ARTICLE
Although the exact mechanism of the pathogenesis of psoriasis is still
unknown, it has been recently suggested that several cytokines released
by activated T cells or keratinocytes may play important roles as mediators
between genetically predisposed keratinocytes and various stimuli of immunological,
neurological, pharmacological and environmental origin [1-3]. The earliest
histological change seen in the lesional skin of psoriasis is an infiltration
of mononuclear cells into the dermis. Several kinds of cytokines such
as tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, or interferon-gamma
(IFN-gamma) released by T cells and monocytes are thought to play an important
role in recruiting lymphocytes and mononuclear cells into lesional skin
by acting on vascular endothelial cells to enhance leukocyte adhesion
[4]. Lesional skin contains activated memory T lymphocytes with production
of messenger RNA (mRNA) for IL-2. IL-2, IL-6, IFN-gamma and TNF-alpha [5-6].
Bacterial infection such as streptococcal infection is a well-known
exacerbating factor in psoriasis guttate [7-8]. In addition. Staphylococcus
aureus (S. aureus) is sometimes detected on the psoriatic plaques
[9]. Actually, we occasionally experience the exacerbation of psoriasis
vulgaris after tonsillitis, in which not only Streptococcus sp.
but also S. aureus is detected. Recently superantigen, a bacterial
product and an extremely potent polyclonal mitogen causing activation
of lymphocytes and monocytes by cross-linking Vß elements of the
T cell with class II molecules on accessory cells [10-13], has been proposed
to be one of the candidates for the induction of psoriasis [14-15]. In
this study, we have analysed the longitudinal response of peripheral blood
mononuclear cells (PBMCs) against staphylococcal superantigens in patients
with psoriasis vulgaris, and the relationship with clinical and laboratory
findings. The kinetics of IL-2, IL-6, and TNF-alpha produced by PBMCs stimulated
by staphylococcal enterotoxin B was also measured in several psoriatic
patients to examine whether these cytokines play a role in the induction
and/or exacerbation of psoriasis.
Patients and methods
Patients
Thirty-seven patients with psoriasis vulgaris (22 males and 15 females
; mean age, 46.5 years) of whom 6 patients had arthralgia, were examined.
All patients had been treated previously with a variety of conventional
therapies such as topical corticosteroid, psoralen-UVA treatment, anthralin,
and retinoids. The psoriasis area and severity index (PASI) score for
each patient was determined according to the method described by Fredriksson
and Petterson [15]; the PASI score of the patients ranged from 0.6 to
31.9 (mean, 12.6). Twenty four age-matched, healthy volunteers (14 males
and 10 females) and 10 patients with atopic dermatitis (6 males and 4
females) were also examined as controls.
Proliferation assay
To measure the lymphocyte proliferative response, PBMCs were isolated
by density gradient centrifugation on a 60% Percoll solution (v/v, density
1.078 g/ml). Cells were resuspended in RPMI 1640 containing 7% fetal calf
serum (FCS), and seeded onto 96-plate microplates (Falcon 3072, Beckton
Dickinson, NJ, USA). In a preliminary study, PBMC, 5 x 103,
5 x 104, and 5 x 105, were cultured for 1, 4, and
7 days respectively. A PBMC concentration of 5 x 104 and a
culture period of 7 days were chosen for the assay. PBMCs were cultured
in the presence or absence of various mitogens (Con A (5 µg/ml, Sigma
Chemical Co. Ltd., St Louis, MO), staphylococcal enterotoxin A (SEA),
SEB, and SEC1 (1 µg/ml, Sigma) for 7 days and pulsed for the last
6 hours with 0.2 µCi/well of 3H-TdR, and harvested. The
incorporated radioactivity in the harvested cells was counted in an automatic
liquid scintillation counter. Stimulation index (SI) was calculated as
follows; DPM (stimulated/DPM (unstimulated). In a separate experiment,
the cell surface marker was characterized by flow-cytometry after stimulation
with SEB for 7 days.
Cytokine assay
Serum samples were obtained from 18 patients (11 males and 7 females,
mean age, 43.6 years) from among the enrolled subjects by veno-puncture
and stored at 20° C, to examine the serum concentration of IL-6.
The mean PASI score was 12.6.
In a separate experiment, PBMCs were isolated by the method described
above, from seven patients with psoriasis vulgaris (4 males and 3 females,
mean age, 48.7 years). They all had active disease and had not been treated
with systemic steroids or immunosuppressive drugs. Their PASI score was
4.0-26.0 (mean; 13.8). Six age- and sex-matched, healthy volunteers served
as controls. On average, the PBMCs consisted of 10-20% monocytes and 80-90%
lymphocytes. Cells (106/ml) were seeded on a 24-well plate
in the presence or absence of SEB (100 ng/ml). Supernatants were harvested
at different times, centrifuged and stored at 20° C until
use. The concentrations of IL-2, IL-6 and TNF-alpha in the supernatants were
determined by commercially available enzyme-linked immunosorbent assay
(ELISA) kits: IL-2 (Otsuka, Tokyo, Japan) ; IL-6 (R & D Systems, Minneapolis,
MN) ; TNF-alpha (R & D).
Statistical analysis
Results were expressed as mean 6 SD triplicate cultures. Statistical
analysis was performed using Student's t-test or the Mann-Whitney U-test.
Pearson's correlation coefficient was also used. A p value < 0.05 was
considered to be significant.
Results
Stimulation of PBMCs by superantigens
PBMCs from patients with psoriasis vulgaris and healthy volunteers were
strongly activated by SEB. In a preliminary experiment, the SI of the
psoriatic patients (n = 3) was 18.5 ± 6 (day 1), 48.4 ± 10 (day
4) and 59.0 ± 14 (day 7). In contrast, that of normal subjects (n
= 3) was 15.6 ± 5, 30.7 ± 9 and 26.6 ± 12 at day 1, 4 and
7, respectively. Thus the 7 day culture period was chosen.
PBMCs from the psoriatic patients (34,468 6 6455) (mean DPM 6 SD) responded
more intensely than those from healthy controls (22,756 ± 5780) (p
< 0.05). The SI of the psoriatic patients (63.9 ± 55) was significantly
higher than that of normal volunteers (26.0 6 23) (p < 0.005) and patients
with atopic dermatitis (40.7 ± 30) (p < 0.005) (Fig.
1). PBMCs from patients with psoriasis vulgaris, atopic dermatitis
and normal subjects also responded strongly to SEA and SEC 1, and the
SI of the PBMCs from psoriatic patients were significantly higher (p <
0.005 in comparison with normal controls against SEA and SEC1, p <
0.05 in comparison with atopic dermatitis patients against SEA and SEC1)
(data not shown). 3H-TdR uptake with Con A was not significantly
different between psoriatic patients (5.688 6 2.743), atopic dematitis
patients (5.274 ± 2,016) and normal subjects (4.984 ± 3.191).
Characterization of lymphocytes stimulated by SEB
The surface markers of SEB-stimulated peripheral blood from 4 psoriatic
patients and 2 normal subjects were determined by flow cytometry analysis
using corresponding antibodies. In all cases, CD4+ cells were
preferentially induced by SEB (Fig.
2).
Relationship between SI and clinical and laboratory
findings
Next we asked whether the SI of SEB-stimulated PBMC correlated with
clinical disease activity as reflected in the PASI score. A weak correlation
was noted between the SI and the PASI score (r = 0.62) (Fig.
3). The SI also weakly correlated with the serum IL-6 level (r
= 0.45) (Fig. 4). Comparison
between the SI of the patients with psoriasis vulgaris with arthralgia
(n = 6) and those without showed no significant difference (data not shown).
Kinetics of PBMC-released
cytokine
In unstimulated cultures of PBMC from psoriatic patients as well as
healthy controls, small amounts of IL-2, below the normal range, were
detectable. After stimulation with SEB, PBMC from healthy controls secreted
increasing concentrations of IL-2 with a maximum after 3 days (272 ±
194 pg/ml) (Fig. 5). PBMCs
from psoriatic patients exhibited similar enhancement of IL-2 secretion
after 3 days, whereas the maximum concentration reached 561 ± 171
pg/ml. PMBCs from psoriatic patients revealed a significantly increased
level of IL-2 on stimulation by SEB after 3 days as compared with normal
controls (p < 0.05).
PBMCs from both healthy controls (196 ± 66 pg/ml) and psoriatic
patients (467 ± 265 pg/ml) (Fig. 5)
spontaneously secreted. Il-6, with a peak level after 2 days. Treatment
with SEB led to an increasing concentration of IL-6 in culture supernatants
of PBMCs from healthy controls (1,416 ± 575 pg/ml) and psoriatic
patients (2 188 ± 1,242 pg/ml) with a maximum after 2 days in both
groups. PBMCs from psoriatic patients revealed a higher increased level
of IL-6 following stimulation by SEB over 1-5 days as compared with normal
controls, however, the difference did not reach significance.
A spontaneous TNF-alpha secretion into the culture supernatants was below
detectable limits in PBMC cultures from both healthy controls and psoriatic
patients. After stimulation by SEB, PBMCs secreted increasing concentrations
of TNF-alpha with a maximum after 3 days in healthy controls (242 ± 114
pg/ml) and psoriatic patients (373 ± 228 pg/ml) (p < 0.05) (Fig.
5).
Discussion
Recent studies have suggested that psoriasis is one immunological skin
disease in which a certain antigen stimulates T cells and then a variety
of cytokines released from keratinocytes and inflammatory cells contribute
to induce and maintain the inflammatory process. Several kinds of cytokines
play important roles in recruiting lymphocytes and mononuclear cells into
lesional skin by acting on vascular endothelial cells to enhance adhesion
of leucocytes. Streptococcal infection is one of the exacerbating factors
in psoriasis [6-8]. It is presumed that the triggering of guttate psoriasis
by Streptococci is immunologically mediated, and the possible mechanisms
include an inherited abnormality in the immune responsiveness to streptococcal
antigens and the cross-reaction between streptococcal components and human
keratinocytes [17-18]. Thus, streptococcal superantigens are thought to
induce psoriasis [19-20]. On the other hand, S. aureus is occasionally
detected on the lesional skin of plaque type psoriasis [9]. In addition,
not only streptococcal but also staphylococcal infection is sometimes
implicated, as can been seen from the occasional correlation between tonsillitis
and the exacerbation of chronic, plaque type psoriasis. Recently, staphylococcal
superantigens are proposed as a possible antigen in psoriasis [14-15].
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 [10-13]. Increased responsiveness
of peripheral blood of patients with psoriasis vulgaris against staphylococcal
superantigen has been recently reported [21].
In this study, we have examined the in vitro proliferation of
PBMCs in response to staphylococcal superantigens. Although there was
a significant increase in PBMC proliferation in response to SEB in patients
with psoriasis vulgaris and normal volunteers, the SI of the former group
was significantly higher. Although it involved only a small number of
subjects, our preliminary data showed that PBMCs from psoriatic patients
responded after 7 days' stimulation with SEB, while PBMCs from normal
subjects showed a peak at 4 days and decreased at 7 days. Thus, in this
study, we examined the longitudinal response of PBMCs from psoriatic patients
against staphylococcal superantigens. Our data suggest that hyper-reactivity
of peripheral blood to superantigens may lead to the exacerbation and
persistence of psoriasis through several inflammatory cytokines. For example,
TNF-alpha induces production of intercellular adhesion molecule-1 (ICAM-1)
by keratinocytes, as well as IL-8 by keratinocytes, dermal fibroblasts,
and endothelial cells [22-24], which is chemotactic for neutrophils and
lymphocytes. TNF-alpha also increases the rate of secretion of pro-inflammatory
cytokines such as IL-1 and IL-6 by keratinocytes [25]. TNF-alpha is thought
to play a key role in the pathogenesis of psoriasis on account of its
immunomodulatory properties. Neuner et al. [26] have shown that
PBMCs from psoriatic patients spontaneously produce significantly increased
amounts of biologically active IL-6. Our results showed increased production
of the T cell-derived cytokines IL-2, IL-6, and macrophage-derived TNF-alpha
and IL-6 in PBMCs from psoriatic patients on incubation with SEB, as compared
with healthy controls. PBMCs from psoriatic patients released significantly
greater amounts of IL-2 and TNF-alpha after a 3 day culture period. IL-6 reached
a peak level at 2 days, and maintained these elevated levels longer than
the other cytokines examined. These results suggest that PBMCs from psoriatic
patients are more activated than normal controls after stimulation with
SEB.
CONCLUSION Our
data raise the possibility that longitudinal hyper-reactivity of PBMCs against
staphylococcal superantigens may reflect activated circulating monocytes,
as well as T lymphocytes, in psoriatic patients, because the spontaneous
and SEB-stimulated production of IL-6 correlates with the SI period. The
exposure to superantigens may be one of many potential mechanisms of T cell
and monocyte activation in psoriasis, and may be associated with the exacerbation
and long persistence of the inflammatory condition, via secreted
cytokines. Further studies are necessary to clarify the role of superantigens
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