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Department of Medicine, Northwestern University Medical School, Chicago, IL 60611;
Veteran’s Administration Chicago Health Care System, Lakeside Division, Chicago, IL 60611;
Departments of Medicine and Pathology and
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University of Michigan Medical Center, Ann Arbor, MI 48109; and
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Theodor Kocher Institute, University of Bern, Bern, Switzerland
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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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ENA-78, a C-X-C chemokine cloned in humans, is a PMN chemotactic factor. It is a 8.3-kDa protein with 78 amino acids containing four cysteines positioned identically to those of IL-8 and its homologues (13). ENA-78 activates PMNs by inducing PMN chemotaxis and promoting cytosolic-free calcium changes and is an angiogenic factor (10, 11, 13, 14). In human disease, similar to other PMN chemotactic factors such as IL-8, C5a, and leukotriene B4, we found ENA-78 to account for a significant portion of RA SF chemotactic activity for PMNs (14). ENA-78 concentrations were significantly greater in RA SFs than SFs from patients with osteoarthritis or other inflammatory and noninflammatory forms of arthritis (14).
Many aspects of inflammation and the molecules involved have not as yet
been examined in RA because of the limitations of obtaining multiple
sequential tissue biopsies in humans. There are a number of
well-established rodent models for human RA, including the rat
adjuvant-induced arthritis (AIA) model (15). AIA is similar clinically
and pathologically to RA. In AIA, 90–100% of rats develop arthritis
within 14 days after adjuvant injection, showing joint histologic
changes including leukocyte invasion preceding joint swelling (16). The
role of several cytokines, such as IL-1 (17, 18), IL-6 (17, 19),
TNF-
(20), and IL-8 (21), has been implicated in the pathogenesis of
various animal arthritis models.
In this study, we examined the involvement of an ENA-78-like protein in AIA. We demonstrated that anti-ENA-78 Ab inhibited rat peritoneal PMN recruitment to recombinant human (rh)ENA-78 as well as to LPS in vivo. We determined the expression of this ENA-78-like protein in the serum and joint homogenates of AIA compared with control rats using ELISAs. We then modulated the progression of AIA by treatment with neutralizing Abs against human ENA-78 either before the onset of clinical symptoms or after establishment of the disease. Immunization after the onset of disease resulted in no significant changes compared with sham-immunized AIA rats. Immunization of the animals with anti-ENA-78 before development of the disease decreased the joint inflammation compared with sham-immunized AIA rats, suggesting that the ENA-78-like protein is an important pathogenic factor in the development of AIA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fourteen Lewis rats were given an i.p. injection of 10 ng rhENA-78 with 1 ml anti-ENA-78 (rabbit serum) or 1 ml control rabbit serum. Rabbit serum and rhENA-78 were mixed together immediately before injection. Animals were sacrificed after 4 h, and the i.p. cavity of each rat was lavaged with 3 ml PBS + 5 mM EDTA. White blood cell counts, percentage of PMNs, and number of PMNs/ml were determined from differential and hemocytometer counts performed on peritoneal lavages.
An additional 14 rats received an i.p. injection of 1 mg LPS with 1 ml anti-ENA-78 or 1 ml control rabbit serum. LPS and rabbit serum were combined immediately before injection. After 6 h, animals were sacrificed and their peritoneal cavities were lavaged, as described above.
Induction of rat AIA
Lewis female rats (100 g) were injected s.c. into the base of the tail with lyophilized Mycobacterium butyricum (0.3 ml; Difco, Detroit, MI) suspended in mineral oil at 5 mg/ml (22). This animal model has been used previously to compare mineral oil control animals with AIA animals (22). In time-course studies, control animals were injected with mineral oil only (0.3 ml).
Arthritis evaluation
The degree of arthritis, indicated by joint swelling, was
quantitated by measuring two perpendicular diameters of the joint using
a caliper (Lange Caliper; Cambridge Scientific Industries, Cambridge,
MA). Joint circumference was calculated using the geometric formula:
circumference = 2
(sqrt(a2 +
b2/2)), where a is the latero-lateral
diameter and b is the antero-posterior diameter, as we have
done previously (22, 23). In the anti-ENA-78 treatment experiments,
severity of arthritis was quantified by scoring each paw on a scale of
0–4 based on joint swelling, erythema, and joint rigidity (24). The
articular index was defined as the sum of the scores of all four paws
from each animal, with a maximal possible score of 16. AIA affects
mostly the hind limbs. For instance, scores on day 24 postadjuvant
injection were 2–3 for hind limbs, while front limb scores were
typically 1. Animals were scored by observers blinded to the
experimental group identity.
Clinical and laboratory follow-up
Body weight evaluation was conducted on a regular basis postadjuvant injection during the time-course studies. At the time of sacrifice, blood was saved for laboratory tests. Erythrocyte sedimentation rate (ESR) was determined by the Westergren method using a Sediplast autozero ESR system (Polymedco, Cortlandt, NY). A total leukocyte count was obtained using a hemacytometer after RBC lysis in 3% acetic acid. Blood smears were prepared, and the percentage of PMNs was evaluated after Diff-Quik (Baxter, Miami, FL) staining. Ankles were obtained, since they were generally the most affected joints. Joints were either embedded in OCT compound (Miles, Elkhart, IN) for immunohistochemical studies or homogenized.
Preparation of joint homogenates
Isolated ankles were placed into a 50-ml conical centrifuge tube containing 3 ml of lysis buffer (2 mM PMSF, 1 µg/ml antipain, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A (all from Sigma, St. Louis, MO) in PBS) and homogenized on ice using a motorized homogenizer, followed by sonication for 30 s. The homogenates were centrifuged at 2000 x g for 10 min. The supernatants were filtered using millipore filters (0.2 µm) and stored at minus 80°C until analyzed.
Treatment of rats with anti-ENA-78
Neutralizing polyclonal anti-human ENA-78 was produced in our laboratory, as previously described (25). F(ab')2 fragments of anti-ENA-78 were generated using an immobilized pepsin system (Pierce, Rockford, IL). Two different experimental groups were employed. In the first group, the animals were injected, as described, with adjuvant on day 0. On days 8, 10, and 12 after adjuvant injection, animals were injected i.p. with either 1 ml of 1 mg/ml neutralizing F(ab')2 anti-ENA-78 or 1 ml of 1 mg/ml F(ab')2 control IgG. Animals were sacrificed on day 14 postadjuvant injection, and serum and joints were collected for investigation. In the second group, animals were injected with adjuvant on day 0. After onset of AIA, animals were injected with either 1 ml of 1 mg/ml neutralizing F(ab')2 anti-ENA-78 or 1 ml of 1 mg/ml F(ab')2 control IgG on days 18, 20, and 22 postadjuvant injection. Animals were sacrificed on day 24 postadjuvant injection, and serum and joints were collected for investigation.
Cytokine ELISA assay and data analysis
The ENA-78 concentration in the serum and homogenate samples was
determined using a sandwich ELISA (25). Ninety-six-well plates (Nunc,
Kamstrup, Denmark) were coated with 50 µl/well rabbit anti-human
ENA-78 (3.2 µg/ml in 0.6 M NaCl, 0.26 M
H3BO4, and 0.08 N NaOH, pH 9.6) for 16 h
at 4°C and then washed in PBS, pH 7.5, 0.05% Tween-20 (wash buffer).
Nonspecific binding sites were blocked with 2% BSA in PBS (200 µl),
and the plates were incubated for 90 min at 37°C. Plates were rinsed
(three times) with wash buffer, and 50 µl of undiluted and 10x
diluted serum or homogenate samples, as well as standard samples were
added to the wells in duplicate, followed by incubation for 1 h at
37°C. Plates were washed (four times), and 50 µl/well biotinylated
rabbit anti-ENA-78 (6 µg/ml in PBS, pH 7.5, 0.05 Tween-20, 2%
FCS) was added for 45 min at 37°C. Plates were washed (four times),
100 µl streptavidin-peroxidase conjugate (100 µg/ml; Dako,
Carpenteria, CA) was added, and the plates were incubated for 30 min at
37°C. The plates were washed (three times), and 100 µl chromogen
substrate (0.67 mg/ml orthophenylenediamine dichloride; Dako) was
added. The plates were incubated at 25°C for 6–10 min, and the
reaction was terminated with 50 µl/well of 3 M sulfuric acid solution
in wash buffer plus 2% FCS. Plates were read at 490 nm. The ELISA
consistently detected ENA-78 concentrations above 50 pg/ml, when
compared against a human ENA-78 standard. The ELISA did not detect
human IL-1, IL-1ß, IL-1R antagonist, IL-4, IL-6, IL-7, IL-8,
granulocyte-chemotactic protein-2, monocyte-chemoattractant protein-1,
TGF-ß, TNF-, growth-related gene product ,
neutrophil-activating peptide-2, 
IP-10, or rat cytokine-induced
neutrophil chemoattractant-1/Gro.
