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Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production.

Horwood NJ, Mahon T, McDaid JP, Campbell J, Mano H, Brennan FM, Webster D, Foxwell BM - J. Exp. Med. (2003)

Bottom Line: We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region.Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production.This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London W6 8LH, United Kingdom.

ABSTRACT
Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

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The effect of M-CSF treatment on LPS-stimulated TNFα production and Tec expression. (A) Adherent monocytes from XLA and normal donors were either stimulated with LPS for 2 h, or incubated with M-CSF for 48 h before LPS stimulation for 2 h. Supernatants were assayed for TNFα production. (B) Adherent monocytes from normal (lanes 1 and 2) and XLA donors (lanes 3 and 4) were either lysed immediately, or after M-CSF treatment for 48 h. Western blot analysis was performed using either α-TecSH3, α-Btk, or α-actin antibody. (C) Elutriated monocytes from normal donors were stimulated with LPS for the indicated time periods. Tec was immunoprecipitated and in vitro autokinase assay was performed. Each study is representative of at least three separate experiments.
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fig6: The effect of M-CSF treatment on LPS-stimulated TNFα production and Tec expression. (A) Adherent monocytes from XLA and normal donors were either stimulated with LPS for 2 h, or incubated with M-CSF for 48 h before LPS stimulation for 2 h. Supernatants were assayed for TNFα production. (B) Adherent monocytes from normal (lanes 1 and 2) and XLA donors (lanes 3 and 4) were either lysed immediately, or after M-CSF treatment for 48 h. Western blot analysis was performed using either α-TecSH3, α-Btk, or α-actin antibody. (C) Elutriated monocytes from normal donors were stimulated with LPS for the indicated time periods. Tec was immunoprecipitated and in vitro autokinase assay was performed. Each study is representative of at least three separate experiments.

Mentions: Although we have demonstrated that Btk is able to modulate TNFα expression, XLA patients do not show a serious impairment in their innate immune function. Treatment of XLA monocytes for 2 d with M-CSF greatly enhanced their production of TNFα in response to LPS to levels similar to those obtained with normal monocytes (Fig. 6 A). Consequently, we examined the mechanism of the increased responsiveness of monocytes after M-CSF treatment. Because members of the Tec family of kinases can functionally complement each other (39), we tested whether M-CSF can regulate the expression of an alternative Tec family kinase to Btk. An obvious candidate was Tec kinase, as this enzyme is expressed in myeloid cells and does, to some degree, complement Btk activity in xid B cells (40). Western immunoblot analysis showed that Tec kinase was expressed at low levels in untreated monocytes from both normal donors and XLA patients (Fig. 6 B), however, after treatment with M-CSF for 48 h the expression of Tec protein was increased. Counter blots confirmed that the expression of Btk was restricted to the normal cells (Fig. 6 B). Next, the response of Tec kinase to LPS in M-CSF–treated monocytes was investigated using autokinase assays of immunoprecipitated enzyme. Like Btk, Tec kinase activity was increased in response to LPS treatment of human M-CSF–treated monocytes (Fig. 6 C).


Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production.

Horwood NJ, Mahon T, McDaid JP, Campbell J, Mano H, Brennan FM, Webster D, Foxwell BM - J. Exp. Med. (2003)

The effect of M-CSF treatment on LPS-stimulated TNFα production and Tec expression. (A) Adherent monocytes from XLA and normal donors were either stimulated with LPS for 2 h, or incubated with M-CSF for 48 h before LPS stimulation for 2 h. Supernatants were assayed for TNFα production. (B) Adherent monocytes from normal (lanes 1 and 2) and XLA donors (lanes 3 and 4) were either lysed immediately, or after M-CSF treatment for 48 h. Western blot analysis was performed using either α-TecSH3, α-Btk, or α-actin antibody. (C) Elutriated monocytes from normal donors were stimulated with LPS for the indicated time periods. Tec was immunoprecipitated and in vitro autokinase assay was performed. Each study is representative of at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193950&req=5

fig6: The effect of M-CSF treatment on LPS-stimulated TNFα production and Tec expression. (A) Adherent monocytes from XLA and normal donors were either stimulated with LPS for 2 h, or incubated with M-CSF for 48 h before LPS stimulation for 2 h. Supernatants were assayed for TNFα production. (B) Adherent monocytes from normal (lanes 1 and 2) and XLA donors (lanes 3 and 4) were either lysed immediately, or after M-CSF treatment for 48 h. Western blot analysis was performed using either α-TecSH3, α-Btk, or α-actin antibody. (C) Elutriated monocytes from normal donors were stimulated with LPS for the indicated time periods. Tec was immunoprecipitated and in vitro autokinase assay was performed. Each study is representative of at least three separate experiments.
Mentions: Although we have demonstrated that Btk is able to modulate TNFα expression, XLA patients do not show a serious impairment in their innate immune function. Treatment of XLA monocytes for 2 d with M-CSF greatly enhanced their production of TNFα in response to LPS to levels similar to those obtained with normal monocytes (Fig. 6 A). Consequently, we examined the mechanism of the increased responsiveness of monocytes after M-CSF treatment. Because members of the Tec family of kinases can functionally complement each other (39), we tested whether M-CSF can regulate the expression of an alternative Tec family kinase to Btk. An obvious candidate was Tec kinase, as this enzyme is expressed in myeloid cells and does, to some degree, complement Btk activity in xid B cells (40). Western immunoblot analysis showed that Tec kinase was expressed at low levels in untreated monocytes from both normal donors and XLA patients (Fig. 6 B), however, after treatment with M-CSF for 48 h the expression of Tec protein was increased. Counter blots confirmed that the expression of Btk was restricted to the normal cells (Fig. 6 B). Next, the response of Tec kinase to LPS in M-CSF–treated monocytes was investigated using autokinase assays of immunoprecipitated enzyme. Like Btk, Tec kinase activity was increased in response to LPS treatment of human M-CSF–treated monocytes (Fig. 6 C).

Bottom Line: We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region.Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production.This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London W6 8LH, United Kingdom.

ABSTRACT
Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

Show MeSH
Related in: MedlinePlus