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Biochimica et Biophysica Acta 1763 (2006) 1051 – 1058
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Differential regulation of lipopolysaccharide and Gram-positive bacteria
induced cytokine and chemokine production in splenocytes by Gαi proteins
Hongkuan Fan a , David L. Williams b , Basilia Zingarelli c , Kevin F. Breuel d , Giuseppe Teti e ,
George E. Tempel a , Karsten Spicher f , Guylain Boulay g , Lutz Birnbaumer h ,
Perry V. Halushka i , James A. Cook a,⁎
a
Department of Neuroscience, 173 Ashley Ave., BSB Room 403, Charleston, SC 29425, USA
Department of Surgery, East Tennessee State University, Johnson City, Tennessee, 37614, USA
c
Division of Critical Care Medicine, Cincinnati ChildrenTs Hospital Medical Center, Cincinnati, OH 45229, USA
Obstetrics and Gynecology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, 37614, USA
e
Department of Experimental Pathology and Microbiology, Medical University of Messina, Messina, Italy
f
Institute for Biochemistry and Molecular Biology, Heinrich-Heine-University, School of Medicine, D-40225 Duesseldorf, Germany
g
Department of Pharmacology, School of Medicine, Sherbrooke University, Sherbrooke, Québec, Canada J1H 5N4
h
Transmembrane Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
i
Pharmacology, and Medicine, Medical University of South Carolina, Charleston, South Carolina, 29425, USA
b
d
Received 12 April 2006; received in revised form 12 July 2006; accepted 1 August 2006
Available online 5 August 2006
Abstract
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), Gαi2 (−/−) or Gαi1/3 (−/−) mice were
investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly
increased (1.2 to 33 fold, p < 0.05) in splenocytes harvested from Gαi2(−/−) mice compared with WT mice. The effect of Gαi protein depletion was
remarkably isoform specific. In splenocytes from Gαi1/3 (−/−) mice relative to WT mice, SA-induced IL-6, IFNγ, 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 Gαi2 (−/−) and Gαi1/3 (−/−) 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 Gαi2 (−/−) and Gαi1/3 (−/−) mice relative to WT mice. The disparate response of splenocytes
from the Gαi2 (−/−) relative to Gαi1/3 (−/−) mice therefore cannot be attributed to major differences in spleen cellular composition. These data
demonstrate that Gi2 and Gi1/3 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.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Gi protein deficient mice; Endotoxin; Staphylococcus aureus; TLR signaling
1. Introduction
Sepsis is the host-derived systemic inflammatory response to
invasive infections that may result in septic shock [1]. Lipopoly-
⁎ Corresponding author. Tel.: +1 843 792 2978; fax: +1 843 792 1066.
E-mail address: [email protected] (J.A. Cook).
0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2006.08.003
saccharide (LPS) from Gram-negative bacteria, and peptidoglycan, lipoteichoic acid, and lipoproteins from Gram-positive
bacteria are potent inducers of the pro-inflammatory responses
in macrophages, monocytes, or other host cells [2]. These
products induce the release of inflammatory mediators that play
a major role in the pathophysiology of septic shock [3,4].
The Toll-like receptor family plays a critical role in
mediating the innate immune response. LPS-induced signaling
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H. Fan et al. / Biochimica et Biophysica Acta 1763 (2006) 1051–1058
is mediated by Toll-like receptor 4 coupled with CD14 and MD2 [5]. The Gram-positive bacteria Staphylococcus aureus (SA)induced signaling pathways are mediated, in part, through
TLR2 and other receptors [6–9]. Stimulation of TLR2 or TLR4
signaling pathways results in activation of a series of signaling
proteins leading to expression of pro-inflammatory cytokine
and chemokine genes. The cytokines include the pro-inflammatory mediators TNFα, IL-1β, IL-6, IL-12, and IFNγ, [10].
The CXC chemokine family includes KC (the murine
homologue of IL-8), IFNγ-inducible protein (IP)-10, and
monokine induced by IFNγ (MIG). The CC chemokine family
includes monocyte chemoattractant protein (MCP)-1, and
macrophage inflammatory protein (MIP)-1α [11,12]. These
cytokines and chemokines contribute to the complex inflammatory milieu of sepsis. However, it remains uncertain to what
extent common or discrete signaling pathways regulate
cytokine and chemokine production.
