1Departments of Physiology and Neuroscience, 4Pharmacology, and 5Medicine, Medical University of South Carolina, Charleston, South Carolina; 2Division of Critical Care Medicine, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio; and 3Department of Experimental Pathology and Microbiology, Medical University of Messina, Messina, Italy
Submitted 10 August 2004 ; accepted in final form 16 March 2005
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ABSTRACT |
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Gi protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors
A complex network of receptors allows for rapid identification of pathogens on the basis of shared invariant molecular structures of a variety of microorganisms. CD14, a 55-kDa glycosylphosphatidylinositol (GPI)-anchored protein, binds LPS with high affinity (26, 29, 41). The family of Toll-like receptors (TLRs), which regulates innate immunity and antimicrobial responses, was discovered several years ago (3, 24). It has been demonstrated that TLR4 plays a critical role in LPS-mediated immune responses (31, 33). The gram-positive bacterium Staphylococcus aureus (SA)-induced signaling pathways are mediated mainly through TLR2 coupled with CD14 (9, 38, 42). The TLR receptors for the gram-positive bacteria group B streptococci (GBS) remain unknown (22). GBS-induced TNF- production is mediated by MyD88, suggesting that TLRs coupled with CD14 are also involved in GBS recognition, but TLR1, TLR2, TLR4, and TLR6 do not recognize GBS (22). Stimulation of TLR2- or TLR4- and MyD88-dependent signaling pathways leads to activation of a series of signaling proteins, leading in turn to expression of pro- and anti-inflammatory genes.
The extent to which gram-negative or gram-positive bacteria use common postreceptor signaling proteins remains uncertain (2). Heterotrimeric guanine nucleotide binding regulatory (G) proteins of the G inhibitory class (Gi) are involved in LPS signaling (37). There are three isoforms of Gi, and their potential individual and/or relative roles in mediating LPS and gram-positive bacterial signaling are unknown. It has been shown that G
i proteins are physically associated with the CD14 receptor (37). Mastoparan, a Gi protein agonist/antagonist, suppressed LPS-induced TNF-
and IL-6 production (37). Studies using pertussis toxin (PTx), which inhibits receptor-Gi coupling by catalyzing the ADP ribosylation of G
i proteins, have demonstrated inhibition of LPS-induced mediator production in different cell types (12, 16, 17, 40, 43). We have demonstrated that PTx inhibited not only LPS- but also SA- and GBS-induced TNF-
production in murine J774A.1 and human THP-1 cell lines (15). Aside from pharmacological approaches, molecular biological approaches have demonstrated that constitutively active TLR4-induced ERK1/2 activation was blocked by dominant-negative Gi protein constructs in human embryonic kidney HEK-293 cells (14).
Although studies have suggested that Gi proteins mediate TLR-induced proinflammatory gene expression, these studies were conducted largely with pharmacological inhibition of Gi proteins, and such an approach does not demonstrate the relative role of the Gi1, G
i2, and G
i3 isoforms. Also, paradoxically targeted genetic deletion of G
i2 proteins in mice induces a predominant proinflammatory phenotype. G
i2-knockout (G
i2/) mice develop an inflammatory bowel disease similar to ulcerative colitis (34), and analysis of their inflamed colons demonstrated increased expression of Th1-type cytokines (23). Augmented thymocyte and splenocyte production of proinflammatory cytokines in response to activation with several microbial stimuli have subsequently been demonstrated in G
i2/ mice (5, 21, 23). Collectively, the latter studies suggest that specific TLR ligands activate Gi protein signaling pathways that are counterinflammatory in lymphoid-rich tissues [i.e., tissues containing T cells, B cells, natural killer (NK) cells, or dendritic cells]. These findings are in contrast to studies demonstrating that TLR stimulation activates Gi protein signaling pathways that upregulate proinflammatory gene expression in peritoneal M
and myeloid cell lines (12, 15, 16, 37, 40, 43).
To further examine potential cellular phenotype specificity of Gi proteins in cytokine expression, the effects of murine G
i deficiency on pro- and anti-inflammatory cytokine and mediator production in splenocytes and peritoneal M
after stimulation with the TLR ligands LPS, SA, and GBS were investigated. Genetic deletion of G
i proteins in G
i2/ mice and G
i1/3-knockout (G
i1/3/) mice affords unique opportunities to investigate which Gi protein isoforms may be involved in LPS and gram-positive bacterial signaling. We hypothesized that genetic deletion of specific Gi protein isoforms would differentially alter inflammatory mediator production in splenocytes and peritoneal M
stimulated by LPS, SA, and GBS.
