Quantitation of IL-4 expression in small numbers of cells from mice.
Interleukin-4 (IL-4) is an important T cell and mast cell product that participates in allergic and cytotoxic responses, as well as functions as a growth factor for B, T, and inflammatory cells. Studies of the expression of IL-4 by T cells present in inflammatory reactions would be facilitated by using polymerase chain reaction (PCR) coupled to reverse transcription of mRNA to amplify the small quantity of mRNA present in these cells. In order to use this method in a quantitative manner, a plasmid was constructed that contained a modified form of mouse IL-4 cDNA. This plasmid was transcribed to produce cRNA for this modified sequence. The cRNA was used as an internal standard for the reverse transcription and amplification of IL-4 transcripts in RNA samples from mouse thymocytes. Amplification of reverse-transcribed native IL-4 mRNA produced a 286 bp PCR product. Amplification of the reverse-transcribed standard RNA produced a 155 bp product, which reflected a deletion introduced into the original IL-4 cDNA sequence. Comparison of the amount of the 286 bp native product to the amount of 155 bp standard product enabled the quantitative determination of IL-4 expression in each sample. This method was used to demonstrate that platelet activating factor increases the expression of IL-4 in mouse thymocytes and in a mouse T cell line. The expression of IL-4 by thymocytes exposed to platelet-activating factor (PAF) may reveal an important link between inflammation and the maturation of T cells in the thymus.
Barthelson
R A
RA
VA Medical Center San Francisco, CA 94121.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D015847:Interleukin-4 / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010972:Platelet Activating Factor / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology*
D009838:Oligodeoxyribonucleotides; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D015847:Interleukin-4