Heterotrimeric guanine nucleotide binding regulatory (G)
proteins of the G inhibitory class (Gi) are involved in signaling
to microbial stimuli [13–17]. It has been shown that Gαi
proteins are co-immunoprecipitated with the CD14 receptor
[17]. Mastoparan, a Gi protein agonist/antagonist, suppressed
LPS-induced cytokine production in human monocytes, human
dermal microvessel endothelial cells, primary murine macrophages, and RAW 264.7 cells [16–18]. Studies utilizing
pertussis toxin (PTx), an inhibitor of receptor-Gi coupling,
have demonstrated inhibition of LPS- and SA-induced mediator
production in different cell types [14,19–23]. The availability
of Gαi deficient mice has provided another approach to
examine Gi proteins as post-receptor signaling proteins for both
TLR4 and TLR2 ligands. Our previous studies have demonstrated that LPS- and SA-induced TNFα, and TxB2 production
were decreased in peritoneal macrophages from Gαi2 (−/−)
mice compared with WT mice [15]. However, despite the
suppressed mediator production in peritoneal macrophages
from Gαi2 (−/−) mice, the in vivo inflammatory response of
Gαi2 (−/−) mice to LPS challenge was significantly augmented
as measured by increased plasma TNFα and lung and liver
leukosequestration relative to WT mice [15]. The latter finding
suggested a predominant pro-inflammatory phenotype in the
Gαi2 knock out mice to LPS challenge. This pro-inflammatory
phenotype paralleled the in vitro response of splenocytes from
the Gαi2 (−/−) mice. LPS- and SA-induced cytokine and
thromboxane (Tx)B2 production were increased in splenocytes
from Gαi2 (−/−) mice compared with WT mice [15]. Therefore
splenocytes appear to be a better predictor of the overall
inflammatory response to microbial stimuli in the Gαi deficient
mice. The potential role of specific Gαi proteins in regulation of
other important cytokines and chemokines induced in immune
cells by LPS and Gram-positive bacteria remain to be
investigated.
To examine potential isoform specificity of Gαi proteins in
cytokine and chemokine expression, the effect of Gαi2 or Gαi1/3
gene deletion on chemokine and pro-inflammatory cytokine
production in splenocytes following stimulation with either LPS
or SA was investigated. Genetic deletion of Gαi2 and Gαi1/3 in
mice and luminex analysis offered a unique opportunity to
investigate the role of Gαi proteins in LPS- and SA-induced
cytokine and chemokine release. We hypothesized that genetic
deletion of specific Gαi protein isoforms differentially alters
cytokine and chemokine production in splenocytes stimulated
by LPS and SA.
2. Materials and methods
2.1. Mice
Gαi2 (−/−) mice and littermate WT mice with 129Sv background were
generated by breeding heterozygous and homozygous knockout mice. Gαi1/3
(−/−) mice were generated by breeding homozygous double knockout mice.
Studies employed 5 to 8 week old Gαi2 (−/−), Gαi1/3 (−/−) and age matched
WT mice for all the experiments. These mice appear healthy with normal
colon. The original knockout mice were obtained from Dr. Lutz Birnbaumer.
(NIH, Research Triangle Park, NC). Western blot analysis has confirmed the
presence of Gαi2 and Gαi3 in splenocytes from WT mice, but absence of Gαi2
in splenocytes from Gαi2 (−/−) mice (Fig. 1A) and Gαi3 in Gαi1/3 (−/−) mice
(Fig. 1B). The investigations conformed to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health and with the
approval of the institutional animal care and use committee.
2.2. Genotyping
PCR was performed with genomic DNA from 4-week-old Gαi2 (−/−) mice
tails. We used the following primer pairs: wild type (+), forward, 5′-GAT CAT
CCA TGA AGA TGG CTA CTC AGA AG-3′; 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′; 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 wild type allele and 509 bp for the targeting construct.
Fig. 1. Gαi2 and Gαi3 protein expression in splenocytes from Gαi2(−/−) and Gαi1/3(−/−) mice. Splenocytes were harvested from WT, Gαi2(−/−) and Gαi1/3(−/−) mice.
Cells were lysed and Western blot analysis was performed using antibodies against Gαi2 and Gαi3 protein in splenocytes from WT, Gαi2(−/−) (A) and Gαi1/3(−/−) (B)
mice. Data are representative of three independent experiments.
H. Fan et al. / Biochimica et Biophysica Acta 1763 (2006) 1051–1058
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2.3. Cell stimulation
2.7. Flow cytometry analysis
Splenocytes were harvested from Gαi2 (−/−), Gαi1/3 (−/−) mice and
littermate WT mice and maintained in RPMI 1640 medium (Cellgro Mediatech
Inc., Herndon, VA), supplemented with heat inactivated 1% fetal calf serum
(FCS) (Sigma, St. Louis, MO), 50 U/ml penicillin, 50 μg/ml streptomycin
(Cellgro Mediatech Inc., VA). Splenocytes (1 × 107 cells/well) in 24-well plates
were stimulated with 10 μg/ml of LPS (from Salmonella enteritidis, Sigma, St.
Louis, MO), 10 μg/ml of heat killed SA (Heat killed SA was prepared as
described previously [24]), protein free S. minnesota R595 LPS (provided by
Dr. Ernst Reitschel, Borstel Germany, 1 μg/ml) or Pam3CysSK4 (Calbiochem,
La Jolla, CA, 1 μg/ml) for 18 h. The concentration of agonist and time point of
stimulation were selected based on our previous time course and dose–response
studies [15]. The supernatants were collected for assay of mediator production.
Splenocytes were prepared for flow cytometry by crushing freshly dissected
tissues between flat forceps in PBS. Cells were passed through a nylon mesh to
get single cell suspension. Red blood cells were removed by adding RBC lysis
buffer (eBioscience, San Diego, CA) for 2 min at room temperature. Cells were
counted and washed with PBS. A total of 106 cells were incubated with primary
antibody for 30 min at 4 °C. The following antibodies were used to characterize
spleen cell subsets: macrophages; FITC-conjugated anti-mouse F4/80 (Caltag,
Burlingame, CA); CD8+ T cells; TC-conjugated anti-mouse CD8a (Caltag,
Burlingame, CA); CD4+ T cells; APC-conjugated anti-mouse CD4 (Caltag,
Burlingame, CA); and dendritic cells; PE-conjugated anti-mouse CD83
(eBioscience, San Diego, CA). PE-conjugated anti-mouse TLR2 and PEconjugated anti-mouse TLR4 (eBioscience, San Diego, CA) were used to
examine cell surface TLR2/TLR4 expression. Cells were washed twice in
staining buffer, resuspended in 500 μl of staining buffer, and analyzed on a
FACSCalibur flow cytometer (BD Pharmingen). Data were analyzed with
CellQuest Pro. software.
2.4. Western blot
Splenocytes harvested from WT, Gαi2 (−/−) and Gαi1/3 (−/−) 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 (PMSF), 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 taken 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 Gαi2 and Gαi3, lysates were added to Laemmli sample
buffer and boiled for 4 min. Subsequently, protein from each sample was
subjected to a 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF)
membrane. The membranes were washed with Tris-buffered saline-Tween 20
(TBST; 20 mM Tris, 500 mM NaCl, 0.1% Tween 20) and blocked with 5% milk
in TBST for 1 h. After washes with TBST, membranes were incubated with
primary antibody (Ai12 antibody from Dr. John D. Hildebrandt (Pharmacology
Dept., Medical University of South Carolina, Charleston, SC) at 1:5000 dilution
for Gαi2 protein; Anti-Gαi3 antibody (Upstate biotechnology, Lake Placid, NY)
at 1:1000) overnight at 4 °C. The blots were washed twice with TBST and
incubated for 1 h with horseradish peroxidase-conjugated donkey anti-rabbitIgG antibody (1:4000 dilution, Amersham Pharmacia Biotech, Inc., Piscataway,
NJ) in blocking buffer. Immunoreactive bands were visualized by incubation
with ECL Plus detection reagents (Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ) for 5 min and development of the exposed ECL hyperfilm
(Amersham Pharmacia Biotech, Inc., Piscataway, NJ).
2.5. Luminex analysis
The samples were stored in liquid nitrogen until assayed. Cytokine levels
were assayed with a Biosource murine10 plex cytokine and 5 plex chemokine
assay (Camarillo, CA) on a Luminex 100 instrument. Specifically, we assayed
the sample for IL-1β (sensitivity 8.7 pg/ml), IL-2 (sensitivity 10 pg/ml), IL-4
(sensitivity 5 pg/ml), IL-5 (sensitivity 6.5 pg/ml), IL-6 (sensitivity 2.7 pg/ml),
IL-12(p40/p70) (sensitivity 11 pg/ml), IL-17 (sensitivity 5 pg/ml), TNFα (sensitivity 4.5 pg/ml), GM-CSF (sensitivity 10 pg/ml), IFNγ (sensitivity 1 pg/ml),
MIP1α (sensitivity 15.2 pg/ml), MCP-1 (sensitivity 4.2 pg/ml), MIG (sensitivity
3 pg/ml), IP-10 (sensitivity 46 pg/ml) and KC (sensitivity 11.5 pg/ml). Cytokine
levels were established by comparison to a standard curve as per the manufacturer's instructions. The luminex analysis results for TNFα, IL-6, IFNγ were
confirmed by ELISA.
2.6. ELISA
In addition to the luminex analysis, we also examined the effect of the
soluble TLR2 ligand Pam3CysSK4 and ultra pure S. minnesota R595 LPS
induced splenocytic TNFα production using an enzyme-linked immunosorbant
assay (ELISA) with mouse TNFα ELISA kits (eBioscience, San Diego, CA).
Salmonella enteritidis LPS and Heat-killed SA induced TNFα, IL-6, IFNγ
were also measured with TNFα (sensitivity 8 pg/ml), IL-6 (sensitivity 4 pg/ml),
IFNγ (sensitivity 15 pg/ml) ELISA kits (eBioscience, San Diego, CA).
2.8. Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM).
Statistical significance was determined by analysis of variance (ANOVA) with
Fisher's probable least-squares difference test using Statview software (SAS
Institute Inc., Cary, NC). p < 0.05 was considered significant.
3. Results
3.1. Gαi2 and Gαi1/3 differentially regulate cytokine
production induced by microbial stimuli in splenocytes
LPS- and SA-induced cytokine production was determined
in splenocytes from Gαi2 or Gαi1/3 deficient mice. Splenocytes
from Gαi2 (−/−) and Gαi1/3 (−/−) and age matched WT mice
were stimulated in vitro with LPS and SA for 18 h and subjected
to luminex analysis for TNFα, IFNγ, IL-1β, IL-4, IL-5, IL-6,
IL-12, IL-17, and GM-CSF. In splenocytes from WT mice, LPS
significantly stimulated TNFα (Fig. 2A), IL-6 (Fig. 2B), and
GM-CSF (Fig. 2F) production but not IFNγ (Fig. 2C), IL-12
(Fig. 2D), or IL-17 (Fig. 2E) production. In splenocytes from
Gαi2 (−/−) mice, LPS-induced TNFα, IL-6, IFNγ, IL-17, and
GM-CSF production was significantly increased 2.6 ± 0.2 fold;
1.9 ± 0.4 fold; 30.8 ± 15.3 fold; 25.6 ± 18.6 fold; 2.8 ± 1.2 fold,
respectively, (n = 3, p < 0.05) compared to WT mice. In
splenocytes from WT mice, SA significantly stimulated
TNFα (Fig. 2A), IL-6 (Fig. 2B), IFNγ (Fig. 2C), IL-12 (Fig.
2D) and GM-CSF (Fig. 2F) production but not IL-17 (Fig. 2E)
production. In splenocytes from Gαi2 (−/−) mice, SA-induced
TNFα, IL-6, IFNγ, IL-12, IL-17, and GM-CSF production was
significantly increased 2.0 ± 0.1 fold; 5.1 ± 1.5 fold; 3.1 ± 0.3
fold; 7.5 ± 1.7 fold; 7.6 ± 2.9 fold; and 2.1 ± 0.5 fold respectively,
(n = 3, p < 0.05) compared to WT mice. IL-1β, IL-4, and IL-5
production were not stimulated in splenocytes by LPS and SA
(data not shown).
