Granulocyte chemotaxis and disease expression are
differentially regulated by GRK subtype in an acute
inflammatory arthritis model (K/BxN)
Teresa K. Tarranta,⁎, Rishi R. Rampersada, Denise Essermanb,
Lisa R. Rothleina, Peng Liua, Richard T. Premontc, Robert J. Lefkowitzc,d,
David M. Leee, Dhavalkumar D. Patela,f
aThurston Arthritis Research Center and the Department of Medicine, Division of Rheumatology, Allergy and Immunology,
University of North Carolina School of Medicine, CB 7280, 3300 Manning Dr., Chapel Hill, NC 27599, USA
bUNC School of Public Health, Chapel Hill, NC 27599, USA
cDuke University, Department of Medicine, Division of Cardiology, Durham, NC 27110, USA
dHoward Hughes Medical Institute, Chevy Chase, MD 20815, USA
eBrigham and Women's Hospital, Division of Rheumatology, Allergy and Immunology, Boston, MA 02115, USA
fNovartis Institutes for BioMedical Research, Basel, Switzerland
Received 22 January 2008; accepted with revision 9 June 2008
Available online 26 July 2008
Objective: Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-
protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK
subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/
Methods: Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−, GRK2+/−, and
wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed
by transendothelial migration assays.
Results: Both GRK6−/− and GRK2+/− mice had increased arthritis disease severity (pb0.001);
whereas GRK5−/− was not different from controls. Acute weight loss was enhanced in GRK6−/−
and GRK2+/−mice (pb0.001, days 3–10). However, GRK6−/−mice uniquely had more weight loss
(N10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro.
Conclusions: GRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the
K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte
trafficking toward chemoattractants.
© 2008 Elsevier Inc. All rights reserved.
⁎ Corresponding author.
E-mail address: firstname.lastname@example.org (T.K. Tarrant).
1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
available at www.sciencedirect.com
Clinical Immunology (2008) 129, 115–122
G-protein coupled receptors (GPCRs) are seven transmem-
brane spanning receptors that represent the largest super-
family of membrane-bound receptors in nature. With respect
to immune function, GPCR–ligand interactions play critical
roles in organ-specific leukocyte trafficking and activation,
inflammatory-mediated chemotaxis, effector cell function,
and cell survival [1,2]. Consequently, the regulation of GPCRs
and their downstream signaling molecules is an attractive
therapeutic target for patients with autoimmune disease.
GPCR-mediated signaling maintains a coordinated balance
betweenreceptoractivation, desensitization,and subsequent
of molecules that play a critical role in the desensitization
process of activated GPCRs [3,4]. Specifically, GRKs phosphor-
ylate only ligand-bound and activated GPCRs, leading to rapid
subsequent homologous desensitization [4,5].
There are 7 different subtypes of GRKs  that have
varying tissue distribution, suggesting some non-overlapping
regulation of cellular functions. GRK1 and -7 are exclusively
expressed in the retinal rods and cones respectively, and
GRK4 has a high level of expression in the testes and a low
level in kidney and cerebellum . GRK subtypes -2, -3, -5,
and -6 are expressed ubiquitously but have particularly high
expression in leukocytes [8,9,10]. Moreover, GRK subtypes
appear to be differently regulated in inflammatory disease
states. Specifically, GRK6 and -2 protein levels are down-
regulated within peripheral blood mononuclear cells of
rheumatoid arthritis patients . In the rat model of
adjuvant arthritis, GRK6 and -2 are decreased N50% in
lymphoid organs at the peak of the inflammatory process,
which subsequently normalized during the remission phase of
the disease . These observations suggest that GRKs play
an important regulatory role in immune function and could
have a more critical role in autoimmune disease states.
