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
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.