Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Mercury in blood cells—Altered elemental profiles

The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure.     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

In vitro microwave effects on human neutrophil precursor cells (CFU-C)

Human marrow cells were irradiated with 2450-MHz CW microwaves in a fluid-filled waveguide irradiation system. Cell exposure was conducted by placing a marrow cell suspension in 20-μl glass microcapillary tubes that were positioned in the exposure chamber, and irradiated at power densities from 31 to 1,000 mW/cm² (with corresponding specific absorption rates of 62 to 2,000 mW/g) for 15 minutes. The temperature of the sample was maintained at a fixed point. Sham-irradiated (SI) and microwave-irradiated (MWI) cells were cultured in a methylcellulose culture system for neutrophil colony proliferation. There was no reduction in neutrophil colony number on days 6–7 or 12–14 in cells exposed at 31 or 62 mW/cm², but as the power density was increased to 1,000 mW/cm², there was a reduction in colony number of MWI cells compared with SI cells. The microwave interaction with the human neutrophil colony-forming cells was apparently not related to temperature rise, or to the state of cell cycle, and was irreversible.     

Effects of a bout of traditional and original sumo training on neutrophil immune function in amateur university sumo wrestlers

In order to examine in detail the influence on the neutrophil immune function in sumo wrestlers of performing traditional and original training we examined changes in the neutrophil immune function in 17 male amateur university sumo wrestlers (aged 20.2 ± 1.5 years), before (‘Pre’) and after the training (‘Post’) for 2.5 h under fasting conditions. Assays included blood leukocyte and neutrophil counts, serum concentration of immunoglobulins, complements, myogenic enzymes and neutrophil oxidative burst activity (OBA) and phagocytic activity (PA). Myogenic enzymes, neutrophil counts, the ratio of neutrophil counts:leukocyte counts significantly increased and immunoglobulins and complements decreased in Post compared with Pre. There was a positive correlation between the change of neutrophil counts before and after the training and the change of creatine kinase (r = 0.667, p < 0.01). The Post OBA significantly increased and PA significantly decreased compared with Pre. It was concluded that sumo training causes muscular damage and an increase in the neutrophil count as a response. In this time, although OBA increased, PA decreased after training. Compensation between PA and reactive oxygen species production may exist to maintain the overall integrity of the neutrophil immune function. Copyright © 2008 John Wiley & Sons, Ltd.     

淋巴细胞功能相关抗原-1在冻融PMN介导的VEC损伤中的作用

目的：探讨组织冻结融化造成血管内皮细胞（VEC）损伤的机理。方法：采用密度梯度离心法分离大鼠外周血嗜中性粒细胞（PMN）,速率冷冻仪冷冻PMN后水浴复温建立冻融PMN模型。冻融后4、12和24h,测定PMN表面淋巴细胞功能相关抗原－1（LFA－1）的表达;将冻融PMN与正常VEC共同孵育后测定培养液中乳酸脱氢酶活性及冻融PMN与VEC的粘附。结果：冻融后24h内,冻融PMN表面LFA－1的表面呈时间依赖性增强。（2）冻融PMN与VEC相互作用后,PMN－VEC粘附明显增强、VEC受到明显损伤。（3）抗LFA－1单克隆抗体明显抑制冻融PMN与VEC粘附的增强、明显减轻VEC损伤。结论：冻融可诱发PMN表面LFA－1的表达,进而增强PMN－VEC粘附而导致VEC损伤。     

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β₂ integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β₂ integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.     

Neutrophil extracellular traps activate lung fibroblast to induce polymyositis‐related interstitial lung diseases via TLR9‐miR‐7‐Smad2 pathway

Excessive neutrophil extracellular trap (NET) formation may contribute to polymyositis (PM)‐associated interstitial lung diseases (ILD), but the underlying mechanism is not fully revealed. In this study, we found that NET accelerated the progression of ILD and promoted pulmonary fibrosis (PF) in vivo. miR‐7 expression was down‐regulated in lung tissue of PM group than control group, and NETs further decreased miR‐7 expression. TLR9 and Smad2 were up‐regulated in lung tissue of PM group than control group, and NETs further increased TLR9 and Smad2 expressions. In vitro experiments showed that PMA‐treated NETs accelerated the proliferation of LF and their differentiation into myofibroblast (MF), whereas DNase I decreased the promotion effect of NETs. Neutrophil extracellular trap components myeloperoxidase (MPO) and histone 3 also promoted the proliferation and differentiation of LF. In addition, we demonstrated that TLR9 involved in the regulation of NETs on LF proliferation and differentiation, and confirmed the interaction between miR‐7 and Smad2 in LF. Finally, miR‐7‐Smad2 pathway was confirmed to be involved in the regulation of TLR9 on LF proliferation and differentiation. Therefore, NETs promote PM‐related ILD, and TLR9‐miR‐7‐Smad2 signalling pathway is involved in the proliferation of LFs and their differentiation into MFs.