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Research Communications |
a Department of Immunology, University Hospital for Children and Youth, `Het Wilhelmina Kinderziekenhuis', 3584 EA Utrecht, The Netherlands;
b Institut für Medizinische Psychologie, 45122 Essen, Germany;
c Department of Rheumatology and Clinical Immunology, University Hospital, 3584 CX Utrecht, The Netherlands; and
d Department of Clinical Immunology and
e Department of Medical Psychology, Hannover Medical School, 36025-Hannover, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by ß2-adrenergic stimulation, suggesting that
reduced GRK activity is associated with increased sensitivity to
ß2-adrenergic activation.—Lombardi, M. S., Kavelaars,
A., Schedlowski, M., Bijlsma, J. W. J., Okihara, K. L., Van de Pol, M.,
Ochsmann, S., Pawlak, C., Schmidt, R. E., Heijnen, C. J. Decreased
expression and activity of G-protein-coupled receptor kinases in
peripheral blood mononuclear cells of patients with rheumatoid
arthritis.
Key Words: immune system • lymphocytes • cytokines • autoimmunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines may also play a role in arthritis. These proteins are known to mediate the chemotaxis and activation of leukocytes (8) . In two murine models of arthritis (MRL-lpr and DBA/1), it has recently been shown that administration of receptor antagonists of MCP-1 (monocyte chemoattractant protein-1) and RANTES (regulated on activation normal T cell expressed and secreted), respectively can have beneficial effects on the disease process 9, 10) . The latter data strongly suggest that these chemokines and their receptors play an important role in this chronic inflammatory disease by initiating and maintaining the local inflammatory process through recruitment of monocytes and lymphocytes in the joints (2) .
ß2-Adrenergic and chemokine receptors belong to
the G-protein-coupled receptor (GPCR) family whose responsiveness is
actively `turned off' by members of the G-protein-coupled receptor
kinase (GRK) family consisting of six known subtypes, GRK-1 to -6 (11)
.
These kinases are responsible for the rapid loss of receptor
responsiveness despite continuous presence of the agonist, a process
known as homologous desensitization. GRKs phosphorylate
serine/threonine residues in the carboxyl-tail and/or intracellular
loops of receptors in an agonist-dependent manner. The phosphorylated
form of the receptors act as substrates for a class of inhibitory
proteins called ß-arrestins, which sterically inhibit further
receptor/G-protein coupling (12)
. Uncoupled receptors are subsequently
removed from the plasma membrane. Recent studies also suggest that both
GRK and arrestins play a key role in this sequestration process 13, 14)
. Receptor substrates for GRKs identified so far are involved in a
wide variety of functions, ranging from neurotransmission to immune
responses (i.e., ß2- and
2 adrenergic receptors, muscarinic cholinergic
receptors, substance P receptor, CCR2B and CCR5 chemokine receptors,
fMLP receptor, etc.), and transduce signals through various
intracellular second messengers (11)
.
Among the six known GRKs, four (GRK-2, GRK-3, GRK-5, and GRK-6) are highly expressed in peripheral blood leukocytes (PBL) and in some myeloid and lymphoid cell lines 15-17) . Alterations in GRK activity have been demonstrated in T cell activation (16) as well as in human diseases like hypertension (18) and heart failure (19) . As the use of antagonists of GPCRs has been proven to produce beneficial effects on the onset and the severity of the arthritis, we wondered whether in RA patients the proinflammatory signal pathways mediated through some G-protein-coupled receptors are less efficiently turned off by the GRK/ß-arrestin desensitization machinery. Therefore, we have assessed GRK activity and expression in human peripheral blood mononuclear cells (PBMC) from RA patients and healthy controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sample preparation
PBMC were isolated from heparin anticoagulated whole blood by
Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) density gradients (21)
.
