CD8+ cell changes in psoriasis associated with roxithromycin-induced clinical improvement

ARTICLE

Psoriasis is an inflammatory skin disease of epidermal keratinocyte hyperproliferation, which is thought to be driven by cytokines and growth factors produced by infiltrating cells. Several studies have suggested that the epidermal hyperproliferation is preceded by infiltration of activated T cells [1-4], which play a critical role in triggering psoriasis [5-8]. Among T cells, CD8⁺ T cells predominate in lesional epidermis of psoriasis [9, 10], suggesting a pathogenetic role for this type of T cell in the development of the skin eruption.

One of the causative or aggravative factors in psoriasis is bacterial infection. Two pathways that bacteria exacerbate psoriasis have been proposed. One is tonsillar or pharyngeal infection with Streptococcus pyogenes (group A-Streptococcus) [11] and possibly Staphylococcus aureus (S. aureus) [12], and the other one is cutaneous colonization of bacteria, in particular S. aureus. In both pathways, superantigenic exotoxins that these bacteria produce stimulate pathogenetic T cells, thereby triggering or aggravating the skin eruption. However, in the former case, superantigens released from focally infecting bacteria seem to activate T cells systemically, while in the latter case, the toxins from colonizing bacteria may stimulate T cells locally in the presence of Langerhans cells (LC) and major histocompatibility complex (MHC) class II-bearing keratinocytes as antigen-presenting cells [13-15]. It is considered more likely that the former systemic pathway operates than the latter.

On analysis of T cell receptor Vbeta (BV) repertoire of T cells concerned with psoriasis, we [16] and the other groups [17, 18] have shown that BV2⁺ and/or BV8⁺ T cells accumulate in skin lesions of guttate and plaque psoriasis. Furthermore, our previous study has demonstrated that BV2- and BV8-bearing CD8⁺ T cell populations are decreased in percentage in peripheral blood mononuclear cells (PBMC) from patients with chronic plaque and guttate psoriasis compared to those from normal subjects, whereas the percentages of CD4⁺ cells possessing these BVs are comparable to those from normal individuals [19]. Since both BV2⁺ and BV8⁺ T cells are reactive with streptococcal superantigens [20, 21], these findings have suggested an antigenic role for the exotoxins in the pathogenesis of psoriasis. Cutaneous lymphocyte-associated antigen (CLA) is a homing receptor involved in selective migration of memory and effector T cells to the skin [22, 23]. Recent reports have indicated that staphylococcal and streptococcal superantigens can induce T cell expression of CLA, by augmenting interleukin (IL)-12 production [24]. This raises the possibility that CD8⁺ T cells bearing BV2 or BV8 become positive for CLA following stimulation with streptococcal superantigen(s) and enter the epidermis to trigger psoriatic lesions.

In addition to their antibacterial action, macrolides have characteristic, immunomodulatory or anti-inflammatory potencies. Roxithromycin (RXM), which belongs to a new generation of macrolide antibiotics, inhibits T cell responses to mitogens and production of cytokines, such as IL-2 and IL-5 [25, 26]. In our previous study, RXM inhibits the ability of LC to present superantigen and hapten, and to produce IL-1beta [27]. This macrolide also suppresses immunologic events in interferon-gamma-treated keratinocytes, including the expression of MHC class II, secretion of IL-1alpha, and superantigen-presenting ability [28]. These findings illustrate the potential therapeutic effectiveness of RXM in T cell-mediated cutaneous diseases via not only its killing action on superantigen-producing bacteria but also its immunomodulatory effect on epidermal LC and keratinocytes.

Considering these immunologic properties of RXM, psoriasis is one of the candidate skin diseases that are successfully treated or well controlled with RXM. In fact, our preliminary study has demonstrated that some of the patients with psoriasis respond well to RXM [29]. In this study, therefore, we administered psoriatic patients with RXM and monitored, before and after the therapy, T cell receptor BV usage and CLA expression by peripheral blood T cells and alteration in their CLA expression promoted by bacterial superantigens or cytokines. Our results demonstrated that after RXM treatment, BV2- and BV8-bearing CD8⁺ T cells were increased in percentage and superantigen-promoted expression of CLA on CD8⁺ cells was decreased with clinical improvement.

Materials and methods

Patients and controls

To select psoriatic patients who respond to RXM, ten patients with chronic plaque psoriasis were advised to take 150 mg RXM (Eisai, Tokyo, Japan) orally twice daily for 1 to 7 weeks. Before starting the administration, blood samples were taken from the patients and subjected to analysis. Six out of the ten patients exhibited a decrease in psoriasis area and severity index (PASI) score (30) and blood samples were again taken and analyzed after cessation of RXM treatment. The remaining four unresponsive or dropped-out patients were then excluded from the study. The six patients (five males and one female) with chronic plaque psoriasis thus enrolled in this study were aged 28-59 years, their baseline PASI score, were 17.3 ± 13.8 (mean ± SD). As contols, we also took blood samples before and after RXM administration from two atopic dermatitis patients (both aged 23 years, both males), two acne patients (aged 19 and 26 years, both females), one patient with pustulosis palmoplantaris (aged 63 years, male) and one patient with pityriasis lichenoides chronica (aged 65 years, male). All psoriatic and control patients had been treated with topical corticosteroids or acne lotion before the study.

Reagents and monoclonal antibodies (mAbs)

Staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin-1 (TSST-1), streptococcal pyrogenic exotoxin (SPE) A and SPEC were obtained from Toxin Technology (Sarasota, FI, USA). Phycoerythrin (PE)-labeled anti-Leu3a (CD4), -Leu2a (CD8), -LeuM3 (CD14), and -Leu16 (CD20), and fluorescein isothiocyanate (FITC)-labeled anti-HLA-DR mAbs were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA, USA). FITC-labeled anti-human CLA mAb was obtained from PharMingen (San Diego, CA, USA), and FITC-labeled anti-T cell-receptor-BV2, -BV3, and -BV8 mAbs were from Immunotech (Marseille, France). Recombinant human IL-2 and IL-12 were purchased from PharMingen.

PBMC isolation and cultures of PBMC

PBMC obtained from patients were isolated from heparinized venous blood by density-gradient sedimentation over a Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and interface cells were collected and washed three times in phosphate-buffered saline (PBS; pH 7.4).

In some experiments, cells (2 x 10⁶/ml) were cultured in 24-well flat-bottom culture plates (Corning Incorporated, Corning, NY, USA) for 72 hrs at 37° C in 5% CO₂ in air. RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 5 x 10^­5M 2-mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin was used as a culture medium (all from Gibco BRL Life Technologies, Grand Island, NY, USA). As stimulants, staphylococcal exotoxins, SEB (final concentration, 1 ng/ml) and TSST-1 (1 ng/ml), streptococcal toxins, SPEA (10 ng/ml) and SPEC (10 ng/ml), and recombinant human IL-2 (20 units/ml) and/or IL-12 (0.2 ng/ml) [24] were added to wells at the beginning of culture.

Flow cytometry

Freshly isolated PBMC were suspended in Hank's balanced salt solution containing 0.1% NaN₃ and 1% heat-inactivated fetal calf serum. Cells (10⁵) were double-stained with PE-labeled anti-CD4, or -CD8 mAbs and FITC-labeled anti-human CLA mAb or anti-T cell-receptor-BV2,
-BV3, or -BV8 mAbs, and with PE-labeled anti-CD14, or -CD20 mAbs and FITC-labeled anti-HLA-DR mAb for 30 min on ice. After three washes, the staining cells were analyzed in a FACSCalibur (Becton Dickinson Immunocytometry Systems). The percentages of positive cells were obtained by counting 10⁴ cells, and mean fluorescence intensity was calculated on a log scale.

PBMC were isolated from the six psoriatic patients, and aliquots of PBMC were cultured for 3 days with the stimulant(s). Both freshly isolated and cultured PBMC were incubated with PE-labeled anti-CD4 or -CD8 and FITC-labeled anti-human CLA mAbs and analyzed by flow cytometry. Augmentation ratio of CLA expression after cultivation with stimulant(s) was calculated using the following formula: % of CLA- and CD4- or CD8-double positive cells in cultured PBMC/% of those cells in freshly isolated PBMC.

Statistical analysis

Wilcoxon signed-ranks test was used to test differences in data of before and after the therapy, and Student's t-test was employed to determine statistical differences between means. p < 0.05 was considered to indicate a significant difference.

Results

Increases in the percentages of CD8⁺ T cells bearing BV2 and BV8 after clinical improvement by RXM

As shown in Figure 1, when administered orally with 150 mg RXM twice daily for 1 to 7 weeks, six out of ten patients with chronic plaque psoriasis exhibited clinical improvement as assessed by PASI score, which was reduced from 17.3 ± 13.8 to 11.5 ± 11.7 (mean ± SD) after RXM administration. Thus, the mean score was reduced to 66.5% of the pretreatment level with statistical significance (Wilcoxon signed-ranks test, p < 0.03). We used blood samples from these RXM-responsive patients in the following study.

We speculated that CD8⁺ T cells bearing BV2 or BV8 enter the epidermis to trigger psoriatic lesions and this migration results in reduction of their numbers in the peripheral blood [19]. BV usage in freshly isolated PBMC from the six patients with psoriasis was examined before and after administration of RXM. As shown in Figure 2, the percentages of CD8⁺ T cells bearing BV2⁺ and BV8⁺ were significantly increased after the therapy, whereas CD8⁺ BV3⁺ cells were not changed. On the other hand, neither CD4⁺BV2⁺ nor CD4⁺BV8⁺ cells were altered in percentage after the therapy. No significant change was found in PBMC from control patients with the other diseases (data not shown). Thus, clinical improvement was associated with increment of percentages of CD8⁺BV2⁺ and CD8⁺BV8⁺ T cells in psoriatic patients.

Superantigen-induced augmentation of CLA expression on CD8⁺ T cells is lowered with clinical improvement
by RXM

The percentages of T cells positive for CD4 or CD8, and CLA were examined before and after RXM treatment in the six patients with psoriasis. The means ± SD of percentage before the therapy were: CD4⁺, 32.7 ± 11.3; CD8⁺, 13.0 ± 6.5; CD4⁺CLA⁺, 5.3 ± 2.8; and CD8⁺CLA⁺, 2.2 ± 1.4; and those after the therapy were: CD4⁺, 29.3 ± 8.1; CD8⁺, 14.8 ± 8.3; CD4⁺CLA⁺, 4.7 ± 1.9; and CD8⁺CLA⁺, 3.1 ± 2.4, indicating no significant change between before and after the therapy. In addition, there was no significant difference in any of these values between patients with psoriasis and those with the other diseases examined (data not shown). However, we cannot necessarily evaluate the effect of the therapy on CLA expression on T cells, by simply enumerating circulating CLA⁺ T cells, because these T cells may emigrate from the blood to lesional skin. Therefore, we compared the ability of T cells to express CLA in stimulation with bacterial superantigens between PBMC taken before and after RXM administration. PBMC were cultured with SEB, TSST-1, SPEA, or SPEC for 3 days and analyzed by flow cytometry. Superantigen augmentations of percentage of CLA⁺ cells were expressed as augmentation ratio as described in Materials and Methods. As shown in Figure 3, when TSST-1, SPEA, and SPEC were used as stimulants, the augmentation ratios of CLA expression on CD8⁺ T cells were significantly reduced after RXM therapy, whereas the augmentation ratios of CD4⁺ T cells were unchanged.

In the parallel experiment, we also examined CLA expression on T cells cultured with cytokines IL-2 and IL-12, instead of bacterial superantigen, since staphylococcal and streptococcal superantigens can stimulate T cells to express CLA by enhancing IL-12 production [24]. As shown in Figure 4, the augmentation ratios of CLA expression on CD8⁺ T cells in PBMC cultured with IL-12 and those with both IL-2 and IL-12 were significantly decreased after RXM treatment to the level comparable to CD4⁺ T cells. When cultured with IL-2, PBMC exhibited no substantial change after the therapy (data not shown). PBMC from control patients with the other diseases did not show such a reduction in the augmented expression of CLA (data not shown). These data suggested that the superantigen-induced augmentation of CLA expression on CD8⁺ T cells is lowered in association with clinical improvement by treatment with RXM.

No change in the percentage or the level of MHC class II expression of superantigen-presenting cells after clinical improvement by RXM

We have previously reported that RXM inhibits MHC class II expression of murine LC [27] and of interferon-gamma-treated human keratinocytes [28]. Therefore, a possibility remained that RXM inhibited T cell responses to superantigens not only directly but also indirectly by blocking class II expression of antigen-presenting cells. HLA-DR expressions of circulating superantigen-presenting cells, monocytes and B cells, were analyzed in PBMC taken from the six psoriatics before and after RXM treatment. There was no significant change in the percentage of CD14⁺ or CD20⁺ cells, or the mean fluorescence intensity of HLA-DR on CD14⁺ or CD20⁺ cells after the therapy compared with those before the therapy (data not shown). Thus, RXM therapy did not change either the number of monocytes or B cells, or the intensity of MHC class II expression, suggesting its direct effect on T cells.

Discussion

Our previous studies have demonstrated that BV2⁺ and BV8⁺ T cells accumulate in skin lesions of plaque psoriasis [16], and that BV2- and BV8-bearing CD8⁺ cells were decreased in percentage in PBMC from chronic plaque and guttate psoriasis [19]. Furthermore, the current study showed that the percentages of CD8⁺BV2⁺ and CD8⁺BV8⁺ T cells are significantly increased after improvement of the disease by RXM treatment compared with those before the therapy. This is in accordance with the previous finding and further suggests that the numbers of these T cell populations are inversely correlated with the disease activity. One can postulate that CD8⁺ T cells bearing these BVs enter the epidermis to trigger psoriatic lesions and this migration results in their numerical reduction in the peripheral blood of active psoriasis. However, there is a considerable debate concerning the number of BV2- and BV8-bearing T cells, as another group found that BV2⁺ cells, including both CD4⁺ and CD8⁺ cells, are increased in number in the CLA-positive peripheral lymphocytes in psoriasis [31].

The migration of leukocytes into tissues is a process mediated by leukocyte-endothelial interactions, in which receptors for adhesion molecules play a crucial role. CLA is expressed by a subset of circulating memory/effector T cells and by the vast majority of skin-infiltrating T cells, and is thought to target skin-associated T cells to inflammatory skin sites by interacting with endothelial cell ligand E-selectin [23]. Throat-infecting or skin-colonizing S. pyogenes and possibly S. aureus exacerbate or trigger psoriasis and their superantigens can induce T cell expression of CLA via stimulation of IL-12 production [24]. Our study suggested that clinical improvement of psoriasis by RXM treatment is associated with inhibited superantigen-induced augmentation of CLA expression on CD8⁺ T cells. This inhibition after the therapy was found when patients' PBMC were cultured with TSST-1, SPEA and SPEC, but not with SEB. A similar inhibited CLA expression was also observed in stimulation with IL-12 or both IL-2 and IL-12. The difference in reactivity to each superantigen may be explainable by the fact that T cells possessing BV2 and BV8, which are responsible for the formation of psoriatic eruption, are reactive with TSST-1, SPEA and SPEC, but not with SEB, while BV3⁺ T cells respond to SEB [20, 21, 32].

We have previously found that RXM inhibits MHC class II expression by LC [27] and interferon-gamma-treated human keratinocytes [28]. In this study, however, the mean fluorescence intensity of HLA-DR on CD14⁺ or CD20⁺ cells was not altered after RXM treatment. In the case of cutaneous bacterial colonization, released superantigens may stimulate infiltrating T cells locally in the skin mileu, where Langerhans cells and class II-bearing keratinocytes are capable of functioning as superantigen-presenting cells [13-15] and we have reported that RXM inhibits superantigen-presenting ability of LC and class II-bearing keratinocytes [27, 28]. On the contrary, the current study suggests that RXM cannot change antigen-presenting cells in the peripheral blood. It is possible that epidermal antigen-presenting cells are different from blood antigen-presenting cells in the susceptibility to RXM.

Our results suggest that RXM exerts a therapeutic effect in the treatment of psoriasis via not only killing bacteria but also changing the ability of psoriasis-pathogenetic CD8⁺ T cells to express CLA following stimulation with bacterial superantigens.

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