In gene deletion studies, deficiencies in GRK6 or -2 have
been shown to specifically affect the migration of leukocyte
subsets both in vitro and in vivo [13,14]. Specifically, GRK6-
deficient (GRK6−/−) mice have increased granulocyte
infiltration in the AA-induced ear inflammation model after
topical arachidonic acid is applied . GRK6−/− mice also
have enhanced granulocyte-mediated inflammation of the
gut in a dextran sodium sulfate (DSS)-induced colitis model
. In contrast, in vitro work has shown reduced lymphocyte
chemotaxis in GRK6−/− T cells to CXCL12 . GRK2
heterozygote mice (GRK2+/−) have been evaluated in the
animal model of multiple sclerosis, (experimental allergic
encephalomyelitis, EAE) since homozygotes are embryonic
lethal due to cardiac dysgenesis . Tcells and macrophages
were increased in the central nervous system of GRK2+/−
mice in the early stages of disease, followed by a reduction in
the inflammatory cell infiltrate at later time points .
Given the aforementioned data supporting GRK involve-
ment in inflammatory disease expression in vivo and in
leukocyte trafficking in vitro, we hypothesized that GRK6 and
-2 may be involved in the pathogenesis of inflammatory
arthritis via effects on chemotaxis. To test this, we performed
studies using the K/BxN serum transfer mouse model of acute
inflammatory arthritis  where the serum from K/BxN mice
can transfer disease to naive recipients , resulting in bony
erosions similar to human rheumatoid arthritis . The K/
BxN serum transfer model is notably independent of both B
and Tcells, whereas granulocytes are critical for disease .
Consequently, this model enables us to examine the regula-
tory effects of GRK2, -5, and -6 gene deletions that are
independent of the lymphocytic induction phase of disease
and more reflective of the effector immune response.
Materials and methods
Animals used in these experiments were backcrossed ≥9
generations onto the C57/Bl6 strain with littermate controls.
All animals were bred, housed, and cared for in DLAM
facilities under the approved IACUC protocol number
03-265.0 in pathogen free specific conditions.
Induction and evaluation of K/BxN arthritis
Male 6 week old mice heterozygous for GRK2 or deficient in
GRK5 or -6 and wildtype littermates were injected on days 0
and 2 with a dose of 5 µl K/BxN serum per gram of mouse
weight intraperitoneally. Mice were measured daily for
endpoints of paw swelling, clinical disease index, and
weight. Paw swelling was represented as a change in the
mean thickness of the mouse's fore- and hindlimbs (mm) from
its baseline average and was obtained by a blinded measurer.
Clinical index was assessed as the consensus of 2 blinded
observers with the following scoring system: 0=normal paw;
1=mild but definite swelling of either the ankle or digits;
2=moderate redness and swelling of an ankle±any number of
digits; 3=maximal redness and swelling of the entire paw and
of 12 per mouse. Results for paw swelling and clinical index
are reported as an average in the total score± SEM for the
group. At experiment termination, hindlimbs were fixed for
24 h in 4% paraformaldehyde, decalcified using formic acid for
24–48 h, and embedded in paraffin for histopathology.
Histopathology sections of the hindlimbs were stained with
hematoxylin and eosin and were scored by a blinded observer
for the severity of inflammatory cell infiltrate and the pre-
sence of erosions using the following scoring system:
0=normal paw; 1=mild inflammatory cell infiltrate with mini-
mal synovial thickening and no architectural distortion; 2=
moderate inflammatory cell infiltrate with enhanced synovial
proliferation without erosions; 3=severe inflammatory cell
infiltrate with erosions.
Transendothelial migration assays
Transendothelial migration (TEM) assays were performed as
previously described . Briefly, 104Ea.hy 926 endothelial
for 7 days. Monolayer integrity was determined by [14C]
mannitol diffusion. For migration studies, the medium in the
bottom chamber was replaced with medium or chemoattrac-
tant (10 nM LTB4, 100 nM C5a; Sigma-Aldrich, St. Louis, MO, or
116 T.K. Tarrant et al.
hIL-8 100 ng/ml; R&D Systems, Minneapolis, MN) and primary
bone marrow cells isolated from individual mice of the
respective GRK genotypes were added to the top chamber.
were harvested and stained with anti-mouse Gr-1 conjugated
granulocytes was determined by flow cytometry.
Serum cytokine analysis
Mouse blood was collected by tail vein nick on days 0, 4, and
8 after immunization with K/BxN mouse serum on days 0 and
2. Cytokine measurements were performed using a Millipore
Beadlyte Multiplex assay (Millipore Corporation, Billerica,
MA), and analyzed on a Luminex 100 Total System v.1.7
(Luminex Corporation, Austin, Texas) according to the
manufacturer's protocol. Data were analyzed using Starsta-
tion v.2.0 (Applied Cytometry Systems, Sacramento, CA).
For clinical index, paw swelling, and % weight loss curves, a
statistical curve-fit was usedtodetermine whether significant
differences existed in the course of the disease over time
between the GRK-deficient mice versus wildtype controls. A
backward selection (α=.05) procedure was used to select a
linear mixed model with the best fit for the individual curves.
Variables considered in the statistical analysis included group,
time, and experiment effect. The overall group effect was
assessed using a likelihood ratio test (LRT) (α=.05). The best
the fixed effects from the models, averaging across the
experiment, which was controlled for if it was a significant
(α=.05) predictor in the model. Statistical analysis was
performed using SAS, v. 9.1. For transendothelial migration
assay results, an unpaired two-tailed T test was used to
compare the means between groups. IL-1 and IL-6 systemic
cytokine production was analyzed using an unpaired single-
tailed T test to compare the means between groups.
GRK6−/− and GRK2+/− mice have more severe arth-
ritis early in the K/BxN model compared to controls
To investigate whether or not the reduction in GRK
expression previously described in rheumatoid arthritis
patients  directly contributes to the pathophysiology of
inflammatory arthritis in our animal model, we examined
GRK6−/−, GRK2+/−, GRK5−/−, and wildtype mice in the
K/BxN serum transfer model. We found that animals with a
targeted genetic deletion of GRK6 had the most pronounced
enhanced arthritis early in their disease compared to their
wildtype littermate controls by two independent measures
of paw swelling (pb0.0001) and clinical disease severity
index (pb0.0003) (Figs. 1A, 2A). Although GRK2+/− mice also
showed enhanced disease compared to controls by paw
swelling (pb0.0001) and clinical disease index (p=0.0003)
and -2, but not -5, compared to wildtype controls. Shown is the
mean difference in mm swelling from baseline thickness of the
days. The data are combined from 3 (GRK6−/−) and 2 (GRK2+/−
most disease severity that is more pronounced earlier in the
disease course (LRT=56, df=3df, pb0.0001; GRK6−/− n=14, WT
n=13). (B) GRK2+/− mice also show enhanced disease
(LRT=32.4, df=3, pb0.0001; GRK2+/− n=12, WT n=13). (C)
GRK5−/− mice do not differ from wildtype controls (LRT=1.3,
df=2, p=0.522; GRK5−/− n=11, WT n=13).
117GRK subtypes differentially regulate granulocyte chemotaxis and disease expression in K/BxN model
(Figs. 1B, 2B), it was not as severe as GRK6−/− animals. In
contrast, GRK5−/− mice had similar disease indices when
compared to wildtypes (Figs. 1C, 2C), suggesting that the
observed effects were specific and differing between the
To investigate whether or not the earlier disease
enhancement observed in the GRK6−/− and GRK2+/− mice
translated into chronic disease differences, we examined the
histopathology of the hindlimbs at experiment termination
(day 22 or 23) by hematoxylin and eosin staining. There were
no substantial differences between the different GRK
genotypes and the wildtype controls with respect to the
severity of inflammation or bony erosions at the later stage
of disease (Fig. 3).
Granulocytes from GRK6−/− mice have enhanced
chemotaxis to LTB4 and C5a, but not to IL-8, in vitro
LTB4 and C5a are potent chemoattractants for granulocytes
and signal through their respective GPCRs (BLT and C5aR).
Disease expression in the K/BxN serum transfer model is
critically dependent on granulocytes  as well as LTB4 
and C5a . Therefore, we hypothesized that the enhanced
disease seen in the GRK6 and -2-deficient mice may be
mediated through interactions with the BLT and C5a
receptors on granulocytes. In agreement with a previous
report , we found that GRK6−/− granulocytes have
enhanced chemotaxis to LTB4 in vitro (p=0.0042) that is
approximately 30% more compared to wildtype cells (Fig. 4).
