Cell culture conditions affect LPS inducibility of the inflammatory mediators in J774A.1 murine macrophages.
Peripheral blood monocytes and tissue macrophages contribute significantly to the inflammatory response. Bacterial lipopolysaccharide (LPS) has profound effects on these cells including, but not limited, to differentiation into macrophages, production of reactive nitrogen and oxygen species and secretion of pro-inflammatory cytokines. Herein, we describe a variant of the J774A.1 murine macrophage line that is reversibly resistant to multiple effects of LPS when cultured in different types of media. J774A.1 cells are adherent and spread out when cultured in DMEM/F12; however, when cultured in RPMI 1640, the cells are rounded and relatively non-adherent. Different types of tissue culture plates, sera, and media supplements were not responsible for these changes. We examined LPS-induced reactive nitrogen species using the Greiss reagent. J774A.1 cells cultured in RPMI exhibit a 5-fold increase in nitrites in culture supernatants after LPS stimulation whereas those in DMEM/F12 do not. Zinc staining of total cellular protein of cells in COHLY ET AL. RPMI and DMEM/F12 electrophoresed on a SDS-PAGE showed noticeable banding differences. LPS-induced cytokine gene expression was studied by RT-PCR. LPS induced TNF-alpha, IL-1alpha, IL-1beta, and sIL-1Ra in cells cultured in RPMI but not those cultured in DMEM/F12 with the exception of TNFalpha. This report shows that environmental factors contained in the culture medium alone can reversibly alter the biochemical nature of monocytes and macrophages.
Cohly
H
H
Department of Surgery, University of Mississippi Medical Center, Jackson, USA. hcohly@surgery.umsmed.edu
Stephens
J
J
Markhov
A
A
Angel
M
M
Campbell
W
W
Ndebele
K
K
Jenkins
J
J
D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D048429:Cell Size; D003470:Culture Media; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D004351:Drug Resistance; D015870:Gene Expression / Q000187:drug effects; D018836:Inflammation Mediators / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D011506:Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide