JEADV
DOI: 10.1111/jdv.12929
SHORT REPORT
Calcitriol exerts anti-inflammatory effects in keratinocytes treated with autoantibodies from a patient with bullous pemphigoid €ner,1 C. Tukaj,2 D. Zillikens,1 M. Kasperkiewicz1,* S. Tukaj,1 D. Gru €beck, Lu €beck, Germany Department of Dermatology, University of Lu sk, Gdan sk, Poland Department of Electron Microscopy, Medical University of Gdan *Correspondence: M. Kasperkiewicz. E-mail: [email protected] 1 2
Abstract Background The hormonally active vitamin D metabolite calcitriol and its analogues exert potent effects on cellular differentiation and regulation of immune responses. Although topical vitamin D analogues are widely used for treatment of psoriasis and vitamin D has been increasingly implicated in prevention and protection from several autoimmune diseases, experimental and clinical data in autoimmune bullous diseases are generally lacking. Objective Here, we investigated the effects of calcitriol on keratinocytes treated by bullous pemphigoid (BP) autoantibodies. Methods Human keratinocyte (HaCaT) cells were treated with purified human BP or normal IgG from one BP patient and healthy subject, respectively, in the absence or presence of calcitriol and effects on (i) cell viability, (ii) IL-6 and IL-8 secretion, (iii) STAT3 and NFjB activation, (iv) heat shock protein 70 (Hsp70) level, and (v) vitamin D receptor (VDR) expression were studied. Results We found that BP IgG-induced IL-6 and IL-8 release from HaCaT cells was reduced in the presence of nontoxic doses of calcitriol. Additionally, calcitriol blunted BP IgG-mediated STAT3 phosphorylation and NFjB activity, whereas Hsp70 and VDR expression were not affected. Conclusion Although the results of this study are based on autoantibodies prepared from a single patient, they show that calcitriol protects from BP IgG-induced inflammatory processes in vitro, thus favouring its potential inclusion into the therapeutic repertoire of BP. Received: 8 May 2014; Accepted: 19 November 2014
Conflicts of interest None declared.
Funding sources This work was supported by Deutsche Forschungsgemeinschaft (DFG) Excellence Cluster “Inflammation at Interfaces” €beck (E22-2013), Focus Program “Autoimmunity” (EXC 306/2), DFG KA 3438/1-1, Medical Faculty of the University of Lu sk (ST-59). €beck, and the Medical University of Gdan at the University of Lu
Introduction The autoimmune blistering disease bullous pemphigoid (BP) is characterized by presence of autoantibodies against the hemidesmosomal proteins BP180 and BP230. The extracellular non-collagenous 16A domain (NC16A) of BP180 is considered to harbour the major pathogenically relevant epitopes recognized by autoantibodies in the majority of BP cases. BP patients are usually treated with topical (class IV) and/or systemic corticosteroids, frequently in combination with immunomodulatory agents like dapsone and tetracyclines or immunosuppressants such as methotrexate and azathioprine.1
JEADV 2015
Vitamin D, in addition to its important role in bone metabolism, can initiate gene transcription and exert immunomodulatory effects after being metabolized into the biologically active form, calcitriol, and bound to the vitamin D receptor (VDR) on keratinocytes and multiple immune cell lineages.2 In dermatology, topical application of vitamin D analogues is a well established safe and effective treatment option for psoriasis. In addition, patients with some other skin diseases like vitiligo and different keratinizing disorders have also been successfully treated using such therapy.3 In contrast, however, experimental and clinical data on possible
© 2015 European Academy of Dermatology and Venereology
Tukaj et al.
2
anti-inflammatory effects of vitamin D analogues in autoimmune bullous diseases are lacking.
Materials and methods IgG purification
Measurement of VDR expression
Equal amounts of HaCaT lysates were separated by 10% SDSPAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked with 3% milk in PBS for 2 h, followed by incubation with rabbit polyclonal antibodies against VDR (1:200; Santa Cruz Biotechnology, Inc.) and b-actin (1 : 1000; Cell Signaling Technology, Frankfurt, Germany) at room temperature for 2 h. Horseradish peroxidase-conjugated gout antirabbit antibodies (1 : 1000; Dako) were used as secondary antibodies. The bands were visualized using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, United Kingdom).
Total serum IgG from a healthy blood donor and a patient with documented BP (based on typical clinical findings as well as on detection of linear deposits of IgG and C3 at the dermal–epidermal junction and serum IgG autoantibodies against BP180 NC16A by direct immunofluorescence microscopy and enzymelinked immunosorbent assay [ELISA; Euroimmun, L€ ubeck, Germany], respectively) was isolated by Protein G Resin (GenScript Corp., Piscataway, NJ) as described previously.4 The collection of blood samples was approved by the Ethics Committee of the University of L€ ubeck, and informed consent was obtained according to the Declaration of Helsinki.
Data were analysed using Student’s t-test or one-way analysis of variance (ANOVA). A P-value
DOI: 10.1111/jdv.12929
SHORT REPORT
Calcitriol exerts anti-inflammatory effects in keratinocytes treated with autoantibodies from a patient with bullous pemphigoid €ner,1 C. Tukaj,2 D. Zillikens,1 M. Kasperkiewicz1,* S. Tukaj,1 D. Gru €beck, Lu €beck, Germany Department of Dermatology, University of Lu sk, Gdan sk, Poland Department of Electron Microscopy, Medical University of Gdan *Correspondence: M. Kasperkiewicz. E-mail: [email protected] 1 2
Abstract Background The hormonally active vitamin D metabolite calcitriol and its analogues exert potent effects on cellular differentiation and regulation of immune responses. Although topical vitamin D analogues are widely used for treatment of psoriasis and vitamin D has been increasingly implicated in prevention and protection from several autoimmune diseases, experimental and clinical data in autoimmune bullous diseases are generally lacking. Objective Here, we investigated the effects of calcitriol on keratinocytes treated by bullous pemphigoid (BP) autoantibodies. Methods Human keratinocyte (HaCaT) cells were treated with purified human BP or normal IgG from one BP patient and healthy subject, respectively, in the absence or presence of calcitriol and effects on (i) cell viability, (ii) IL-6 and IL-8 secretion, (iii) STAT3 and NFjB activation, (iv) heat shock protein 70 (Hsp70) level, and (v) vitamin D receptor (VDR) expression were studied. Results We found that BP IgG-induced IL-6 and IL-8 release from HaCaT cells was reduced in the presence of nontoxic doses of calcitriol. Additionally, calcitriol blunted BP IgG-mediated STAT3 phosphorylation and NFjB activity, whereas Hsp70 and VDR expression were not affected. Conclusion Although the results of this study are based on autoantibodies prepared from a single patient, they show that calcitriol protects from BP IgG-induced inflammatory processes in vitro, thus favouring its potential inclusion into the therapeutic repertoire of BP. Received: 8 May 2014; Accepted: 19 November 2014
Conflicts of interest None declared.
Funding sources This work was supported by Deutsche Forschungsgemeinschaft (DFG) Excellence Cluster “Inflammation at Interfaces” €beck (E22-2013), Focus Program “Autoimmunity” (EXC 306/2), DFG KA 3438/1-1, Medical Faculty of the University of Lu sk (ST-59). €beck, and the Medical University of Gdan at the University of Lu
Introduction The autoimmune blistering disease bullous pemphigoid (BP) is characterized by presence of autoantibodies against the hemidesmosomal proteins BP180 and BP230. The extracellular non-collagenous 16A domain (NC16A) of BP180 is considered to harbour the major pathogenically relevant epitopes recognized by autoantibodies in the majority of BP cases. BP patients are usually treated with topical (class IV) and/or systemic corticosteroids, frequently in combination with immunomodulatory agents like dapsone and tetracyclines or immunosuppressants such as methotrexate and azathioprine.1
JEADV 2015
Vitamin D, in addition to its important role in bone metabolism, can initiate gene transcription and exert immunomodulatory effects after being metabolized into the biologically active form, calcitriol, and bound to the vitamin D receptor (VDR) on keratinocytes and multiple immune cell lineages.2 In dermatology, topical application of vitamin D analogues is a well established safe and effective treatment option for psoriasis. In addition, patients with some other skin diseases like vitiligo and different keratinizing disorders have also been successfully treated using such therapy.3 In contrast, however, experimental and clinical data on possible
© 2015 European Academy of Dermatology and Venereology
Tukaj et al.
2
anti-inflammatory effects of vitamin D analogues in autoimmune bullous diseases are lacking.
Materials and methods IgG purification
Measurement of VDR expression
Equal amounts of HaCaT lysates were separated by 10% SDSPAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked with 3% milk in PBS for 2 h, followed by incubation with rabbit polyclonal antibodies against VDR (1:200; Santa Cruz Biotechnology, Inc.) and b-actin (1 : 1000; Cell Signaling Technology, Frankfurt, Germany) at room temperature for 2 h. Horseradish peroxidase-conjugated gout antirabbit antibodies (1 : 1000; Dako) were used as secondary antibodies. The bands were visualized using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, United Kingdom).
Total serum IgG from a healthy blood donor and a patient with documented BP (based on typical clinical findings as well as on detection of linear deposits of IgG and C3 at the dermal–epidermal junction and serum IgG autoantibodies against BP180 NC16A by direct immunofluorescence microscopy and enzymelinked immunosorbent assay [ELISA; Euroimmun, L€ ubeck, Germany], respectively) was isolated by Protein G Resin (GenScript Corp., Piscataway, NJ) as described previously.4 The collection of blood samples was approved by the Ethics Committee of the University of L€ ubeck, and informed consent was obtained according to the Declaration of Helsinki.
Data were analysed using Student’s t-test or one-way analysis of variance (ANOVA). A P-value
