Peripheral blood mononuclear cell proliferative response against staphylococcal superantigens in patients with psoriasis arthropathy

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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