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MATERIALS AND METHODS |
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Gi2/ mice and their wild-type (WT) littermates with C57BL6/129Sv background were generated by breeding heterozygous and homozygous knockout animals. G
i1/3/ mice were generated by breeding homozygous double-knockout mice. In all experiments, we used 5- to 8-wk-old G
i2/, G
i1/3/, and age-matched WT mice. The original knockout mice were obtained from Dr. Lutz Birnbaumer (National Institute of Environmental Health Sciences, Research Triangle Park, NC). Western blot analysis of M
and splenocytes confirmed the absence of G
i2 in the G
i2/ mice, with no observed effect on G
i3 (Fig. 1). The investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and commenced with the approval of the Institutional Animal Care and Use Committee.
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PCR was performed with genomic DNA from 4-wk-old Gi2-knockout mouse tails. We used the following primer pairs: WT (+) forward, 5'-GAT CAT CCA TGA AGA TGG CTA CTC AGA AG-3', and WT reverse, 5'-CCC CTC TCA CTC TTG ATT TCC TAC TGA CAC-3'; knockout () forward, 5'-CAG GAT CAT CCA TGA AGA TGG CTA C-3', and knockout reverse, 5'-GCA CTC AAA CCG AGG ACT TAC AGA AC-3'. The reactions were run for 35 cycles. Amplified sequences were 805 bp for the WT allele and 509 bp for the targeting construct.
Cell Stimulation
Splenocytes and peritoneal M were harvested from G
i2/, G
i1/3/ mice, and their WT littermates and maintained in RPMI 1640 medium (CellGro Mediatech, Herndon, VA) supplemented with heat-inactivated 1% fetal calf serum (Sigma, St. Louis, MO), 50 U/ml penicillin, 50 µg/ml streptomycin (CellGro Mediatech). Splenocytes and peritoneal M
were stimulated with different concentrations of LPS (from Salmonella enteritidis; Sigma) or heat-killed SA or GBS [heat-killed SA and GBS were prepared as described previously (10)] for different lengths of time. Also, 10 µg/ml LPS, SA, and GBS stimulation for 18 h was used for specific experiments. Stimulation studies with S. enteritidis LPS in G
i2/ and WT splenocytes and M
were repeated with protein-free Salmonella minnesota R595 LPS (provided by Dr. Ernst Reitschel, Borstel, Germany). The supernatants were collected for assays of mediator production.
Assays for TNF-, IL-10, IFN-
, and Thromboxane B2 Production
TNF-, IL-10, and IFN-
production were measured using an enzyme-linked immunosorbent assay (ELISA) with mouse TNF-
, IL-10, or IFN-
ELISA kits (eBioscience, San Diego, CA). Thromboxane B2 (TxB2) production was measured in cell culture medium via radioimmunoassay as previously described (20).
In Vivo LPS Shock Studies
Plasma TNF- measurement.
G
i2/ and WT mice were challenged with either saline or LPS (125 mg/kg) via intraperitoneal (IP) administration. Four hours after challenge, the mice were bled from the vena cava using a syringe containing 0.02 ml of heparin (10,000 U/ml). The plasma was collected for TNF-
and IFN-
assays as described above.
Measurement of myeloperoxidase activity. Myeloperoxidase activity was determined in gut, liver, and lung as an index of neutrophil accumulation as previously described (44). Tissues were homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0) and were centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was allowed to react with a solution of teramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured by performing spectrophotometry at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol hydrogen peroxide/min at 37°C and was expressed in units per 100 milligrams of tissue.
Western Blot Analysis
Peritoneal M and splenocytes harvested from G
i2/ and WT mice were washed and lysed with ice-cold RIPA lysis buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A). Cells were kept on ice for 30 min, sonicated for 3 s, and centrifuged for 10 min at 4°C at 10,000 g. An aliquot was obtained for protein determination using the Bio-Rad protein assay (Bio-Rad, Hercules, CA), and the remaining supernatant was stored at 20°C until Western blot analysis.