In contrast to Gαi2 protein, Gαi1/3 differentially regulated
LPS- and SA-induced cytokine production. Although LPSinduced TNFα production was increased 1.5 ± 0.2 fold (n = 3,
p < 0.05) in the splenocytes from Gαi1/3 (−/−) mice compared
to WT mice, SA-induced TNFα production was not significantly
different compared to WT mice. SA-induced IL-6 (Fig. 2B),
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Fig. 2. Effect of deletion of Gαi2 or Gαi1/3 gene on LPS- and SA-induced TNFα, IL-6, IFNγ, IL-12, IL-17, and GM-CSF production in splenocytes. Splenocytes were
isolated from Gαi2(−/−), Gαi1/3(−/−) and age matched C57BL6/129sv WT mice and stimulated in vitro with LPS or SA (10 μg/ml). LPS- and SA-induced TNFα (A),
IL-6 (B), IFNγ (C), IL-12 (D), IL-17 (E), and GM-CSF (F) production in splenocytes were studied. 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.
IFNγ (Fig. 2C), and GM-CSF (Fig. 2F) production were
decreased (59 ± 5%, 86 ± 6% and 77 ± 2% respectively, n = 3,
p < 0.05) in the splenocytes from Gαi1/3 (−/−) mice compared to
WT mice. LPS- and SA-induced IL-12, IL-17, and LPS-induced
IL-6, INFγ, and GM-CSF production were unchanged in the
Gαi1/3(−/−) mice compared to WT mice (Fig. 2).
Luminex analysis of LPS and SA-induced TNFα, IL-6 and
INFγ production in splenocytes from Gαi2 (−/−), Gαi1/3 (−/−)
and WT mice were confirmed by ELISA assay (data not
shown). Additionally, stimulation of TNFα of Gαi2 (−/−) and
WT splenocytes were repeated with protein free S. minnesota
R595 LPS and Pam3CysSK4. Similar results were found with
these TLR4 or TLR2 ligands as with Salmonella enteritidis LPS
and SA (data not shown).
3.2. Gαi2 and Gαi1/3 differentially regulate chemokine
production induced by microbial stimuli in splenocytes
In order to determine the effect of deletion of Gαi2 or Gαi3
genes on chemokine production in response to LPS and SA
stimulation, splenocytes from Gαi2 (−/−), Gαi1/3 (−/−) and age
matched WT mice were stimulated in vitro with LPS, and SA
for 18 h and subjected to luminex analysis for MIP-1α, MCP-1,
KC and IP-10 and MIG production. In Gαi2 (−/−) mice, LPS-
H. Fan et al. / Biochimica et Biophysica Acta 1763 (2006) 1051–1058
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Fig. 3. Effect of deletion of Gαi2 or Gαi1/3 gene on LPS- and SA-induced MIP-1 α, MCP-1, IP-10, and MIG production in splenocytes. Splenocytes were isolated from
Gαi2(−/−), Gαi1/3(−/−) and age matched C57BL6/129sv WT mice and stimulated in vitro with LPS or SA (10 μg/ml). LPS- and SA-induced MIP-1 α (A), MCP-1 (B),
IP-10 (C), and MIG (D) production in splenocytes were studied. 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.
induced CC chemokine MIP-1α (Fig. 3A) and MCP-1 (Fig. 3B)
was significantly increased 3.1 ± 1.1 fold and 7.3 ± 3.7 fold,
respectively (n = 3, p < 0.05) compared to WT mice. SA-induced
MIP-1α and MCP-1 were significantly increased 7.3 ± 3.4 fold
and 5.4 ± 2.2 fold, respectively (n = 3, p < 0.05) compared to WT
mice. LPS-induced CXC chemokine IP-10 (Fig. 3C) and MIG
(Fig. 3D) were significantly increased 9.6 ± 5.7 fold and 3.8 ±
1.5 fold respectively (n = 3, p < 0.05) in Gαi2(−/−) mice
compared to WT mice. SA-induced IP-10 and MIG were
significantly increased 17.0 ± 3.3 fold and 6.1 ± 1.9 fold
respectively (n = 3, p < 0.05) in Gαi2(−/−) mice compared to
WT mice. Although LPS and SA stimulated KC production, its
Fig. 4. Effect of deletion of Gαi2 or Gαi1/3 gene on splenocytes subsets population change. Splenocytes were isolated from Gαi2(−/−), Gαi1/3(−/−) and age matched
C57BL6/129sv WT mice and analyzed with flow cytometry. Percentage of total number of macrophages (MΦ), CD4+ T cells, CD8+ T cells and dendritic cells (DC)
were illustrated. Data represent means ± SE from three independent experiments. *, p < 0.05 compared to WT cells; #, p < 0.05 compared to cells from Gαi2(−/−) mice.
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H. Fan et al. / Biochimica et Biophysica Acta 1763 (2006) 1051–1058
synthesis was not significantly different among WT, Gαi2 and
Gαi1/3 deficient mice (data not shown).
In contrast to Gαi2 deficiency, Gαi1/3 deficiency had no
effect on LPS- and SA-induced MIP-1α (Fig. 3A), MCP-1 (Fig.
3B), and MIG (Fig. 3D) production and LPS-induced IP-10
(Fig. 3C) production compared to WT mice. However, SAinduced IP-10 (Fig. 3C) production was decreased (54 ± 1%,
n = 3, p < 0.05) in splenocytes from Gαi1/3(−/−) mice compared
to WT mice.
3.3. Characterization of splenocytes subsets and cell surface
TLR2 and TLR4 expression in WT, Gαi2 or Gαi1/3 deficient
mice
To determine whether different responses of the splenocyte
stimulation are due to changes in cell population of the
knockout mice, spleen cell subsets from WT, Gαi2(−/−), Gαi1/3
(−/−) mice were analyzed by flow cytometry. Macrophages
from both Gαi2 and Gαi1/3 deficient mice were increased (1.9 ±
0.2 fold and 1.8 ± 0.1 fold respectively, n = 3, p < 0.05)
compared to WT mice. CD4+ T cells from Gαi2(−/−) mice
were decreased (28 ± 10% and 22 ± 10% respectively, n = 3,
p < 0.05) compared to WT and Gαi1/3(−/−) mice. CD8+ T cells
from Gαi1/3(−/−) mice were decreased (49 ± 15%, n = 3,
p < 0.05) compared to WT mice. There were no significant
differences in CD8+ T cells between Gαi2(−/−) mice and WT
mice. Dendritic cells from Gαi1/3(−/−) mice were increased
(1.8 ± 0.2 fold and 1.9 ± 0.2 fold respectively, n = 3, p < 0.05)
compared to WT and Gαi2(−/−) mice (Fig. 4).
We also examined the possibility that Gαi2 and Gαi1/3
deficiency have altered the expression of TLR2 and TLR4 in
splenocytes. However, flow cytometry analysis of TLR2 and
TLR4 expression in Gαi deficient mice were not different from
WT mice (data not shown).
4. Discussion
Our studies demonstrate that Gαi isoforms differentially
regulate cytokine and chemokine production in murine
splenocytes in response to microbial stimuli. LPS- and SAinduced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GMCSF, MIP-1α, MCP-1, MIG and IP-10 were increased in
splenocytes harvested from Gαi2(−/−) mice compared to WT
mice. Remarkably, splenocytes from Gαi1/3(−/−) mice did not
exhibit this augmented mediator response to LPS or SA. Indeed,
SA-induced IL-6, IFNγ, GM-CSF, and IP-10 production were
suppressed in splenocytes from Gαi1/3(−/−) mice suggesting
that Gαi1/3 and Gαi2 reciprocally regulate the latter cytokine and
chemokine responses. LPS- and SA-induced splenocyte
production of KC were unchanged in both Gαi protein deficient
groups. Because splenocytes are composed of multiple cell
types, i.e., macrophages, T cells, B cells, dendritic cells, and NK
cells, it is possible that changes in cellular composition could
affect the outcome. Analysis of splenic cellular composition
demonstrated an increase in dendritic cells in Gαi1/3(−/−) mice,
but there was an otherwise comparable increase in macrophages
and reduction in CD4 T cells in both groups of Gαi deficient
mice. Different splenic cellular compositions do not appear to
account for the isoform specificity in cells from Gαi2(−/−) and
Gαi1/3(−/−) mice. Splenocytes surface TLR2 and TLR4
expression were also examined by flow cytometry analysis.
TLR2 and TLR4 expression in splenocytes were unaffected by
Gαi2 and Gαi1/3 deficiency, demonstrating that different
response of splenocytes stimulation is not due to up-regulation
of TLR2 or TLR4 expression. Western blot also confirmed that
different response of splenocytes stimulation are not due to upregulation of other Gαi protein isoforms. Collectively, the data
suggest that Gi proteins play isoform specific positive and
negative roles in the production of cytokines and chemokines in
response to microbial stimuli. A summary of the major effects
of Gαi2 and Gαi1/3 deficiency on LPS and SA induced
cytokines and chemokines is presented in Table 1.
The augmented splenocyte response in mice with a targeted
genetic deletion of Gαi2 proteins is consistent with previous
studies suggesting a predominant pro-inflammatory phenotype.
Gαi2(−/−) mice develop an inflammatory bowel disease similar
to ulcerative colitis [25] and analysis of the inflamed colons
demonstrated increased expression of Th-1 type cytokines [26].
Augmented thymocyte and splenocyte production of proinflammatory cytokines in response to activation with several
microbial stimuli have subsequently been demonstrated in Gαi2
(−/−) mice [26–28]. We have recently shown there is cellular
phenotype specificity in macrophages vs. splenocytes in the role
of Gi proteins in TLR signaling [15]. In peritoneal macrophages, Gi proteins appear to positively regulate cytokine and
chemokine production, and Gαi1/3 depletion had a more
predominant effect than Gαi2 depletion. However in splenocytes, as shown by present data, Gi proteins appear to
negatively regulate cytokine and chemokine production, and
Gαi2 depletion clearly has predominant effects. The Gi isoform
specificity in splenocytes was manifested in the form of
reciprocal regulation of specific cytokines and chemokines.
The only notable exception was KC, which was unaffected by
Table 1
Summary of effects of Gαi2 and Gαi1/3 deficiency on LPS- and SA-induced
cytokine and chemokines in splenocytes
Stimuli
G protein
deficiency
A. Cytokine production a
LPS
Gαi2 (−/−)
SA
Gαi2 (−/−)
LPS
Gαi1/3 (−/−)
SA
Gαi1/3 (−/−)
Stimuli
G protein
deficiency
B. Chemokine production
LPS
Gαi2 (−/−)
SA
Gαi2 (−/−)
LPS
Gαi1/3 (−/−)
SA
Gαi1/3 (−/−)
a
TNFα
IL-6
IFNγ
IL-12
IL-17
GM-CSF
+
+
+
=
+
+
=
−
+
+
=
−
=
+
=
=
+
+
=
=
+
+
=
−
MIP-1α
MCP-1
KC
IP-10
MIG
+
+
=
=
+
+
=
=
=
=
=
=
+
+
=
−
+
+
=
=
Based upon results with splenocytes from Gαi2(−/−) and Gαi1/3(−/−) mice.
(+):increased production relative to wild type response; (−): reduced production
relative to wild type response; =: no difference from wild type response.
H. Fan et al. / Biochimica et Biophysica Acta 1763 (2006) 1051–1058
Gαi2 or Gαi1/3 deficiency. This is the first evidence of such
remarkable Gi isoform specificity in immune cell activation.
The Gi isoform specificity may underlie the propensity of Gαi2
(−/−) mice to develop inflammatory bowel disease and
expression of Th1 cytokines in inflamed colons [25]. This
phenotype is noticeably absent in the Gαi1/3(−/−) mice [25].
Previous studies have shown differences in TLR2 and TLR4
ligand stimulated cytokine and chemokine expression [29–32].
