Overexpression of D-dopachrome tautomerase increases ultraviolet B irradiation-induced skin tumorigenesis in mice.

Ultraviolet irradiation (UV) exposure is the leading factor underlying the development of skin malignancies. D-dopachrome tautomerase (D-DT), a functional homolog of macrophage migration inhibitory factor (MIF), has functional similarities to MIF. However, its role, unlike the role of MIF in photocarcinogenesis, is unknown. We therefore explored the role of D-DT in photocarcinogenesis by developing D-DT transgenic (D-DT Tg) mice and provided a research model for future studies targeting D-DT. Chronic UVB exposure accelerated tumor development in D-DT Tg mice compared with wild-type (WT) mice, with a higher incidence of tumors observed in D-DT Tg mice than in WT mice. In D-DT Tg irradiated mouse keratinocytes, the p53, PUMA, and Bax expression was lower than that in WT mice. These results indicate that D-DT Tg overexpression confers prevention against UVB-induced apoptosis in keratinocytes. Taken together, these findings support D-DT as a functionally important cytokine in photocarcinogenesis and potential therapeutic target for the prevention of photocarcinogenesis.

Berberine induces anti-atopic dermatitis effects through the downregulation of cutaneous EIF3F and MALT1 in NC/Nga mice with atopy-like dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with severe pruritus. Berberine, a naturally occurring isoquinoline alkaloid, has anti-inflammatory effects. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In this study, NC/Nga mice with atopy-like dermatitis (dermatitis mice), fibroblast and mast cells were used. In dermatitis mice, intermittent oral administrations of berberine 3 times a week for 12 days inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the expression of cutaneous eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also attenuated IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip® microarray showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The siRNAs for them inhibited the expression of MIF and IL-4 in antigen-stimulated mast cells. These results suggest that berberine improves AD-like symptoms through the inhibition of the eotaxin and pro-inflammatory cytokine expression and the related inflammatory cell recruitment. It is also suggested that the downregulation of EIF3F and MALT1 by berberine is involved in suppressing the cytokine expression. Taken together, berberine or berberine-containing crude drugs are expected to contribute to the improvement of AD symptoms.

Trichohyalin-like 1 protein plays a crucial role in proliferation and anti-apoptosis of normal human keratinocytes and squamous cell carcinoma cells.

Epidermal differentiation is a complex process that requires the regulated and sequential expression of various genes. Most fused-type S100 proteins are expressed in the granular layer and it is hypothesized that these proteins may be associated with cornification and barrier formation. We previously identified a member of the fused-type S100 proteins, Trichohyalin-like 1 (TCHHL1) protein. TCHHL1 is distributed in the basal layer of the normal epidermis. Furthermore, the expression is markedly increased in cancerous/non-cancerous skin samples with the hyperproliferation of keratinocytes. We herein examined the role of TCHHL1 in normal human keratinocytes (NHKs) and squamous cell carcinoma (SCC). The knockdown of TCHHL1 by transfection with TCHHL1 siRNA significantly inhibited proliferation and induced the early apoptosis of NHKs. In TCHHL1-knockdown NHKs, the level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was markedly decreased. In addition, the slight inhibition of v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation and upregulation of forkhead box-containing protein O1(FOXO1), B-cell lymphoma2 (BCL2) and Bcl2-like protein 11 (BCL2L11) was observed. Skin-equivalent models built by TCHHL1-knockdown NHKs showed a markedly hypoplastic epidermis. These findings highlight that TCHHL1 plays an important role in homeostasis of the normal epidermis. TCHHL1 was expressed in the growing cells of cutaneous SCC; therefore, we next examined an association with the cell growth in HSC-1 cells (a human SCC line). In HSC-1 cells, the knockdown of TCHHL1 also suppressed cell proliferation and induced apoptosis. These cells showed an inhibition of phosphorylation of ERK1/2, AKT and signal transducers and activator of transcription 3, and the significant upregulation of FOXO1, BCL2, and BCL2L11. Accordingly, TCHHL1 is associated with survival of cutaneous SCC. In addition, we hypothesize that TCHHL1 may be a novel therapeutic target in cutaneous SCC.

The regulation of protein kinase casein kinase II by apigenin is involved in the inhibition of ultraviolet B-induced macrophage migration inhibitory factor-mediated hyperpigmentation.