We also show that GRK6−/− granulocytes have 30% more
chemotaxis toward C5a (p=0.0074) (Fig. 4). Interleukin-8
(IL-8 or CXCL8) is also a potent chemoattractant of
granulocytes and signals through the GPCR, CXCR1. However,
migration to IL-8 was not different in any of the GRK-
deficient subtypes compared to wildtype controls (Fig. 4).
While statistical significancewasnotreached(p=0.4074),
GRK2+/− granulocytes had a trend towards increased migra-
tion to LTB4 with approximately 15% enhanced migration
compared to wildtype cells. The migration of GRK2+/−
granulocytes toward C5a (p=0.8753) was similar to controls
to increased granulocyte migration, may enhance inflamma-
tion through a failure to desensitize signaling of the LTB4 and
C5a GPCRs (BLT1&2 and C5aR respectively), and partial GRK2
deficiency may also play a role in BLT1&2 receptor signaling.
GRK6−/− mice have enhanced systemic effects in
the K/BxN model
In the K/BxN model, wildtype mice typically experience a
reversible weight loss approximating b5% of total body
weight that begins during the acute inflammatory phase
level of weight loss compared to wildtypes, GRK6−/− mice
had a profound weight loss of N10% of their total body weight
compared to that of controls (pb0.0001) (Fig. 5A). We
hypothesized that this finding may result from increased
inflammation leading to enhanced systemic production of
proinflammatory cytokines. Indeed, GRK6−/− mice have
animals deficient in GRK6 and -2, but not -5, compared to wild-
type controls. Shown is the mean clinical index±SEM over timein
days as determined by the summation of each paw scored on a
scale of 0–3 and averaged for the group. The data are combined
from 3 (GRK6−/−) and 2 (GRK2+/− and GRK5−/−) separate ex-
periments. (A) GRK6−/− mice have the most disease severitythat
is more pronounced earlier in the disease course (LRT=19, df=3,
pb0.0003; GRK6−/− n=14, WT n=13). (B) GRK2+/− mice also
show enhanced disease (LRT=18.9, df=3, p=0.0003; GRK2+/−
n=12, WT n=13). (C) GRK5−/− mice do not differ from wildtype
controls (LRT=6.1, df=3, p=0.1068; GRK5−/− n=11, WT n=13).
Clinical index of disease activity is increased in
118T.K. Tarrant et al.
increased IL-6 detectable in the serum (Fig. 6A), which
correlates temporally with the nadir weight loss occurring
between days 3 and 10. Systemic production of IL-1β was
detected in the serum but was not significantly different
between GRK6-deficient animals and controls (Fig. 6B).
Although GRK2+/− animals had an average 5% more weight
loss than wildtypes that was significant (pb0.0001) (Fig. 5B),
it was less pronounced than GRK6−/− and the systemic
cytokine profile did not differ significantly from that of
wildtype controls (data not shown). We attempted to
measure TNF-α in the serum in addition to the IL-1β and IL-
6 of the GRK-deficient and wildtype mice, but it was rarely
measured above the limit of detection in the majority of
samples for an analysis to be reliable (data not shown).
model. Shown is representative H&E histology at experiment termination (day 22 or 23) of (A) wildtype, (B) GRK6−/−, (C) GRK2+/−,
and (D) GRK5−/− mice. The severity of inflammatory infiltrate and erosions did not differ between groups;↑ localize the erosions on
hematoxylin and eosin stained sections.
Late stage inflammatory disease and erosions do not differ between the GRK genotypes and wildtype controls in the K/BxN
controls. The migration of granulocytes in response to 10 nM LTB4, 100 nM C5a, or 100 ng/ml IL-8 was determined by anti-Gr-1 staining
and flow cytometry of migrated primary bone marrow cells of the different murine GRK genotypes compared to wildtype. Data are
represented as % migration of the total input (i.e. total number of anti-Gr-1 positive cells harvested from the lower chamber divided
by the total number of anti-Gr-1 positive cells in the upper chamber)±SEM, n=6. Only GRK6−/− granulocytes show statistically
increased migration to LTB4 (§, p≤0.01) and C5a (⁎, p≤0.01). There was no difference between migration of the different GRK
genotypes compared to wildtypes in response to IL-8.