ELISAs detecting the quantity of rat IL-1ß and rat TNF- in joint
homogenates were purchased commercially (R&D Systems, Minneapolis, MN,
and Biosource International, Camarillo, CA, respectively). Each was
used in accordance with the recommendation of the manufacturer.
Immunohistochemistry
Rats were sacrificed on day 14 or day 24, and their joints were
embedded in OCT (Miles, Elkhart, IN). Sections (8-µm) were cut using
a D-profile knife suitable for bone cutting (Leica, Nussloch, Germany).
Immunostaining was performed using a Vector ABC Kit (Vector
Laboratories, Burlingame, CA) and diaminobenzidine (Kirkegaard & Perry,
Gaithersburg, MD) as a chromogen (22). Sections were fixed in cold
acetone for 20 min. Endogenous peroxidase activity was quenched by
incubating slides for 30 min in 0.3% hydrogen peroxidase in methanol.
All subsequent incubations were performed for 15 min at 37°C in a
moist chamber. The tissue sections were pretreated with 50 µl diluted
goat serum; incubated with either rabbit anti-rat IL-1ß (Cytokine
Science, Boston, MA), rabbit anti-mouse TNF- (Genzyme,
Cambridge, MA), or nonimmune serum (negative control); and washed
(twice) in PBS. Slides were not incubated with anti-ENA-78 since
this Ab did not react immunohistologically with ST in our hands. The
slides were incubated with a 1/400 dilution of anti-rabbit
biotinylated Ab in PBS/BSA, washed (twice) with PBS, incubated with
avidin/biotinylated HRP complex, and washed (twice) with PBS. Slides
were then stained with diaminobenzidine tetrachloride substrate for 5
min at room temperature, rinsed in tap water for 2 min, counterstained
with Harris’ hematoxylin, and dipped in saturated lithium
carbonate solution for bluing. Immunohistochemical staining was
accomplished in batches that were run with positive and negative
controls as well as experimental comparison groups simultaneously.
Microscopic analysis
Various cell types in the ST, including lining cells, sublining macrophages, PMNs, lymphocytes, and blood vessels, were identified by immunohistochemical staining reactions and/or morphologic features. The number of PMNs, lymphocytes, macrophages, and blood vessels was recorded as the mean ± SE obtained in any of three high power fields (x400) examined per slide.
Immunostaining was graded by a frequency of staining scale (0–100%), in which 0% indicated no staining and 100% showed that all of the cells were immunoreactive, for each of the ST components. Each slide was evaluated by a single observer without knowledge of the animal’s experimental group or sacrifice date. Selected sections were analyzed by two additional observers. The percentage of reactive cells for each Ab was analyzed for different time points within each group and compared between experimental and control groups.
Statistical analysis
Data were analyzed using a Student’s t test. The log
values of data were used for analysis on Fig. 1
data due to high
interanimal variation. Correlations were performed using a Pearson
correlation coefficient. When significant, p values are
indicated by an asterisk (p < 0.05) (26).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether anti-ENA-78 Ab affected inflammation in
rats, 10 ng rhENA-78 was used to induce PMN influx into the peritoneal
cavity. Upon sacrifice, the peritoneal cavity was lavaged and the
number of cells in the lavage was compared between groups. Anti-ENA-78
significantly inhibited the number of PMNs/ml recruited to the
peritoneum in lavages when compared with controls (Fig. 1
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Next, we induced rat peritoneal PMN recruitment using LPS, a known
stimulus for the production of human ENA-78 protein. LPS (1 mg) was
administered via an i.p. injection to rats, and their peritoneal
cavities were lavaged at different time points ranging from 4–18 h
(data not shown). The greatest infiltration of white blood cells into
the rat peritoneum occurred 12 h after LPS injection. Therefore,
we next determined whether a portion of this cellular influx could be
due to ENA-78-like protein and if rat PMN recruitment induced by LPS
could be inhibited by anti-ENA-78. LPS was introduced concomitant
with anti-ENA-78 or control rabbit serum, and after 6 h,
peritoneal lavages were collected to determine cell counts (Fig. 2). Anti-ENA-78 significantly inhibited
LPS-induced rat PMN recruitment, suggesting the neutralization of an
endogenous rat ENA-78-like protein that acts to recruit PMNs in vivo.
