The Regulation of Procalcitonin Production in Human Peripheral Blood Mononuclear Cells
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목 적 : 프로칼시토닌은 중증 전신성 세균 감염의 새로운 표지자로 이용되고 있다. 프로칼시토닌의 구조는 감상선에서 분비되는 칼시토닌의 전구체로, 카타칼신과 칼시토닌으로 알려져 있으나, 패혈증의 발병에서 프로칼시토닌의 합성장소와 그 역할은 아직 분명하지 않다. 본 연구는 어떤 말초혈액세포가 내독소와 PMA로 자극시에 PCT분비를 유도할 수 있는지와 어떤 기전에 의해 PCT 생성이 조절되는지를 보고자 한다.재료 및 방법 : 말초혈액 단핵구세포를 분리하여, 각각 LPS (1 ug/mL)나 PMA (5 ng/mL)가 들어있는 배지에 하룻 밤 배양시킨 뒤 다음 날 세포내 프로칼시토닌을 FITC-항 카타칼신 항체를 이용하여 측정하였다. 단핵구와 림프구는 각각 phycoerthrin-CD14 항체와 Cychrome- CD3 항체를 이용하였다. 기전에 대한 분석실험은 여러 자극제를 말초혈액 단핵구세포에 넣기 1시간 전 수용성 TNF 항체 10 ug/mL로 세포를 미리 처리한 뒤 유세포분석기를 이용하여 분석하였다.결 과 : LPS는 건강한 공여자에서 얻은 말초혈액 단핵구에서 대조군의 프로칼시토닌 생성을 10.0%에서 27.2%로 증가시켰고 PMA는 10.0% 에서 40.8%로 더 많은 PCT생성의 증가를 보였다(n=8 중 대표). CD3 양성 림프구에서는 LPS는 PCT생성을 시키지 못하였으나 PMA는 프로칼시토닌 생성을 2.35배까지 증가시켰다(P<0.05, n= 8). 같은 공여자의 말초혈액 단핵구에서 수용성 TNF 항체는 LPS로 자극된 PCT생성을 감소(대조군 10.0%, LPS군 27.2%, 수용성 TNF 항체 12.3%)시켰으나, PMA자극으로 생성된 프로칼시토닌에는 영향을 주지 못하였다. 패혈증 환자에서는 건강한 군에 비해 PMA가 LPS보다 더 많은 PCT생성을 보였다.
Abstract
Background : Procalcitonin (PCT) is a new marker of severe systemic bacterial infection. PCT consists of fragments katacalcin and calcitonin, which are precursors of calcitonin in thyroid. The source and role of PCT in pathogenesis of sepsis remains clarified. This study was focused on which subsets of peripheral blood mononuclear cells (PBMC) can induce PCT when they are stimulated with endotoxin or phorbol myristate acetate (PMA), and how the PCT production is controlled. Materials and Methods : PBMC were isolated and incubated overnight in each media containing 1 ug/mL lipopolysaccharide (LPS), or 5 ng/mL PMA. Intracellular PCT was detected using fluorescein isothiocyanate (FITC) labeled anti-katacalcin antibody (Ab). Monocytes and lymphocytes were identified by phycoerythrin-conjugated CD14 Ab and CyChrome-conjugated CD3 Ab, respectively. Ten micrograms of soluble TNF receptor (sTNFR) were pretreated in PBMC 1 hr prior to adding the stimuli. Then, PBMC were analyzed using a flow cytometer. Results : LPS increased intracellular PCT from 10.0 % to 27.2% in CD14-positive monocytes from healthy donors, but PMA induced more PCT production from 10.0% to 40.8%. (one representative, n=8). For CD3-p ositive lymphocytes, LPS did not stimulate PCT, but PMA increased PCT production by 2.35 fold (P<0.05, n=8). In the PBMC from the same donor, sTNFR highly decreased LPS-stimulated PCT (control 10.0%, LPS 27.2%, sTNFR 12.3%), but it did not significantly affect PMA-stimulated PCT. For sepsis patients, PMA stimulated more PCT than LPS did and PCT was more expressed compared with healthy donors. Conclusion : The PCT was produced in both monocytes and lymphocytes. PMA stimulated more PCT production than LPS did. The LPS-induced PCT production is partly mediated through TNF-α production.
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