Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils.
In vitro studies of neutrophil adhesion generally utilise purified populations and are often performed at 37 degrees C. This study determines the effects of temperature changes and neutrophil separation procedures on the expression of cell adhesion molecules on neutrophils. We found that neutrophil separation procedures involving erythrocyte sedimentation and hypotonic lysis are associated with a significant increase in the expression of both a structural and functional epitope of the beta 2 integrin CD11b, an increase in the expression of sialyl Lewisx (CD15s) and the hyaluronate receptor (CD44) as well as a significant decrease in L-selectin (CD62L) expression. Separated neutrophils are also more resistant than unseparated neutrophils to PMA induced upregulation of a functional epitope of CD11b. Incubating neutrophils at 37 degrees C is associated with increases in the expression of structural and functional epitopes of CD11b. Neutrophil separation is also associated with increases in the expression of both structural and functional epitopes of CD11b which is greater when neutrophil separation is performed at room temperature compared with neutrophil separation at 0-4 degrees C. However, this difference is lost when the latter are incubated at 37 degrees C. Furthermore, neutrophil separation at both 0-4 degrees C and room temperature is associated with a significant increase in CD15s expression. This increase is less when separation is performed at room temperature. These findings suggest that neutrophil separation should be performed at room temperature unless the cells are going to be used at 0-4 degrees C. Researchers using purified neutrophil populations need to be aware of these significant structural and functional changes when extrapolating in vitro results to in vivo situations.
Youssef
P P
PP
Rheumatology Unit, Flinders Medical Centre, Adelaide, South Australia, Australia.
Mantzioris
B X
BX
Roberts-Thomson
P J
PJ
Ahern
M J
MJ
Smith
M D
MD
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000235:genetics; D007038:Hypotonic Solutions / Q000494:pharmacology; D019041:L-Selectin / Q000096:biosynthesis / Q000235:genetics; D016256:Lewis X Antigen / Q000096:biosynthesis / Q000235:genetics; D016177:Macrophage-1 Antigen / Q000096:biosynthesis / Q000235:genetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013048:Specimen Handling; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D018960:Hyaluronan Receptors; D007038:Hypotonic Solutions; D016256:Lewis X Antigen; D016177:Macrophage-1 Antigen; D019041:L-Selectin; D013755:Tetradecanoylphorbol Acetate