For preparation of cytosolic fractions, PBMC were lysed in ice-cold
lysis buffer (10 mM Tris, 5 mM EDTA, 7.5 mM
MgCl2, 0.1 mM PMSF, 10 µg/ml leupeptin, 5
µg/ml pepstatin, 10 µg/ml benzamidine, at pH 7.4), using a polytron
tissue disrupter (Janke and Kundel, Staufen, Germany) at low speed for
40 s on ice. Unbroken cells and nuclei were pelleted by
centrifugation (800 x g for 5 min) and discarded. The
supernatant was then centrifuged (48,000 x g for 20
min at 4°C) to separate plasma membrane from the cytosol. The latter
was centrifuged at 300,000 x g for 30 min at 4°C.
The supernatant was collected and frozen in liquid nitrogen. Membrane
preparation was washed once in cell lysis buffer and recentrifuged at
48,000 x g for 20 min at 4°C; the resultant membrane
pellet was resuspended in cell lysis buffer and sonicated for 5 s
before protein determination. In addition to assay membrane-associated
GRK activity, membranes were washed once in cell lysis buffer,
centrifuged at 48,000 x g for 20 min at 4°C, and the
resultant membrane pellet was resuspended in cell lysis buffer with 250
mM NaCl (to detach membrane-bound GRKs) (22)
. The suspension was
sonicated briefly, incubated for 30 min at 4°C and centrifuged as
above. Membrane-detached proteins (2–5 µg) were used in the
phosphorylation assay. Protein concentration was determined with a
Bio-Rad protein assay reagent, using bovine serum albumin as standard.
Assessment of G-protein-coupled receptor kinase activity
GRK enzymatic activity was assessed using light-dependent
phosphorylation of rhodopsin (23)
. We purified rod outer segment
membranes (ROS) from dark-adapted bovine retinas by stepwise sucrose
gradient centrifugation and subsequent treatment with 5M urea to
inactivate endogenous rhodopsin kinase activity. The resulting
preparation contained ~ 95% rhodopsin (23)
and showed
negligible endogenous kinase activity.
Four GRK subtypes are highly expressed in PBL and their ability to phosphorylate ROS, when transiently expressed in COS7 cells, shows the following relative order of potency: GRK-2 >> GRK-3 = GRK-5 >> GRK-6. In this paper, therefore, we refer to the ROS phosphorylation assay as GRK activity, although it mainly measures GRK-2 activity (16) .
GRK-dependent phosphorylation was determined by incubating 50 µg of
cytosolic protein with ~ 300 pmol of rhodopsin in a buffer
containing 65 µM [ 
-33P] ATP (2–5
cpm/fmol, Amersham, Buckinghamshire, U.K.), 20 mM Tris, 8 mM
MgCl2, 3 mM EDTA, 5 mM NaF, 12 mM NaCl at pH 7.4
in a final reaction volume of 100 µl. The reactions were carried out
at 30°C for 30 min in presence (or absence) of light. The incubations
were terminated by the addition of 40 µl of sodium dodecyl sulfate
(SDS) sample buffer (8% SDS, 20% glycerol, 5% ß-mercaptoethanol,
250 mM Tris-HCl pH 6.8 and 0.003% bromphenol blue). Samples were then
electrophoresed on 10% SDS-polyacrylamide gel electrophoresis (PAGE)
(24)
. After electrophoresis, the gel was stained with Coomassie blue,
dried, and phosphorylated rhodopsin was visualized by autoradiography.
Bands corresponding to rhodopsin (~ 38 kDa) were cut from the gel and
quantitated via liquid scintillation spectroscopy. The GRK-dependent
phosphorylation was confirmed by adding the protein kinase A inhibitor
PKI (1 µM, Sigma Chemical Co., St. Louis, Mo.) and heparin (10
µg/ml, Leo chemicals, Netherlands) to the reaction and the ability to
phosphorylate rhodopsin was determined. Heparin inhibited
phosphorylation but PKI did not. All results were confirmed in at least
three separate experiments, using cells obtained from different
individuals.