Ultraviolet (UV) radiation elicits melanogenesis and pigmentation in the skin. Apigenin (4',5,7-trihydroxyflavone [AGN]) is a plant flavone contained in various herbs, fruits, and vegetables. We herein investigated antimelanogenic properties of AGN and the molecular mechanisms of the action of AGN. In UVB-treated mice, AGN inhibited cutaneous hyperpigmentation and macrophage migration inhibitory factor (MIF) expression as a melanogenesis-related key factor. In mouse keratinocytes, AGN inhibited the expression of MIF and also the related factors (e.g., stem cell factor and proteinase-activated receptor 2) induced by MIF. In addition to ellagic acid as a casein kinase II (CK2) inhibitor, AGN suppressed CK2 enzymatic activity and UVB-induced CK2 expression and subsequent phosphorylation of IκB and MIF expression. These results suggest that AGN inhibits UVB-induced hyperpigmentation through the regulation of CK2-mediated MIF expression in keratinocytes.

Mechanisms of itching in mycosis fungoides: grade of itching correlates with eosinophil infiltration and kallikrein 5 expression.

BACKGROUND: Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a major symptom for patients with CTCL, however, itching associated with MF is not relieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen. Therefore, the underlying mechanisms of itching in MF patients remain unclear. OBJECTIVES: To investigate the clinical and histopathological features associated with MF-related itching. MATERIALS AND METHODS: Skin sections from MF patients and healthy subjects were used for pathophysiological analysis and evaluation of protease activity. These results were compared with the degree of itching. RESULTS: Of the MF patients, 40% did not report itching and 60% reported itching (moderate itching: 40%; strong itching: 20%). The number of eosinophils, but not mast cells, that infiltrated into skin was increased in the group with strong itching. In the skin of patients, both serine protease activity and immunoreactivity to kallikrein 5 (KLK5), a known itch mediator, increased relative to the grade of itching. CONCLUSION: These results suggest that KLK5 and eosinophil infiltration may be involved in itching in patients with MF.

Involvement of α-Melanocyte-Stimulating Hormone-Thromboxane A2 System on Itching in Atopic Dermatitis.

α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptide hormone involved in cutaneous pigmentation in atopic dermatitis (AD) with severe itching. α-MSH elicits itch-related responses in mice. We, therefore, investigated whether α-MSH was involved in itching in AD. In the skin of AD patients and mice with atopy-like dermatitis, α-MSH and the prohormone convertase 2, which is the key processing enzyme for the production of α-MSH, were distributed mainly in keratinocytes. In the skin of mice with dermatitis, melanocortin receptors (MC1R and MC5R) were expressed at the mRNA level and were distributed in the dermis. In the dorsal root ganglion of mice with dermatitis, mRNAs encoding MC1R, MC3R, and MC5R were also expressed. MC1R antagonist agouti-signaling protein inhibited spontaneous scratching in mice with dermatitis. In healthy mice, intradermal α-MSH elicited itch-associated responses, which were inhibited by thromboxane (TX) A2 receptor antagonist ONO-3708. In mouse keratinocytes, α-MSH increased the production of TXA2, which was inhibited by adenylyl cyclase inhibitor SQ-22536 and Ca2+ chelator EGTA. In mouse keratinocytes treated with siRNA for MC1R and/or MC5R, α-MSH-induced TXA2 production was decreased. α-MSH increased intracellular Ca2+ ion concentration in dorsal root ganglion neurons and keratinocytes. These results suggest that α-MSH is involved in itching during AD and may elicit itching through the direct action of primary afferents and TXA2 production by keratinocytes.

Inflammatory cytokine-mediated induction of serine racemase in atopic dermatitis.

Serine racemase (SR) is an enzyme that catalyses the synthesis of d-serine, an endogenous coagonist for N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild-type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune-reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d-serine in stratum corneum in AD patients and healthy controls using a tape-stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d-serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor -α or macrophage migration inhibitory factor. Taken together, these findings suggest that d-serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.

Role of macrophage migration inhibitory factor in heat-induced apoptosis in keratinocytes.

