The Expression of Perforin, Fas-ligand, and Granzyme B in Peripheral Blood Lymphocytes of Renal Allograft Recipients

Abstract

Background: Previous findings demonstrated that the expression of cytotoxic effector molecules is increased in acute rejection of renal allografts. In the present study, we serially examined the gene expression of perforin, granzyme B and Fas ligand(FasL) in peripheral blood lymphocytes(PBLs) of renal allograft recipients to assess the potential of their expression as a marker of acute rejection. Methods: PBLs were isolated from blood samples taken on days 2, 4, 6, 8, 10 and 12 after transplantation. Competitive PCR was performed to evaluate the abundance of mRNA of perforin, granzyme B and FasL. The mean value of each molecule plus 2 SD for the control group was set as a discriminatory level. Results: When all measured samples were compared, perforin expression was significantly higher in patients with acute rejection than in the control group(1.84±3.01 versus 0.71±0.48, p=0.01). The percentage of perforin expression exceeding the discriminatory level was also significantly higher in patients with acute rejection (p=0.0003). Five patients in the rejection group(5/7, 71.4%) showed perforin expression exceeding the discriminatory level, while only 1 patient in the control group did so(1/8, 12.5%)(p=0.02). Perforin expression of days 0 and 1 of rejection crisis was the highest over the study period. No consistent pattern of granzyme B and FasL expression was identified in relation to rejection crisis. Conclusions: Gene expression of perforin by PBLs was upregulated in accordance with acute rejection, thus offering the possibility that it may be utilized as a marker of acute rejection.

배경: 최근의 여러 연구를 통하여 신이식 환자의 급성거부반응 시에 세포독성 물질들의 발현이 증가한다고 알려져 있다. 본 연구에서 저자 등은 신이식 환자의 말초 혈액 림프구에서 perforin, granzyme B와 Fas ligand(FasL)의 mRNA 발현을 순차적으로 검사하여, 이들이 급성거부반응을 진단할 수 있는 지표로서 유용한지 평가하고자 하였다. 방법: 말초혈액 림프구를 신이식후 2, 4, 6, 8, 10, 12일에 환자의 혈액으로부터 분리하여, perforin, granzyme B, FasL의 mRNA에 대한 발현 정도를 competitive polymerase chain reaction으로 평가하였다. 결과는 β-Actin의 값으로 나누어 보정하였다(fg/pg). 대조군에서 perforin, granzyme B, FasL 각각의 평균값＋2×표준편차를 기준상한치로 설정하였다. 결과: Perforin mRNA 발현의 평균치는 대조군(8명, 47개)에 비해 급성거부반응 군(7명, 41개) 에서 현저히 높게 나타났다(1.84±3.01 vs 0.71±0.48, p=0.01). 기준상한치(1.67)를 초과하는 perforin mRNA 발현의 수도 급성거부반응군에서 현저히 높았다(12/41 vs. 1/47, p=0.0003). 급성거부반응 군에서 5명이 기준상한치를 초과하는 perforin mRNA 발현을 보인 반면(5명/7명, 71.4%), 대조군에서는 한 명만이 이러한 결과를 보였다(1명/8명, 12.5%, p=0.02). 거부반응 0-1일째의 perforin mRNA 발현이 연구 기간 중 가장 높게 나타났다. Granzyme B와 FasL mRNA 발현은 급성거부반응 시기와 일정한 연관성을 보이지 않았다. 결론: 본 연구에서 말초혈액 림프구의 perforin mRNA 발현이 급성거부반응시에 유의하게 증가되었으며, perforin mRNA 발현을 급성거부반응의 진단에 이용할 가능성을 제시하였다.