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*
Department of Comparative Medicine, University of Alabama, Birmingham, AL 35294;
Cell Biology and Inflammation Research, Pharmacia & Upjohn, Kalamazoo, MI 49001;
Department of Molecular and Human Genetics, Baylor College of Medicine and Howard Hughes Medical Institute, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. L-selectin is
expressed on the majority of leukocytes and is shed from the cell
surface following activation. The contribution of P-selectin to leukocyte rolling and emigration has been well documented. Inhibition or loss of P-selectin results in a significant reduction of neutrophil rolling and acute emigration in many inflammatory models (4, 5, 6, 7, 8, 9). In addition, in vitro flow chamber studies have shown that P-selectin also participates in both lymphocyte and monocyte rolling on endothelial cells (10, 11, 12, 13). The functional role of P-selectin in mediating leukocyte recruitment during chronic inflammatory diseases has only recently been investigated (14, 15, 16, 17, 18). Several studies provide evidence that P-selectin expression is important in the development of joint inflammation during rheumatoid arthritis (RA)5 (19, 20, 21, 22). P-selectin is expressed on both cultured synovial microvascular endothelial cells and on synovial tissue endothelium, and increased levels of soluble P-selectin have been reported in synovial fluid from RA patients (23, 24, 25, 26). Also, Grober et al. (27) showed that monocyte adherence to microvascular endothelium in RA synovial tissue can be blocked with anti-P-selectin Abs using the Stamper-Woodruff assay, suggesting that P-selectin may promote monocyte rolling and emigration from the synovial vasculature.
Murine collagen-induced arthritis (CIA) is a well-known model of chronic inflammatory and erosive joint disease with a number of features in common with RA (28). In this report, we present data on the role of P-selectin in both the initiation and progression of CIA using mutant mice lacking the P-selectin protein. Interestingly, we found that the onset of arthritis was significantly accelerated, and the severity increased in P-selectin mutant mice, compared with wild-type. These studies show that P-selectin-mediated leukocyte adhesion is not required for the development of CIA, but P-selectin may play a regulatory role in controlling early leukocyte-dependent processes in the initiation and maintenance of immunologically driven joint inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice containing a null mutation for P-selectin were generated by gene targeting in 129/Sv embryonic stem cells, as previously described (5). The P-selectin mutation was then backcrossed onto the DBA/1 strain background for five generations. Control wild-type inbred DBA/1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice used were 12–16 wk of age, and routine serology tests were consistently negative for common viral pathogens.
Induction and assessment of arthritis
Arthritis was induced with chick type II collagen (Genzyme, Boston, MA), as previously described (29). Collagen was dissolved in 0.01 N acetic acid overnight at 4°C and emulsified with an equal volume of CFA (Difco, Detroit, MI) to give a final concentration of 1 mg/ml. Each mouse was injected intradermally at the base of the tail with 100 µl of emulsified collagen on day 0. For analysis of incidence and severity of arthritis, three separate experiments were performed, and experimental groups contained approximately equal ratios of male and female mice. Mice were identified by ear punch code, and arthritis was scored on a daily basis by an observer blinded to the P-selectin genotypes. Swollen digits were noted, but paws were only considered arthritic when the entire paw was inflamed. Each paw was inspected and assessed for the severity of erythema and swelling using scores of 0 (normal appearance), 1 (mild), 2 (moderate), and 3 (severe; maximum score of 12 for each mouse). During the chronic phase of the arthritis, paws and digits were inspected for distortion and manipulated to identify loss of flexion (ankylosis). Paws received an additional score of 3 for the presence of distortion and 3 for the presence of ankylosis (maximum score of 6 per paw). Daily severity scores were calculated from the combined inflammation, distortion, and ankylosis scores.
Measurement of anti-type II collagen IgG
Blood was obtained at various times by retro-orbital puncture under metofane anesthesia (Pitman-Moore, Mundelein, IL). Serum levels of anti-type II collagen IgG were measured by ELISA (29). Microtiter plates (96-well; Corning Costar, Cambridge, MA) were coated with 1 µg/well of chick type II collagen in bicarbonate buffer (pH 9.6) overnight at 4°C. After washing and blocking with 1% BSA, serum samples diluted 1/250 were added for 60 min at 37°C. This dilution was based on a titration of pooled serum from collagen-immunized DBA/1 mice, which gave submaximal color development in the assay. After washing, wells were incubated with goat anti-mouse IgG conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL) for 60 min at 37°C and developed with phosphatase substrate (p-nitrophenyl phosphate disodium; Sigma, St. Louis, MO) dissolved in diethanolamine buffer for 8 min. Color development was stopped with 3 M NaOH and absorbencies read at 405 nm using a microplate reader (Molecular Devices, Palo Alto, CA). Sera from nonimmunized DBA/1 mice were used as negative controls.
Isolation and culture of splenocytes for cytokine production
Spleens were removed from nonimmunized wild-type and P-selectin
mutant mice and from type II collagen-injected mice 14 days after
immunization. Single cell suspensions were made and erythrocytes lysed
with Tris-ammonium chloride. Cells were resuspended in RPMI 1640
containing 5% FBS, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate
(Life Technologies, Rockville, MD). Splenocytes were cultured at
1.0 x 106/well in 200-µl volumes for
72 h at 37°C in quadruplicate wells and were either unstimulated
or stimulated with plastic-adherent anti-mouse CD3 Ab (1 µg/well
145-2C11; PharMingen, San Diego, CA). Cell culture supernatants were
collected and levels of IL-2, IFN-
, IL-4, IL-5, and IL-10 measured
by ELISA assays using anti-cytokine Abs and protocols supplied by
PharMingen.
Histological evaluation of joint tissue
Joint tissue was collected from mice displaying the initial clinical signs of joint inflammation (0–5 days postonset, six mice each for wild-type and mutant groups) and from all mice at the end of each experiment, and fixed in 10% buffered formalin. Mouse paws were cut in half longitudinally, embedded in OCT (Miles, Kankakee, IL), and snap frozen in liquid nitrogen chilled iso-pentane. For histochemical staining, cryostat sections (10 µm) were air dried and run through a Diff-Quik staining procedure (Baxter, McGaw Park, IL). Briefly, slides were fixed with fixation solution (5 dips), washed in tap water to remove excess OCT, stained with solution 1 (eosin; 6 dips), tapped dry, stained with solution 2 (Thiazine Dye mixture, 6 dips), tapped dry, washed in tap water, air dried, and coverslipped with Permount (Fisher, Fair Lawn, NJ) for histopathological evaluation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In three separate experiments, wild-type and P-selectin mutant
DBA/1 mice were immunized with type II collagen and inspected daily for
the development of arthritis. Surprisingly, the lack of expression of
P-selectin did not suppress the development of arthritis, but actually
led to an acceleration of arthritis. By day 28, a significant
difference in the incidence of arthritis (as determined by
2 analysis; p < 0.001) was
observed, as only 1/55 (2%) of wild-type mice showed clinical signs of
arthritis, whereas 16/69 (23%) of P-selectin mutant mice had already
developed arthritis (Fig. 1
). On day 40,
12/55 (22%) of wild-type mice were arthritic, compared with 37/69
(54%) of P-selectin mutant mice (p < 0.001),
and on day 70, the incidence was 28/55 (51%) and 51/69 (74%),
respectively (p < 0.01). By the end of the
studies, however, no significant differences were observed in the
incidence of arthritis between the two groups (69% for wild-type, 77%
for P-selectin mutants; p > 0.05). The day of onset in
wild-type mice ranged from day 27 to day 91 with a median of 49 days
postimmunization (mean = 52.9 ± 3.0 SEM). In contrast, the
onset of arthritis in the P-selectin mutant mice was as early as day 14
(range 14–84), and the median onset, day 34, was 15 days earlier than
the controls (mean = 37.9 ± 2.0).
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In all experiments, arthritic P-selectin mutant mice had a
consistently greater number of affected paws than the corresponding
controls (data not shown), and the severity of arthritis was also
increased in P-selectin mutant mice. As shown in Fig. 2, the severity scores in P-selectin
mutant mice with arthritis were consistently higher than those in
wild-type mice. The greatest differences were observed on days 50–91,
where distortion and ankylosis progressed more rapidly in P-selectin
mutant mice than in the wild-type controls.
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Serum IgG anti-type II collagen Ab levels were measured by ELISA
at several time points postimmunization (Table I). No significant levels of Ab were
detected on day 7 in either group. By day 14, however, Ab levels were
readily detectable and were significantly higher in P-selectin mutant
mice, compared with wild-type mice. This increase was not due to a
difference in the relative incidence of arthritis, since none of the
mice in these test groups had developed arthritis at the time of
analysis. Significantly higher levels of Ab were also observed in
nonarthritic P-selectin mutant mice on days 14 and 40, when compared
with nonarthritic controls. A similar increase was observed
when the Ab levels in arthritic P-selectin mutant mice were
compared with arthritic controls on days 40 and 91. Ab levels
on day 28 in arthritic P-selectin mutant and wild-type mice could
not be statistically compared, since only one wild-type mouse was
arthritic.
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Since Ig synthesis is driven by Th2-dependent cytokines, we
determined whether the increased levels of anti-collagen IgG
observed in both nonarthritic and arthritic P-selectin mutant mice were
due to a shift in the relative balance of Th1 and Th2 cytokines. Spleen
cells from both normal and type II collagen-immunized mice were
cultured with or without anti-mouse CD3 Ab. The levels of IL-2,
IFN-, IL-4, IL-5, and IL-10 were measured in the culture
supernatants (Fig. 3). In contrast to
wild-type mice, splenocytes from P-selectin mutant mice produced
significantly less IL-2 (Fig. 3A). This difference was most
pronounced in splenocytes from P-selectin mutant mice that had been
immunized with type II collagen (a decrease of 84.8% of
anti-CD3-stimulated IL-2 production, compared with wild-type mice).
Production of IFN-, however, was comparable between the two groups
of mice (data not shown). Spontaneous levels of IFN- were
2.1–1.2 ng/ml, and anti-CD3-stimulated production ranged from
3.9 to 3.5 ng/ml. Interestingly, both IL-10 and IL-5 synthesis induced
by anti-CD3 were significantly increased in P-selectin mutant mice
(Fig. 3, B and C). Augmentation of IL-10
production in both nonimmunized and immunized P-selectin mutant mice
was increased 1.8- to 2.2-fold, and anti-CD3-induced IL-5 synthesis
was elevated 1.9- to 2.8-fold, when compared with corresponding
wild-type mice. No significant levels of spontaneous IL-10 or IL-5
synthesis were observed in any group. In contrast to the IL-10 and IL-5
data, IL-4 production in P-selectin mutant mice was decreased by 65%
(nonimmunized) and 74% (type II collagen-immunized), compared with
wild-type mice (Fig. 3D).
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Joint tissue from arthritic wild-type and P-selectin mutant mice
was assessed for the degree of leukocyte infiltration in forepaws and
hindpaws. In mice displaying initial clinical signs of joint
inflammation (0–5 days postonset), an intense inflammatory response
was observed in periarticular soft tissues, but integrity of the joints
remained intact, regardless of the severity of the inflammatory
reaction. In both groups of mice, no obvious differences in the overall
intensity of leukocytic infiltration between P-selectin mutant (Fig. 4A) and wild-type mice (data
not shown) were observed. Gross inflammation of the fore and hindpaws
(as assessed by clinical scoring) correlated well with marked
infiltration of granulocytes (Gr-1+), the major
cell type present, and macrophages (F4/80+) as
defined by immunocytochemistry (data not shown). T lymphocytes
(CD4+, CD8+ cells) were
rarely seen in the lesions of either group (data not shown). Histologic
examination of joint tissue from arthritic mice sacrificed at the end
of each experiment revealed significant leukocytic infiltration, joint
capsule thickening, bone destruction and/or bone remodeling, and
ankylosis (Fig. 4B). No obvious differences in these
parameters were observed between groups. We concluded that the
P-selectin mutant mice exhibit the pathology expected in early and
late CIA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the development of CIA was not inhibited in P-selectin mutant mice and that loss of P-selectin expression actually resulted in an acceleration and increased severity of the disease. These observations contrast to those reported in two different rat arthritis models. P-selectin Ab treatment of rats immunized with streptococcal cell wall proteins was shown to reduce both ankle edema and the number of infiltrating neutrophils into joints (33). In addition, anti-P-selectin Abs were found to inhibit both early (2 h) neutrophil and monocyte emigration into joint tissue, following induction of rat adjuvant arthritis (34). Our findings in the mouse CIA model suggest that either P-selectin expression on the synovial vasculature is not necessary for rolling and subsequent emigration of proinflammatory leukocytes into the joint in this model, or that other adhesion molecules can compensate for the loss of P-selectin expression and mediate leukocyte rolling. The absence of methods to directly quantitate leukocyte rolling in synovial vessels does not allow discrimination between these two possibilities.
E- and L-selectin-mediated leukocyte rolling has been documented in
cremaster muscle venules in P-selectin mutant mice following
TNF- treatment, and these adhesion molecules may be important for
mediating proinflammatory leukocyte emigration into the synovium in
this model (7, 35). Other adhesion molecules including
VLA-4, VCAM-1, or CD44 may also mediate leukocyte rolling in synovial
vessels. Treatment of mice with inhibitory Abs against CD44 has been
shown to reduce joint swelling and synovial leukocyte emigration
in established CIA and in proteoglycan-induced arthritis
(36). VLA-4 Ab treatment of rats with adjuvant-induced
arthritis also resulted in a reduction of leukocyte recruitment into
joints (37, 38, 39, 40). However, in both of these studies, it was
not determined whether Ab treatment inhibited leukocyte rolling, firm
adhesion, and/or transendothelial migration. Further studies of all
adhesion molecules in both the CIA and other inflammatory models will
be necessary to determine their role in the initial
leukocyte/endothelial interactions within the synovial vasculature.
The accelerated arthritis and increased severity observed in P-selectin
mutant mice was unexpected, and the mechanism(s) accounting for this
observation is not clear. It is also not immediately obvious why
P-selectin deficiency in this model results in increased production of
anti-type II collagen Ab. Accelerated development of CIA has
been reported in mice treated with anti-IL-10 Abs, IL-12, and in
mice containing a null mutation in the IFN- receptor gene,
suggesting that these cytokines play key roles in the initiation of
joint inflammation in the CIA model (41, 42, 43, 44, 45). However,
these studies did not determine the mechanisms by which loss or
increased production of these cytokines led to the acceleration of
arthritis.
Loss of P-selectin expression may selectively alter the trafficking
patterns of specific lymphocyte subpopulations to peripheral lymphoid
organs during the development of the immune response against type II
collagen. This may result in differential production of cytokines,
which may then directly or indirectly influence the inflammatory
response in the joint. In our studies, we observed increased
circulating titers of anti-type II collagen Abs in both arthritic
and nonarthritic P-selectin mutant mice at several time points
throughout these experiments (Table I
). This correlated with
significant increases in the production of the Th2 cytokines IL-10 and
IL-5 from splenic lymphocytes in P-selectin mutant mice (Fig. 3). It is
possible that the higher titers of circulating anti-type II
collagen Ab in P-selectin mutant mice may contribute to the
acceleration and significant increase in arthritis severity by
augmenting immune complex deposition in the joint, thus leading to
severe inflammation and joint destruction.
Another contributory factor to the accelerated onset and/or increased severity may be altered trafficking of a regulatory T cell subset into the joint during the initiation and/or progression of inflammation in P-selectin mutant mice. T lymphocytes play critical roles in the pathogenesis of both RA and CIA, and are thought to promote and inhibit the inflammatory response in the joint capsule through selective cytokine production (46, 47, 48). P-selectin has been shown to mediate rolling and emigration of specific T cell subtypes into tissue under various stimulation conditions. For example, P-selectin preferentially mediates the emigration of Th1 lymphocytes over Th2 lymphocytes into the skin during 2,4-dinitro-1-fluorobenzene (DNFB)-induced delayed-type hypersensitivity (49, 50). In addition, studies using in vitro flow chambers have shown that CD45RA-RO+ (memory) T cells roll and bind more efficiently to Chinese hamster ovary cells expressing P-selectin than do CD45RA+RO- (naive) T cells, suggesting that P-selectin may influence their in vivo trafficking patterns (51, 52). Our data suggest that the role of P-selectin in T cell trafficking may be complex, since the acceleration of CIA cannot be easily explained by the inhibition of memory or Th1 regulatory cells into the joint.
It is also possible that the absence of P-selectin expression on synovial endothelium inhibits the recruitment of a suppressive monocyte population into the joint (53, 54). This could result in reduced levels of antiinflammatory mediators, such as TGF-ß and IL-1ra, leading to accelerated inflammation (48, 55, 56). Inhibition or loss of P-selectin expression has been previously shown to reduce monocyte adhesion and emigration both in in vivo and in vitro models of arthritis, as well as other inflammatory models (8, 14, 15, 27, 34). Although no obvious differences in the inflammatory infiltrates between arthritic P-selectin mutant and nonmutant mice were observed in swollen joints during the early phases of arthritis, alterations in the infiltrating leukocyte populations and/or cytokine production during the initiation of joint inflammation (before the onset of obvious joint swelling) may occur.
Our observations that P-selectin expression plays a regulatory role in the development of arthritis suggest that pharmacologic approaches designed to inhibit P-selectin might be detrimental for the treatment of inflammatory diseases, such as RA. These findings also suggest that variation in P-selectin expression among individuals could be a predisposing factor for the development of severe forms of inflammatory joint disease. Further studies of P-selectin in both RA and animal models of arthritis will be necessary to more specifically define its role in the initiation and progression of joint inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel C. Bullard, Department of Comparative Medicine, 413A Volker Hall, University of Alabama, 1670 University Boulevard, Birmingham, AL 35294-0019. E-mail address:
3 Current address: Parke Davis Pharmaceutical Research, Ann Arbor, MI 48105.
4 Current address: Searle Pharmaceuticals, Skokie, IL 60077.
5 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis.
Received for publication February 23, 1999. Accepted for publication June 25, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated vascular endothelium under flow. J. Exp. Med. 181:1179.-activated vascular endothelium under flow in vitro. J. Immunol. 156:326.
receptors. J. Immunol. 158:5501.[Abstract]
receptor-deficient mice. J. Immunol. 158:5507.[Abstract]
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