| Summary: | Mononuclear phagocyte activation enhances the functional properties of these cells, and
endotoxin (bacterial lipopolysaccharide) is one of the most potent agonists known to cause an
activated state. This model was used to investigate signaling pathways that regulate macrophage
activation. Exposure of mononuclear phagocytes to lipopolysaccharide resulted in rapid and
dose-dependent increases In tyrosine phosphorylation of a set of proteins. This was due, at least
in part, to increased tyrosine kinase activity. In particular, compared to untreated cells,
lipopolysaccharide-treated monocytes showed increased activity of the Src-family tyrosine kinase
p53/56lyn. In addition, both tyrosine kinase activity, as well as tyrosine phosphorylated
proteins without endogenous kinase activity, associated with the lipopolysaccharide receptor
CD 14 in an inducible manner.
Lipopolysaccharide treatment of monocytes also activated the lipid kinase,
phosphatidylinositol 3-kinase in a dose and time dependent manner. Activation of this enzyme
resulted in increased levels of D3-phosphorylated phosphoinositides and this response was
mediated through CD 14. Activation of phosphatidylinositol 3-kinase also resulted in its
association with activated p53/56lyn without leading to a detectable change in the tyrosine
phosphorylation state of the p85 regulatory subunit of the enzyme.
Increased protein kinase activity is not restricted to tyrosine kinases, as
lipopolysaccharide also activated serine/threonine kinases. Anion exchange chromatography of
endotoxin-treated mononuclear phagocytes showed increased myelin basic protein-kinase
activity eluting as two major peaks. Based on immunoreactivity and substrate preference, the
earliest eluting peak, peak "one" was identified as p42/p44 mitogen-activated protein kinases.
Tyrosine phosphorylation of both p42 and p44 mitogen-activated protein kinases increased in
response to lipopolysaccharide. The second peak of myelin basic protein-kinase activity, peak
"two", was identified as protein kinase C-ζ, an atypical isoform of protein kinase C. This
conclusion is based on immunoreactivity, substrate preference, and cofactor independency of
the enzyme.
The cellular mechanisms regulating the activity of protein kinase C-ζ were investigated.
Protein kinase C-ζ activation by lipopolysaccharide occurred downstream of phosphatidylinositol
3-kinase. This conclusion is based on two findings: (i) phosphatidylinositol 3-kinase inhibitors
blocked activation of protein kinase C-ζ, and (ii) transfection of cells with a dominant negative
mutant of phosphatidylinositol 3-kinase impaired activation of protein kinase C-ζ by
lipopolysaccharide.
Functional responses of mononuclear phagocytes dependent on signaling pathways
involving phosphatidylinositol 3-kinase were examined. Phosphatidylinositol 3-kinase inhibitors
abrogated lipopolysaccharide-induced adherence, but not adherence induced in response to
phorbol 12-myristate 13-acetate in the monocytic cell line THP-1. In contrast, induction of
transcription of cytokine genes by lipopolysaccharide was independent of phosphatidylinositol
3-kinase.
In conclusion, examination of lipopolysaccharide signaling in mononuclear phagocytes
showed that endotoxin activates both protein and lipid kinases including p53/56lyn, p42 and
p44 isoforms of mitogen activated protein kinase and phosphatidylinositol 3-kinase through a
CD-14 dependent mechanism. Activation of PKC-ζ is phosphatidylinositol 3-kinase dependent
and signaling through this lipid kinase regulates monocyte adherence, but not cytokine
production induced by lipopolysaccharide. Given the requirement that monocytes must adhere
to endothelium, to other leukocytes and to extracellular matrix proteins in order to mediate
functional responses, it may be possible to modify macrophage responses to lipopolysaccharide
and perhaps other agonists by targeting phosphatidylinositol 3-kinase. [Scientific formulae used in this abstract could not be reproduced.]
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