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Splenectomy attenuates streptococcal cell wallinduced arthritis and alters leukocyte activation.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 12, December 2003, pp 3557–3567
DOI 10.1002/art.11424
© 2003, American College of Rheumatology
Splenectomy Attenuates Streptococcal Cell Wall–Induced
Arthritis and Alters Leukocyte Activation
Donald Kimpel, Tim Dayton, John Fuseler, Laura Gray, Krishnaswamy Kannan,
Robert E. Wolf, and Matthew Grisham
Objective. To investigate the role of the spleen in
the pathogenesis of streptococcal cell wall (SCW)–
induced arthritis and determine the impact of splenectomy on monocytes and T cells involved in the arthritis.
Methods. Female Lewis rats were separated into 4
groups: 1) saline-injected, sham-operated; 2) salineinjected, splenectomized; 3) peptidoglycan–
polysaccharide (PG-PS)–injected, sham-operated; and
4) PG-PS–injected, splenectomized. After a 10-day recovery period, rats received a single intraperitoneal
injection of saline or PG-PS (25 ␮g rhamnose/gm body
weight). We evaluated the effect of splenectomy on joint
inflammation, histopathology, leukocyte subtypes in
blood and lymph nodes, cytokines, and cell surface
expression of CD44 and CD45RC in the chronic phase
of the disease (day 28).
Results. Splenectomy dramatically decreased
chronic joint inflammation and histopathologic damage
as well as altered cell types in lymph nodes and peripheral blood, as analyzed by flow cytometry. Nitric oxide
(NO) production, levels of interleukin-1␤ (IL-1␤), IL-6,
tumor necrosis factor ␣, and a biomarker of Th1 cell
predominance correlated with the level of joint inflammation. Surprisingly, in splenectomized animals, increased expression of adhesion molecules thought to
track T cells to inflamed tissue were observed in lymph
nodes.
Conclusion. The result of splenectomy was attenuation of SCW-induced arthritis and changes in mediators of inflammation, including T cell subsets, proinflammatory cytokines, and NO production. Splenectomy
may remove an important antigen reservoir and alter
immune cell activation in the SCW-induced arthritis
model.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint inflammation and
joint destruction, but encompassing a wide spectrum of
features, which may cause joint deformity and extraarticular damage. It is currently held that genetic and
environmental factors pattern this inflammatory response (1). Current models for understanding the pathogenesis of RA highlight interactions between T cells and
professional antigen-presenting cells in initiating responses to antigens found in synovial tissue (2). Clinical
evidence also indicates that the presence of articular
cartilage is essential for the perpetuation of arthritis,
although specific autoantigens have not yet been identified or characterized (3). Moreover, there is evidence
that cartilage degradation is associated with the development of cartilage-responsive T cells (4).
Therapies targeted to the immune system are
effective in the treatment of RA, both in humans and in
animal models (5–7). In the case of streptococcal cell
wall (SCW)–induced arthritis, joint inflammation is suppressed by cyclosporin A, FK-506, or depletion of T cells
(8–10). Much evidence from animal models of arthritis
and human studies suggests that a Th1 mechanism is
involved in inflammatory arthritis (11). However, evidence also suggests important roles for other cell types,
such as mononuclear phagocytes (12). Recently, we have
shown that significant changes occur in both T cells and
mononuclear phagocytes during the development of
Supported by the Center of Excellence for Arthritis and
Rheumatology. Dr. Kimpel’s work was supported by a research grant
from the NIH (R01-AR-46976) and by a Louisiana Education Quality
Support Fund grant from the State of Louisiana Board of Regents
(1988-01-RD-A-11).
Donald Kimpel, MD (current address: University of Virginia,
Charlottesville), Tim Dayton, MS, John Fuseler, PhD, Laura Gray, BS,
Krishnaswamy Kannan, PhD, Robert E. Wolf, MD, PhD, Matthew
Grisham, PhD: Louisiana State University Health Sciences Center,
Shreveport.
Address correspondence and reprint requests to Donald
Kimpel, MD, Associate Professor of Medicine, Division of Rheumatology and Immunology, PO Box 800412, Charlottesville, VA 229080412. E-mail: dlk9t@hscmail.mcc.virginia.edu.
Submitted for publication December 3, 2002; accepted in
revised form September 4, 2003.
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KIMPEL ET AL
SCW-induced chronic inflammatory arthritis (13).
These cellular changes were most evident in the spleen
but also occurred in lymph nodes and peripheral blood.
Together, these findings highlight the fact that an
autoimmune disease such as RA is a systemic inflammatory process that uses several lymphoid organs in addition to the inflamed tissue itself. Use of these widely
distributed lymphoid organs, such as the spleen and
peripheral lymph nodes, requires a highway network
(blood and lymphatics) for lymphocytes and other inflammatory cells, as well as appropriate traffic signals for
them to reach their target. The adhesion molecules
expressed on the inflammatory cell and on the endothelium of vessels provide these signals, which guide the
trafficking and development of cells such as lymphocytes
as they mature and mediate the inflammation process.
The complex sequence of events in inflammatory arthritis as it transitions from an acute to a chronic process is
poorly defined at present. However, the spleen as a
central organ in the immune system may play a prominent role in the development of this systemic inflammatory process.
Splenectomy has been used as an effective therapy in several autoimmune diseases, including idiopathic
thrombocytopenic purpura (ITP) and Felty’s syndrome
(FS) (14–17). Because the results of our previous studies
(13,18) suggested a central role for the spleen, we
hypothesized that splenectomy may alter the course of
SCW-induced chronic arthritis. We set out to test this
hypothesis and at the same time determine the impact of
splenectomy on the cell populations we previously
showed to be altered by the development of SCWinduced arthritis. In this study, we showed that splenectomy indeed reduces the chronic phase of arthritis in
association with changes in cellular and cytokine parameters. This is the first study to show quantifiable
immunologic changes and clinical effects of splenectomy
on the development of chronic inflammatory arthritis.
MATERIALS AND METHODS
Reagents. The monoclonal antibodies used in this
study were as follows: fluorescein isothiocyanate (FITC)–,
phycoerythrin (PE)–, or biotin-conjugated mouse anti-rat antibodies to CD3, CD4, CD8b, CD11b/c, CD44, CD45R,
CD45RC, and NKR-P1A (all from PharMingen, Mountain
View, CA). Second-step reagent (streptavidin–allophycocyanin
[APC]) was obtained from Biomeda (Foster City, CA). Appropriate murine isotype-matched negative controls were used
to establish background fluorescence for each fluorochrome.
The 10S fraction of peptidoglycan–polysaccharide (PG-PS)
was purchased from Lee Laboratories (Grayson, GA). The
material was briefly sonicated before use. All other chemicals
and reagents used in this study were purchased from Sigma (St.
Louis, MO) unless specified otherwise.
Splenectomy. Female Lewis rats weighing a mean ⫾
SD of 100 ⫾ 10 gm were purchased from Harlan SpragueDawley (Indianapolis, IN). Rats were anesthetized by inhalation of isoflurane (fraction of inspired oxygen 0.35 1/1 isoflurane [IsoFlo] Abbott Laboratories, North Chicago, IL). Under
sterile conditions, a midline laparotomy was performed. For a
splenectomy, the spleen was gently mobilized, exteriorized,
and the vascular supply was cut off by 2 ligatures (4-0 resorbable suture) placed around the vessels on the upper and lower
poles of the spleen. The abdominal wall was closed in 2 layers,
each by a running suture (4-0 resorbable suture). In shamoperated animals, the spleen was exteriorized after a midline
laparotomy and was gently mobilized before closing the abdominal wall. Rats were clinically monitored during the immediate recovery phase following inhalation of anesthesia. During postoperative monitoring, rats were observed daily. All
animal surgical procedures, including administration of anesthesia, laparotomy, and splenectomy, were approved by the
Institutional Animal Care and Use Committee (IACUC).
Groups of animals used in the study were as follows: 1)
saline-injected, sham-operated (C/Sh); 2) saline-injected, splenectomized (C/Spl); 3) PG-PS–injected, sham-operated (PG/
Sh); and 4) PG-PS–injected, splenectomized (PG/Spl). In each
experiment, 16 animals were randomly divided into 4 groups
composed of 4 animals/group. For histology, joint tissues were
processed from 1 or 2 representative animals per group.
Splenectomy studies were repeated 4 times with various combinations of antibodies. The arthritis responses and cellular
changes between experiments were similar. Unless stated
otherwise, the data reported here are from a single experiment
using a single lot of PG-PS. During the surgical procedure, 1
rat from the C/Sh group died; thus, the C/Sh group had only 3
rats.
Induction of rat arthritis by PG-PS injection. The
standard protocol for SCW-induced arthritis was followed as
previously described (8,19). Briefly, experimental rats were
given a single intraperitoneal (IP) injection of PG-PS (25 ␮g
rhamnose/gm body weight). Control animals were injected
with an equal volume of saline. Rats were observed daily, and
the development of arthritis was assessed by objective and
histopathologic criteria.
For all splenectomy experiments, surgical procedures
involving sham operation and splenectomy were performed
using sterile techniques as applicable. Animals were allowed to
recover from surgery for 10 days. At that point, controls
received saline only, whereas PG-PS groups received a single
IP injection of PG-PS. The day of PG-PS injection was day 0,
and all rats were killed on day 28. Animals were fasted
overnight prior to killing to allow for measurement of endogenous nitrate and nitrite (NOx). All animal protocols described
in this study were approved by the IACUC.
Evaluation of arthritis. Paw edema was measured by
immersion of the shaved paw to a marked line above the ankles
in a water plethysmometer, as previously described (8,13). The
mean swelling in both hind paws was added together to yield a
single data point. The change from the initial paw volume
(preinjection or day 0) was calculated. Data are presented as
the mean change in paw volume per group.
Joint swelling was scored (arthritis index) according to
IMPACT OF SPLENECTOMY ON SCW-INDUCED ARTHRITIS
a standardized method by an experienced observer (TD).
Briefly, a score of 0–4 was assigned as follows: 0 ⫽ no evidence
of hyperemia and/or inflammation; 1 ⫽ hyperemia with little
or no paw swelling, 2 ⫽ swelling confined predominantly to the
ankle region, with modest hyperemia, 3 ⫽ increased paw
swelling and hyperemia of the ankle and metatarsal regions,
and 4 ⫽ maximal paw swelling and hyperemia involving the
ankle, metatarsal, and tarsal regions. The scores for each paw
were summed, for a maximum possible score of 16.
For histologic evaluation, ankle (hind paw) joints were
fixed at 4°C in Zamboni’s fixative and decalcified in an
extraction buffer, as previously described (8). The joints were
split, placed in cryomolds containing tissue-freezing medium
(Triangle Biomedical Sciences, Durham, NC), and slow-frozen
in liquid nitrogen. Serial sections (10–12 ␮m thick) were cut
parallel to the long axis of the joint. The sections were stained
with Masson’s trichrome stain (Sigma). Images were captured
using a Nikon E600 microscope equipped with a SynSys digital
camera (Photometrics, Tucson, AZ). Exposure times were
automatically determined using the Trichrome Image dialog of
MetaMorph software (Universal Imaging, Downingtown, PA).
Flow cytometry analysis. Peripheral blood and lymph
node cells were isolated as previously described (13). For
immunofluorescence staining, cells were washed twice in phosphate buffered saline supplemented with 1% bovine serum
albumin (fluorescence-activated cell sorter [FACS] buffer),
incubated on ice for 30 minutes with saturating concentrations
of appropriate monoclonal antibodies or isotype controls,
washed 3 times in FACS buffer, fixed in 2% paraformaldehyde,
and resuspended in FACS buffer. When appropriate, cells
were incubated with biotin-conjugated monoclonal antibodies,
washed 3 times, incubated for 30 minutes with the relevant
streptavidin conjugate, washed, and then fixed in paraformaldehyde. Cell fractions were gated on viable cells, and samples
were analyzed using a FACSVantage flow cytometer (Becton
Dickinson, Franklin Lakes, NJ). Fluorescence was detected at
525 nm (FITC), 590 nm (PE), and 660 nm (APC); data were
analyzed using CellQuest software (Becton Dickinson). Typically, 10,000 cells were analyzed based on forward versus side
scatter gating.
Phenotype analysis. Lymph node and blood cells were
analyzed in the lymphocyte gate using forward and side scatter
patterns. After gating, fluorescence was analyzed for single-,
dual-, or 3-color analysis using a FACSCalibur flow cytometer
(Becton Dickinson) to detect the cell surface expression of
various markers. Immunostaining results were expressed in
most cases as the percentage of positive cells in blood and
lymph nodes. Cell types were defined in lymph nodes and
peripheral blood by flow cytometry using well-established
lineage-specific markers. All T cells were analyzed in the
lymphocyte gate for the presence of CD3⫹, CD4⫹, and CD8⫹
cells, and T cell subsets were evaluated for CD44 and CD45RC
expression. B cells were identified by the expression of pan–B
cell marker CD45R (B220). Natural killer (NK) cells were
identified by the expression of NKR-P1A. Monocytes in the
lymphocyte gate were identified by CD11b/c positivity and by
CD3 negativity; the identity of the monocytes in the lymphocyte gate was confirmed by backgating, by comparison with the
CD3⫺ population, and by analysis of cells in the monocyte
gate. Leukocyte counts and differential counts were confirmed
morphologically by automated cell counting and by light
microscopy.
3559
Figure 1. Effect of splenectomy on hind paw volume in streptococcal
cell wall (SCW)–induced arthritis. Experimental arthritis was induced
by a single intraperitoneal injection of peptidoglycan-polysaccharide
(PG-PS) in Lewis rats, as outlined in Materials and Methods. The
increase in hind paw volume (joint swelling) is characteristic of
SCW-induced experimental arthritis in Lewis rats during a 28-day
period. Rats in the PG-PS ⫹ splenectomy group had significantly
reduced chronic arthritis compared with sham-operated, PG-PS–
injected rats. Values are the mean ⫾ SD (n ⫽ 15).
Cytokine measurement. Concentrations of tumor necrosis factor ␣ (TNF␣) and interleukin-1␤ (IL-1␤) in plasma
samples from 2 experiments were determined using rat
enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s recommendations. Plasma IL-6 levels were measured by 7TD1 bioassay, as
previously described (20,21). Both negative and positive controls were included in each run. TNF␣ and IL-1␤ were
expressed in pg/ml per million cells, whereas IL-6 levels were
expressed in units/ml.
Measurement of nitrate and nitrite. Rats were fasted
overnight before collecting plasma samples from individual
rats. All samples were stored at ⫺70°C until analyzed. Levels
of NOx were measured by Griess reaction, in which plasma
nitrate was converted to nitrite by nitrate reductase (22,23).
Statistical analysis. Using GraphPad Prism software
(GraphPad Software, San Diego, CA), each data set was
subjected to analysis of variance followed by post hoc analysis
with Bonferroni adjustment for multiple comparisons. Individual results are expressed as the mean ⫾ SD. Differences
between groups were considered statistically significant when
the P value was less than 0.05.
RESULTS
Effect of splenectomy on the development of
SCW-induced arthritis. SCW-induced arthritis was produced in female Lewis rats by a single IP injection of
PG-PS, as described previously (8). All rats in the PG/Sh
group developed acute and chronic joint inflammation.
Over a period of 28 days, the joint inflammation fol-
3560
KIMPEL ET AL
Figure 2. Reduction of the arthritis index in peptidoglycan–polysaccharide (PG-PS)–injected,
splenectomized (PG/Splenect.) rats. Splenectomy significantly reduced the arthritis index in rats in
the PG/Splenect. group compared with that in the PG-PS–injected, sham-operated (PG/Sh) group.
Initially, there was acute inflammation both in PG/Sh and in PG/Splenect. rats. However, from day
13 onward, the arthritis index and paw edema were stable in the PG/Splenect. rats. The arthritis
index was 0 in all saline-treated controls. Values are the mean ⫾ SD (n ⫽ 15).
lowed a typical pattern, progressing from the initial
acute phase to the remission phase followed by spontaneous reactivation of persistent chronic arthritis (8,13).
PG/Sh rats were compared with PG/Spl rats. Splenectomy had a striking effect on the development of arthritis. The data in Figure 1 clearly show that after day 11
post–PG-PS injection, paw joint volume was significantly
reduced in PG/Spl rats. Similar changes were observed
in the arthritis index, as shown in Figure 2.
Effect of splenectomy on joint histopathology.
Figure 3 shows the effect of splenectomy on PG-PS–
induced joint histopathology. In the normal joint (Figure
3A), the articular cartilage was characterized by an
intact smooth surface and exhibited metachromatic (a
mixture of red and green) staining with Masson’s
trichrome stain. Minimal bone remodeling was present
at the articular cartilage–synovium interface, and the
synovial fluid was clear and acellular. The synovial
membrane was typically composed of ⬃2 cell layers
(synoviocytes), which stained intensely red. The synovial
membrane was supported by the connective tissue of the
capsule, which stained green.
Synovitis, extensive pannus formation, and erosion of the articular cartilage and subchondral bone
characterized the histopathology of chronic joint inflammation in this model of polyarthritis (Figure 3B). Numerous small, red-staining inflammatory cells were
densely localized in the synovium, the synovial space,
Figure 3. Joint histology evaluated on day 28 in sham-operated and
splenectomized rats injected with saline or PG-PS. A, Sham-operated,
saline-treated animal with normal joint morphology. B, PG/Sh rat
showing typical PG-PS–induced damage to the joint, characterized by
synovitis, pannus development, and erosion of the articular cartilage
and subchondral bone. C, Saline-injected, splenectomized control rat.
The morphology of the joint in this group has the same appearance as
that of the normal control animals. D, PG/Splenectomy animal. Joint
histopathology is predominantly characterized by synovitis accompanied by some erosion of the marginal bone and articular cartilage.
Development of pannus is absent, and there is minimal erosion of the
subchondral bone. a ⫽ articular cartilage; sb ⫽ subchondral bone; p ⫽
pannus; sv ⫽ synovial villus. See Figure 2 for other definitions.
(Original magnification ⫻ 200.)
IMPACT OF SPLENECTOMY ON SCW-INDUCED ARTHRITIS
3561
and in and along the trailing edge of pannus. Additionally, there were large multinucleated red-staining cells
(presumed to be osteoclasts), which appeared to be
closely associated with erosion of marginal bone and
cartilage at the synovium–bone interface and especially
with erosion of the subchondral bone (Figure 3B). The
pannus also contained green-staining loose connective
tissue fibers.