Assessment of GRK and arrestin expression
Assessment of GRK and arrestin protein expression was determined
by immunoblotting. GRK-2, GRK-5, and GRK-6 protein expression was
determined using a 1:200 dilution (in all cases) of a rabbit polyclonal
antibodies raised against amino acids 675–689 of human GRK-2, amino
acids 571–590 of human GRK-5, and amino acids 525–544 of human GRK6,
respectively. All antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, Calif.).
Expression of arrestins was determined using a 1:2,000 dilution of the arrestin mouse monoclonal antibody F4C1, which recognizes the epitope DGVVLVD, identical in human ß-arrestin 1, arrestin 3 (ß-arrestin 2), and arrestin (25) .
Samples containing 30 µg of proteins were suspended in sample buffer (as above) by gentle shaking for 10–15 min at 37°C and electrophoresed on 10% SDS-PAGE. Proteins were transferred to presoaked nitrocellulose membranes (Hybond-C, Amersham) by electroblotting at 100 V for 1 h Efficiency of transfer was verified by Ponceau red staining. Membranes were soaked in a blocking buffer solution of 5% non-fat dry milk in TTBS (10 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.05% Tween 20) for 1 h (2 h in the case of arrestin) at room temperature, then incubated with antibodies (anti GRKs or arrestin) diluted in TTBS for 1 h (2 h in the case of arrestin) at room temperature. Membranes were washed three times with Tris-buffered saline and incubated for 1 h at room temperature with either peroxidase-conjugated donkey anti-rabbit IgG (Amersham) at 1:10,000 dilution (for all GRKs antibodies) or peroxidase-conjugated sheep anti-mouse IgG (Boehringer Mannheim GmbH, Germany) at 1:10,000 dilution (for arrestin antibody). Immunoreactivity was detected with an enhanced chemiluminescence detection system (ECL, Amersham Int.) and bands were visualized after exposing blots to X-ray film. In some cases the same membrane was used for subsequent reprobing with specific antibodies after being stripped by incubating for 20 min in 0.1M glycine pH 2.9 at room temperature. Autoradiographs were scanned using a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, Calif.). All results were confirmed in at least three separate experiments, using cells obtained from different individuals.
Northern blot analysis
Total RNA was isolated from PBMC using RNAzol (CAMPRO Sci.,
Veenendaal, Netherlands). The integrity and purity of the RNA was
assessed by gel electrophoresis and ultraviolet absorbance ratio; the
sample was rejected if the ratio was < 1.6 or if visual
inspection of the photographed gel suggested degradation. Then 15
µg/lane were fractionated on a 1% agarose-formaldehyde gel,
transferred to a Hybond N+ membrane (Amersham), and immobilized with a
Stratalinker UV light source. Northern blot analysis was performed
using the random primed cDNA fragment (bp 955-2007) of GRK-2 as probe
(15)
. Hybridization was for 20 h at 42°C in 50% formamide, 10%
dextran sulfate, 1% SDS, 5.8% NaCl, and denatured herring sperm DNA
(100 µg/ml). The blot was then washed twice in 2X SSC for 5 min,
twice in 2X SSC/1% SDS at 60°C for 15 min, once in 0.2X SSC/0.1%
SDS at 42°C for 15 min, then rinsed briefly in 0.1X SSC at room
temperature and subjected to autoradiography at -80°C for 24–72 h.
GRK-6 mRNA expression was determined on the same filter, after being
stripped with boiling 0.5% SDS, using a random primed cDNA fragment
(bp 1114–2030) of GRK-6 (26)
. Hybridization and washings were
performed as described above. All results were confirmed in at least
two separate experiments, using cells obtained from different
individuals.