GRK6-deficient mice have increased granulocyte migration to LTB4 and C5a, but not IL-8, in vitro compared to wildtype
119GRK subtypes differentially regulate granulocyte chemotaxis and disease expression in K/BxN model
GPCR-mediated leukocyte trafficking is critical in inflamma-
tory disease states, and the GRK regulatory system has been
implicated in animal models of autoimmune disease.
Recently, GRK6-deficient animals have been shown to have
increased neutrophil migration to the gut and impaired
regulatory Tcell responses in a DSS-induced colitis model of
inflammatory bowel disease . In the EAE model of
multiple sclerosis, GRK2 heterozygote animals exhibit earlier
inflammation that correlates with T cell and macrophage
acute weight loss in the K/BxN model that reverses with disease
improvement. Data is represented as a mean of the change in %
weight from an individual animal's baseline=1.0 of wildtype and
GRK-deficient animals (±SEM) over time in days. The data are
combined from 3 (GRK6−/−) and 2 (GRK2+/− and GRK5−/−)
separate experiments. (A) There is a significant group effect
indicating more weight loss in the GRK6−/− mice (LRT=102;
df=3; p-valueb0.0001; GRK6−/− n=14, WT n=13). (B) There is
also a significant group effect in the GRK2+/− mice (LRT=35.2;
df=4; p-valueb0.0001; GRK2+/− n=12, and WT n=13). (C)
There is no significant difference between GRK5−/− animals and
controls (LRT=2.7; df=2; p-value=0.2592; GRK5−/− n=11, and
GRK6-deficient animals demonstrate maximum
levels of IL-6 compared to wildtype controls in the K/BxN model
of inflammatory arthritis. Data represent the mean serum
cytokine production+SEM in pg/ml of (A) IL-6, and (B) IL-1β on
days 0, 4, and 8 after i.p. injection with K/BxN serum. Results
presented are from 2 separate experiments; GRK6−/− n=8, WT
n=9. Animals were bled by tail vein nick, and serum cytokine
analysis was performed using an Upstate BeadLyte assay and
interpreted on a Luminex 100 instrument. GRK6−/− had sta-
tistically increased levels of systemic IL-6 on day 4 (⁎, p=0.05)
compared to wildtype animals. Limit of detection of the assay
was 18.7 pg/ml for IL-6 and 4.8 pg/ml for IL-1β.
GRK6-deficient animals produce higher systemic
120 T.K. Tarrant et al.
infiltration into the CNS, but then subsequently have less
disease and inflammatory cell infiltrate than wildtype
controls at later time points . Given that GRK6 and -2
levels are reduced in disease flares of adjuvant-induced
arthritis  and in the PBMC of rheumatoid arthritis
patients , we investigated how and which GRK subtypes
play a role in leukocyte migration to the joint in an acute
model of inflammatory arthritis, the K/BxN serum transfer
model. This model system closely mirrors the acute flares of
rheumatoid arthritis and allowed us to evaluate mononuclear
cell contribution toward disease independent of the lym-
The in vitro studies further support a role for GRK6 and -2
in granulocyte-mediated chemotaxis to LTB4 and C5a (Fig.
3). LTB4 is a potent neutrophil chemoattractant  and
critical for disease in the K/BxN model . One of its high
affinity receptors (BLT1) is a GPCR that has been shown to
have GRK6 and -2-mediated receptor internalization  and
desensitization  respectively. Previous work by Kavelaars
et al. has demonstrated enhanced chemotaxis in GRK6-
deficient granulocytes in vitro to LTB4 , and our data
here support this finding. We also show that GRK6-deficient
granulocytes have enhanced chemotaxis to C5a compared to
controls. The C5a anaphylatoxin receptor (C5aR) is a GPCR
important in the chemotaxis and activation of myeloid cells
 and necessary for K/BxN disease expression . Over
expression ofwildtype or catalytically inactive forms of GRK6
and -2 in COS-7 cells does not change agonist-mediated
phosphorylation of C5aR . However, Milcent et al. noted
that transfection of COS-7 cells with wildtype GRK6
consistently increased the expression of C5aR by two fold
and that GRK6 appeared to be autophosphorylated ,
suggesting a biologic relevance not apparent in this in vitro
model system. Our data suggests that GRK6 may have
receptor-kinase interactions mediated through the BLT and
C5aR receptors on granulocytes that are physiologically
relevant as it pertains to inflammatory disease.