The joint morphology of the C/Spl animals (Figure 3C) showed no difference from that in the C/Sh
animals (Figure 3A). In the PG/Spl animals, joint inflammation appeared to be confined to the synovium–
bone interface at the joint margins (Figure 3D). The
inflammation seen was predominantly synovitis. There
was some marginal bone erosion and loss of articular
cartilage, but these did not appear to be extensive.
Interestingly, there was suppression of the development
of pannus, and subchondral bone erosion was also
conspicuously absent.
Effect of splenectomy on lineage markers of
leukocytes. Peripheral blood. In PG-PS–treated rats, a
decrease in the percentage of CD3⫹ cells was previously
Figure 5. Effect of splenectomy on the CD11⫹ cell population in
peripheral blood (top) and lymph nodes (LN) (bottom). On day 28
after peptidoglycan–polysaccharide (PG-PS) injection, the monocyte
population was analyzed using CD11b/c monoclonal antibodies (see
Materials and Methods). Values are the mean and SD (n ⫽ 15).
C/Sh ⫽ saline control, sham-operated; C/Spl ⫽ saline control, splenectomized; PG/Sh ⫽ PG-PS–injected, sham-operated; PG/Spl ⫽
PG-PS–injected, splenectomized.
Figure 4. Effect of splenectomy on T cell subsets in peripheral blood
(top) and lymph nodes (LN) (bottom). Lewis rats were sham operated
or splenectomized 10 days prior to injection of saline or
peptidoglycan–polysaccharide (PG-PS). On day 28, rats were killed
and cells were analyzed using lineage-specific markers (see Materials
and Methods). T cells were identified as CD3⫹, and the T cell subset
identified as CD3⫹,CD4⫹ was analyzed as a percentage of the CD3⫹
population. Values are the mean ⫾ SD (n ⫽ 15).
observed due to the large increase in CD11⫹ cells, some
of which were captured in the lymphocyte gate. Splenectomy also produced a nonsignificant decrease in CD3⫹
cells; however, subsequent subset analysis did not show
any differences in the percentage of CD3⫹,CD4⫹ (Figure 4) or CD3⫹,CD8⫹ (data not shown) cells. There
was no difference in the percentage of B cells or NK cells
(data not shown). One of the striking changes in peripheral blood was the increase in CD11⫹ cells in both
PG/Spl and PG/Sh rats (Figure 5). Between the PG-PS
groups, PG/Spl rats had a lower percentage of CD11⫹
cells than did PG/Sh rats (P ⬍ 0.05).
Lymph nodes. In lymph nodes, the percentage of
total CD3⫹ cells was lower in both of the splenectomized groups (C/Spl and PG/Spl) compared with the
sham-operated comparator groups. When the percentage of CD3⫹,CD4⫹ cells was analyzed, there were no
significant differences between groups (Figure 4). Further subset analysis did not reveal changes in the percentages of CD3⫹,CD8⫹ or NK cells (data not shown).
3562
KIMPEL ET AL
Figure 6. Expression of CD45RC in peripheral blood and lymph nodes. Expression of CD45RC
was analyzed using Ox-22 monoclonal antibodies, and mean channel fluorescence was determined.
CD45RChigh-expressing cells are associated with a Th1 phenotype (see Results). Values are the
mean ⫾ SD (n ⫽ 15). PG-PS ⫽ peptidoglycan–polysaccharide; Splenect. ⫽ splenectomized.
However, B cell levels were increased in C/Spl and
PG/Spl rats compared with their respective controls
(P ⬍ 0.01) (data not shown). A significant decrease was
also found in the CD11⫹ monocyte population in PG/
Spl rats compared with PG/Sh rats (P ⬍ 0.05), although
the total percentage of CD11⫹ cells was very low, as
expected (Figure 5).
Effect of splenectomy on T lymphocyte subsets.
The role of Th1 lymphocytes in RA pathogenesis is one
of the most intensely researched areas (24); however,
the characterization of the particular subset of T cells
that drives the inflammatory process is a subject of
controversy. In this regard, characterization of CD45RC
expression by CD4⫹ T cells deserves mention because
there is a direct correlation between these cells and the
pathogenic process in several autoimmune models (25).
To better understand the role of these cells in SCWinduced arthritis, CD3⫹,CD4⫹ T cells were analyzed
for the expression of CD45RC. PG/Sh animals demonstrated increased CD45RC in association with SCWinduced arthritis in both blood and lymph nodes (Figure
6). Splenectomy resulted in decreased CD45RC expression in T cells isolated from peripheral blood and lymph
nodes in both saline- and PG-PS–treated rats (Figure 6).