For detection of Gi2 and G
i3, lysates were added to Laemmli sample buffer and boiled for 4 min. Subsequently, protein from each sample was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membranes were washed with Tris-buffered saline-Tween 20 (TBST; 20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) and blocked with 5% milk in TBST for 1 h. After being washed with TBST, membranes were incubated with primary antibody (anti-G
i2 antibody provided by Dr. John D. Hildebrandt, Dept. of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC) at 1:5,000 dilution for G
i2 protein and with anti-G
i3 antibody (1:1,000 dilution; Upstate Biotechnology, Lake Placid, NY) overnight at 4°C. The blots were washed twice with TBST and incubated for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (1:4,000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ) in blocking buffer. Immunoreactive bands were visualized using incubation with ECL Plus detection reagents (Amersham Pharmacia Biotech) for 5 min and development of the exposed ECL Hyperfilm (Amersham Pharmacia Biotech).
Statistical Analysis
Data are expressed as means ± SE. Statistical significance was determined using ANOVA with Fishers probable least-squares difference test using StatView software (SAS Institute, Cary, NC). P < 0.05 was considered significant.
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RESULTS |
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Western blot analysis was performed to examine Gi2 and G
i3 protein expression in the M
and splenocytes from G
i2/ mice. It was shown that in M
and splenocytes, G
i2 protein was not expressed and G
i3 protein expression was unchanged in G
i2/ mice (Fig. 1).
Gi2 Deficiency Augments Mediator Production Induced by Microbial Stimuli in Splenocytes
Because previous studies have shown augmented mediator production in splenocytes isolated from Gi2 deficiency mice in response to SA stimulation (5), we sought to determine whether deletion of the G
i2 gene also would have a significant effect on inflammatory mediator production in response to LPS and GBS stimulation. Splenocytes were harvested from G
i2/ and age-matched WT mice and stimulated in vitro with different concentrations of LPS, SA, or GBS for different lengths of time. LPS, SA, and GBS significantly induced TNF-
production in splenocytes in a concentration-dependent (Fig. 2A) and time-dependent (Fig. 3A) manner. All three agonists (10 µg/ml) and 18-h stimulation were used for the following experiments. TNF-
production stimulated by all three agonists was significantly increased 3.5 ± 0.2-fold, 2.5 ± 0.03-fold, and 3.3 ± 0.2-fold, respectively, in splenocytes from G
i2/ mice compared with WT mice (Fig. 4A). There was no significant difference between LPS- and GBS- induced IL-10 production in G
i2/ and WT mice. However, SA-induced IL-10 production was significantly increased 2.3 ± 0.2-fold in splenocytes from G
i2/ mice compared with WT mice (Fig. 4B). LPS-, SA-, and GBS-induced TxB2 production in splenocytes was also investigated. LPS- and SA-induced TxB2 production was significantly increased 2.0 ± 0.1-fold and 1.7 ± 0.1-fold, respectively, in splenocytes from G
i2/ mice compared with WT mice (Fig. 4C). However, GBS-induced TxB2 production in splenocytes was not significantly different between G
i2/ and WT mice. IFN-
production in splenocytes was stimulated by all three agonists and was significantly increased 4.9 ± 0.2-fold, 10.2 ± 1.0-fold, and 3.5 ± 0.2-fold, respectively, from G
i2/ mice compared with WT mice (Fig. 4D).
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To determine the effect of deletion of Gi2 on inflammatory mediator production in response to LPS, SA, and GBS in M
, peritoneal M
were harvested from G
i2/ and age-matched WT mice and stimulated in vitro with different concentrations of all three agonists for different lengths of time. All three bacterial products stimulated TNF-
production in a concentration-dependent (Fig. 2B) and time-dependent manner (Fig. 3B). In contrast to splenocytes, TNF-
production stimulated by LPS, SA, or GBS was decreased 51 ± 7%, 42 ± 7%, and 66 ± 2%, respectively, compared with WT M
(Fig. 5A). IL-10 production was stimulated by all three agonists. As with TNF-
production, in the G
i2/ M
, IL-10 production stimulated by LPS, SA, or GBS was decreased 64 ± 12%, 75 ± 4%, and 40 ± 11%, respectively, compared with WT M
(Fig. 5B). Similar to TNF-
and IL-10 production in G
i2/ M
, TxB2 production stimulated by LPS, SA, or GBS was decreased 24 ± 4%, 39 ± 3%, and 59 ± 2%, respectively, compared with WT M
(Fig. 5C). LPS-, SA-, and GBS-induced IFN-
production in M
was also investigated. All three bacterial products did not induce any IFN-
production in M
(data not shown). Because it could be argued that the S. enteritidis LPS may contain trace amounts of protein, which could alter its TLR4 specificity, similar studies were conducted in M
from G
i2/ and WT mice stimulated with protein-free S. minnesota R595 LPS. These studies demonstrated a similar suppression of TNF-
in G
i2/ M
that was observed with the S. enteritidis LPS (data not shown).
