Detection of neutrophil-activating peptide NAP/IL-8 and NAP/IL-8 mRNA in human recombinant IL-1 alpha- and human recombinant tumor necrosis factor-alpha-stimulated human dermal fibroblasts. An immunocytochemical and fluorescent in situ hybridization study.
A neutrophil-activating peptide (NAP)/IL-8 produced by LPS-stimulated human peripheral blood monocytes was biochemically purified and functionally characterized by different investigators. Work conducted in our laboratory showed that NAP/IL-8 as well as variants of this peptide are produced by a variety of cells (e.g., monocytes, T lymphocytes, endothelial cells) and that lesional psoriatic scales contain large amounts of biologically active NAP/IL-8. We now investigated human dermal fibroblasts for production of NAP/IL-8. The peptide was detected by immunocytochemistry by using the mAb 46E5. NAP/IL-8 mRNA was visualized by high resolutive fluorescent in situ hybridization with biotinylated antisense/sense RNA probes. Among the various stimuli used [human (h)rIL-1 alpha, hrTNF-alpha, hrIL-3, hr-granulocyte-macrophage-CSF, LPS, FMLP, and platelet-activating factor (PAF)] only hrIL-1 alpha (100 U/ml) and hrTNF-alpha (100 ng/ml) induced the transcription and translation of NAP/IL-8. In contrast to monocytes, LPS was without effect in cultured human dermal fibroblasts. Both NAP/IL-8 and NAP/IL-8 mRNA were found in the cytoplasm adjacent to the nucleus, but interestingly NAP/IL-8 mRNA was not restricted to the cytoplasm. In positive cells only two small bright spots were randomly distributed in the nucleus. Most likely these spots represent transcription sites where NAP/IL-8 mRNA is accumulated during gene expression. Our observations show that stimulation of dermal fibroblasts with the cytokines hrIL-1 alpha and hrTNF-alpha results in expression of IL-8.
Mielke
V
V
Department of Dermatology, University of Kiel, Federal Republic of Germany.
Bauman
J G
JG
Sticherling
M
M
Ibs
T
T
Zomershoe
A G
AG
Seligmann
K
K
Henneicke
H H
HH
Schröder
J M
JM
Sterry
W
W
Christophers
E
E
D002451:Cell Compartmentation; D002630:Chemotactic Factors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D016209:Interleukin-8; D007378:Interleukins / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000502:physiology; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012335:RNA, Ribosomal / Q000378:metabolism; D011994:Recombinant Proteins; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D002630:Chemotactic Factors; D007375:Interleukin-1; D016209:Interleukin-8; D007378:Interleukins; D010455:Peptides; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha