Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model

Abstract

There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-kappa B for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-kappa B activation by blocking the degradation of its inhibitory protein I kappa B. We report that proteasome inhibition by PS-519 reduces superantigen-mediated T cell-activation in vitro and in vivo. Proliferation was inhibited along with the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vitro. Finally, PS-519 proved to be therapeutically effective in a SCID-hu xenogeneic psoriasis transplantation model. We conclude that inhibition of the proteasome, e.g., by PS-519, is a promising means to treat T cell-mediated disorders such as psoriasis.

Figure 1

The PHA/IL-2/TGF-β–induced NF-κB DNA complex is suppressed by the proteasome inhibitor PS-519. Human T cells were stimulated with PHA/IL-2/TGF-β for 4 hours. PS-519 (1–10 μg/ml) suppressed NF-κB DNA-binding activity. The NF-κB DNA complex is indicated by a filled arrowhead. The open arrowhead shows the position of the unbound DNA probe. Binding mixture without cell extract was applied on lanes 1 and 10. The antioxidant N-acetylcysteine (NAC; 25 mM), which is known to inhibit IκB kinase, served as control (lane 4). The results are representative of three electrophoretic mobility shift assays from three independent donors.

Figure 2

PS-519 inhibits TSST-1–induced T cell proliferation. PBMCs (2 × 10⁶/ml) obtained from five healthy volunteers were stimulated with TSST-1 (100 ng/ml) in the absence or presence of PS-519 (0.25–2.5 μg/ml) for 4 days and thereafter pulsed with ³H-thymidine. Incorporation of ³H-thymidine into DNA was calculated using a liquid scintillation counter. Stimulation index was calculated by the ratio: decays per minute of experimental group/decays per minute of control group. Open symbols represent resting PBMCs, filled symbols TSST-1–stimulated PBMCs. A significant reduction in proliferation was observed starting at 0.25 μg/ml PS-519 (*P < 0.001). Values represent mean ± SD of five healthy donors.

Figure 3

PS-519 inhibits TSST-1–induced expression of T cell activation molecules. PBMCs were stimulated with TSST-1 (100 ng/ml) in the presence or absence of PS-519 (0.25–2.5 μg/ml). CD69⁺ CD3⁺ (a), CD25⁺ CD3⁺ (b), and HLA-DR⁺ CD3⁺ (c) surface expression was measured at days 1, 3, 5, 7, and 9 (days 7 and 9 not shown) by flow cytometry. Appropriate isotype Ig’s served as controls to set gates for positive and negative staining. For CD69 expression, significant reduction was observed on day 1 starting at 1.0 μg/ml (P < 0.05), and on days 3 and 5 starting at 0.5 μg/ml PS-519 (P < 0.001 and P < 0.05, respectively). For CD25 expression, significant reduction was observed on day 3 starting at 1.0 μg/ml (P < 0.001), and on day 5 starting at 0.25 μg/ml PS-519 (P < 0.001). For HLA-DR expression, significant reduction was observed on day 1 at 2.5 μg/ml (P < 0.05), on day 3 starting at 1.0 μg/ml (P < 0.05), and on day 5 starting at 0.25 μg/ml PS-519 (P < 0.001). Data represent means of five experiments ± SD.

Figure 4

PS-519 inhibits TSST-1–induced expression of T cell adhesion molecules. PBMCs were stimulated with TSST-1 (100 ng/ml) in the presence or absence of PS-519 (0.25–2.5 μg/ml). CLA⁺CD3⁺ (a) and CD15s⁺CD3⁺ (b) surface expression and binding of CD3⁺ cells to E-selectin (c) was measured at days 1, 3, 5, 7, and 9 (days 1 and 9 not shown) by flow cytometry. Appropriate isotype Ig’s served as controls to set gates for positive and negative staining. Staining in the absence of fusion proteins and in the presence of anti-CD3–FITC together with secondary anti-human IgG-phycoerythrin demonstrated absence of unspecific binding reactivity. For CLA expression, significant reduction was observed on day 3 at 2.5 μg/ml (P < 0.05), and on days 5 and 7 starting at 0.25 μg/ml PS-519 (P < 0.001). For CD15s expression, significant reduction was observed on days 5 and 7 starting at 0.25 μg/ml PS-519 (P < 0.001). For E-selectin binding, significant reduction was observed on day 5 starting at 0.25 μg/ml (P < 0.001), and on day 7 starting at 0.5 μg/ml PS-519 (P < 0.05). Data represent means of five independent experiments ± SD.

Figure 5

PS-519 suppresses hallmarks of psoriasis in a xenogeneic transplantation model. Grafts from lesional psoriatic skin were transplanted onto SCID mice as outlined in Methods. After 2 weeks, mice were treated with PS-519 (1 mg/kg body weight), dexamethasone (0.2 mg/kg body weight), or vehicle for 4 weeks. Subsequently, epidermal thickness (a), proliferation of basal keratinocytes measured by Ki-67 reactivity (b), and leukocytic infiltration (c) were determined by a blinded investigator. All parameters showed a marked reduction (*P < 0.05, **P < 0.001). Data represent means of four independent experiments ± SD.

Figure 6

PS-519 suppresses hallmarks of psoriasis in a xenogeneic transplantation model. Grafted skin in PS-519–treated mice (1 mg/kg body weight) showed normalization of epidermal architecture, loss of papillomatosis, and marked reduction of acanthosis (c, hematoxylin-and-eosin stain) as compared with vehicle-treated mice (a, hematoxylin-and-eosin stain). In PS-519–treated (d, Ki-67 stain) as compared with vehicle-treated mice (b, Ki-67 stain), proliferation of basal keratinocytes was markedly reduced. Treatment with dexamethasone (0.2 mg/kg body weight; e and f) was as effective as PS-519 treatment. The sections show one representative of four experiments. ×200.

Figure 7

20S proteasome activity is reduced in PS-519 as compared with vehicle treated mice. Peripheral blood from vehicle- and PS-519–treated mice was drawn 2 hours after the final injection at days 4, 8, 14, and 28. Thereafter, 20S proteasome activity was determined as described above. PS-519–treated animals showed an 85.7% ± 8.6% (mean ± SD) inhibition of the 20S proteasome activity as compared with vehicle-treated mice at day 28 (**P < 0.0001). Already after 4 days, 81.7% ± 1.6% inhibition as compared with controls was achieved (*P < 0.01). The 20S proteasome activity in vehicle-treated mice remained unchanged when measured at 4, 8, and 14 days as compared with day 28 (data not shown). The 20S proteasome activity is given in pmol/s/mg protein. Data represent mean ± SD; n = 6, days 4–14; n = 8, day 28.