However, in PG-PS–treated rats, the difference between
sham-operated and splenectomized groups was very
pronounced (P ⬍ 0.01). These results are consistent with
Figure 7. Effect of splenectomy on CD44 expression in T cells in
peripheral blood (top) and lymph nodes (LN) (bottom). Intensity of
CD44 antigen expression was analyzed as the mean channel fluorescence in T cells (CD3⫹), CD4⫹ T cells (CD3⫹,CD4⫹), and CD8⫹ T
cells (CD3⫹,CD4⫺). CD44 is an adhesion and homing molecule that is
expressed at high levels on lymphocytes in joint fluid (see Results). Values
are the mean ⫾ SD (n ⫽ 15). PG-PS ⫽ peptidoglycan–polysaccharide.
IMPACT OF SPLENECTOMY ON SCW-INDUCED ARTHRITIS
our understanding that CD45RChigh expression is associated with proinflammatory Th1 potential and demonstrate a correlation between the degree of arthritis and
CD45RC expression (26,27).
Effect of splenectomy on CD44 expression. Several studies have suggested that CD44high phenotype is a
marker of activation, is associated with aggressive inflammatory phenomena in various autoimmune models,
and has a propensity to traffic to joints (28–30). We had
previously seen that CD4⫹ T cells isolated from the
joints of PG-PS–treated animals expressed high levels of
CD44 compared with those from the lymph nodes (13).
Here we evaluated the effect of an arthritis-suppressing
therapy (splenectomy) on the cell surface expression of
CD44 by various T cell subsets in lymph nodes and
peripheral blood. In the blood, there was no difference
in CD44 mean channel fluorescence in any T cell subsets
(Figure 7). However, in the lymph nodes, the expression
of CD44 on T cell subsets (CD3⫹, CD3⫹,CD4⫹, and
CD3⫹,CD4⫺) increased significantly in PG/Spl rats
compared with PG/Sh rats. In saline-treated rats, the
same trend was present but reached significance only in
the CD3⫹,CD4⫺ population. In both cases (saline and
PG-PS treatment), splenectomy appeared to result in
accumulation of T cells expressing high levels of CD44 in
the lymph nodes, although these rats had no arthritis
(saline) or had attenuated arthritis (PG-PS).
Splenectomy suppression of nitrate and nitrite
production in SCW-induced arthritis. NOx in plasma
and serum samples served as a measure of nitric oxide
(NO) production in vivo (21–23). As previously observed, increased NO production was demonstrated in
PG/Sh rats, the most profoundly arthritic group (P ⬍
0.01) (Figure 8). Compared with the C/Sh rats, C/Spl rats
showed a small elevation of NOx levels, which was not
statistically significant. However, splenectomy resulted
Figure 8. Alteration of nitrate and nitrite (NOx) levels in plasma by
PG-PS and by splenectomy (Splenect.). Plasma was obtained from all
fasted animals on day 28, and NOx was measured (see Materials and
Methods). NOx levels correlated with the severity of arthritis. Values
are the mean and SD (n ⫽ 15). See Figure 5 for other definitions.
3563
Figure 9. Changes in plasma cytokine levels in sham-operated and
splenectomized rats after PG-PS treatment. Plasma concentrations of
interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) were
measured by enzyme-linked immunosorbent assay (n ⫽ 32). The IL-6
level was determined by 7TDI bioassay (n ⫽ 15). Cytokine levels
correlated with the arthritis severity. Values are the mean and SD. See
Figure 5 for other definitions.
in significant reductions in NOx levels in PG/Spl rats,
down to the level in the saline controls (P ⬍ 0.01). Thus,
NO production paralleled the severity of the arthritis.
Effect of splenectomy on cytokines. Several studies have shown that proinflammatory cytokines play a
pivotal role in driving the inflammatory activity in
arthritis (31,32). To elucidate the underlying mechanisms involved in the attenuation of chronic inflammation in PG/Spl rats, plasma levels of TNF␣, IL-1␤, and
IL-6 were measured. As shown in Figure 9, the PG/Sh
group had significantly elevated plasma levels of TNF␣,
IL-1␤, and IL-6 compared with those in the C/Sh group.
In PG/Spl animals, TNF␣, IL-1␤, and IL-6 concentra-
3564
KIMPEL ET AL
tions dropped to the basal levels similar to those in the
C/Sh group.
DISCUSSION
This is the first study to show a suppression of
chronic joint inflammation as measured by changes in
the arthritis index and paw volume in Lewis rats when
splenectomy was performed prior to the induction of
experimental arthritis. Joint histopathology showed significantly reduced pannus and conspicuously absent
subchondral bone erosion. Our previous studies of cellular changes in arthritis showed that PG-PS induced an
increase in CD11⫹ monocytes with a concomitant decrease in T cells, while activation markers increased in
both cell types, most obviously in the spleen (13).
Activated neutrophils, monocytes, and lymphocytes are
present in inflamed joint tissues, but their journey may
be initiated by events at distant sites, such as the lymph
nodes and spleen.
Splenectomy, however, induced cellular changes
in PG-PS–treated animals, including a reduction in
CD11⫹ cells, a reduction in T cells, and an increase in B
cells. Our previous time course studies showed that both
monocytes and T cells in the spleen are activated by day
5 after injection of PG-PS (13). It is therefore difficult to
determine if one cell type leads to activation of the other
or if there is synergistic coactivation of T cells and
CD11⫹ cells. In any case, splenectomy decreased the
numbers and the activation of both T cells and CD11⫹
cells. Splenectomy also led to a reduction in plasma
levels of NOx and proinflammatory cytokines compared
with those in the PG/Sh animals.
The spleen, a secondary lymphoid organ, is a
major site of immune surveillance, antigen recognition,
activation, and clonal proliferation. We hypothesized
that splenectomy may alter the dynamics of the cellular
and humoral elements that play a vital role in joint
inflammation (14,15). Splenectomy as a therapeutic
intervention has been used in autoimmune diseases such
as chronic ITP and FS (14–17). Unlike these primary
antibody–mediated disorders, antibodies in SCWinduced arthritis are generally not considered the primary driving force (13). Anecdotally, patients with sickle
cell disease have a low incidence of autoimmune diseases, and RA coexistent with sickle cell disease has
rarely been reported (33). Although it is difficult to
separate the role of genetic differences, this observation
indirectly suggests that the autosplenectomy that occurs
in patients with sickle cell disease may protect against
the development of autoimmune disorders.
Splenectomy could be beneficial to arthritis patients, based on our data and the beneficial effects
specific to FS, but clinical evidence is contradictory. A
recent literature search found one case of long-term
improvement in arthritis following splenectomy in a
patient with FS (34). However, in another case, a patient
who had features of FS but without arthritis who was
treated with splenectomy later developed inflammatory
arthritis (35). It is possible that selected patients with
RA could benefit from splenectomy.
Schwab and coworkers investigated the role of
the spleen as a site of antigen deposition in SCWinduced arthritis by demonstrating the presence of polysaccharide complex within splenic macrophages, synovial macrophages, and neutrophils during various stages
of inflammation in SCW-induced arthritis (36–39).
These studies suggest that splenic macrophages and
Kupffer cells of the liver can act as antigen reservoirs for
PG-PS. It has been shown that degradation of PG-PS by
mutanolysin reduced the amount of complexes in the
spleen and the liver, and that the smaller-sized complexes did not induce chronic arthritis (38). More recently, Schrijver and other investigators (11,40) have
proposed that PG, a major component of the cell wall of
gram-positive bacteria, plays a role in the pathogenesis
of RA. In the studies described here, it is likely that
splenectomy resulted in the loss of antigen reservoirs
and a loss of cell populations that are very important for
joint inflammation, thus resulting in altered immune
kinetics and attenuation of joint damage in the PG/Spl
group.
To evaluate for gross hematologic alterations, we
performed a total white blood cell (WBC) count, total
lymphocyte count, and hematocrit in all 4 groups of rats.
Splenectomy in Lewis rats did not cause any major
changes in the total WBC count, total lymphocyte count,
and hematocrit in saline- and PG-PS–injected rats.
In RA, neutrophils and mononuclear phagocytes
play a prominent role in joint destruction (5,12), but the
predominance of T cells in synovial tissue with an
activated/memory phenotype has led to the conviction
by many that T cells play a prominent role in the disease
process. In fact, T cells, neutrophils, and monocytes all
appear to play a role (5,41). This view is supported in the
SCW-induced arthritis model in which the evidence for
a T cell role is strong (18,42), while neutrophils and
monocytes are also associated with disease activity
(2,36,43,44). Consistent with this view, we have quantitated changes in both T lymphocytes and monocytes
during the course of development of SCW-induced
arthritis (13).
IMPACT OF SPLENECTOMY ON SCW-INDUCED ARTHRITIS
The percentage of CD3⫹ T cells in the lymph
nodes of both saline and PG-PS groups decreased after
splenectomy. One possible explanation for this is altered
homeostasis in peripheral compartments, leading to
accumulation in other secondary lymphoid tissues after
such as Peyer’s patches. A second possibility is that
altered regulation leads to an overall reduction in the T
cell repertoire. In contrast, the B cell population was
found to be increased in splenectomized rats. Analysis of
the monocyte population produced 2 observations. As
compared with the C/Sh rats, the PG/Sh rats showed a
significant increase in the percentage of monocytes in
both blood and lymph nodes, a finding consistent with
our previous observations. However, upon splenectomy,
there was a significant drop in the monocyte population;
this may be influenced by the changes in T cells or may
be due to other factors, such as the loss of the splenic
PG-PS reservoir.
