Takeko Ishikawa

Human β-defensin-2 enhances IFN-γ and IL-10 production and suppresses IL-17 production in T cells

Psoriasis is an inflammatory dermatosis with enhanced expression of hBD‐2 in keratinocytes and infiltration of cytokine‐producing T cells, which in turn, up‐ or down‐regulate hBD‐2 expression. We determined the serum levels of hBD‐2 and cytokines in psoriasis patients and analyzed the effects of hBD‐2 on cytokine production in human peripheral blood T cells. Serum hBD‐2 levels in patients were higher than those in controls and correlated with PASI, serum IFN‐γ, and IL‐10 levels and correlated inversely with serum IL‐17 levels. IFN‐γ, IL‐17, IL‐22, TNF‐α, IL‐1β, and IL‐6 enhanced, and IL‐10, IL‐4, and IL‐13 suppressed hBD‐2 secretion from keratinocytes. hBD‐2 enhanced secretion and mRNA levels of IFN‐γ, TNF‐α, IL‐10, IL‐1β, IL‐6, and IL‐22 and reduced those of IL‐17 in CD3/28‐stimulated T cells. These effects of hBD‐2 were counteracted by PTX. hBD‐2 induced phosphorylation of JNK, ERK, and Akt in T cells. Inhibitors of these signals attenuated hBD‐2‐induced production of IFN‐γ, TNF‐α, IL‐10, IL‐1β, IL‐6, and IL‐22. hBD‐2 suppressed phosphorylation of STAT3 and enhanced expression of SOCS3 in CD3/28‐stimulated T cells. siRNA against SOCS3 reversed hBD‐2‐induced suppression of IL‐17 production and STAT3 phosphorylation. JNK and MEK inhibitors suppressed hBD‐2‐induced expression of SOCS3. In conclusion, hBD‐2 may bind PTX‐sensitive GPCR(s) on T cells and act as a stimulator by enhancing IFN‐γ, TNF‐α, IL‐1β, IL‐6, and IL‐22 production via JNK, MEK/ERK, and PI3K/Akt and as a regulator by suppressing IL‐17 production via SOCS3 or by stimulating IL‐10 production.

Histamine induces human β-defensin-3 production in human keratinocytes

BACKGROUND The antimicrobial peptide human beta-defensin-3 (hBD-3) is produced by epidermal keratinocytes, and promotes cutaneous antimicrobial defense, inflammation, and wound repair. hBD-3 induces histamine release from mast cells. We previously showed that histamine enhanced transcriptional activity of activator protein-1 (AP-1) in human keratinocytes by inducing the expression of AP-1 component c-Fos via the activation of extracellular signal-regulated kinase (ERK) through H1 receptors. OBJECTIVE To examine in vitro effects of histamine on hBD-3 production in normal human keratinocytes. METHODS The hBD-3 production was examined by enzyme-linked immunosorbent assays and reverse transcription-polymerase chain reaction. The transcriptional activities were analyzed by dual luciferase assays. The phosphorylation of proteins was examined by Western blotting. RESULTS Histamine enhanced hBD-3 secretion and mRNA expression in keratinocytes. The histamine-induced hBD-3 production was suppressed by H1 antagonist pyrilamine and antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) and AP-1 components c-Jun and c-Fos. Histamine enhanced STAT3 transcriptional activity and induced tyrosine and serine phosphorylation of STAT3. The former was suppressed by Janus kinase 2 (JAK2) inhibitor AG490, while the latter was suppressed by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059; both were suppressed by pyrilamine. AG490 and PD98059 suppressed histamine-induced hBD-3 production and STAT3 activity. Histamine induced tyrosine phosphorylation of JAK2, and pyrilamine suppressed the phosphorylation. CONCLUSION It is suggested that histamine induces hBD-3 production in human keratinocytes through H1 receptors by activating STAT3 and AP-1 via JAK2 and MEK/ERK. Histamine may promote cutaneous antimicrobial defense, inflammation, and wound repair through hBD-3.

Increased serum leucine, leucine-37 levels in psoriasis: Positive and negative feedback loops of leucine, leucine-37 and pro- or anti-inflammatory cytokines

Expression of leucine, leucine-37 (LL-37) is enhanced in keratinocytes of skin lesions with psoriasis. We examined serum LL-37 levels in patients with psoriasis vulgaris. Serum LL-37 levels in patients were higher than in normal controls, and were reduced after cyclosporine A treatment. In both groups, LL-37 and interleukin (IL)-17 levels inversely correlated. In patients, LL-37 levels correlated with interferon (IFN)-γ and IL-10 levels. In controls, LL-37 levels inversely correlated with tumor necrosis factor (TNF)-α, IL-6, IL-1β, and IL-22 levels. IFN-γ, IL-17, IL-22, TNF-α, IL-6, and IL-1β enhanced, and IL-10, IL-4, IL-13, and cyclosporine A suppressed, LL-37 secretion from keratinocytes and neutrophils. LL-37 enhanced IFN-γ, IL-4, IL-13, and TNF-α secretion from CD3/CD28-stimulated T cells, suppressed TNF-α, IL-1β, IL-6, and IL-10 secretion from lipopolysaccharide-stimulated monocytes, and IL-17, IL-22, IL-1β, IL-6, and IL-10 secretion from CD3/CD28-stimulated T cells. LL-37 may sustain its production by enhancing IFN-γ or reducing IL-10 production, while suppressing its production by reducing IL-17, IL-22, TNF-α, IL-1β, or IL-6 and enhancing IL-4 or IL-13 production. In patients, systemic LL-37 production is enhanced, and an IFN-γ/LL-37-positive feedback loop may exist. In controls, negative feedback by LL-37 on TNF-α, IL-1β, IL-22, and IL-6 may exist. In both groups, negative feedback by LL-37 on IL-17 may exist. LL-37 may act as an effector and regulator.

Prostaglandin D2 induces the production of human β-defensin-3 in human keratinocytes

The antimicrobial peptide human beta-defensin-3 (hBD-3) is produced by epidermal keratinocytes and protects the skin from infections. This peptide induces the release of a lipid mediator, prostaglandin D(2) from dermal mast cells. Prostaglandin D(2) binds to cell-surface G protein-coupled receptors, D prostanoid receptor, and chemoattractant receptor-homologous molecule expressed on T helper cell type 2 (CRTH2). Both receptors are detected on epidermal keratinocytes. It is reported that prostaglandin D(2) is involved in cutaneous allergy, however, its role in antimicrobial defense is unknown. We examined the in vitro effects of prostaglandin D(2) on hBD-3 production in normal human keratinocytes. Prostaglandin D(2) enhanced hBD-3 secretion and mRNA expression in human keratinocytes. Prostaglandin D(2)-induced hBD-3 production was suppressed by the CRTH2 antagonist ramatroban and by antisense oligonucleotides against c-Jun and c-Fos, components of a transcription factor, activator protein-1 (AP-1). Prostaglandin D(2) enhanced the transcriptional activity and DNA binding of AP-1, expression, phosphorylation, and DNA binding of c-Fos proteins in keratinocytes. Prostaglandin D(2)-induced hBD-3 production, AP-1 activity, and c-Fos expression and phosphorylation were suppressed by U0126, PP2, and pertussis toxin, which are inhibitors of mitogen-activated protein kinase kinase (MEK), src, and G(i) proteins, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK), downstream kinase of MEK, was induced by prostaglandin D(2), and suppressed by ramatroban, pertussis toxin, PP2, and U0126. These results suggest that prostaglandin D(2) induces hBD-3 production in human keratinocytes by activating AP-1 through the expression and phosphorylation of c-Fos via the CRTH2/G(i)/src/MEK/ERK pathway. Prostaglandin D(2) may promote cutaneous antimicrobial activity via hBD-3.

The antimycotic drugs itraconazole and terbinafine hydrochloride induce the production of human β-defensin-3 in human keratinocytes.

The antimicrobial peptide human β-defensin-3 (hBD-3) is produced by epidermal keratinocytes, and exhibits broad killing activity against bacteria or fungi. Prostaglandin D(2) enhances hBD-3 production in human keratinocytes by stimulating a transcription factor, activator protein-1 via chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2). Prostaglandin H(2), a precursor of prostaglandin D(2) can be converted to thromboxane A(2). Certain antimycotic drugs act on keratinocytes and modulate their production of chemokines. In this in vitro study, we examined the effects of antimycotics on hBD-3 production in human keratinocytes. Antimycotics itraconazole and terbinafine hydrochloride increased hBD-3 secretion and mRNA levels in parallel to the enhanced activity of activator protein-1, expression and phosphorylation of activator protein-1 component, c-Fos, but fluconazole was ineffective. These effects were abrogated by CRTH2 antagonist. Itraconazole and terbinafine hydrochloride increased prostaglandin D(2) release from keratinocytes and reduced the release of thromboxane B(2), a thromboxane A(2) metabolite. The conditioned medium from itraconazole or terbinafine hydrochloride-treated keratinocytes inhibited the growth of Candida albicans dependently on hBD-3. These results suggest that itraconazole and terbinafine hydrochloride may enhance c-Fos expression and phosphorylation, activator protein-1 activity and hBD-3 production by increasing prostaglandin D(2) release from keratinocytes. These antimycotic drugs may suppress thromboxane A(2) synthesis and redirect the conversion of prostaglandin H(2) towards prostaglandin D(2). The induction of hBD-3 in keratinocytes is another possible mechanism for the antimycrobial effects of these drugs, which may augment the cutaneous defense activity against infection.

Decrease in eosinophils infiltrating into the skin of patients with dipeptidyl peptidase-4 inhibitor-related bullous pemphigoid

Bullous pemphigoid (BP) is an acquired autoimmune blistering disease in which autoantibodies against epitopes in the basement membrane zone of the skin such as BP180 or BP230 are produced. Dipeptidyl peptidase (DPP)‐4 inhibitors have become commonly used to treat diabetes. As DPP‐4 inhibitors are more commonly prescribed for diabetes, BP related to DPP‐4 inhibitors has been reported and has attracted attention. Therefore, we retrospectively investigated patients who were diagnosed with BP in order to examine characteristics of DPP‐4 inhibitor‐related BP (nine patients; median age, 85 years) in comparison with non‐DPP‐4 inhibitor‐related BP (21; median age, 85 years). There was no significant difference in Bullous Pemphigoid Disease Area Index between DPP‐4 inhibitor‐related BP patients and non‐DPP‐4 inhibitor‐related BP patients, except for erosions/blisters score in mucosa. Laboratory tests revealed no significant differences between DPP‐4 inhibitor‐related BP patients and non‐DPP‐4 inhibitor‐related BP patients in total white blood cell count, eosinophil count, neutrophil count and the titer of anti‐BP180 antibody. The number of eosinophils infiltrating into the skin was significantly lower in patients with DPP4 inhibitor‐related BP than in patients with non‐DPP4 inhibitor‐related BP. Our results showed that DPP‐4 inhibitor‐related BP has some distinct pathological characteristics from BP not associated with DPP‐4 inhibitor.

Open-label pilot study to evaluate the effectiveness of topical bimatoprost on rhododendrol-induced refractory leukoderma

Rhododendrol (RD), 4‐(4‐hydroxyphenyl)‐2‐butanol, inhibits melanin synthesis and had been used in skin‐whitening cosmetic products until 2013. However, some individuals developed leukoderma on the skin where RD had been applied and have suffered from refractory depigmentation even after discontinuing RD application. Bimatoprost is a prostaglandin F2α analog and is often used for eyelash growth for cosmetic reasons as well as in the treatment of glaucoma. It was reported that bimatoprost induced skin pigmentation in addition to iris pigmentation as adverse effects. Therefore, we conducted an open‐label single‐center pilot study to evaluate the effectiveness of bimatoprost on refractory RD‐induced leukoderma. Eleven Japanese female patients with skin type III who developed leukoderma on the exact or slightly extended area of skin where RD had been applied and gained a halt of enlargement of leukoderma or repigmentation on a part of the affected skin after discontinuation of RD were enrolled. Bimatoprost 0.03% solution was applied on the leukoderma once daily for 3 months, and then the frequency of application was increased to twice daily for the subsequent 3 months. Ten patients completed the 6‐month course of bimatoprost application. In four patients, bimatoprost application brought slight improvement in RD‐induced refractory leukoderma by dermatologists’ evaluation. Because the number of enrolled patients was limited, further larger studies are necessary to better assess the effectiveness of bimatoprost in inducing repigmentation in patients with RD‐induced refractory leukoderma.

Cutaneous sarcoidosis in a patient with rheumatoid arthritis receiving tocilizumab

Dear Editor, Tocilizumab, an anti-interleukin (IL)-6 receptor antibody, is effective in treating rheumatoid arthritis (RA). Serious adverse events of tocilizumab are mainly infections, and cutaneous adverse effects have rarely been reported. We herein report a patient with RA receiving tocilizumab who developed cutaneous sarcoidosis. A 65-year-old Japanese woman with RA had received treatment with corticosteroids, bucillamine, methotrexate and salazosulfapyridine for over 20 years. Despite such systemic therapies, her condition gradually deteriorated. Then, she began to be treated with tocilizumab, which induced remission. However, 14 months after the first administration of tocilizumab, reddish nodules and erythemas appeared on her knees and left forearm (Fig. 1a,b). Laboratory tests demonstrated that the serum angiotensin-converting enzyme level and the serum calcium level were within normal limits. Rheumatoid factor was positive. Chest radiograph revealed the absence of bilateral hilar lymphadenopathy. An eye examination performed by an ophthalmologist did not show any indication of sarcoid uveitis. Electrocardiograms and echocardiography showed no abnormalities. A cutaneous biopsy from a reddish papule on her left forearm was performed. Histopathology revealed non-caseating epithelioid granulomas with Langhans giant cells in the upper dermis surrounded by a paucity of lymphocytes (Fig. 1c). Polarizing microscopy revealed refracted objects in the giant cells (Fig. 1d). Based on these findings, she was diagnosed with cutaneous sarcoidosis. Because tocilizumab was effective for RA, she continued to receive tocilizumab even after being diagnosed with sarcoidosis. As for cutaneous sarcoidosis, a topical steroid was administrated and almost all skin lesions disappeared. Regarding cutaneous adverse events during tocilizumab treatment, 12 cases have been previously reported, as shown in Figure 1(e). Sarcoidosis is characterized by the formation of granulomas involving various organs. Although the pathogenesis of sarcoidosis is poorly understood, it is thought to be an exaggerated immune response to killed or partly degraded bacteria such as mycobacteria or propionibacteria. Several papers reported increased IL-6 and tumor necrosis factor (TNF)-a in bronchoalveolar lavage fluid of patients with sarcoidosis. Although these evidences demonstrate that IL-6 is important for maintaining the formation of granuloma, Yimin and Kohanawa reported that in granuloma formation and the inflammatory response to Rhodococcus aurantiacus infection in mice, TNF-a was essential and anti-IL-6 treatment increased TNF-a production in vitro. They also demonstrated upregulation of granulomatous inflammation in IL-6-deficient mice infected with R. aurantiacus. These data suggest that in our case inhibition of the IL-6 pathway by tocilizumab might have enhanced TNFa production at least locally and contributed to initial granuloma formation of localized cutaneous sarcoidosis in addition to unknown triggers. Furthermore, sarcoidosis was successfully treated with only a topical corticosteroid in our case, which could suppress enhanced TNF-a production. Additionally, because IL-6 is essential for maintenance of sarcoidosis, blockage of the IL-6 pathway by tocilizumab might have hindered the maintenance of sarcoidosis in this case. We herein reported a patient who developed sarcoidosis during tocilizumab treatment for RA. Sarcoidosis is a possible adverse effect of tocilizumab, although there is a possibility that it might have occurred coincidentally. Further accumulation of cases is required to properly assess the adverse effects of tocilizumab.