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Because there are three isoforms of Gi, we sought to determine the effect of the deletion of the genes for G
i1 and G
i3 on LPS-induced mediator production. Splenocytes and M
from G
i1/3/ and WT mice and stimulated in vitro with LPS (10 µg/ml). LPS-induced TNF-
production was significantly increased in splenocytes harvested from G
i1/3/ mice (6.0 ± 0.3-fold, n = 3; P < 0.05) compared with WT mice. However, LPS-induced IL-10 production remained unchanged between WT and G
i1/3/ mice and TxB2 production was not stimulated by LPS in splenocytes from either G
i1/3/ or WT mice (Fig. 6A). In M
, TNF-
, IL-10, and TxB2 production was stimulated by LPS. LPS-induced TNF-
production was not significantly different between WT and G
i1/3/ mice. However, LPS-induced IL-10 and TxB2 production was significantly decreased in M
harvested from G
i1/3/ mice (81 ± 4% and 35 ± 3%, respectively, n = 3; P < 0.05) compared with WT mice (Fig. 6B).
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Because there was a differential regulation of TNF- production in splenocytes and M
derived from G
i2/ mice, we sought to determine the effect on LPS-stimulated plasma levels of TNF-
. Mice were challenged with LPS (125 mg/kg) or IP vehicle for 4 h. Plasma TNF-
levels were significantly increased in G
i2/ mice (2.8 ± 0.7-fold, n = 3; P < 0.05) compared with WT mice (Fig. 7).
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We next examined myeloperoxidase activity as an index of neutrophil accumulation in gut, liver, and lung harvested from Gi2/ and WT mice. In mice, which were subjected to LPS (125 mg/kg) IP administration, LPS-induced myeloperoxidase activity was significantly increased (1.32 ± 0.03-fold and 1.44 ± 0.11-fold, respectively) in the gut (Fig. 8 A) and lung (Fig. 8C) from G
i2/ mice rendered endotoxemic compared with WT mice. However, the myeloperoxidase activity was not significantly changed in the liver from G
i2/ mice compared with WT mice (Fig. 8B).
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DISCUSSION |
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The predominant role of Gi proteins in TLR signaling appears to be anti-inflammatory because bacterial stimuli increased cytokine and TxB2 production in splenocytes from Gi/ mice. The increased plasma TNF-
levels in LPS-challenged G
i2/ mice further suggest a predominant proinflammatory tissue phenotype as a consequence of G
i deficiency. These findings extend previous studies demonstrating the enhanced capacity of splenocytes from G
i2/ mice to produce IL-1
, IL-6, IL-12, IFN-
, and TNF-
production in response to microbial and nonmicrobial stimuli (5, 21, 23, 34). It is evident from our present findings that there are obvious cellular phenotype differences in splenocytes vs. M
in the regulatory role of Gi proteins in TLR signaling. Other studies have demonstrated cell-specific effects of Gi protein deletion. G
i2/ mice displayed a blunted myocardial inhibition of adenylyl cyclase (35). However, in adipocytes, adenylyl cyclase activity was less affected in G
i2/ mice (35).
