Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2 - PubMed (original) (raw)
Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2
Seung Hyun Han et al. J Immunol. 2006.
Erratum in
- J Immunol. 2006 Feb 15;176(4):2671
Abstract
NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.
Figures
FIGURE 1.
PnLTA induces NO and TNF-α production. A and B, Cultures of the murine macrophage cell line RAW 264.7 were stimulated with PnLTA for the indicated time periods. The culture supernatants were analyzed for (A) nitrite levels or for (B) TNF-α levels. All error bars in the figures indicate SD. C, The cells were also stimulated with PnLTA, StLTA, or E. coli LPS at the indicated concentrations for 48 h, and the supernatants were analyzed for NO2 levels.
FIGURE 2.
A, LTA induces mRNA synthesis for iNOS and TNF-α but not for IFN-β. RAW 264.7 cells were stimulated with PnLTA, StLTA, or E. coli LPS for 3 h at the indicated concentrations. RT-PCR products of IFN-β, iNOS, TNF-α, and β-actin mRNA were separated in an agarose gel and visualized by ethidium bromide staining. B, NO production by the cells stimulated with PnLTA can be suppressed with a NO synthase inhibitor (
l
-NAME, ■) but not with an inert control molecule (
d
-NAME, □). ***, Significant reduction (p < 0.001) in nitrite levels by inhibitors. C, NO production by the cells stimulated with PnLTA can be suppressed with 2-IPD (iminopiperidine), an iNOS-specific inhibitor.
FIGURE 3.
PAFR signaling is necessary for LTA induction of NO but not of TNF-α. RAW 264.7 cells were pretreated with varying concentrations of a PAFR antagonist for 1 h and then stimulated with (A and D) PnLTA, (B and E) StLTA, or (C and F) E. coli LPS for 16 or 48 h for assessment of TNF-α production or of NO production, respectively. PAFR antagonists were (A-C) CV6209 and (D-F) CV3988. At the end of stimulation, the amounts of nitrite (bar) and TNF-α (line) in the culture media were quantified. *, p < 0.05 and ***, p < 0.001 when the levels produced in the presence of an inhibitor are compared with those without an inhibitor.
FIGURE 4.
A synthetic lipopeptide, Pam3CSK4, induces NO production, and NO production requires PAFR signaling. A, RAW 264.7 cells were stimulated with Pam3CSK4 at the indicated concentrations for 16 or 48 h for assessment of TNF-α production (○) or of NO production (●), respectively. B and C, The cells were pretreated with varying concentrations of CV6209 or ABT-491 for 1 h and then stimulated with the Pam3CSK4 for 16 or 48 h for assessment of TNF-α production (lines) or of NO production (bars), respectively. ***, Significant (p < 0.001) reduction in NO production by an inhibitor.
FIGURE 5.
LTA signaling for NO or TNF-α production does not involve G proteins or EGFR. RAW 264.7 cells were incubated with (A) pertussis toxin or (B) AG1478 for 1 h and then stimulated with StLTA, PnLTA, or E. coli LPS for 48 h for the NO assay and 15 h for the TNF-α assay. At the end of the culture period, the media were analyzed for the levels of TNF-α (lines) and nitrite (bars).
FIGURE 6.
Intracellular signal transduction for LTA-induced NO vs TNF-α production. A, RAW 264.7 cells were stimulated with PnLTA (50 μg/ml), StLTA (1 μg/ml), or E. coli LPS (0.1 μg/ml) in the presence of varying concentration of a Jak2 inhibitor (AG490). Culture supernatants were harvested after 16 h for the TNF-α determination or after 48 h for the nitrite determination. B, RAW 264.7 cells were pretreated with specific inhibitors for p38 kinase (SB203580), SAPK/JNK (SP600125), or Erk (PD98059) for 1 h and then stimulated with PnLTA (50 μg/ml) for 16 h for the TNF-α determination or 48 h for the NO determination. At the end of the culture period, the media were analyzed for TNF-α and nitrite levels. ***, A significant (p < 0.001) change in TNF-α or nitrite levels due to an inhibitor.
FIGURE 7.
Effect of PAFR inhibitor on LTA-induced phosphorylation of STAT1 and Jak2. A, RAW 264.7 cells were treated with (lane 1) none, (lanes 2, 4, and 6) PnLTA, or (lanes 3, 5 and 7) StLTA. Cells were treated with (lanes 4 and 5) a PAFR inhibitor or with (lanes 6 and 7) DMSO alone (0.01%). The PAFR inhibitor is CV6209 (1 μM) in DMSO (0.01%). Phosphorylated serine at position 727 is shown at the top, phosphorylated tyrosine at position 701 in the middle, and the “Total p38” at the bottom. B, RAW 264.7 cells were treated with (lanes 1) none, (lanes 2-4) StLTA (5 μg/ml), or (lanes 5-7) both StLTA (5 μg/ml) and CV6209 (1 μM) for the indicated time periods. Jak2 phosphorylation is shown at the top, and the “Total p38” is at the bottom.
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