ß2-Adrenergic receptor binding on PBMC
ß2-Adrenergic receptors were quantitated
using the ligand [125I]iodocyanopindolol
([125I]ICYP) (Amersham). The assay was carried
out in triplicate in Eppendorf tubes. The incubations were performed in
a total volume of 125 µl of PBS/0.5% bovine serum albumin. Total
binding was determined by incubating the cells (2.5 x
105cells/well) with 8 concentrations
[125I]ICYP (range 10–150 pM) at 37°C for 30
min Nonspecific binding was determined under the same conditions in the
presence of 1 mM (-) propranolol (Sigma Chemical Co.). After the
incubation, 100 µl of the mixture was pipetted in to Scatchard tubes
(Sarstedt), containing 150 µl of an oil-phthalate mixture (20% oil,
80% phthalate). Subsequently, the tubes were centrifuged for 90 s
at 13,000 x g in a Microfuge and the part of the tube
containing the cell pellet was cut off. Both the radioactivity bound to
the cells as well as the amount of free label was determined in a gamma
counter.
TNF- production
Tumor necrosis factor alpha (TNF-) production was induced by
culturing 100 µl of diluted whole blood (1:10 in RPMI 1640
supplemented with antibiotics) with 50 µl of lipopolysaccharide (LPS)
(Escherichia coli, Difco, Detroit, MI) in a final
concentration of 2 ng/ml and 50 µl of medium or the
ß2-adrenergic receptor agonist terbutaline
(Sigma Chemical Co.) in the concentrations 0–500 nM. After 18 h
of culture at 37°C, supernatants were harvested and stored at
-80°C until analysis. TNF- levels in the supernatants were
determined by ELISA (Pelikine, CLB, Amsterdam, The Netherlands).
cAMP accumulation
PBMC were isolated as described above and resuspended in RPMI
containing 20 mM HEPES at a density of 10 x
106 cells/ml. Cell viability, as assessed by
trypan blue exclusion, was higher than 90% in all samples. The
stimulation of cAMP accumulation (27)
was performed (in duplicate) by
adding 0.1 ml of cell suspension to 0.9 ml DMEM (prewarmed at 37°C)
containing 1 mM isobutyl-methylxanthine (IBMX, to inhibit
phosphodiesterase activity) and 1 µM (-) isoproterenol (Sigma,
Chemical Co.) for different times or no agonist (basal) at 37°C.
Isoproterenol was protected against oxidation in these incubations by
adding 20 µg/ml each of superoxide dismutase and catalase (both from
Sigma). The reactions were terminated by centrifugation for 20 s.
The supernatant was quickly removed and the pellet was resuspended in
250 µl Tris-EDTA (50 mM) buffer at pH 7.5. The samples were placed in
a boiling water bath for 5 min, then frozen. After thawing, samples
were sonicated and the protein flocculate was pelleted by
centrifugation. Aliquots (50 µl) of the resulting supernatants were
assayed using a cAMP[125I] scintillation
proximity assay system kit (Amersham Pharmacia Biotech) following
manufacturer's instructions.
Cytokine treatment of PBMC
PBMCs from healthy donors were isolated as described above and
resuspended in RPMI-1640 culture medium (Life Technologies, Inc. Life
Technologies, Inc., Grand Island, N.Y.) supplemented with 5% fetal
calf serum (Life Technologies, Inc.), 2 mM L-glutamine, 100 U/ml
penicillin and 100 µg/ml streptomycin. PBMCs (2 x
106/ml) were then cultured for 6, 24, or 48 h in the absence or presence of 10 ng/ml of IL-6 (PreproTech, Inc.,
Rocky Hill, N.J.) or 20 ng/ml of rhIFN- (recombinant human
interferon-, R&D Systems, Minneapolis, Minn.) in 5%
CO2 at 37°C. Cells were then harvested and
tested for viability by trypan blue exclusion. In all samples viability
was higher than 90%. Cytosolic fractions were isolated and analyzed by
Western blot, as described above. All results were confirmed in at
least two separate experiments, using cells obtained from different
individuals.
Statistical analysis
Data are expressed as a mean value ± SE.