Interleukin-8 (IL-8 or CXCL8) is also a known chemoat-
tractant of granulocytes and is thought to have pathophy-
siologic significance in rheumatoid arthritis through its
chemoattractant  and angiogenic  properties. How-
ever, we did not observe differences in granulocyte migra-
tion to IL-8 in the animals with gene defects in GRK2, -5, or -6
compared to controls (Fig. 4). The role of IL-8 in the K/BxN
model in specific is largely unknown, whereas LTB4 and C5a
in this model have been found to be of critical importance to
disease pathogenesis [22,23].
Our data also suggest that the enhanced inflammatory
disease seen in GRK2 heterozygote mice may be mediated
through one or both of the BLT GPCRs. GRK2+/− granulocytes
have slightly increased chemotaxis to LTB4 in vitro, albeit
our results did not achieve statistical significance. This could
be partially explained by the heterozygous expression of
GRK2 that may have reduced this phenotype. Further support
that GRK2 plays an important role in BLT1 signaling has been
suggested in transfection model systems . LTB4-induced
internalization of BLT1 can be blocked by dominant negative
GRK2 co-expression, and GRK2 co-localizes with the BLT1
receptor through the C terminus .
We also show that in addition to increased organ-specific
inflammation, GRK6-deficient animals have enhanced sys-
temic effects in the K/BxN model. Weight loss is observed in
inflammatory disease states such as rheumatoid arthritis and
has been linked to elevated cytokine production, particularly
TNF-α . We were unable to reliably detect TNF-α in the
serum of our arthritic animals to investigate its role in the
cachexia, but we do show that GRK6−/− animals have
elevated systemic levels of IL-6 that coincide with the
observed weight loss. IL-6 can be elevated in rheumatoid
arthritis patients and may correlate with disease activity
. In addition, IL-6 has been associated with profound
cachectic states such as malignancy [33,34]. Importantly, IL-
6 is not elevated chronically in the K/BxN model in either
wildtypes or GRK6-deficient animals (Fig. 6A and unpub-
lished observations), and the presence of malignancy has not
been observed in the K/BxN model in either controls or GRK-
Unlike the DSS-colitis model studied by Kavelaars et al.,
we did not observe significant differences in systemic
production of IL-1β between groups . This could be
explained by the different types of inflammatory responses
being observed (i.e. DSS, chronic v. K/BxN, acute) or by the
different pathophysiologic mechanisms regulating the two
disease models. Although we did observe increased weight
loss in the GRK2+/− mice, we did not detect any significant
differences in systemic cytokine production, possibly
because their overall weight loss and disease was less severe.
In conclusion, the GRK system is an important regulatory
pathway in the K/BxN serum transfer model of arthritis,
particularly as it pertains to the early infiltration of
granulocyte-mediated acute inflammatory responses. Gran-
ulocytes, which are critical to disease expression in the
K/BxN model, have increased chemotaxis to LTB4 and C5a,
but not IL-8, and GRK6−/− mice have more arthritis, weight
loss, and IL-6 production. These data highlight the unique
and subtype-specific effects of the GRKs on leukocyte
trafficking and inflammatory disease and underscore the
important regulatory role that GRK molecules may play inthe
acute versus chronic phases of autoimmunity. Although
specific gene polymorphisms in the GRKs have not been
described in the human rheumatoid arthritis population, an
increased understanding of these regulators as they pertain
to acute versus chronic inflammatory cell trafficking may
lead to targeted therapies or diagnostics for patients with
rheumatoid arthritis or other autoinflammatory diseases.
The authors would like to thank James Ellinger for technical
assistance in the arthritis disease models, Dr. M.G. Caron for
providing the GRK2 heterozygote animals for these studies,
and Dr. David Siderovski for critical review of the manuscript.
Arthritis Research Center at UNC, the American College of
tist Award, and the National Institutes of Health (grants K12
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