SIGNAL-TRANSDUCING PROPERTIES OF THE N-FORMYL PEPTIDE RECEPTOR EXPRESSED IN UNDIFFERENTIATED HL-60 CELLS

Citation
Er. Prossnitz et al., SIGNAL-TRANSDUCING PROPERTIES OF THE N-FORMYL PEPTIDE RECEPTOR EXPRESSED IN UNDIFFERENTIATED HL-60 CELLS, The Journal of immunology, 151(10), 1993, pp. 5704-5715
Citations number
49
Categorie Soggetti
Immunology
Journal title
The Journal of immunology
ISSN journal
00221767 → ACNP
Volume
151
Issue
10
Year of publication
1993
Pages
5704 - 5715
Database
ISI
SICI code
0022-1767(1993)151:10<5704:SPOTNP>2.0.ZU;2-H
Abstract
Differentiated HL60 cells respond to challenge with ligand by mobilizi ng intracellular second messengers, resulting in superoxide Production , degranulation, and actin polymerization with subsequent chemotaxis a nd phagocytosis. The functional capabilities of undifferentiated HL60 cells have not been similarly characterized due to the absence of the cell surface receptors required to initiate these processes. To invest igate these properties, undifferentiated HL60 cells were transfected w ith one of the better characterized neutrophil chemotactic receptors, the N-formyl peptide receptor (FPR). Expression of the recombinant FPR gene product in FPR-transfected HL60 cells and the absence of the end ogenous FPR in vector-transfected HL60 cells was demonstrated by North ern blot and flow cytometric analyses. FPR-transfected HL60 cells reta ined their ability to undergo granulocytic differentiation with dibuty ryl cAMP, as determined by FMLP- and PMA-stimulated superoxide product ion. Furthermore, incubation of FPR-transfected HL60 cells for 5 days in the presence of FMLP resulted in limited differentiation as evidenc ed by the expression of functional C5a receptors. Binding studies of F PR-transfected HL60 cells demonstrated the presence of two binding aff inities with dissociation constants of 0.6 and 33 nM, similar to dibut yryl cAMP differentiated HL60 cells and human neutrophils but contrast ing the single high affinity state of the FPR expressed in mouse L cel l fibroblasts. FPR-transfected HL60 cells displayed FMLP-dependent cal cium mobilization with an EC50 of 3 nM and actin polymerization with a n EC50 of approximately 10 nM. Actin polymerization was not observed i n FPR-transfected L cell fibroblasts or undifferentiated vector-transf ected HL60 cells. Both calcium mobilization and actin polymerization w ere sensitive to treatment with pertussis toxin, indicating the requir ement for a G(i)-like protein. Stimulation of either undifferentiated or differentiated HL60 cells with ATP resulted in pertussis toxin-inse nsitive calcium mobilization but was ineffective in producing actin po lymerization. The results described herein show for the first time tha t undifferentiated HL60 cells can respond to chemoattractant receptor stimulation with many of the properties of the mature neutrophil. Tran sfected HL60 cells will provide an excellent system to study the chara cteristics of chemotactic receptors as well as the functional properti es of myeloid cells.