oreactive plasma cells by activation of the UPR and of protecting mice from lupuslike disease. We therefore hypothesized that, similar to blocking the disposal of misfolded immunoglobulins by bortezomib, disrupting clientchaperone interactions using Hsp90 inhibitors would result in an inability to handle immunoglobulin production STAT Signaling Pathway and death of not only malignant but also autoreactive plasma cells. In the present study, we show that pharmacologic blockade of Hsp90 by the geldanamycin derivative 17dimethylaminoethylamino 17demethoxygeldanamycin or the nontoxic peptide derivative TCBL145 is associated with amelioration of type VII collagen–induced EBA in mice, and that T cells, rather than plasma cells, represent targets of antiHsp90 treatment in this autoimmune disease.
Methods Mice Six to 8weekold SJL mice were purchased from Charles River Laboratories. The experiments were approved by local authorities of the Animal Care and Use Committee and performed by certified personnel. Production of autoantigen The recombinant fragment of murine type VII collagen was prepared as described previously.2022 Recombinant Rivaroxaban tagged fragments GSTmCVIIC and HismCVIIC were produced using a prokaryotic expression system and purified by glutathione and metallochelate affinity chromatography, respectively.20,22 Induction of EBA and phenotype analysis Experimental EBA was induced in mice by active immunization, as described previously.22 Briefly, mice were injected subcutaneously in the hind footpads with a single injection of 100 L of emulsion containing 60 g of GSTmCVIIC in TiterMax .
Mice were examined every week for their general condition and for cutaneous lesions . Disease severity was calculated as the percentage of the tissues body surface area affected by skin lesions. Treatment of mice We treated mice intraperitoneally with 30 mg kg of 17DMAG or 3 mg kg of TCBL145 . We injected control mice with an equivalent volume of the solvent that is, water or water with 5% ethanol, respectively. For prophylactic treatment, mice received a total of 2 injections with 17DMAG or vehicle 1 day before and 1 day after immunization or with a total of 14 daily injections with TCBL145 or vehicle starting 1 day before immunization, and were followed for 6 weeks.
In a second experimental paradigm, if 2% of the body surface area was affected by EBA lesions, we analyzed the therapeutic effect of 17DMAG compared with vehicle 3 times a week or TCBL145 compared with vehicle once a day, each given over a 6week treatment period. One day after the last injection, mice were killed for clinical and immunologic evaluation. In a third set of experiments, we gave immunized mice 17DMAG or vehicle twice with an interval of 36 hours between injections.We analyzed mice 48 hours after the first injection.To investigate whether Hsp90 inhibitors eliminate not only malignant but also normal plasma cells, B220 CD138 plasma cells from spleen and bone marrow were investigated by flow cytometry 24 hours after the last injection of 17DMAG and TCBL145 given over 6 weeks. Cytofluorimetric quantification revealed no difference in the number of splenic or bone marrow plasma cells in vehicle and 17DMAG–treated mice . B220PNA germinal center B cells in the spleen were also not significantly affected .