Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Galphai proteins.
Heterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS- or SA-induced production of TNFalpha, IL-6, IFNgamma, IL-12, IL-17, GM-CSF, MIP-1alpha, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Galpha(i2)(-/-) mice compared with WT mice. The effect of Galpha(i) protein depletion was remarkably isoform specific. In splenocytes from Galpha(i1/3) (-/-) mice relative to WT mice, SA-induced IL-6, IFNgamma, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice relative to WT mice. The disparate response of splenocytes from the Galpha(i2) (-/-) relative to Galpha(i1/3) (-/-) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that G(i2) and G(i1/3) proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli.
Fan
Hongkuan
H
Department of Neuroscience, 173 Ashley Ave., BSB Room 403, Charleston, SC 29425, USA.
Williams
David L
DL
Zingarelli
Basilia
B
Breuel
Kevin F
KF
Teti
Giuseppe
G
Tempel
George E
GE
Spicher
Karsten
K
Boulay
Guylain
G
Birnbaumer
Lutz
L
Halushka
Perry V
PV
Cook
James A
JA
D000818:Animals; D002478:Cells, Cultured; D018925:Chemokines / Q000096:biosynthesis*; D016207:Cytokines / Q000096:biosynthesis*; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics / Q000502:physiology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D020033:Protein Isoforms; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000502:physiology*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism
D018925:Chemokines; D016207:Cytokines; D008070:Lipopolysaccharides; D020033:Protein Isoforms; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go