophils are factors which predispose neonates to neutrophil reserve exhaustion during bacterial sepsis. Our objective is to try to improve in utero the myelopoietic function of the fetus before delivery. In the first series, recombinant human (rh) granulocyte colony-stimulating factor (G-CSF) (rhG-CSF; 100 μg/kg) was injected subcutaneously into rat fetuses at the indicated times to assess drug absorption and fetal response. In the second series, rhG-CSF (100 μg/kg) or saline (control) was injected into the fetuses once every other day to investigate the effect of repeated injections of rhG-CSF on enhancing fetal myelopoiesis preceding birth. Delivery was performed by cesarean section on embryonic day 21. The plasma concentration of G-CSF was determined by ELISA. The effect of rhG-CSF injection on granulopoiesis was assessed by measurement of the neutrophil count in the fetal peripheral blood and by histological examination of the fetal bone marrow, spleen, and liver. Fetally administered rhG-CSF enhanced fetal myelopoiesis preceding birth.

Significance of fetography in prenatal diagnosis of limb-body wall complex (LBWC)

The diagnosis of limb body wall complex (LBWC) by ultrasonography does not usually present a problem. The major difficulty we found was severe oligohydramnios or a very complex relationship between the herniated viscera and the deformities. We performed fetography in a pregnant woman with fetal anterior abdominal wall defect at the 28th gestational week to show a peculiar structure of intrauterine membranes of LBWC, and we could obtain a successful prenatal diagnosis of LBWC.