The finding that mice deficient in Gi protein exhibited decreased LPS-, SA-, and GBS-induced mediator production in peritoneal M
is consistent with the findings in our previous studies that pretreatment with PTx, an inhibitor of Gi proteins, inhibited LPS-, SA-, and GBS-induced TNF-
production in J774A.1 cells (15). Also, previous studies have shown that PTx inhibits the production of a variety mediators, including TNF-
, TxB2, PGE2, nitric oxide, and IL-1
in human promonocytic THP-1 cells, cultured rat glomerular mesangial cells, the promonocytic cell line U937, and murine M
(12, 16, 40, 43). Adib-Conquy et al. (1) demonstrated that heat-killed, SA-induced IL-10 production in whole blood samples from trauma patients was suppressed by PTx pretreatment. In human monocytes, the Gi protein agonist/antagonist mastoparan also inhibited LPS-induced cytokine production and signaling (37). The latter data are similar to the observations of Böcker et al. (6), who found that mastoparan inhibited SA-induced IL-10, IL-12, and IL-18 production in peripheral blood mononuclear cells. In addition to peritoneal M
, Gi protein signaling may also upregulate cytokine production in B cells, because studies have shown that SA-induced IL-10 production is suppressed in B cells from G
i2/ mice (11).
Although our results demonstrate differences in cellular phenotype responses between splenocytes and peritoneal M, the spleen consists of multiple cell types (i.e., M
, dendritic cells, NK cells, B cells, and different T-cell subtypes), and the role of Gi signaling pathways in specific cellular phenotypes may have been masked by other subpopulations of cells. It is also possible that autocrine or paracrine mediators may affect splenocytic responses to TLR activation that are not present in peritoneal M
. Indeed, NK cells and T-helper cells produce IFN-
in response to LPS stimulation (25). IFN-
has been shown in many studies to amplify LPS-induced M
proinflammatory gene expression (30). Increased local production of IFN-
by lymphoid cells could indirectly amplify the proinflammatory response of splenic M
and other TLR ligand-responding subpopulations. In support of this notion, we found that LPS-stimulated splenocytes from G
i2/ mice produced greatly augmented IFN-
production compared with the WT mice, whereas LPS-induced IFN-
production was not detectable in peritoneal M
. These findings may explain in part the different response to bacterial products in peritoneal M
and splenocytes. Identification of specific cell-type responses in the spleen will be the focus of subsequent investigations.
In addition to potential cellular specific effects of Gi proteins, another finding was that there are apparent differences in the role of the Gi isoforms Gi2 vs. G
i1/3. While G
i2/ mice displayed suppressed TNF-
production in M
in response to the TLR ligands, the G
i1/3/ mice exhibited no difference in TNF-
production in response to LPS. A difference in the role of Gi isoforms in TLR signaling was also apparent in splenocytes. The G
i1/3/ mice exhibited TNF-
responses to LPS that were almost twice as high as those of the G
i2/ mice. To our knowledge, this is the first observation of Gi isoform specificity in regulating inflammation.
The finding that Gi-deficient mice exhibit a predominant proinflammatory phenotype in vivo raise questions regarding how Gi proteins participate in TLR signaling. One possibility is that Gi proteins are coupled to TLR or TLR-coupled signaling proteins that regulate counterinflammatory signaling pathways. An example of a counterinflammatory signaling pathway is the phosphatidylinositol 3-kinase pathway, which has been proposed as a braking mechanism for LPS-induced proinflammation (13, 19). Indeed, in other receptor-coupled systems, it has been shown that G protein - and/or
-subunits can regulate phosphatidylinositol 3-kinase activity (7). Another possibility is that the TLR signaling pathway may transactivate other receptors activated by LPS that are Gi coupled. Of interest in this context is that Triantafilou et al. (39) proposed that LPS interacts with a cluster of receptors in lipid rafts, including Gi protein-coupled receptors, e.g., CXCR4 (27), growth factor receptors (28), and
2-integrins (36). Finally, an alternative possibility is that Gi proteins are not coupled directly to TLR or to TLR postreceptor signaling proteins but rather to heptahelical receptors that are activated by autocrine mediators that are counterinflammatory (32). Potential candidates for the latter mediators include prostaglandins, purinergic agonists, and chemokines. Thus one intriguing idea is that in primary murine cells, proinflammatory responses to microbial stimuli are negatively regulated by autocrine mediators that bind and signal through surface Gi-coupled heptahelical receptors.
Despite the phenotypic differences in the pattern of Gi-regulated expression of proinflammatory and anti-inflammatory mediators in M and splenocyte responses, it is important that responses to the three distinct TLR ligands (LPS, SA, and GBS) were similarly affected (9, 22, 30). These findings support a common convergent role of Gi proteins in TLR signaling. Understanding the role of Gi proteins in the regulation of TLR activation in response to gram-negative and gram-positive microbial stimuli will provide important insights into the regulation of innate immunity.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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