Synovial T cells from RA patients have been
described as CD44bright, and CD44 is thought to be
important in trafficking to inflamed joints (29). In the
SCW-induced arthritis model, we have also seen that
CD4⫹ T cells in inflamed joints express high levels of
CD44, and expression in blood and the spleen is increased by PG-PS treatment (13). Surprisingly, expression of CD44 was increased even more dramatically in
lymph nodes after splenectomy, although the arthritis
was attenuated. There are several possible explanations
for this. CD44 may be one of several adhesion molecules
necessary for entry into joints, or it may be an inexact
marker of adhesion capability. In any case, the role of
CD44 in the trafficking of cells to sites of inflammation
may be more complex than previously appreciated.
To further characterize the T cells involved in this
arthritis model, we used an antibody that recognizes one
epitope of CD45, CD45RC, the expression of which
defines two subpopulations of functionally distinct, mature CD4⫹ T cells. The CD45RChigh cells produce a
predominance of interferon-␥ and IL-2, provide help to
B cells during primary immune responses, are active in
graft-versus-host responses, can cause autoimmunity,
and are the precursors of the CD45RClow population
(25,45). In contrast, the CD45RClow population contains
the majority of T helper cells for secondary humoral
responses, produces more IL-4 than the CD45RChigh
subpopulation, proliferates to recall antigens, and contains cells that suppress some autoimmune manifestations (27,46,47). Similar cell populations have been
described in a mouse model of inflammatory bowel
disease (48–50).
Consistent with this pattern, PG/Sh rats exhibited
3565
high expression of CD45RC antigen in concert with
active inflammation. In comparison, we observed a
reduction in CD45RC mean fluorescence in PG/Spl rats
to the levels in saline-treated rats. Taken together, our
data also support the current belief that CD45RC is an
important marker of inflammation in the rat model and
an indicator of a Th1-type inflammatory response.
To examine noncellular indicators of inflammation, we measured levels of NO, IL-1␤, IL-6, and TNF␣.
Although characterization of NO as pro- or antiinflammatory is often difficult, the involvement of NO in
immune activation and inflammatory processes is now
well established (for review, see ref. 51). TNF␣ and IL-1
are well-established proinflammatory cytokines in RA
and are important targets of current treatment strategies, while IL-6 is a pluripotent inflammatory cytokine
produced by numerous cell types during inflammation
(31,52,53). Furthermore, proinflammatory cytokines
such as TNF␣ and/or IL-1 can potentiate the production
of NO in cultured synoviocytes, synovial fibroblasts, and
articular chondrocytes (8,54). Our results demonstrated
that levels of NOx, IL-1␤, IL-6, and TNF␣ correlate with
the degree of arthritis and are decreased in parallel with
the arthritis in PG/Spl animals. We have also observed
that there is a clear difference between splenocytes and
lymph node cells isolated from naive rats when cultured
in the presence of 10 ␮g/ml PG-PS. Lymph node cells
responded minimally, whereas the splenocytes produced
⬃50-fold more TNF␣ (Kimpel D, et al: unpublished
observations).
Joint histopathology of the PG/Sh rats demonstrated extensively developed pannus and the associated
erosion of articular cartilage and subchondral bone, as
seen previously in SCW-induced arthritis (8). In PG/Spl
rats, joint histopathology revealed that there was a
suppression of pannus formation and minimal erosion of
the articular cartilage and subchondral bone. The results
of the histopathologic evaluation strongly correlated
with the clinical, serologic, and cellular inflammatory
changes that occurred in response to splenectomy.
In summary, splenectomy prior to induction of
arthritis has a pronounced effect on the development of
chronic inflammation, and several humoral (NOx, IL1␤, IL-6, and TNF␣) and cellular (CD45RC, CD11⫹)
biomarkers correlate with the degree of inflammation.
The impact of splenectomy is likely mediated by removal
of a critical antigen reservoir of PG-PS, with diminished
immune cell activation and attenuated joint inflammation. Finally, an important role of secondary lymphoid
organs in the systemic process of inflammation in RA is
3566
KIMPEL ET AL
emphasized, with the spleen playing an important role in
T cell–dependent arthritis induced by PG-PS.
ACKNOWLEDGMENTS
We thank Dr. Robert Chervenak and Ms Deborah
Chervenak of the Core Facility for Flow Cytometry for technical assistance and thoughtful discussion.
16.
17.
18.
19.
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