Specific measurements were compared using the Student's t
test for unpaired (unless otherwise stated) data. Data for inhibition
of TNF- production and cAMP accumulation were analyzed using two-way
analysis of variance (ANOVA). A value of P<0.05 on a
two-tailed test was used as a minimum level of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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o such phosphorylation was observed when the cytosolic fraction was
omitted from the ROS incubation, indicating the absence of rhodopsin
kinase (GRK-1) in the ROS preparation (data not shown). The
light-dependent ROS phosphorylation was completely inhibited by the
addition of heparin (10 µg/ml) and was not blocked by 1 µM protein
kinase A inhibitor PKI (data not shown).
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In cytosolic fractions of PBMC from RA patients, GRK activity was
significantly decreased (109 ± 9
fmol·min-1·mg cytosolic
protein-1, P<0.001) as compared with
GRK activity in samples from healthy donors (216 ± 14
fmol·min-1·mg cytosolic
protein-1) (Fig. 1C
).
Although GRKs are essentially cytosolic proteins, a significant amount
of kinase activity is associated with the plasma membrane 16, 28)
. To
rule out the possibility that the decrease in GRK activity found in
PBMC from RA patients was simply due to GRKs translocation from cytosol
to membrane fractions, we also assessed membrane-associated kinase
activity. We found a similar decrease (46 ± 6%) in
membrane-associated GRK activity in PBMC from RA patients in comparison
to healthy donors (Fig. 1B
).
GRK-2 protein expression
To determine whether the decrease observed in GRK-mediated
phosphorylation of ROS in PBMC from RA patients was associated with a
decrease in immunodetectable GRK-2 protein, we performed quantitative
Western blotting analysis. The anti-GRK-2 antibody recognizes a protein
of an apparent molecular mass of ~ 80 kDa that comigrates with
recombinant GRK-2 protein (Fig. 2
A). Immunodetectable GRK-2 (Fig. 2B
) was
significantly reduced in cytosolic fractions from PBMC of RA subjects
(191 ± 10 ng/mg cytosolic protein, P<0.001) compared
with healthy donors (453 ± 24 ng/mg cytosolic protein).
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GRK-5 and GRK-6 protein expression
We assessed levels of immunodetectable GRK-5 and GRK-6 by Western
blotting to determine whether the decrease observed in GRK activity was
due to a selective decrease in GRK-2 expression or to a more general
decrease in the expression of other GRKs. Western blotting of
immunodetectable GRK-5 (Fig. 3
A) showed that there were no significant differences (RA:
91 ± 4.5% of expression in healthy donors) in cytosolic
fractions of PBMC from RA patients when compared with healthy controls
(Fig. 3B
). Similar results were obtained with
membrane-extracted fractions (29)
(data not shown).
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In PBMC from RA patients and healthy controls, two GRK6 immunoreactive
bands of ~ 65 and ~ 67 kDa were detected in cytosolic
fractions (Fig. 4
A). We found a decrease in GRK-6 protein levels in cytosolic
fractions from PBMC from RA patients compared with healthy controls.
When both bands of the 65/67 kDa protein doublet were analyzed by
densitometry, the net decrease observed in RA subjects was ~
66% (Fig. 4B, P
<0.001). Moreover, when the
bands were analyzed separately, the 65 kDa bands showed a ~ 90%
decrease in PBMC from RA subjects while the decrease of the 67 kDa
bands was ~ 53% when compared with healthy controls. A similar
decrease (~ 40%) in RA patients compared with healthy donors was
observed in membrane fractions (Fig. 4C
).
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Assessment of ß-arrestin expression by immunodetection
We assessed whether the decrease in GRK-2 and GRK-6 protein was
associated with a change in ß-arrestin immunodetectable levels. The
anti-ß-arrestin antibody used for Western blot recognizes two
proteins of ~ 55 and ~ 52 kDa corresponding to
ß-arrestin-1 and arrestin 3, respectively (Fig. 5
A). Figure 5B
shows that immunodetectable levels
of the arrestins were not significantly different in cytosolic samples
of PBMC from RA patients in comparison to healthy controls (94 ±
5%, P=0.25).
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Northern blot analysis of GRK-2 and GRK-6
We next determined whether GRK-2 and GRK-6 mRNA expression levels
were associated with the decrease observed in protein expression in
PBMC from RA patients. GRK-2 mRNA levels (~ 3.8 kb transcript) in
PBMC from RA patients were not significantly different from that of
healthy donors (102 ± 3% of controls) (Table 1)
. The latter suggests that the decrease in GRK-2 protein is caused by a
mechanism of posttranscriptional regulation and/or alteration in
protein stability. The same gel was stripped and hybridized again with
a GRK-6 cDNA probe, which detects two transcripts of ~ 3 and
~2.4 kb, respectively (26)
. Recently it has been shown that these two
mRNA species can arise from alternative splicing in the 3' untranslated
region and/or be due to alternative polyadenylation of transcripts from
a single gene in human chromosome 5 (30)
. We observed a slight decrease
of the upper transcript in RA patients (16.5 ± 1.4%,
P=0.054) whereas the lower mRNA species is not significantly
changed (94 ± 6.5% of healthy donors) (Table 1)
.
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Determination of ß2-adrenergic receptors on PBMC
The density (Bmax) and
Kd of ß2-adrenergic
receptors on PBMC from RA patients (n=7) and healthy donors
(n=8) showed no significant differences between the two
groups (Table 2
).
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ß2-Adrenergic receptor function
To investigate whether the reduced GRK activity is associated with
an alteration in ß2-adrenergic receptor
function, we tested the ability of the
ß2-adrenergic receptor agonist terbutaline to
inhibit the production of TNF- by PBL of RA patients and healthy
donors in a dose-dependent manner (31)
. For this purpose, whole blood
was diluted (1:10 in RPMI 1640) and cultured in the presence of LPS (2
ng/ml) to induce TNF- production and terbutaline in a dose range of
0–500 nM. The results of Fig. 6
A show clearly that the dose-dependent inhibition of TNF-
production by terbutaline is significantly increased in RA patients
compared with healthy controls (two-way ANOVA, P<0.0001).
We also studied the kinetics of cAMP accumulation in PBMC from RA
patients and healthy donors in the presence of a phosphodiesterase
inhibitor (IBMX). Figure 6B
shows that the production of
cAMP in PBMC from RA patients was significantly increased (two-way
ANOVA, P<0.001).
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Effects of cytokine treatment on GRK-2 protein expression
To investigate a possible mechanism responsible for the GRK-2
down-regulation observed in PBMC from RA patients, we tested whether
proinflammatory cytokines, which are known to be increased in RA, can
affect GRK-2 protein expression. PBMCs from healthy donors were
cultured in the absence or presence of 10 ng/ml of IL-6 or 20 ng/ml of
rhIFN- for 6, 24, or 48 h and GRK-2 protein levels were
assessed by Western blot analysis. No significant changes in GRK-2
protein were observed for rhIFN--treated cells after 6 h, but
after 24 h and 48 h we observed a marked decrease of 57% and
80%, respectively, when compared with the expression of the respective
untreated controls, which was considered 100% (Fig. 7
A). An even more pronounced effect was observed in IL-6
treated cells. A decrease of ~ 40% was already evident after
6 h; after 48 h, the GRK-2 protein was barely detectable
(Fig. 7B
).
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DISCUSSION |
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production by a ß2-adrenergic agonist
stimulation. Moreover, treatment of PBMC from healthy donors with
proinflammatory cytokines like IL-6 and IFN- induces a
down-regulation of GRK-2 protein levels. The possibility that drug treatment of patients is responsible for the observed down-regulation of GRKs in RA patients is very unlikely. We demonstrated in an in vitro model (32) that GRK activity is not altered by NSAIDs (M. S. Lombardi, unpublished observations). Moreover, treatment of PBMC with 1 µM dexamethasone for 24–48 h does not induce changes in GRK activity and GRK-2 protein expression levels (M. S. Lombardi et al., unpublished observations). Fifty percent of our patients received methotrexate. However, we did not observe any difference in GRK activity and expression between patients who did or did not receive this antimetabolite drug. Moreover, it seems unlikely that this interference with cellular metabolism has differential effects on the expression of the various GRK proteins.
The Northern blot analysis of GRK-2 and GRK-6 suggests that the reduction in GRK activity and GRK-2 and GRK-6 protein may be due to changes at the posttranscriptional level and/or on the level of protein stability. Previous sequence analysis has shown that there is a high degree of secondary structure formation at the 5' region of GRK-2 mRNA due to a high GC content. This observation suggests an argument for posttranscriptional regulation to occur (33) . Western blot analysis of cytosolic GRK-6 revealed a differential expression of the two immunodetectable bands (a ~53% decrease of the 67 kDa bands and a ~ 90% decrease of the 65 kDa bands in RA subjects compared with healthy donors). The significance of this phenomenon is unclear. However, two GRK-6 splice variants were recently found in the rat. They encode for two isoforms of GRK-6: GRK-6a and GRK-6b, of 576 aa and 589 amino acids, respectively, differing in their COOH-terminal domain (34) . It may be that the observed ~ 65/67 kDa doublet reflects the homologous human GRK-6 splice variants. Elucidation of the physiological role of the two GRK-6 isoforms and the identification of more specific substrates for GRK-6, which are expected to be distinct from those identified for GRK-2 as suggested by in vitro studies (35) , may provide more insight on this issue. For both kinases, a selective and cell-specific differential expression at both mRNA and protein levels has been recently described in myelomonocytic and lymphoid cells 16, 36) . These data suggest that these kinases play an important role in modulating several G-protein-coupled receptors expressed in immune cells.
It has been described that T cells in the peripheral circulation of RA patients are activated due to the inflammatory process (37) . However, the decrease of GRK activity and expression in PBMC of RA patients is apparently not due to already in vivo activated T cells, since activation of PBMC by mitogen or anti-CD3 leads to a clear increase of GRK-2 and GRK-6 activity and expression 16, 36) .
Why do we observe a decrease in GRK activity and GRK-2 and GRK-6
expression? To approach this issue, we investigated whether the
proinflammatory cytokines IL-6 and IFN- are able to modulate GRK-2
expression in cells of healthy control donors. We show here for the
first time that these cytokines indeed down-regulate the expression of
GRK-2 in PBMC from healthy donors. The production of proinflammatory
cytokines is known to be increased in RA patients, and the TH1 cytokine
IFN- has been shown to play a central role in maintaining the
inflammatory process (11)
More recently, a role for IL-6 in a murine
model of antigen-induced arthritis has been more clearly established
(38)
. On the basis of our results, we hypothesize that the increased
production of proinflammatory cytokines causes the decrease observed in
GRK-2 expression and activity in RA. Via this mechanism, cytokines may
contribute to enhanced reactivity of PBMC to agonists that signal via
G-protein-coupled receptors. In heart failure, a sustained activation
of the sympathetic nervous system is associated with an increase of
both GRK activity and GRK-2 mRNA 19, 39)
. The concept that GRK
activity may be related to the degree of sympathetic stimulation
in vivo also arises from the observation that an increase of
GRK activity occurs in the liver of neonate rats after the transient
physiological increase of catecholamines (40)
. On the other hand,
diminished activity of the autonomic nervous system has recently been
demonstrated in both established and recent onset RA patients and
associated with decreased sympathetic activity (41)
. From this data, we
may hypothesize that reduced sympathetic activity could be responsible
for the observed reduction in GRK activity and expression in PBMC from
RA patients. Whether down-regulation of GRKs may be interpreted as an
adaptive response of the adrenergic receptor system remains to be
elucidated. It is, however, remarkable that
ß2-adrenergic receptors on PBL from RA patients
have an increased signaling capacity when compared with
ß2-adrenergic receptors from healthy
individuals and that the production of cAMP in PBMC from RA patients
was significantly increased (Fig. 6A, B
). The fact that the
number and the Kd of
ß2-adrenergic receptors are not significantly
different between patients and controls indicates a reduced homologous
desensitization consistent with the decreased levels of GRK activity
observed in RA patients. Therefore, we suggest that the reduced GRK
activity is associated with an increased sensitivity to
ß2-adrenergic stimulation.