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We performed a time-course study of ENA-78-like protein expression
in the serum and joints of control normal and AIA animals. Most of the
animals (90%) injected with adjuvant developed AIA by day 14 after
injection, as determined by joint circumference, ESR, and total
peripheral leukocyte count (data not shown). ENA-78-like protein levels
in serum were significantly higher in arthritic rats compared with
controls starting at day 7 (p < 0.05) and
remained significantly increased through day 41 (Fig. 3A). The highest level of
ENA-78-like protein in AIA animals by comparison with control was found
on day 18 (Fig. 3A).
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B). ENA-78-like
protein levels were significantly increased on day 18 postadjuvant
injection (p < 0.05) (Fig. 3B). The
peak level of ENA-78-like protein over controls was reached on day 41
in AIA joints (Fig. 3B). On day 47 of the study, ENA-78-like
protein levels remained high in the joints of AIA animals and were
significantly higher than in the joints of control animals
(p < 0.05) (Fig. 3B). ENA-78-like
protein serum levels were positively correlated with the ENA-78-like
protein levels in the joints (r = 0.87;
p < 0.05, n = 7). Significant positive
correlations were determined between joint circumference and both joint
ENA-78-like protein (r = 0.78; p <
0.05, n = 7) and serum ENA-78-like protein
concentrations (r = 0.96; p < 0.05,
n = 7). Assessment of anti-ENA-78 treatment before onset of AIA
Animals were injected with anti-ENA-78 or control IgG on days 8, 10, and 12 postadjuvant injection. Based on the evaluation of ENA-78-like protein expression during the development of the disease, anti-ENA-78 was administered to adjuvant-injected animals before ENA-78-like protein was significantly increased in the joint, but was already being significantly expressed in the serum. When the animals were sacrificed on day 14 postadjuvant injection, the number of peripheral blood leukocytes, the percentage of peripheral blood PMNs, and ESR were determined in the serum. There were no significant differences detected in peripheral blood leukocyte counts, percentage of PMNs, ESR, or body weight between control IgG- and anti-ENA-78-treated animals (data not shown).
There was a decrease in severity of arthritis, as determined by
measurement of the joints in anti-ENA-78-treated animals. The four
joints (front and hind limbs) were measured as described and their sum
totaled. The anti-ENA-78-treated animals had a significantly lower
sum circumference than did control IgG-treated animals (168 mm ±
10 for control IgG vs 157 mm ± 5 for anti-ENA-78;
p < 0.05) (Fig. 4A). The articular index for
anti-ENA-78 animals trended toward a decrease compared with
IgG-injected controls, but did not attain statistical significance
(3.9 ± 2.3 for control IgG vs 2.2 ± 1.5 for
anti-ENA-78) (Fig. 4B). We found a significant positive
correlation between joint circumference and articular index
(r = 0.86; p < 0.05, n
= 10).
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The joints from anti-ENA-78-treated animals displayed decreased
mean numbers of PMNs compared with control IgG-treated animals (13
± 6 vs 2 ± 10 cells/3 high power fields), although these values
did not attain statistical significance. There was no significant
difference detected for these cell counts between
anti-ENA-78-treated and control IgG-treated animals.
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(Figs. 5 and 6).
Synovial lining cells, which are composed of macrophages and
fibroblasts, contained many IL-1ß-immunopositive cells (40 ±
9.1% for control IgG), and this reactivity was reduced with
anti-ENA-78 treatment (20.4 ± 12.5%) (Fig. 5A).
Expression of IL-1ß on sublining synovial macrophages was minimal for
both control IgG (2.8 ± 0.7%) and anti-ENA-78 treatment
(1.5 ± 0.2%) (Fig. 5A). ELISAs performed on ankle
homogenates for rat IL-1ß demonstrated a mean 3-fold reduction with
anti-ENA-78 treatment, from 68.5 ± 37.5 pg/ml in IgG-treated
control animals to 18.8 ± 8.7 pg/ml in animals treated with
anti-ENA-78 Ab (Table II). Synovial
lining cells expressed TNF- (21.3 ± 16.4% for controls), and
this reactivity was decreased with anti-ENA-78 treatment (0.5
± 0.2%) (Fig. 5B). Expression of TNF- on sublining
synovial macrophages, like IL-1ß, was found in the minority of ST
macrophages for control IgG (1.8 ± 0.7%) and anti-ENA-78
treatment (1.5 ± 0.7%) (Fig. 5B). TNF- levels in
joint ankle homogenates, as detected by ELISA, were not decreased by
anti-ENA-78 treatment when compared with IgG-treated animals
(n = 7; Table II).
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Animals were injected with anti-ENA-78 or control IgG on days 18, 20, and 22 postadjuvant injection. When the animals were sacrificed on day 24 postadjuvant injection, number of peripheral blood leukocytes, percentage of PMNs, and ESR were determined in the serum. There were no significant differences detected in the peripheral blood leukocyte counts, percentage of PMNs, ESR, or body weight between control IgG- and anti-ENA-78-treated animals (data not shown).
Control and anti-ENA-78-injected joints were analyzed by measuring
circumference, articular index, and by histopathology. Each rat’s four
joints were measured as described, and their sum totaled. The
anti-ENA-78 and control IgG animals had similar sum circumferences
(181 ± 16 mm for control IgG vs 178 ± 15 mm for
anti-ENA-78) (Fig. 7A).
The articular index also showed no significant difference between the
two groups (6.1 ± 4.3 for control IgG vs 4.8 ± 2.8 for
anti-ENA-78) (Fig. 7B). A significant positive
correlation was found between joint circumference and articular index
(r = 0.93; p < 0.05, n
= 10). Histologic analysis was performed on sections of the hind limb
ankle joints for control and treated groups to determine the number of
macrophages, lymphocytes, PMNs, and blood vessels. No significant
differences between control IgG-treated ST sections and
anti-ENA-78-treated ST sections were detected (Table III).
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was determined (Figs. 8 and
9). IL-1ß reactivity of ST lining cells
was reduced significantly in anti-ENA-78-treated ankles compared
with the control IgG ankle sections (74 ± 8.9% for control IgG
vs 5.8 ± 1.8% for anti-ENA-78; p < 0.05)
(Fig. 8A). In addition, rat IL-1ß was determined in ankle
homogenates by ELISA (Table II). Anti-ENA-78 treatment resulted in a
slight decrease from a mean of 114.7 ± 43.8 pg/ml for IgG-treated
animals to a mean of 97.6 ± 59.5 pg/ml with anti-ENA-78 Ab
(n = 7). While mean numbers of TNF--immunopositive
lining cells decreased with anti-ENA-78 treatment (31.8 ±
14.7 vs 13.3 ± 8.3), these results did not attain statistical
significance (Fig. 8B). Analysis of rat TNF- by ELISA
assay in ankle homogenates demonstrated a mean 10-fold decrease when
anti-ENA-78 Ab was administered (Table II). Values of TNF- in
the control IgG-treated ankle homogenates dropped from a mean of
34.2 ± 16.2 pg/ml to a mean of 3.9 ± 2.1 pg/ml with
anti-ENA-78 treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found a significant increase of ENA-78-like protein levels in the serum in AIA animals compared with control animals starting at day 7 postadjuvant injection. Serum ENA-78-like protein levels continued to increase as the disease developed. This increase in expression as AIA developed was also seen for ENA-78-like protein expression in the joint homogenates. It should be noted that ENA-78-like protein was found in control and AIA animals on day 0, indicating that rats have a low-level constitutive expression of this protein in their joints. ENA-78-like protein expression in the joint as well as in the serum correlated with the increase in joint circumference of the animals as the disease developed.
Administering neutralizing anti-ENA-78 to AIA animals resulted in a
decrease in the severity of arthritis. The timing of the
anti-ENA-78 treatment was critical for inflammation to be affected.
It was necessary to treat animals with anti-ENA-78 before the onset
of disease. We have noted in a previous study (22) (data not shown)
that the sum joint circumference of nonarthritic mineral oil-injected
rats did not change in a 25-day period by more than 0.6%. Therefore,
the initial sum joint circumference on day 0, taken before adjuvant was
injected, may serve as a reference for determining how much
anti-ENA-78 treatment has actually altered AIA. The initial sum
joint circumference of animals used in this study was 151.2 mm. In our
preventative model, as shown in Fig. 4, the sum joint circumference was
reduced from a mean of 167.9 mm in control animals to a mean of 156.9
mm with anti-ENA-78 treatment, a 66% reduction of the induced
swelling. Thus, anti-ENA appears beneficial when given in a
preventative manner. However, when anti-ENA was given after AIA
development, Fig. 7 shows that the sum joint circumference was reduced
from a mean of 180.9 mm in control animals to a mean of 177.6 mm in
anti-ENA-treated animals. Thus, giving the Ab after disease
development only alleviated the induced swelling by 11%.
Other AIA treatment protocols have shown a similar dependency on the timing of treatment. Williams and coworkers, using an AIA model, found that when methotrexate treatment was initiated on the day of arthritis induction, the drug suppressed development of joint inflammation (30). However, when giving methotrexate on day 11 postadjuvant, no significant suppression in joint inflammation was observed (30). Conversely, Bradbeer and coworkers demonstrated treatment of AIA with the compound SK&F 106615 on either days 0–16 postadjuvant injection (prophylactically) or days 10–23 (therapeutically) resulted in significant improvement in disease for both protocols (31). These studies show that the progression and establishment of AIA depend not only on the presence or absence of certain factors, but also suggest a disease-stage specificity for their actions. Thus, our results support a role for ENA-78-like protein in the early stages of the disease and as an initial cytokine in the complex network of cytokines functioning in the inflamed joint. While it does not appear to have these effects through altering the number of cells, it is possible that the activation state of the cells may be a contributing factor to the degree of arthritis in the anti-ENA-78-treated animals. Once the disease is established, the cells in the joint are producing numerous cytokines with overlapping functions that are capable of maintaining chronic inflammation. It is likely that due to the redundancy of the cytokines, the removal of ENA-78-like protein at this stage no longer has an effect.
AIA and murine type II collagen-induced arthritis (CIA) demonstrate the
complicated interactions of cytokines and chemokines involved in the
arthritic process. Issekutz and coworkers investigated the role of
endogenous IL-1, IL-1ß, and TNF- in the leukocyte recruitment
and the inflammatory responses in the joints of AIA rats (18). They
observed that anti-TNF- Ab treatment significantly improved
clinical score, local inflammation, and the infiltration of PMNs, and
to a lesser extent T cells, into the joint. In contrast,
anti-IL-1 or anti-IL-1ß treatment alone was ineffective,
although when combined with anti-TNF-, a significant effect on T
cell migration was observed. Thorbecke and coworkers showed that
induction of CIA in DBA/1 mice was reduced significantly if TGF-ß or
anti-TNF- were administered systemically, whereas
anti-TGF-ß or TNF- increased the incidence of CIA (32). Kasama
and coworkers analyzed the expression of IL-10 and the chemokines
MIP-1 and MIP-2 and their contribution to the evolution of CIA (33).
They found anti-IL-10 treatment increased the expression of
MIP-1 and MIP-2, as well as myeloperoxidase activity and leukocyte
infiltration in the inflamed joints. These studies as well as ours
demonstrate the complexity of the cellular interactions within the AIA
synovium.
ENA-78-like protein may be involved in the progression of AIA in
concert with other cytokines. Expression of ENA-78-like protein in the
joint during the progression of AIA correlated with the expression of
several proinflammatory cytokines, namely TNF-, IL-6, JE, and
MIP-1 (manuscript in preparation). Furthermore, ENA-78-like protein
expression is inducible by both the proinflammatory cytokines, TNF-
and IL-1ß. When the human type II epithelial cell line, A549, was
stimulated with either TNF- or IL-1ß, ENA-78 release was
increased, as was the release of several other chemokines (13). RA
synovial tissue fibroblasts stimulated with TNF- in vitro increased
their secretion of ENA-78 (14). In addition, human ENA-78 is an
ELR-containing CXC chemokine that is chemotactic for endothelial cells
as well as angiogenic in vivo (34). These are biological functions that
may be shared with the ENA-78-like protein that is recognized by
anti-ENA-78.
A sequential rather than simultaneous expression of TNF- and
IL-1ß, with TNF- being expressed first, has been suggested for CIA
development in mice (35, 36). Marinova-Mutafchieva and coworkers found
progressive increases in the number of TNF-- and IL-1ß-positive
cells in the joints of CIA mice as arthritis developed (35). They also
reported TNF- expressed in the joints early in the study (day 1),
while IL-1ß was not expressed in the joints until day 3 (35).
Furthermore, Joosten and coworkers found anti-TNF- treatment of
CIA to be effective only if administered early after onset of CIA and
not in late stages of CIA, whereas anti-IL-1ß was effective when
administered early or late in CIA (36).
It is possible that ENA-78 may also modulate the expression of IL-1ß
and/or TNF-. To address this possibility, we performed ELISAs and
immunohistochemistry on joint tissue. When anti-ENA-78 was
administered before the onset of AIA, mean IL-1ß levels decrease
73%, as determined by ELISA. In agreement with this finding, the
percentage of IL-1ß-immunopositive lining cells decreases, which most
likely contributes to the overall decrease, as demonstrated by ELISA.
In contrast, anti-ENA-78 Ab treatment before AIA onset did not
decrease TNF- levels in ankle homogenates, as determined by ELISA,
although the mean percentage of immunopositive lining cells decreased.
TNF- in joint homogenates, which include cell extracts from
cartilage and bone, may show TNF- levels reflective of these other
nonsynovial cell types in the joint milieu, thus accounting for the
disparate immunohistologic and ELISA results.
When anti-ENA-78 was administered after the development of AIA,
ELISA data demonstrate that IL-1ß levels decrease slightly (15%).
This decrease was most likely supported by a decrease of IL-1ß
produced by synovial lining cells, since the percentage of
immunopositive IL-1ß lining cells was significantly decreased.
Furthermore, anti-ENA-78 treatment after AIA onset decreased the
quantity of TNF- by 89%, as determined by ELISA.
Immunohistochemical data suggest that lining cells, but not sublining
macrophages, may have played a role in this overall reduction in
TNF-. These results suggest that a cytokine network involving
IL-1ß, TNF-, and ENA-78-like protein is present in the AIA joint.
In rat peritoneal inflammation, anti-ENA-78 clearly reduces the PMN
influx in response to rhENA-78 or LPS. In rat AIA, however, treatment
with anti-ENA-78 Ab before AIA onset decreased the mean number of
PMNs by 85%, as determined histologically in the joint, but this
decrease was not statistically significant. A number of possible
explanations could account for this result. These include: 1) imperfect
cross-reactivity and/or neutralization of the rat ENA-78-like protein
with the anti-human ENA-78-like protein; 2) ineffective injection
times, dosage, and/or method of administration (i.p. injection) of the
Ab for an efficacious neutralization of the ENA-78-like protein; and 3)
variability in the data with a large SE accounting for lack of
statistical significance. It is also possible that different homing
pathways exist for PMNs to be recruited into the joint versus the
peritoneum. In both rats and humans, distinct organ-specific homing
mechanisms have been identified for inflammatory leukocytes (37, 38, 39).
Hence, PMNs may respond to ENA-78-like protein differently in the rat
peritoneum and joint. Finally, ENA-78-like protein may recruit cell
types other than PMNs, as is suggested in Table I.
In summary, in this study we demonstrated that anti-ENA-78 inhibits LPS-induced peritoneal recruitment of rat PMNs, and that use of this Ab can modify the development of AIA. We found that anti-ENA-78 treatment before establishment of disease led to a decrease in the severity of inflammation, as measured by joint circumference. When anti-ENA-78 was administered after clinical onset of AIA, there was no modification of the disease. An ENA-78-like protein appears to be an important chemokine in the progression of AIA.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alisa E. Koch, Northwestern University Medical School, 303 E. Chicago Avenue, Ward Building 3-315, Chicago, IL 60611. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; AIA, adjuvant-induced arthritis; CIA, collagen-induced arthritis; ENA-78, epithelial neutrophil-activating peptide-78; ESR, erythrocyte sedimentation rate; MIP, macrophage-inflammatory protein; PMN, neutrophil; rh, recombinant human; SF, synovial fluid; ST, synovial tissue.
Received for publication February 9, 1998. Accepted for publication March 30, 1999.
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, and interleukin-6 in cartilage proteoglycan metabolism and destruction. Arthritis Rheum. 38:164.[Medline]
and IL-1 in polymorphonuclear leukocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis. Clin. Exp. Immunol. 97:26.[Medline]
and transforming growth factor ß during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89:7375., anti-IL-1/ß, and IL-1Ra. Arthritis Rheum. 39:797.[Medline]
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