ARTICLE
Bacterial infections including streptococcal and staphylococcal infections
are thought to be triggers of induction and/or exacerbation of psoriatic
lesions. In particular, streptococcal infection is suggested to play an
important role as a triggering factor in psoriasis guttate, because deterioration
of small-sized psoriatic lesions is occasionally observed following respiratory
upper tract infection.
Psoriasis arthropathy (PA) is a well-defined inflammatory joint disease
that develops in approximately 5% of patients with cutaneous psoriasis.
We have occasionnally observed the improvement of cutaneous or joint manifestations
following the treatment of focal infections such as tonsillitis or periodontal
bacterial infections, however, the precise role of focal infections in
PA is still not fully understood. Staphylococcus aureus (S.
aureus) is the most common cause of non-gonococcal infectious arthritis
in humans [1]. Recently, superantigen, a bacterial product and an extremely
potent polyclonal mitogen for human T cells, has been proposed as a possible
antigen for the induction of psoriasis [2-4]. Some bacterial proteins
have been shown to stimulate T cells in a Vß-specific fashion. We
have recently observed that the peripheral blood response to staphylococcal
superantigens (staphylococcal enterotoxin A (SEA), SEB and SEC1) in patients
with psoriasis vulgaris (PV) is significantly higher than that of normal
controls [5]. In this study, we have further analyzed the association
of staphylococcal superantigens with clinical and laboratory findings
in patients with PA.
Materials and methods
Patients
Eleven patients with PA were examined (6 men and 5 women, mean age;
43.7 years), who had been diagnosed according to the criteria of Moll
and Wright [6]. The mean disease duration was 5.8 years. All patients
had been previously treated with a variety of conventional therapies such
as topical corticosteroids, anthralin, and psoralen-UVA treatment (PUVA).
Only one patient had been treated with etretinate for some years. The
psoriasis area and severity index (PASI) score for each patient was determined
according to the method described by Fredrikson and Petterson [7]; the
PASI score of the patients ranged from 0.6 to 31.9 (mean 7.6). Normal
controls comprised 19 healthy volunteers (10 men and 9 women, mean age;
45.2 years). As the mean PASI score was not as high in patients with peripheral
type PA, the peripheral blood response of PA patients was compared with
that of 15 PV patients with a PASI score lower than 10 (mean 7.0) (9 men
and 6 women, mean age; 46.5 years).
Proliferation assay
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient
centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were
resuspended in RPMI 1640 containing 7% fetal calf serum (FCS), and seeded
on 96-well microplates (Falcon 3072, Beckton Dickinson, NJ, USA). PBMC
(5 x 104) were cultured in the presence or absence of concanavalin
A (Con A) (5 µg/ml, Sigma Chemical Co. Ltd., St Louis, MO), SEA,
SEB and SEC1 (1 µg/ml, Sigma) for 7 days and pulsed for the last
6 hrs with 0.2 µCi/well of 3H-TdR, as previously described
[5]. The incorporated radioactivity in the harvested cells was counted
in an automatic liquid scintillation counter. The stimulation index (SI)
was calculated as follows; DPM (stimulated)/DPM (unstimulated). 3H-TdR
uptake by cells was expressed as the mean DPM ± SD of triplicate
cultures.
Serum cytokines
Serum samples were obtained by veno-puncture when the patients were
not being treated with systemic immunosuppresive therapy. Aliquots of
serum samples were immediately frozen at 20° C, prior to use.
Serum levels of cytokines, interleukine-1ß (IL-1ß), IL-2,
IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) were examined using
an enzyme-linked immunosorbent assay (ELISA) kit (IL-1ß; R&D
Systems, Minneapolis, MN, IL-2; Otsuka Pharmaceutical Co. Tokyo, IL-6;
Toray-Fuji, Tokyo, IL-8; Toray-Fuji, TNF-alpha; R&D).
T cell receptor PCR
Total RNA was extracted from peripheral blood lymphocytes (PBL) (1 x
106) and fifty 5-µm cryostat sections of synovial tissue
from two patients with PA, using RNA zol (Chinna/Biotex, Houston, TX,
USA) and then reverse transcribed to cDNA by RAV-2 reverse transcriptase
(Takara Co. Ltd., Tokyo, Japan). Polymerase chain reaction (PCR) analysis
of the T cell receptor (TCR) Vß repertoire was accomplished in 30
µl of mixture containing 1.5 U Taq polymerase (Perkin Elmer, Cetus,
CT, USA), 200 M dNTP, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl
buffer, pH 8.3, and 20 pmol of oligonucteotide primers to amplify specific
Vß gene segments [8], which were synthesized by a DNA synthesizer
(Applied Biosystems model 3919). Each reaction mixture contained 1 of
22 oligonucleotides, specific for a particular Vß family or subfamily,
paired with a consensus ß-chain constant region (Cß) primer.
As internal controls, a pair of 5' sense Calpha-specific primers and 3'
antisense Calpha-specific primers was also amplified, as described previously
by Choi et al. [9]. PCR amplification was performed with an initial
denaturation step at 94° C, followed by a 35-cycle profile that consisted
of 94° C denaturation (1 min), 57° C annealing (1 min), and
72° C extension (1 min and 5 sec). The PCR was completed with a final
extension step of 7 min at 72° C. Twenty microliters of PCR products
were electrophoresed with 1% ethidium bromide and visualized under ultraviolet
light. In preliminary experiments, PCR amplification was performed at
27, 30 and 35 cycles, and it was ensured that the amplification occurred
in a linear range quantified by a densitometer (EPA-3000, Chemiway, Tokyo).
Each intensity was assessed by the ratio with C alpha which was expressed
almost identically in all experiments, and the relative intensities of
each TCR Vß band. Reproductibility of the method was confirmed in
preliminary experiments, showing that two separate assays of the same
cDNA from a healthy donor resulted in almost identical data.
Statistical analysis
Data were expressed as means ± SD. Statistical analysis was performed
using Student's t-test. A p value < 0.05 was considered as significant.
Results
Superantigen-pulsing studies
There was no significant difference in the unstimulated PBMC response
between PV patients, PA patients and normal controls (data not shown).
The differences between the Con A-stimulated PBMC responses also did not
reach significance in PV patients (5,267 ± 2,316 dpm), PA patients
(5,475 ± 1,870) and normal controls (4,980 ± 3,019). The PBMC
responses against SEA (31,819 ± 812 dpm), SEB (38,175 ± 719)
and SEC1 (29,740 ± 633) in PA patients were significantly higher
than that in normal controls (21,989 ± 427 dpm against SEA, 23,708
± 466 against SEB, 20,291 ± 582 against SEC1; p < 0.05 in
all comparisons), however, there was no significant difference between
PA patients and PV patients (28,065 ± 580 dpm against SEA, 33,428
± 467 against SEB, and 26,671 ± 389 against SEC1). Among the
11 patients with PA, 8 patients showed the most intense response to SEB
rather than either SEA or SEC1, 2 showed the highest response to SEA,
and another responded mainly to SEC1. We then calculated the SI for the
PBMC response to SEB. The SI of PA patients (50.2 ± 41.4) was significantly
higher than that of normal controls (30.9 ± 23.8) (p < 0.05),
however, the difference did not reach significance as compared with that
of PV patients (42.8 ± 30.6) (Fig.
1). We noticed that there were episodes of severe disabling lumbago
among several PA patients, which suddenly occurred following throat soreness,
and which persisted for only a few days. Thus, we classified PA patients
into two groups, those with a past history of such an episode (group I)
and those without (group II), and compared the PBMC response against SEB.
Two PV patients with such an episode were added to group I (patient No.
7 and 8 in Table I). Both
of these PV patients had chronic tonsillitis. S. aureus was isolated
from the tonsils of 4 patients (patient No. 2, 4, 7, 8 in group I) among
7 patients examined. Several immune abnormalities such as positive anti-nuclear
antibody and rheumatoid factor were observed in group I patients (Table
I). The difference in SI between group I (73.7 ± 39.7) and
group II (42.6 ± 18.1) did not reach significance.
Serum cytokines
High levels of IL-6, IL-8, and TNF-alpha were observed in some of the
PA patients (Fig. 2).
The lower limit for detection was 40.6 pg/ml for IL-6, 10.0 pg/ml for
IL-8, and 7.0 pg/ml for TNF-alpha. Patients showing high levels of IL-8
also displayed severe joint deformity. The two PV patients with past episodes
of severe lumbago (patients No. 7 and No. 8) showed increased levels of
IL-6 (O in Fig. 2).
On the other hand, IL-6, IL-8 and TNF-alpha levels were all within normal
limits in the 15 PV patients described above.
TCR Vß expression
With the PCR, each Vß-Cß primer yielded a band of 250-300
bp as visualized with ethidium bromide. Results of densitometric analysis
are shown in Figure 3.
Preferential expression of Vß 17 was commonly detected in the lymphocytes
in the synovial tissue. In one case, Vß 10, 15 and 19 were strongly
expressed, and in another case, Vß 12 was preferentially expressed.
In one case, Vß 17 was also highly expressed in the infiltrating
lymphocytes in the involved skin of psoriasis. Results for the TCR Vß
repertoire in the lesional skin of PV showed that Vß 2, 6, 7 and
17 were also detected in two patients, as has been previously reported
[10]. As a control, 3 samples of normal skin obtained from the back during
surgery showed that Vß 1, 6 and 14 were relatively strongly expressed
(data not shown). Diverse expression was observed on PBL from 5 healthy
controls.
Discussion
Recent studies have suggested that psoriasis is an immunological skin
disease, and that streptococcal and staphylococcal superantigens are proposed
as possible antigens [2-4]. They bind to MHC class II molecules without
prior internalization or processing, and stimulate the development of
large quantities of T cells bearing particular T cell receptor Vß
gene products [11-13]. We have recently observed that the response of
PBMC from PV patients, to staphylococcal enterotoxins, is significantly
higher than that of normal controls [5]. In this study, we have further
investigated the response of PBMC from PA patients against Con A, SEA,
SEB and SEC1. Our results showed that there was no significant difference
in the PBMC response against Con A among PA patients, PV patients and
normal controls, while PBMC from psoriatic patients responded significantly
greater to staphylococcal superantigens than those from normal controls.
However, there was no significant difference between PV and PA patients.
In addition, comparison of psoriatic patients with past episodes of severe
lumbago and those without showed that patients who had experienced severe
lumbago showed a higher response to SEB, although the difference did not
reach significance.
Increased IL-8 levels in the synovial tissue of rheumatoid arthritis
(RA) [14, 15], and IL-6 in the synovial fluid and serum in patients with
RA [16, 17] have been reported. It was speculated that IL-6 and IL-8,
which are released from T cells, macrophages or B cells, may be important
contributors to inflammatory events in the joints, inducing arthralgia.
In our study, several patients showed changes in serum levels of IL-6,
IL-8 and TNF-alpha, which was considered to reflect the persisted activation
of circulating lymphocytes or monocytes. Group A streptococcal antigens
are capable of inducing autoimmune arthritis in humans [18], and Vasey
et al. [19] reported elevated levels of antibody to the streptococcal
exotoxin in patients with PA. On the other hand, Migita et al.
[20] have recently reported that stimulation of rheumatoid synovial cells
with SEA selectively induced stromelysin secretion, which degrades extracellular
matrix leading to cartilage destruction. In our study, S. aureus
was actually detected in the tonsils of 2 PA patients and 2 PV patients
who had experienced an episode of sudden-onset, short-lasting severe,
disabling lumbago which followed a sore throat. It is unclear whether
the sore throat was caused by a streptococcal infection or S. aureus
in our patients, because the examination could not be performed at that
time of the onset of the severe lumbago. Our study raises the possibility
that the exposure to staphylococcal superantigens may have generalized,
systemic, superantigenic effects leading to the induction of arthralgia
in some groups of patients, although the correlation between staphylococcal
enterotoxins and the onset of arthralgia is obscure at present. It has
been speculated that staphylococcal superantigens, and streptococcal superantigens,
may be a factor triggering induction or exacerbation of PA in patients
with an abnormal immunological background.
Recent evidence suggests the selective expansion of the TCR Vß
repertoire in the cellular infiltrates of psoriatic plaques [21-23]. In
this study, the TCR Vß repertoires of PBL and synovial infiltrating
T cells were also examined using the RT-PCR method. Vß 17 was preferentially
expressed in PBL and synovial tissue in both PA patients, and Vß
17 was also highly detected in the involved skin in one case examined
(Fig. 3). Vß 12
was commonly expressed in synovial tissue. The Vß 17 element is
stimulated by SEB, and Vß 12 interacts with SEC in human [24]. Superantigens
have been implicated in RA, and restricted T cell clonality has been suggested
to reflect activity of bacterial superantigens [25]. Schwab et al.
[26] showed that superantigens participate in an experimental model of
recurrent arthritis. It has been reported that selective expansion of
Vß 2, 3, 6, 14 and 17-positive T cell clones can be detected in
synovial fluid from patients with RA [27-29], suggesting a Vß-specific
mechanism infiltrating T cell growth. Vß 17 may play a role in the
inflammation joint in PA as well as in RA, although only two cases were
examined in this study. Further studies on the association of TCR in PA
are necessary.
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