The lower GRK activity and expression in RA subjects may also have consequences for the function of other GPCR that play a role in the disease process. The use of several experimental approaches, including reconstitution studies in vitro, receptor mutagenesis, and GRK/ß-arrestin overexpression in some cell lines, have demonstrated that the GRK/ß-arrestin desensitization machinery is involved in the regulation of a large number of GPCR. One example is the substance P (SP) receptor, which is a substrate for GRK-2 and -3 (42) . SP has many proinflammatory effects in vivo and has been demonstrated to play an important role in the pathogenesis of RA. It is significantly increased in the synovial fluid of RA patients (43) and can be released from primary afferent nerve fibers into the joints. If, indeed, the SP receptor signaling system is less efficiently turned off because of the decrease of GRK-2 activity, one would expect RA patients to be more sensitive to this proinflammatory peptide. The latter may be true in view of enhanced SP signaling during inflammation leading to spinal hyperexcitability, which is responsible for the hyperalgesia characteristic of RA. Another group of important mediators in RA that signal via G-protein-coupled receptors includes the chemokines. In murine models of arthritis, inhibition of chemokine function by specific receptor antagonists for MCP-1 and RANTES can prevent the onset and/or reduce the symptoms once the disease had developed. The monocyte chemoattractant protein-1 MCP-1 is produced at high levels by both infiltrated monocytes and synovial cells in RA 44, 45) , and RANTES and MIP-1ß (macrophage inflammatory protein-1ß) are more abundantly expressed in both circulating PBL and in synovial fluid T cells of RA patients (46) . It has recently been demonstrated that the MCP-1 chemokine receptor CCR2B is a substrate for GRK-3 and GRK-2 when they are coexpressed in Xenopus oocytes (47) or HEK293 cells (48) , respectively. Moreover, the CCR-5 chemokine receptor, which interacts with MIP-1ß (macrophage inflammatory protein-1ß) and RANTES, is a substrate for GRK-2 and GRK-3 when overexpressed in HEK-293 cells (49) . Therefore, we suggest that the lower GRK activity will increase the sensitivity for chemokines in cells from RA patients.
In conclusion, although the precise mechanism responsible for the GRK decrease in RA remains to be fully defined, we have shown here that proinflammatory cytokines decrease GRK-2 expression. Moreover, our results suggest that, in RA patients, the proinflammatory signal pathways mediated through some G-protein-coupled receptors (i.e., ß2-adrenergic, substance P, CCR5) are less efficiently turned off by the GRK/ß-arrestin desensitization machinery. The latter may explain the reported beneficial effects on the onset and the severity of joint injury after treatment with some G-protein-coupled receptor antagonists. Furthermore, these data demonstrates that the GRK/ß-arrestin desensitization machinery can be selectively altered during chronic inflammatory diseases and may open up a field for investigating possible therapeutic strategies, including modulation of GRK activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Abbreviations: GPCR, G-protein-coupled receptor; GRK,
G-protein-coupled receptor kinase; IBMX, isobutyl-methylxanthine;
rhIFN-, recombinant human interferon-; LPS,
lipopolysaccharide(s); MCP-1, monocyte chemoattractant protein-1;
NSAIDs, nonsteroidal antiinflammatory drugs; PAGE, polyacrylamide gel
electrophoresis; PBL, peripheral blood leukocytes; PBMC, peripheral
blood mononuclear cells; RA, rheumatoid arthritis; RANTES, regulated on
activation normal T cell expressed and secreted; ROS, rod outer segment
membranes; SDS, sodium dodecyl sulfate; SP, substance P; TNF, tumor
necrosis factor.
Received for publication August 24, 1998.
Revision received November 16, 1998.
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