The kinetics of apoptosis and the apoptosis-regulating gene
p53 in adjuvant arthritis (AA) were investigated to assess the value
of the AA rat model for testing apoptosis-inducing therapies. Very few terminal
deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick
end-labeling (TUNEL)-positive cells were detected during the early phases of
AA, but on day 23 (chronic arthritis) the percentage of TUNEL-positive cells
was significantly increased. Expression of p53 in synovial tissue
gradually increased from days 5-23, which was markedly higher than p53
levels in rheumatoid arthritis (RA) synovium. Significant apoptosis only occurs
late in rat AA and is concordant with marked p53 overexpression,
making it useful model for testing proapoptotic therapies, but rat AA is not
the best model for p53 gene therapy because dramatic p53
overexpression occurs in the latter stages of the disease.
RA is a chronic inflammatory disorder that is characterized by
inflammation and proliferation of synovial tissue. The amount of DNA
fragmentation is significantly increased in rheumatoid synovium. Only low
numbers of apoptotic cells are present in rheumatoid synovial tissue, however.
The proportion of cells with DNA strand breaks is so great that this disparity
suggests impaired apoptosis. Therefore, the development of novel therapeutic
strategies that are aimed at inducing apoptosis in rheumatoid synovial tissue
is an attractive goal.
Although animal models for arthritis only approximate RA, they
provide a useful test system for the evaluation of apoptosis-inducing
therapies. AA in rats is among the most commonly used animal models for RA. For
the interpretation of such studies, it is essential to characterize the extent
to which apoptosis occurs during the natural course of the disease. Therefore,
we evaluated the number of apoptotic cells and the expression of p53 in various
phases of AA.
Materials and methods:
In order to generate the AA rat model, Lewis rats were immunized
with Mycobacterium tuberculosis in mineral oil on day 0. Paw swelling
usually started around day 10. For the temporal analysis rats were sacrificed
on days 0, 5 (prearthritis), 11 (onset of arthritis), 17 (accelerating
arthritis), or 23 (chronic arthritis).
For the detection of apoptotic cells, the hind paws were harvested
on days 0(n=6),5 (n=6), 11 (n=6), 17 (n=6),
or 23 (n=4). The right ankle joints were fixed in formalin,
decalcified in ethylenediaminetetra-acetic acid, embedded in paraffin, and
sectioned. The TUNEL method was applied. The percentage of TUNEL-positive cells
of the total inflammatory cell infiltrate was noted.
For Western blot analysis, hind paws were harvested on days
0 (n=2), 5 (n=3), 11 (n=4), 17 (n=4), or 23
(n=4). In addition, hind paws of normal rats (n=2) were
studied. The right ankle joints were snap frozen and pulverized. Synovial
tissue was also obtained by arthroscopy of three patients with longstanding
(>5 years) RA. After protein extraction in lysis buffer, equal amounts of
protein samples from lysates were pooled and examined by Western bolt analysis
using anti-p53 monoclonal antibody D07, which recognizes wild-type and mutant
p53 from rodents and humans.
For immunohistochemical analysis, six rats were sacrificed on day
23 after immunization and synovial tissue of the right ankle joints was snap
frozen and evaluated by immunohistochemistry using anti-p53-pan. The sections
were evaluated semi-quantitatively using a 0-4 scale.
The kruskal-Wallis test for several group means was used to
compare the percentage of TUNEL-positive cells at different time points.
The percentages of TUNEL-positive cells were strongly dependent on
the stage of the disease. Very few TUNEL-positive cells were detected in normal
rats or in the early phases of AA; the number of TUNEL-positive cells was 1% or
less of the total cell infiltrate, including neutrophils, from days 0-17 (Table
1). On day 23, however, the percentage of TUNEL-positive
cells was significantly increased [15.8±5.1% (mean ± standard error
of the mean); P=0.01]. TUNEL-positive cells were observed in the
intimal lining layer and synovial sublining of the invasive front, as well as
in the articular cartilage (Fig. 1).
Subsequently, we examined expression of the tumor suppressor gene
p53, because this is a key regulator of apoptosis. Expression of p53
in pooled rat AA joint extracts gradually increased from day 0 (6 arbitrary
units) to day 23 (173 arbitrary units), which was markedly higher than p53
levels in RA synovium (32 arbitrary units; Table 1).
Overexpression of p53 protein on day 23 was confirmed by immunohistochemistry
in a separate experiment in six rats with AA. Overexpression of p53 was
observed in the intimal lining layer and synovial sublining in all rats on day
23. In all cases a semiquantitative score of 4 was assigned, indicating that
51% or more of the cells were positive, whereas control sections were
The results presented here reveal that the number of
TUNEL-positive cells remained very low until chronic arthritis developed. This
indicates that, although there was sufficient DNA damage to cause an increment
in p53 expression in the early phases, DNA strand breaks that can be detected
by TUNEL assays only occurred in chronic AA. The observation that
TUNEL-positive cells were nearly absent in early AA clearly indicates that only
very few cells were undergoing programmed cell death. This is an important
observation, which makes it possible to study the effects of apoptosis-inducing
therapies in situ in early and accelerating AA. An effective therapy
would obviously increase the number of TUNEL-positive cells.
There is already some overexpression of p53 in the preclinical
phase and during the onset of the arthritis, with an additional increment in
p53 expression during accelerating and chronic arthritis. Presumably, this is
wild-type p53, because the disease duration is likely too short to allow for
the development of p53 mutations. Transcription of p53 is probably
increased in response to the toxic environment of the inflamed joint. The
increased expression of p53 in the joints of rats with chronic AA was even
greater than that observed in synovial tissue of RA patients with long-standing
Overexpression of p53 and increased numbers of apoptotic cells did
not occur simultaneously in this model; rather p53 overexpression preceded
increased apoptosis. Activation of p53 leads to induction of cell
growth arrest, allowing time for DNA repair. It appears that DNA damage is only
extensive enough to induce apoptosis in the latter stages of AA. Factors other
than p53 may also play an important role in the actual induction of
Taken together, significant apoptosis only occurs late in AA and
it follows marked p53 overexpression, making it a useful model for
testing proapoptotic therapies. AA is not the best model for p53 gene
therapy, however, because dramatic p53 overexpression occurs in the latter
stages of the disease.