Simple Washing Compared to Dithiothreitol (DTT) Method as a Better Method to Preserve Neutrophil Activity in Induced Sputum

痰液以 Dithiothreitol (DTT)處理法是廣泛用作呼吸道發炎時, 痰液及其細胞的可靠測量標識。然而，DTT的細胞毒性會影響會損壞痰液細胞膜，然後妨礙細胞膜表面生物化學標識或者細胞內的新陳代謝。本研究則是探討dithiothreitol（DTT）法對嗜中性白血球活性影響程度，並比較用簡單重複沖洗法的好處。簡單重複沖洗法是重複用3毫升Hank's buffer salt solution+10%小牛胎兒血清（HBSS）重複沖洗引痰痰液3次，然後透過一個尼龍網來分離嗜中性白血球的一個簡單方法。我們利用3%食鹽水，從5個支氣管擴張病人呼吸道引痰，一部份引痰痰液以dithiothreitol（DTT）法來取得痰液及其細胞，一部份引痰痰液以則以簡單重複沖洗法處理。然後計算引痰痰液細胞計數及其生存能力，並用流動細胞分析儀（flowcytometry）測嗜中性白血球之表面粘著分子，CD11b，CD18和neutrophils的細胞內的氧化新陳代謝的表現。在兩個方法之間，eosinophils，neutrophils，lymphocytes，或者macrophages的比例中沒有重要差別。DTT明顯的降低引痰痰液和血液中嗜中性白血球CD11b和CD18的表現，而簡單重複沖洗法則較佳的保存嗜中性白血球CD11b和CD18的表現。DTT也較簡單重複沖洗法明顯的降低lipopolysaccharide引起的嗜中性白血球細胞激素的分泌。在兩個處理在細胞內的氧化新陳代謝中沒有重要差別。我們得出如下結論，HBSS的簡單重複沖洗法是比dithiothreitol（DTT）法能維持引痰痰液中之嗜中性白血球表面分子和細胞活性的一個更好的方法。

關鍵字

簡單重複沖洗法；引痰；呼吸道發炎

並列摘要

Treatment of sputum with dithiothreitol (DTT) is widely used as a reliable measurement of cellular and fluid-phase biochemical markers of airway inflammation. However, the cytotoxic effect of DTT may damage the cell membrane and then interfere with surface biochemical markers or intracellular metabolism. We investigated the extent to which DTT treatment influences neutrophil activities as compared with a method by simple washing with 3 aliquots of 9 ml of Hank’s buffer salt solution+10% fetal calf serum (HBSS) through a nylon mesh to retrieve cells in the sputum samples. Hypertonic (3%) saline-induced sputa, collected from 5 bronchiectasis subjects, were examined within 2 h. All portions which looked more solid (less fluidy) than saliva were collected from the expectorate. The selected sputum was then divided into two portions: one treated with one volume of DTT plus one volume of phosphate buffered saline (PBS), the other treated with HBSS. The filtrates were assessed blind for total and differential cell count, as well as viability. The expression of surface molecules, CD11b, CD18 and intracellular oxidative metabolism of neutrophils were determined by flow-cytometry. Sputum treated with DTT in a concentration greater than 1% had lower proportions of viable cells compared with HBSS. In contrast, there were more cellular counts retrieved by DTT-treatment. There was no significant difference in the proportion of eosinophils, neutrophils, lymphocytes, or macrophages between two methods. DTT depressed the levels of CD11b, CD18 expression on neutrophils retrieved from either sputum [78.2 ±15.7 mean fluorescence intensity (MFI) and 73.5±17.5 MFI, respectively, n=5, P＜0.05] or peripheral blood (26.4±2.0 and 20.9±1.0 MFI, respectively, n=6, P＜0.05) compared with those by simple washing method (250.2±30.4 and 220.0±22.6 MFI, respectively for sputum; and 50.6±2.8 and 51.1±2.0 MFI, respectively, for peripheral blood). DTT, but not simple washing method, also depressed the lipopolysaccharide-induced cytokines release from neutrophils. There was no significant difference in intracellular oxidative metabolism between two treatments. We conclude that simple washing with HBSS compared with DTT method preserves cell membrane surface molecules and cellular activity in retrieving neutrophils from induced sputum samples.

並列關鍵字

dithiothreitol ； simple washing method ； induced sputum ； airway inflammation

國際替代計量

Simple Washing Compared to Dithiothreitol (DTT) Method as a Better Method to Preserve Neutrophil Activity in Induced Sputum

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