Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin.
OBJECTIVE
To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion.


METHODS
A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct.


METHODS
Basic science research laboratory.


METHODS
Secreted TNF-alpha protein concentration.


RESULTS
After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct.


CONCLUSIONS
The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.
Department of Surgery, University of Minnesota, Minneapolis, USA.
P S
PS
Dahlberg
R D
RD
Acton
M E
ME
Uknis
H G
HG
Klaerner
J W
JW
Johnston
C D
CD
Levelle
B H
BH
Gray
D L
DL
Dunn
D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides; D001770:Blood Bactericidal Activity / Q000276:immunology; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004731:Endotoxins / Q000009:adverse effects*; D016905:Gram-Negative Bacterial Infections / Q000517:prevention & control; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000009:adverse effects; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013530:Surgical Wound Infection / Q000517:prevention & control; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D004731:Endotoxins; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C021623:bactericidal permeability increasing protein