Failure to detect interleukin (IL)-3 mRNA or protein in human keratinocytes: antibodies to granulocyte macrophage-colony-stimulating factor or IL-6 (but not IL-3) neutralize "IL-3" bioactivity.
Interleukin (IL)-3-like bioactivity has been found in culture supernatants from human and murine keratinocytes. However, there is controversy as to the presence of IL-3 mRNA in human keratinocytes. Using highly sensitive reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay techniques, we examined human keratinocytes from four different donors (neonatal foreskins) and were unable to detect IL-3 mRNA or IL-3 protein. Despite successful amplification of DNA from an IL-3 cDNA, no product could be obtained by amplification of keratinocyte RNA treated with reverse transcription-polymerase chain reaction. Analysis of concentrated (up to 50-fold) supernatants failed to detect IL-3 protein by enzyme-linked immunosorbent assay. Because ultraviolet radiation up-regulates many cytokines, we irradiated human keratinocytes with 300 J/m2 ultraviolet B and collected supernatants 24 h post-irradiation. Supernatants concentrated 50-fold were also negative for IL-3 protein by enzyme-linked immunosorbent assay. When assayed on the IL-3-responsive M-07e cell line, unirradiated supernatants stimulated M-07e proliferation 22-fold over background levels. Irradiated supernatants stimulated M-07e proliferation 128-fold. Neither the unirradiated nor the irradiated supernatant activity could be neutralized with antibody to human IL-3. However, incubation of irradiated supernatants with antibody to granulocyte macrophage-colony-stimulating factor (GM-CSF) reduced the M-07e proliferation by 90%. Antibodies against GM-CSF and IL-6 completely abrogated proliferation. Reverse transcription-polymerase chain reaction confirmed a concomitant elevation of IL-6 (2.6- to 5.6-fold) and of GM-CSF mRNA (2.7- to 4.3-fold) at 6 and 24 h after ultraviolet B irradiation in keratinocytes, but no IL-3 amplification products could be detected. IL-3 mRNA was also not detected in adult keratinocytes. Even after stimulation by IL-1 alpha, tumor necrosis factor-alpha, or phobol myristate acetate, IL-3 mRNA was not detected in either neonatal or adult human keratinocytes. We have been unable to detect IL-3 mRNA or IL-3 protein in human keratinocytes. The IL-3-like activity in human keratinocytes is mainly due to GM-CSF, with a small contribution from IL-6.
Kondo
S
S
Division of Dermatology, Sunnybrook Health Science Centre, University of Toronto, Ontario, Canada.
Ciarletta
A
A
Turner
K J
KJ
Sauder
D N
DN
McKenzie
R C
RC
D000906:Antibodies; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology; D006801:Humans; D007231:Infant, Newborn; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D015603:Keratinocytes / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays
D000906:Antibodies; D007377:Interleukin-3; D015850:Interleukin-6; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate