Intragraft cytokine mRNA levels in human liver allograft rejection analysed by reverse transcription and semiquantitative polymerase chain reaction amplification.
Cytokine gene expression was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of RNA from 27 human liver allograft specimens diagnosed as acute (n = 19) or chronic (n = 8) rejection and from 12 normal human livers. In initial screening experiments, mRNA for cytokines interleukin (IL)-1 beta, IL-6, IL-10 and gamma-interferon (IFN-gamma) was expressed in all normal livers and almost all allograft specimens tested. IL-2 mRNA was expressed at barely detectable levels in four of 12 normal livers screened and in 20 of 26 liver allograft specimens with rejection. This constitutive expression of cytokine mRNA required semiquantitative PCR analysis to differentiate levels of cytokine mRNA expression between specimens. Titration of cDNA prior to PCR amplification was initially used and showed significantly more IL-2 (p = 0.02) and IFN-gamma (p = 0.03) in acute rejection compared to normal liver. There was also significantly less IL-10 in chronic rejection compared to acute rejection (p = 0.02) or normal liver (p = 0.01) and less IL-6 in acute rejection compared to chronically rejecting liver (p = 0.05). IL-1 beta (p = 0.04) and IL-6 (p = 0.01) were reduced in acute rejection compared to normal liver. The slight increase of IL-2 in acute rejection and the slight decrease of IL-10 in chronic rejection was confirmed by a second semiquantitative analysis which involved removal of aliquots of PCR reaction at successive cycles followed by dot-blotting and hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)
Bishop
G A
GA
AW Morrow Department of Gastroenterology, Royal Prince Alfred Hospital, Camperdown, NSW Australia.
Rokahr
K L
KL
Napoli
J
J
McCaughan
G W
GW
D000208:Acute Disease; D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002675:Child, Preschool; D002908:Chronic Disease; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D006084:Graft Rejection / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase
D016207:Cytokines; D007375:Interleukin-1; D015850:Interleukin-6; D012333:RNA, Messenger; D016753:Interleukin-10; D007371:Interferon-gamma; D012194:RNA-Directed DNA Polymerase