In human skin, keratinocytes are constantly challenged by adverse influences, such as hot and cold temperatures; however, the effects of heat on apoptosis induction in keratinocytes are not well understood. Macrophage migration inhibitory factor (MIF) is a potent cytokine that overcomes p53 function by suppressing its transcriptional activity. Here, we evaluated the effects of MIF on hyperthermia (HT)-induced apoptosis in MIF-deficient [knockout (KO)] and MIF-transgenic (Tg) mouse keratinocytes. Cells were exposed to HT at 44°C, and increased apoptosis was observed in MIF-KO and wild-type (WT) cells compared with MIF-Tg cells. To determine the mechanism, MIF-mediated changes in the cellular p53 level and its effects on p53-dependent death signaling (Bax and p21) and JNK signaling (p-JNK, JNK, p-Bad, and Bad) were investigated. MIF-Tg cells exhibited substantially decreased levels of p53 after HT treatment compared with WT and MIF-KO cells. In addition, HT treatment caused decreased expression of p-JNK and p-Bad in MIF-Tg cells; however, no such changes were observed in MIF-KO and WT cells. These results showed that the activation of JNK (p-JNK and p-Bad) and p53 may be involved in HT-induced apoptosis in keratinocytes and that enhanced endogenous MIF expression suppressed apoptosis.-Yoshihisa, Y., Rehman, M. U., Kondo, T., Shimizu, T. Role of macrophage migration inhibitory factor in heat-induced apoptosis in keratinocytes.

Efficacy of Astaxanthin for the Treatment of Atopic Dermatitis in a Murine Model.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease associated with various factors, including immunological abnormalities and exposure to allergens. Astaxanthin (AST) is a xanthophyll carotenoid that has recently been demonstrated to have anti-inflammatory effects and to regulate the expression of inflammatory cytokines. Thus, we investigated whether AST could improve the dermatitis and pruritus in a murine model of AD using NC/Nga mice. In addition to a behavioral evaluation, the effects of AST on the AD were determined by the clinical skin severity score, serum IgE level, histological analyses of skin, and by reverse transcription-PCR and Western blotting analyses for the expression of inflammation-related factors. AST (100 mg/kg) or vehicle (olive oil) was orally administered once day and three times a week for 26 days. When compared with vehicle-treated group, the administration of AST significantly reduced the clinical skin severity score. In addition, the spontaneous scratching in AD model mice was reduced by AST administration. Moreover, the serum IgE level was markedly decreased by the oral administration of AST compared to that in vehicle-treated mice. The number of eosinophils, total and degranulated mast cells all significantly decreased in the skin of AST-treated mice compared with vehicle-treated mice. The mRNA and protein levels of eotaxin, MIF, IL-4, IL-5 and L-histidine decarboxylase were significantly decreased in the skin of AST-treated mice compared with vehicle-treated mice. These results suggest that AST improves the dermatitis and pruritus in AD via the regulation of the inflammatory effects and the expression of inflammatory cytokines.

Histamine released from epidermal keratinocytes plays a role in α-melanocyte-stimulating hormone-induced itching in mice.

Sunburn, wound repair, and chronic renal failure with hemodialysis are usually accompanied by both pigmentation and itching. Proopiomelanocortin-derived α-melanocyte-stimulating hormone (α-MSH) is produced in response to external stimuli, such as UV irradiation, and is involved in cutaneous pigmentation. However, it is unclear whether α-MSH is also involved in the itching. We therefore investigated whether α-MSH elicited itch-related responses in mice. We found that an intradermal injection of α-MSH induced hind-paw scratching, an itch-related response, in mice. The α-MSH-induced scratching was inhibited by the µ-opioid receptor antagonist naltrexone and the H1 histamine receptor antagonist terfenadine. In mast cell-deficient mice, α-MSH also elicited scratching, which was inhibited by terfenadine. The immunoreactivity for l-histidine decarboxylase, a key enzyme required for the production of histamine, histamine, and the melanocortin 1 and 5 receptors were shown in not only mast cells but also keratinocytes in murine skin. In addition to the expression of l-histidine decarboxylase and melanocortin 1 and 5 receptors, the mouse keratinocyte cell lines (Pam212) also showed immunoreactivity for l-histidine decarboxylase, histamine, and melanocortin 1 and 5 receptors. The application of α-MSH induced the release of histamine from Pam212 cells. These findings indicate that α-MSH may play an important role in the itching associated with pigmented cutaneous lesions and that the histamine released from keratinocytes is involved in this α-MSH-induced itching.

In vitro effects of zinc on the cytokine production from peripheral blood mononuclear cells in patients with zinc allergy.

Metals, such as nickel, cobalt, chromium and zinc, are ubiquitous in the environment. Systemic reactions, including hand dermatitis and generalized eczematous reactions, can be caused by the dietary ingestion of metals. In this study, we aimed to determine whether the cytokine production from peripheral blood mononuclear cells (PBMCs) obtained from zinc allergy patients can be used as a sensitive marker to investigate zinc-allergic contact dermatitis. The diagnosis of sensitivity to metal was made based on the results of a metal patch test. The PBMCs were stimulated with various concentrations (5-100 µM) of zinc sulfate (ZnSO4) for 24 h. The culture supernatants were collected and analyzed using ELISA for measurement of the cytokine production. The levels of IFN-γ, TNF-α, IL-1ß, IL-5, IL-13 and MIF were significantly higher in the zinc-allergic patients (n = 5) than in the healthy controls (n = 5) at 100 µM of ZnSO4 stimulation. Although, patch testing is considered as standard test to diagnose metal allergy but false-positive and -negative reactions may limit its use in conditions of existing dermatitis. Therefore, this study suggest that in support of patch testing the determination of cytokine production using PBMCs cultures would be helpful for making an early diagnosis of such conditions.

Role of macrophage migration inhibitory factor (MIF) in pollen-induced allergic conjunctivitis and pollen dermatitis in mice.

Pollen is a clinically important airborne allergen and one of the major causes of allergic conjunctivitis. A subpopulation of patients with atopic dermatitis (AD) are also known to have exacerbated skin eruptions on the face, especially around the eyelids, after contact with pollen. This pollen-induced skin reaction is now known as pollen dermatitis. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in allergic inflammation. Recent findings suggest that MIF is involved in several allergic disorders, including AD. In this study, MIF knockout (KO), MIF transgenic (Tg) and WT littermate mice were immunized with ragweed (RW) pollen or Japanese cedar (JC) pollen and challenged via eye drops. We observed that the numbers of conjunctiva- and eyelid-infiltrating eosinophils were significantly increased in RW and JC pollen-sensitized MIF Tg compared with WT mice or MIF KO mice. The mRNA expression levels of eotaxin, interleukin (IL)-5 and IL-13 were increased in pollen-sensitized eyelid skin sites of MIF Tg mice. An in vitro analysis revealed that high eotaxin expression was induced in dermal fibroblasts by MIF combined with stimulation of IL-4 or IL-13. This eotaxin expression was inhibited by the treatment with CD74 siRNA in fibroblasts. These findings indicate that MIF can induce eosinophil accumulation in the conjunctiva and eyelid dermis exposed to pollen. Therefore, targeted inhibition of MIF might result as a new option to control pollen-induced allergic conjunctivitis and pollen dermatitis.

Spiruchostatin A and B, novel histone deacetylase inhibitors, induce apoptosis through reactive oxygen species-mitochondria pathway in human lymphoma U937 cells.

Spiruchostatin A (SP-A) and spiruchostatin B (SP-B) are the potent histone deacetylase inhibitors (HDACi), that has the potential for chemotherapy of leukemia but the exact mechanism of these compounds remains unclear. In the present study, the role of reactive oxygen species (ROS) production and the mechanism involved in the apoptosis was investigated in human lymphoma U937 cell. When the U937 cells were treated with SP-A and SP-B for 24h at different concentrations, evidence of apoptotic features, including increase in DNA fragmentation and changes in nuclear morphology, were obtained. SP-B showed maximum potency to induce apoptosis, while SP-A was less potent. Apoptosis was also determined by increase in the fraction of sub-G1 cells and Annexin V-FITC staining cells. SP-A and SP-B induced apoptosis was accompanied by significant increase in the formation of intracellular reactive oxygen species (ROS). Pre-treatment with N-acetyl-l-cysteine (NAC), significantly inhibited the SP-A and SP-B mediated apoptosis, suggesting a vital role of ROS involved in the lethality of both agents. Moreover, SP-A and SP-B treatment resulted in the loss of mitochondrial membrane potential (MMP), and Fas, caspase-8 and caspase-3 activation. In addition Bid activation and the release of cytochrome-c to the cytosol was also observed. In this study, we suggest that a marked induction of intracellular ROS mediated mitochondrial pathway and the Fas plays a role in the SP-A and SP-B induced apoptosis. Taken together, our data provides further insights of the mechanism of action of SP-A and SP-B and their potential application as novel chemotherapeutic agents.

Macrophage migration inhibitory factor (MIF) in the stratum corneum: a marker of the local severity of atopic dermatitis.

