Expression of interleukin-1 beta in a human keratinocyte cell line.
The human keratinocyte cell line COLO-16 expresses mRNA homologous to human IL-1 alpha and IL-beta (transcript sizes 2.3 and 1.6 kb, respectively). A 1.2 kbp cDNA was selected with a human IL-1 beta probe from a lambda gt11 library constructed using poly A+ RNA from COLO-16 cells. Sequence analysis revealed that this cDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1 beta. When this cDNA was expressed in COS cells using a mammalian expression vector, IL-1 activity was detected in the cell conditioned supernatants using assays for D10-T-cell, thymocyte and fibroblast proliferation. Western analysis of lysates from COS cells transfected with this clone revealed the presence of a -17 kDa protein which reacted with antisera to human IL-1 beta. This protein was the same size as the processed form of IL-1 beta present in COLO-16 cells suggesting that this cDNA encodes the mature form of IL-1 beta. COLO-16 cells contain proteins of -30 kDa and 17 kDa which are immunoreactive with specific antisera for human IL-1 alpha and human IL-1 beta. Despite the presence of four-fold greater amounts of immunoreactive IL-1 beta protein than IL-1 alpha in cell lysates, all the IL-1 activity in the lysate could be neutralized by antisera to IL-1 alpha. IL-1 beta comprised only 25% of the IL-1 activity in the cell-conditioned media, all remaining activity was neutralized by antisera to IL-1 alpha. Whereas IL-1 alpha protein in both cell lysates and conditioned supernatants was predominantly in the processed -17 kDa form, IL-1 beta proteins were primarily of the processed and inactive 30kDa species. This apparent inability of keratinocytes to process IL-1 beta may explain our observations that the IL-1 activity secreted by COLO-16 cells is principally due to IL-1 alpha.
McKenzie
R C
RC
Department of Medicine, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada.
Arsenault
T V
TV
Sauder
D N
DN
Harley
C B
CB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D015603:Keratinocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics
D007375:Interleukin-1; D012333:RNA, Messenger; D004247:DNA