Motoshi Wakugawa

Interleukin-12 p40 gene (IL12B) 3'-untranslated region polymorphism is associated with susceptibility to atopic dermatitis and psoriasis vulgaris.

Interleukin-12 (IL-12) is believed to play an important role in inducing Th1-type cytokine profiles. Atopic dermatitis (AD) and psoriasis vulgaris (PsV) are considered to be Th2 and Th1 type disease, respectively. The IL-12 p40 subunit gene (IL12B) is located at chromosome 5q31-33 and linkage findings of AD on 5q31 were reported. Recently single nucleotide polymorphism (SNP) (1188A/C) of IL12B has been reported. In function, it has been reported that this SNP is associated with IL12B mRNA expression levels. To learn whether this SNP is associated with susceptibility to AD or PsV, we investigated the genotype and allele frequencies of the SNP in AD patients, in PsV patients and in controls, examining 164 AD patients, 143 PsV patients and 100 healthy individuals in Japanese population. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism method. The A allele was decreased in AD patients (40.9%, p = 0.031) and increased in PsV patients (60.1%, p = 0.035) compared with controls (50.5%). This suggests that IL12B SNP is associated with susceptibility to AD and PsV, presumably by affecting the Th1/Th2 balance.

Serum macrophage-derived chemokine (MDC) levels are closely related with the disease activity of atopic dermatitis

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of T cells, eosinophils and macrophages in lesional skin. Recently, macrophage‐derived chemokine (MDC)/CCL22, a CC chemokine, was identified as a selective chemoattractant for CC chemokine receptor 4 (CCR4)‐expressing cells, in addition to thymus and activation‐regulated chemokine (TARC). We have previously reported that serum TARC levels correlate with the severity of AD. In this report, we investigated the participation of MDC in AD. First, we measured serum MDC levels in 45 patients with AD, 25 patients with psoriasis vulgaris and 25 healthy controls. Serum MDC levels in AD patients were significantly higher than those in healthy controls and psoriasis patients. Furthermore, the increases in serum MDC levels in AD patients were greater in the severely affected group than in the moderate or mild groups. We compared serum MDC levels in 11 AD patients, before and after treatment, and observed a significant decrease after treatment. Moreover, the serum MDC levels significantly correlated with the Scoring AD (SCORAD) index, serum soluble (s) E‐selectin levels, serum soluble interleukin‐2 receptor (sIL‐2R) levels, serum TARC levels and eosinophil numbers in peripheral blood. Our study strongly suggests that serum MDC levels have a notable correlation with disease activity and that MDC, as well as the CC chemokine TARC, may be involved in the pathogenesis of AD.

Interleukin-13 gene polymorphism G4257A is associated with atopic dermatitis in Japanese patients

Interleukin (IL)-13 plays an important role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD). We investigated the allele and genotype frequencies of three IL-13 single nucleotide polymorphisms (SNPs) (A704C and C1103T in the promoter region and G4257A in exon 4) in Japanese patients with AD. For A704C and C1103T SNPs, there were no significant differences in allele or genotype frequencies between AD patients and controls. For G4257A SNP, A allele was significantly increased in AD patients (39.5%) compared with controls (29.4%) (P = 0.016). The same proportion of each genotype and allele was observed in the patient subgroup with and without asthma. Serum IgE levels and peripheral eosinophil counts were not significantly different among genotypes in G4257A SNP. There was also no significant difference in allele or genotype frequencies between AD patients with mild disease and those with severe disease, between those with family history of AD and those without it, or between those with family history of atopic disorders and those without it. This result suggests that 4257A allele is associated with susceptibility to AD and that it may function in the pathogenesis of AD itself, presumably by other mechanisms than inducing IgE production.

Elevated levels of eotaxin and interleukin‐5 in blister fluid of bullous pemphigoid: correlation with tissue eosinophilia

Background Bullous pemphigoid (BP) often provokes blood and tissue eosinophilia, which suggests that some chemoattractants modulate the eosinophil infiltration in BP. Eotaxin, a CC chemokine, strongly attracts eosinophils, and interleukin (IL)‐5 induces eosinophil differentiation, proliferation and colony formation in vitro. Objectives To examine the correlation between levels of eotaxin and IL‐5 and the number of lesional eosinophils, and the expression of eotaxin in BP lesions. Patients/methods In this study we measured eotaxin and IL‐5 levels in blister fluid of BP by enzyme‐linked immunosorbent assay. We also examined the expression of eotaxin in BP lesions by immunohistochemistry. Results Both eotaxin and IL‐5 were detected at high levels in BP blister fluid. Blister fluid eotaxin, but not IL‐5 levels, correlated significantly with the number of dermal infiltrating eosinophils. By immunohistochemistry, eotaxin was strongly expressed in epidermal keratinocytes around BP blisters. Conclusions These findings suggest that eotaxin and IL‐5 are strongly associated with the tissue eosinophilia of BP. Therapies which aim to inhibit production of eotaxin and IL‐5 may improve the inflammation and blister formation in BP.

High level of thymus and activation-regulated chemokine in blister fluid and sera of patients with bullous pemphigoid

Summary Background Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by eosinophilia and high serum IgE levels. The accumulated evidence suggests that various cytokines are involved in the lesional skin of patients with BP. Recently, thymus and activation‐regulated chemokine (TARC/CCL17), a CC chemokine, was identified as a selective chemoattractant for CC chemokine receptor 4 (CCR4)‐expressing cells.

Chronologic analysis of in situ cytokine expression in mite allergen-induced dermatitis in atopic subjects

Patch testing with crude dust mite extracts, after removal of horny epidermal layers, was performed on normal-appearing skin of nine adult patients with atopic dermatitis who had high mite-specific IgE antibody levels. Positive skin reactions were observed in seven subjects. Skin biopsy specimens were obtained from positive reaction sites at 2, 6, 12, 24, and 48 hours after allergen challenge and subjected to histologic and immunohistochemical studies and extraction of RNA. Perivascular infiltration of small mononuclear cells began at 2 hours, followed by eosinophilic infiltration at 6 hours, which peaked at 24 and 48 hours. Increased expression of IL-4 messenger RNA was detected with reverse-transcription polymerase chain reaction at 12 and 24 hours, whereas immunohistochemical staining with anti-IL-4 antibody showed positive reactions in connective tissue around infiltrating cells after 2 hours. Expression of IL-5 and tumor necrosis factor-alpha mRNA was upregulated 2 hours after an application of allergen. Interferon-gamma mRNA was not detected. These findings suggest the crucial role of TH2-type cytokines in initiating eosinophil infiltration of mite allergen-induced dermatitis in patients with atopic dermatitis.

Eotaxin gene single nucleotide polymorphisms in the promoter and exon regions are not associated with susceptibility to atopic dermatitis, but two of them in the promoter region are associated with serum IgE levels in patients with atopic dermatitis

Eotaxin is believed to play an important role in atopic dermatitis (AD) as a potent chemoattractant and activator of eosinophils and Th2 lymphocytes. The eotaxin gene is located at chromosome 17q21.1-q21.2, and linkage findings of AD on chromosome 17 were reported. Recently we have identified single nucleotide polymorphisms (SNPs) of eotaxin gene (-426C > T, -384A > G, 67G > A). To learn whether eotaxin gene SNPs are associated with susceptibility to AD or phenotypes of AD, we investigated the genotype frequencies at each SNP of the gene in AD patients and in controls. We examined 140 Japanese AD patients and 140 healthy Japanese individuals. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism method. No significant difference was observed in allele or genotype frequencies of any SNP between AD patients and controls. Serum immunoglobulin E (IgE) levels were significantly lower in CT and TT genotype than in CC (P = 0.038) in -426C > T SNP, and lower in GG than in AA and AG with borderline significance (P = 0.053) in -384A > G SNP in AD patients. Eotaxin gene SNPs in the promoter and exon regions are not associated with susceptibility to AD, but two of them in the promoter region are associated with phenotype of AD.

Interferon-Gamma-Induced RANTES Production by Human Keratinocytes Is Enhanced by IL-1β, TNF-α, IL-4 and IL-13 and Is Inhibited by Dexamethasone and Tacrolimus

Background and Methods: Recent studies have shown that RANTES plays a role in the pathogenesis of inflammatory skin diseases. We examined the production of RANTES by human keratinocytes (KCs) when cultured with various cytokines. Results: IFN-γ (100 ng/ml) or IL-1β (100 ng/ml) significantly induced RANTES production by KCs in 48-hour culture. These cytokines synergistically increased RANTES production by KCs. TNF-α (100 ng/ml), IL-4 (100 ng/ml) or IL-13 (100 ng/ml) markedly enhanced the RANTES production by KCs induced by IFN-γ (100 ng/ml) although none of those cytokines significantly enhanced that induced by IL-1β (100 ng/ml) in 48-hour culture. Dexamethasone (10–8 M) strongly inhibited RANTES production by KCs induced by the combination of IFN-γ and IL-4, while tacrolimus (FK-506, 10–8 and 10–6 M) showed partial inhibition. Conclusions: These results revealed that RANTES production by KCs is regulated by inflammatory cytokines, such as IFN-γ, IL-1β, TNF-α, IL-4 and IL-13, and can be modulated by immunosuppressive drugs. Our data suggest that RANTES is involved in skin inflammation.

Evaluation of mite allergen-induced Th1 and Th2 cytokine secretion of peripheral blood mononuclear cells from atopic dermatitis patients : association between IL-13 and mite-specific IgE levels

There have been several reports about Th1/Th2 imbalances in atopic dermatitis (AD), but there have been few precise investigations about the differences between Th1 and Th2 cytokine secretion patterns of peripheral blood mononuclear cells (PBMCs) in such patients. We cultured PBMCs, taken from AD patients and healthy subjects, with dust mite extract (DME) and measured subsequent immunoreactive interferon (IFN)-gamma (Th1 cytokine), interleukin (IL)-4, IL-5, and IL-13 (Th2 cytokines) levels in the supernatants by ELISA assays. There is a difference between IL-4 and IL-13 secretion patterns by DME-stimulated PBMCs in AD subjects; immunoreactive IL-4 levels were detectable maximally within 24-h cultures, while IL-13 levels increased time-dependently within 7-day cultures. IL-13 levels were significantly elevated in AD subjects compared to healthy subjects, while IFN-gamma levels did not significantly differ between the two groups. IL-13 levels were significantly higher in AD patients who had high levels (>100 U/ml) of Dermatophagoides pteronyssinus-specific IgE (Dp-IgE) than in those AD patients who had low levels (<10 U/ml) of Dp-IgE. Tacrolimus (FK-506), at a concentration of 10(-8) M, significantly inhibited DME-induced IL-13 production from PBMCs. These findings suggest that IL-13 produced by Th2 cells are involved in IgE overproduction in AD subjects.

The -431C>T polymorphism of thymus and activation-regulated chemokine increases the promoter activity but is not associated with susceptibility to atopic dermatitis in Japanese patients.

Background:  Thymus and activation‐regulated chemokine (TARC) plays an important role in the pathogenesis of atopic dermatitis (AD). We recently detected the single nucleotide polymorphism (SNP) (−431C>T) in the 5′‐flanking region of TARC gene.