With the exception of IL-12, there were no major differences in
Gαi protein regulation of LPS- and SA-induced cytokines and
chemokines production, which suggest that Gαi protein play a
common role in signaling to both microbial stimuli. The reason
why SA induced a greater response than LPS in splenocytes is
not readily apparent. However, SA was a more potent stimuli
than LPS for the cytokine, e.g., TNFα and IL-6. This may be a
result of the fact that the intact SA cell components may activate
other receptors, e.g., CD36 and specific scavenger receptors in
addition to TLR2 [6,7]. Another issue of specificity is that
possible trace proteins in Salmonella enteritidis LPS could
potentially lead to cross reaction with TLR2 [33,34]. To further
examine TLR ligand specificity, we stimulated splenocytes with
protein free S. minnesota R595 LPS [15] and the soluble TLR2
ligand Pam3CysSK4 [16]. We found that both the latter TLR
ligands induced TNFα production were increased in splenocytes
from Gαi2(−/−) mice relative to WT mice (data not shown).
Thus although Salmonella enteritidis LPS and particularly SA
may activate other receptors, the studies with pure TLR ligands
suggest that Gi protein modulate TLR function.
Our recent studies have demonstrated that Gαi2(−/−) mice
manifest increased plasma TNFα and lung and gut leukosequestration compared to WT mice [15]. The up-regulation of
cytokines and chemokine expression in splenocytes from Gαi2
(−/−) mice may be a significant factor in potentiated
inflammatory innate immune response. The present findings
in splenocytes from Gαi2(−/−) mice suggest that TLR- activated
Gαi2 signaling pathways are counter-inflammatory. It is
possible that autocrine or paracrine mediators affect splenocyte
responses to TLR activation. Indeed, NK cells and T helper cells
produce IFNγ in response to LPS stimulation [35]. IFNγ has
been shown in many studies to amplify LPS-induced proinflammatory gene expression [36]. Increased local production
of IFNγ by lymphoid cells could indirectly amplify the proinflammatory response of splenic macrophages and other TLR
ligand responding sub-populations. In support of this possibility, we found that LPS- and SA-stimulated splenocytes from
Gαi2(−/−) mice produced 3 fold or greater increases in IFNγ
levels compared to WT mice. In contrast, LPS- and SA-induced
IFNγ production in splenocytes from Gαi1/3(−/−) mice were
unaffected or inhibited respectively. It is therefore possible that
TLR activation of Gαi2 signaling pathway may normally downregulate IFNγ production.
The findings that Gαi2 deficient mice exhibit a predominant
pro-inflammatory phenotype in vivo raise questions concerning
the mechanism whereby Gi proteins participate in TLR
signaling. Gi proteins may be directly or indirectly coupled to
TLR signaling proteins that regulate counter-inflammatory
signaling pathways. An example of a counter-inflammatory
1057
signaling pathway is the phosphatidylinositol-3 kinase (PI3
kinase) pathway, proposed as a braking mechanism for LPSinduced inflammation [37,38]. It has been shown that Gi protein
Gα and/or βγ subunits can regulate PI3 kinase activity [39].
However, recent studies by Lentshant et al. [16] demonstrated
that the Gi protein inhibition with mastoparan failed to inhibit
TLR4 or TLR2 activation of AKT, a downstream signaling
protein of PI3 kinase. It is also possible that the TLR signaling
pathway may trans-activate other receptors activated by LPS that
are Gi coupled. It is of interest in this context that Triantafilou et
al. [40] have proposed that LPS interacts with a cluster of
receptors in lipid rafts including Gi protein coupled receptors that
could be immunosuppressive [41–43]. Finally, an alternative
possibility is that Gi proteins are not coupled directly to TLR or
TLR post-receptor signaling proteins, but rather to heptahelical
receptors activated by autocrine mediators that are counterinflammatory [44]. Potential candidates for the latter mediators
include eicosanoids, purinergic agonists or chemokines.
Our findings support a convergent role of Gi proteins in
regulation of LPS- and SA-induced expression of cytokines and
chemokines. This study also provides the first evidence of
remarkable Gi isoform specificity in response to microbial
activation. Understanding the role of Gi protein isoforms in
regulation of TLR activation and other receptors activated by
Gram-negative and Gram-positive microbial stimuli will
provide important insights into regulation of innate immunity.
Acknowledgments
This work was supported by NIH GM27673, NIH GM53522
and NIH GM67202 and also supported in part by NIH
DK19318 (GB, KS and LB) and the Intramural Research
Program of the NIH, NIEHS (LB).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bbamcr.2006.08.003.
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