A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1.
Lipopolysaccharide (LPS) triggers a wide range of cellular responses in mammalian cells. Several proteins, including CD14, have been reported to possess LPS binding capacity. However, the signal transduction molecule(s) and pathway(s) through which LPS induces cellular responses have not been identified. A rabbit antiserum (5299), which had been made against the N-terminal 20-amino-acid sequence of the cytokine, soluble immune response suppressor, unexpectedly activated the human monocyte cell line THP-1 as assessed by tumor necrosis factor (TNF alpha) production. Normal rabbit serum or IgG caused no TNF production, while antiserum 5299 induced TNF at a 1:10(4) dilution. The activity of antiserum 5299 was not due to endotoxin contamination, since antiserum 5299 contained less endotoxin than required for activation of THP-1 and the TNF-inducing activity disappeared when the antiserum was boiled. Moreover, the activity was recovered in purified immunoglobulin G fraction from the antiserum (5299 IgG). These results suggest that antiserum 5299 contains an antibody which recognizes a signal-transducing molecule. THP-1 desensitized by incubation with LPS for 24 hr could not respond to LPS or antiserum 5299 while responsiveness to phorbol ester remained, suggesting that the signals induced by the antiserum 5299 and LPS are transduced through at least a partially common pathway. Western blotting with antiserum 5299 showed a 69-kDa protein which rapidly appeared in Triton X-100-insoluble fraction after stimulation with LPS or 5299 IgG, suggesting an association of 69-kDa protein with the cytoskeleton. Furthermore, two mutant cell lines were isolated (T-15 and T-25) from gamma-irradiated THP-1 which lack responsiveness to LPS. In these mutant cell lines, the 69-kDa protein was not observed in Triton X-100-insoluble fraction after stimulation with LPS. It is proposed that this protein may play an important role in LPS-stimulated signal transduction.
Fukuse
S
S
Center for Inflammation Research, Syntex, Inc., Palo Alto, California 94304, USA.
Maeda
T
T
Webb
D R
DR
Devens
B H
BH
D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D007158:Immunologic Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008970:Molecular Weight; D009000:Monocytes / Q000502:physiology*; D019000:Phosphotyrosine; D011971:Receptors, Immunologic / Q000737:chemistry*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D008070:Lipopolysaccharides; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D014409:Tumor Necrosis Factor-alpha; D019000:Phosphotyrosine; D014443:Tyrosine