Different biomarkers are used to evaluate the severity of atopic dermatitis (AD); however, it remains difficult to determine the severity of localized skin lesions. MIF plays an essential role in the pathophysiology of skin inflammation. To establish whether the MIF level in the stratum corneum (SC) serves as a marker of the severity of AD lesions, we examined the SC MIF (scMIF) levels in AD patients. The SC of the cheek, neck and upper arm skin was collected using tape stripping, and the scMIF levels were measured. Consequently, the scMIF levels were found to be significantly higher in the involved skin lesions than the uninvolved areas within the same patient. Moreover, the scMIF levels were significantly correlated with the severity of local skin lesions. These findings suggest that the scMIF level can be used as an effective marker for evaluating the local severity of AD.

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells.

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O2(-)) and hydrogen peroxide (H2O2), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H2O2), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O2(-) formation.

Involvement of MIF in basement membrane damage in chronically UVB-exposed skin in mice.

Solar ultraviolet (UV) B radiation is known to induce matrix metalloproteinases (MMPs) that degrade collagen in the basement membrane. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in the pathophysiology of skin inflammation induced by UV irradiation. This study examined the effects of MIF on basement membrane damage following chronic UVB irradiation in mice. The back skin of MIF transgenic (Tg) and wild-type (WT) mice was exposed to UVB three times a week for 10 weeks. There was a decrease in intact protein levels of type IV collagen and increased basement membrane damage in the exposed skin of the MIF Tg mice compared to that observed in the WT mice. Moreover, the skin of the MIF Tg mice exhibited higher MIF, MMP-2 and MMP-9 expression and protein levels than those observed in the WT mice. We also found that chronic UVB exposure in MIF Tg mice resulted in higher levels of neutrophil infiltration in the dermis compared with that observed in the WT mice. In vitro experiments revealed that MIF induced increases in the MMPs expression, including that of MMP-9 in keratinocytes and MMP-2 in fibroblasts. Cultured neutrophils also secreted MMP-9 stimulated by MIF. Therefore, MIF-mediated basement membrane damage occurs primarily through MMPs activation and neutrophil influx in murine skin following chronic UVB irradiation.

Astaxanthin, a xanthophyll carotenoid, inhibits ultraviolet-induced apoptosis in keratinocytes.

Intra-cellular reactive nitrogen/oxygen species and apoptosis play important roles in ultraviolet (UV)-induced inflammatory responses in the skin. Astaxanthin (AST), a xanthophyll carotenoid, exhibits diverse clinical benefits. The protective effects of AST against UV-induced apoptosis were investigated in the present study. Astaxanthin (5 µm) caused a significant decrease in the protein content and the mRNA levels of inducible nitric oxide (iNOS) and cyclooxygenase (COX)-2, and decreased the release of prostaglandin E2 from HaCaT keratinocytes after UVB (20 mJ/cm(2) ) or UVC (5 mJ/cm(2) ) irradiation. No significant protective effects against UV-induced reactive oxygen species (ROS) were observed in AST-pretreated cells. Astaxanthin caused a significant inhibition of UV-irradiation-induced apoptosis, as evidence by a DNA fragmentation assay. Furthermore, we found that the treatment with AST caused a reduction in the UVB- or UVC-induced protein and mRNA expression of macrophage migration inhibitory factor (MIF), IL-1ß and TNF-α in HaCaT keratinocytes. These results suggest that AST effectively protects against UV-induced inflammation by decreasing iNOS and COX-2, and thereby inhibiting the apoptosis of keratinocytes.

Localization of serine racemase and its role in the skin.

D-serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.

In vitro cytokine expression by peripheral mononuclear cells in herbal drug-induced skin eruption.

Herbal medicine is widely used worldwide and is associated with side-effects such as skin eruptions. Herbal drugs are often produced by combining multiple crude drugs, mostly of plant origin. Determining which medi-cinal plants are associated with the herbal drugs that induce skin eruptions can therefore be difficult. This study investigated mRNA expression of several cytokines in peripheral mononuclear cells (PBMCs) from two patients with herbal drug-induced skin eruptions; one reacted to keishi-bukuryo-gan (KBG), composed of 5 medicinal plants, and the other patient reacted to senna. PBMCs (1×106) from the 2 patients were cultured for 24 h with the supernatant from the medicinal plants from KBG or senna in various concentrations, and a reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed. A high mRNA level of interleukin (IL)-4 and IL-5 was detected in PBMCs stimulated by KBG and two of its components. Senna stimulated a high level of IL-4 and IL-5 mRNA levels in PBMCs from patient with senna-induced drug reaction.