Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-gamma, and GM-CSF and the necessity to overcome the action of endogenous proteases.
Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases.
McDonald
P P
PP
Department of General Pathology, University of Verona, Italy.
Bovolenta
C
C
Cassatella
M A
MA
D002458:Cell Fractionation; D004268:DNA-Binding Proteins / Q000378:metabolism; D003902:Detergents; D010450:Endopeptidases / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016328:NF-kappa B / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects / Q000235:genetics; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D003902:Detergents; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011480:Protease Inhibitors; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010450:Endopeptidases