FAILURE TO DETECT INTERLEUKIN (IL)-3 MESSENGER-RNA OR PROTEIN IN HUMAN KERATINOCYTES - ANTIBODIES TO GRANULOCYTE MACROPHAGE-COLONY-STIMULATING FACTOR OR IL-6 (BUT NOT IL-3) NEUTRALIZE IL-3 BIOACTIVITY

JOURNAL OF INVESTIGATIVE DERMATOLOGY

卷:104

Article

KONDO, S ; CIARLETTA, A ; TURNER, KJ ; SAUDER, DN ; MCKENZIE, RC

关键词: BIOASSAY;    RT-PCR;    UVB;    GM-CSF;   

DOI  :  10.1111/1523-1747.ep12665370

来源: Elsevier

【 摘 要 】

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/m(2) ultraviolet B and collected supernatants 24 h post-irradiation. Supernatants concentrated 50-fold were also negative for IL-3 protein by enzyme-linked immnunosorbent 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, tuner 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.

【 授权许可】

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