Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in macrophages by Galpha(i) proteins

Abstract

Heterotrimeric G(i) proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of G(i) proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS induced production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 and interferon-gamma-inducible protein-10 (IP-10); SA induced TNF-alpha, and IL-1beta production; and GBS induced TNF-alpha, IL-6, IL-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. In contrast to the role of G(i) proteins as a positive regulator of mediators, LPS-induced production of MIP-1alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Galpha(i1/3) (-/-) mice, and SA-induced MIP-1alpha production was increased in both groups of Galpha(i) protein-depleted mice. LPS-induced production of KC and IL-1beta, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. These data suggest that G(i2) and G(i1/3) proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

Figure 1

Effect of deletion of Gα_i2 or Gα_i1/3 genes on LPS-, SA- and GBS-induced TNF-α, IL-6, IL-10, IL-1β, and GM-CSF production in macrophages. Peritoneal macrophages were isolated from Gα_i2^–/– or Gα_i1/3^–/– and age-matched 129sv WT mice and stimulated in vitro with LPS, SA or GBS (10 μg/ml). LPS-, SA- and GBS-induced TNF-α (a), IL-6 (b), IL-10 (c), IL-1β (d), and GM-CSF (e) production were determined. Data represent means ± SE from three independent experiments. *P < 0·05 compared to basal; ^#P < 0·05 compared to stimulated WT group; ND, not detectable.

Figure 2

Effect of deletion of Gα_i2 or Gα_i1/3 gene on LPS-, SA- and GBS-induced MIP-1α, KC and IP-10 production in macrophages. Peritoneal macrophages were isolated from Gα_i2^–/– or Gα_i1/3^–/– and age-matched 129sv WT mice and stimulated in vitro with LPS, SA or GBS (10 μg/ml). LPS-, SA- and GBS-induced MIP-1α (a), KC (b) and IP-10 (c) production was studied. Data represent means ± SE from three independent experiments. *P < 0·05 compared to basal; ^#P < 0·05 compared to stimulated WT group.