Growth factors G-CSF and GM-CSF differentially preserve chemotaxis of neutrophils aging in vitro.
OBJECTIVE
The ability of human neutrophils to migrate was studied during culture in vitro.


METHODS
Neutrophils were isolated from human blood and cultured at 37 degrees C. Apoptosis was determined by Annexin-V fluorescein isothiocyanate binding. Receptor expression was measured by fluorescence in situ hybridization analysis with monoclonal antibodies. Migration was assessed with Transwell Fluoroblock inserts and calcein-stained neutrophils. Extracellular signal-regulated kinase 1/2 (ERK-1/2) activation was determined with monoclonal antibody against phosphorylated ERK-1/2.


RESULTS
Upon culture, untreated neutrophils downregulated the chemotaxin receptors FPR, CXC chemokine receptor 1, and CXC chemokine receptor 2 and lost the ability to migrate to formyl-methionyl-leucyl-phenylalanin, interleukin 8 (IL-8), and C5a. In contrast, expression of CXCR4 was induced; this receptor was able to signal (increase in intracellular free calcium ions [Ca(2+)](i), ERK-1/2 activation) but was nonfunctional (no chemotaxis to stromal cell-derived factor-1alpha). The myeloid growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) retarded the process of functional decay during cell culture. However, while preserving chemotaxis of neutrophils toward formyl-methionyl-leucyl-phenylalanin or C5a, GM-CSF-in contrast to G-CSF-did not preserve chemotaxis toward IL-8, with a corresponding downregulation of the IL-8 receptors. The decay in neutrophil chemotaxis occurred prior to detectable phosphatidylserine (PS)-exposure. In contrast, the induction of [Ca(2+)](i) rises and ERK-1/2 activation correlated with chemotaxin receptor expression unless the cells were truly apoptotic.


CONCLUSIONS
Neutrophils aging in vitro lose their chemotactic capacity. Functional decay starts prior to PS exposure and can be partially prevented by G-CSF and GM-CSF, in a differential fashion. These growth factors act by increasing the number of viable neutrophils, by altering the levels of chemotaxin receptor expression, and-independently-by affecting signaling cascades.
Wolach
Baruch
B
Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. baruchw@clalit.org.il
van der Laan
Luc J W
LJ
Maianski
Nikolai A
NA
Tool
Anton T J
AT
van Bruggen
Robin
R
Roos
Dirk
D
Kuijpers
Taco W
TW
D017209:Apoptosis; D015153:Blotting, Western; D020013:Calcium Signaling; D016922:Cellular Senescence / Q000187:drug effects*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005456:Fluorescent Dyes; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology
D003470:Culture Media; D005456:Fluorescent Dyes; D016209:Interleukin-8; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D048049:Extracellular Signal-Regulated MAP Kinases