A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist.
BACKGROUND
Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors.


METHODS
We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses.


RESULTS
In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients.


CONCLUSIONS
Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.
Nicholson
Grant C
GC
National Heart and Lung Institute (NHLI), Clinical Studies Unit, Imperial College London, Royal Brompton Hospital, Fulham Road, London SW3 6HP, UK.
Tennant
Rachel C
RC
Carpenter
Donald C
DC
Sarau
Henry M
HM
Kon
Onn Min
OM
Barnes
Peter J
PJ
Salmon
Michael
M
Vessey
Rupert S
RS
Tal-Singer
Ruth
R
Hansel
Trevor T
TT
D015703:Antigens, CD / Q000032:analysis; D039481:CD11b Antigen / Q000032:analysis*; D048430:Cell Shape; D054371:Chemokine CXCL11; D019743:Chemokines, CXC / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D058851:GPI-Linked Proteins; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D029424:Pulmonary Disease, Chronic Obstructive / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D017452:Receptors, IgG / Q000032:analysis; D023063:Receptors, Interleukin-8B / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D015854:Up-Regulation / Q000187:drug effects
D015703:Antigens, CD; D039481:CD11b Antigen; C515202:CXCL11 protein, human; D054371:Chemokine CXCL11; D019743:Chemokines, CXC; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D017452:Receptors, IgG; D023063:Receptors, Interleukin-8B