Detection of cytokine mRNA-expressing cells in peripheral blood of patients with IgA nephropathy using non-radioactive in situ hybridization.
IgA nephropathy (IgA-N) is considered to be an immune-mediated disorder and several immunological abnormalities have been observed. In the present study, we optimized non-radioactive in situ hybridization and applied this technique to evaluate the degree of expression of various cytokine mRNAs in peripheral blood mononuclear cells (PBMC) taken from patients with IgA-N on cytospin preparation. Using this method, together with image analysis, we examined the expression of mRNA in cells which secrete cytokines, such as IL-2, interferon-gamma (IFN-gamma), IL-4, IL-5 and IL-6. The expression of these mRNAs was clearly observed in monocytes and lymphocytes at a single-cell level. Compared with healthy adults, the expression of IL-4 mRNA, IL-5 mRNA and IL-6 mRNA, but not IL-2 mRNA or IFN-gamma mRNA, was significantly enhanced. Our results indicate that non-radioactive in situ hybridization method is a powerful technique for analysis of cytokine mRNAs in PBMC at a cellular level. Our results also suggest that mRNA expression of IL-4, IL-5 and IL-6, produced by Th2 cells, is increased in patients with IgA-N. The abnormal regulatory process involved in cytokine expression may play an important immunopathologic role in IgA-N.
Ichinose
H
H
Second Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
Miyazaki
M
M
Koji
T
T
Furusu
A
A
Ozono
Y
Y
Harada
T
T
Shin
M
M
Nakane
P K
PK
Hara
K
K
D000328:Adult; D016207:Cytokines / Q000235:genetics*; D004076:Digoxigenin; D005404:Fixatives; D005922:Glomerulonephritis, IGA / Q000097:blood*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D015850:Interleukin-6 / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000096:biosynthesis*
D016207:Cytokines; D005404:Fixatives; D015850:Interleukin-6; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004076:Digoxigenin