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

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. OBJECTIVE: In this study, we examined the involvement of TARC in patients with BP. METHODS: We determined the fluid and serum TARC levels in patients with BP by enzyme-linked immunosorbent assay and compared the serum TARC levels with the eosinophil numbers in peripheral blood. We also compared the serum TARC levels in five patients with BP before and after they were treated. Moreover, we examined TARC, CCR4 and CXC chemokine receptor 3 (CXCR3) expression in the lesional skin of patients with BP by immunohistochemical procedures. Furthermore, we measured CCR4 positivity in CD4+ CD45RO+ cells of peripheral blood mononuclear cells (PBMCs) in patients with BP and healthy control subjects. RESULTS: The fluid TARC levels in patients with BP were significantly higher than those in blisters from burn patients or suction blisters of healthy control subjects. The serum TARC levels in patients with BP were also significantly higher than those in pemphigus vulgaris (PV) patients and healthy control subjects, and decreased after the treatment. The serum TARC levels in patients with BP significantly correlated with the eosinophil numbers in peripheral blood (r = 0.72, P < 0.002). Immunohistochemistry showed a strong reactivity of TARC in the epidermal keratinocytes (KCs) of BP. Moreover, both CCR4 and CXCR3 were expressed on the dermal infiltrating CD4+ T cells mainly beneath the bullae of patients with BP. Fluorescence-activated cell sorting analysis showed a higher percentage of CCR4 positivity in CD4+ CD45RO+ cells of PBMCs in patients with BP than that in healthy control subjects, while there was no significant difference of CXCR3 positivity in CD4+ CD45RO+ cells of PBMCs between patients with BP and healthy control subjects. CONCLUSIONS: These findings strongly suggest that TARC may be one of the important chemokines that are involved in the pathogenesis of BP.

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.

CC chemokine receptor 4 expression on peripheral blood CD4+ T cells reflects disease activity of atopic dermatitis.

Recent studies indicate that Th1 and Th2 cells differ in their chemokine receptor expression and their responsiveness to various chemokines. Therefore, selective Th2 cell recruitment in Th2-predominant inflammatory diseases such as atopic dermatitis may be under the influence of some chemokines. It is reported that CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells whereas CXC chemokine receptor (CXCR) 3 is selectively expressed on Th1 cells. In this study we examined CCR4 and CXCR3 expression on peripheral blood CD4+ and CD8+ T cells obtained from adult atopic dermatitis subjects, and compared the results with those from patients with psoriasis vulgaris and healthy controls. CCR4 was preferentially expressed on CD4+ T cells from atopic dermatitis subjects and CXCR3 was preferentially expressed on CD4+ T cells from psoriasis vulgaris subjects. This CCR4 expression was prominent especially in severe atopic dermatitis subjects. CCR4 expression on CD4+ T cells in severe atopic dermatitis subjects decreased on improvement of disease activity. CD25 was preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. Cutaneous lymphocyte-associated antigen was also preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. CD4+ T cells in atopic dermatitis skin lesions were predominantly CCR4+ cells. Taken together, this study strongly indicates that CCR4+CD4+ T cells reflect disease activity and suggests that CCR4 expression is important for T cell infiltration into atopic dermatitis lesions. Thus, CCR4 may be a possible target for therapy of atopic dermatitis in the future.

Interferon-gamma-induced RANTES production by human keratinocytes is enhanced by IL-1beta, TNF-alpha, 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-gamma (100 ng/ml) or IL-1beta (100 ng/ml) significantly induced RANTES production by KCs in 48-hour culture. These cytokines synergistically increased RANTES production by KCs. TNF-alpha (100 ng/ml), IL-4 (100 ng/ml) or IL-13 (100 ng/ml) markedly enhanced the RANTES production by KCs induced by IFN-gamma (100 ng/ml) although none of those cytokines significantly enhanced that induced by IL-1beta (100 ng/ml) in 48-hour culture. Dexamethasone (10(-8) M) strongly inhibited RANTES production by KCs induced by the combination of IFN-gamma 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-gamma, IL-1beta, TNF-alpha, IL-4 and IL-13, and can be modulated by immunosuppressive drugs. Our data suggest that RANTES is involved in skin inflammation.

Expression of CC chemokine receptor 3 on human keratinocytes in vivo and in vitro--upregulation by RANTES.

CC chemokines and their ligands, CC chemokine receptors (CCRs), play an important role in the process of inflammation such as trafficking and activating inflammatory cells. CCR3 is known to be a ligand for CC chemokines such as RANTES, eotaxin and monocyte-chemotactic protein-3 (MCP-3). In this study we examined the expression of CCR3 in cultured normal human keratinocytes (KCs). CCR3 protein and mRNA expressions were detected in cultured normal KCs by flow cytometric (FACS) analysis and reverse-transcription-polymerase chain reaction (RT-PCR) analysis. FACS analysis demonstrated that CCR3 expression on KCs was significantly upregulated when the cells were cultured with RANTES, but not with eotaxin, IL-4 or interferon-gamma. RT-PCR analysis revealed that CCR3 mRNA was detectable in normal KCs. We also examined the immunoreactivity of CCR3 in normal skin and inflammatory skin lesions. CCR3 was detected weakly in epidermis of normal skin, while strong immunoreactivity for CCR3 was seen in epidermis of inflammatory skin lesions such as atopic dermatitis. These results suggest that CCR3 is constitutively expressed on KCs and is involved in inflammatory modulation. RANTES may regulate the function of KCs through CCR3.

Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is closely related with disease activity.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of TH2-type cells in lesional skin. Thymus and activation-regulated chemokine (TARC/CCL17) is a chemokine that attracts CC chemokine receptor 4-positive (CCR4+) or CCR8+ cells. OBJECTIVE: The purpose of this study was to investigate the participation of TARC in AD. METHODS: We measured serum TARC levels in 40 patients with AD, 20 healthy control subjects, and 20 patients with psoriasis. We also examined disease activity by using SCORAD score; serum soluble E-selectin, soluble IL-2 receptor, IgE, and GM-CSF levels; and eosinophil numbers in peripheral blood, as well as correlations between TARC levels and these factors. The positivity of CCR4 of CD4+CD45RO+ cells in PBMCs was examined by using FACS analysis. Immunohistochemical staining of TARC and GM-CSF was performed in the lesional skin of patients with AD. RESULTS: The serum TARC levels of patients with AD were significantly higher than those of healthy control subjects and patients with psoriasis. The serum TARC levels significantly correlated with eosinophil number (r = 0.61), SCORAD score (r = 0.60), and serum soluble E-selectin levels (r = 0.58) and weakly correlated with serum soluble IL-2 receptor levels (r = 0.34) in patients with AD. The TARC levels of patients with AD decreased after the treatment in accordance with the improvement of clinical symptoms. The CCR4 positivity of CD4+CD45RO+ cells in PBMCs of patients with AD was also higher than that of healthy control subjects. Immunohistochemical staining revealed that TARC was positive in keratinocytes in the epidermis and in vascular endothelial cells, T cells, and dendritic cells in the dermis. CONCLUSION: Serum TARC levels are associated with disease activity of AD, and TARC may play an important role in the pathogenesis of AD.

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.

Antibodies to molluscum contagiosum virus in the general population and susceptible patients.

BACKGROUND: Since many attempts to cultivate molluscum contagiosum virus (MCV) in vitro have been unsuccessful, it is difficult to prepare a large quantity of antigens. To assess the seroprevalence of antibodies against MCV in 508 subjects with or without clinical MCV infection, a truncated recombinant protein from open-reading frame MC133L was synthesized using Sendai virus expression system and applied to enzyme-linked immunosorbent assay as an antigen. OBSERVATIONS: Antibodies to MCV were present in 7 (58%) of 12 patients with molluscum contagiosum, 7 (6%) of 108 healthy controls, 7 (9%) of 76 with atopic dermatitis, and 7 (18%) of 39 patients with systemic lupus erythematosus, although no clinical MCV infection was observed in the latter 3 groups. Of 7 human immunodeficiency virus (HIV)-positive patients with molluscum contagiosum, 1 (14%) was antibody positive, compared with 5 (2%) of 266 HIV-positive patients without molluscum contagiosum. Serum samples from patients with atopic dermatitis and systemic lupus erythematosus showed a higher reactivity (P<.001) than those from healthy controls, while serum samples from HIV-positive subjects showed a lower reactivity (P<. 001). CONCLUSION: The humoral immune response to MCV is usually confined to patients with molluscum contagiosum and may be affected by the immunological condition of the host.

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.

Exacerbation of pustulosis palmaris et plantaris after topical application of metals accompanied by elevated levels of leukotriene B4 in pustules.

BACKGROUND: Pustulosis palmaris et plantaris (PPP) is a chronic inflammatory disease consisting of polymorphonuclear leukocyte infiltration, and is often exacerbated by focal infections such as tonsillitis. In some cases, metal allergy has been reported. OBJECTIVE: The purpose of this study was to evaluate (1) the significance of metal allergy in the formation of pustules, and (2) the participation of leukotriene (LT) B(4) in the formation of pustules of PPP. METHODS: Patch tests with metals were performed on 7 patients with PPP, and both pustular and plasma levels of LTB(4) were measured in these 7 patients before and 48 hours after metal patch tests. RESULTS: Palmoplantar pustules were exacerbated after the metal patch tests in all 7 patients. The mean levels of LTB(4) in plasma and pustules of the volar surface at 48 hours after the metal patch tests were significantly higher than those before the metal patch tests. CONCLUSION: Metals can be important in the pathogenesis of PPP by contributing to the induction of high LTB(4) concentration in the pustules.

Changes in eosinophil and leukocyte infiltration and expression of IL-6 and IL-7 messenger RNA in mite allergen patch test reactions in atopic dermatitis.

Patch testing with crude dust mite extracts after removal of homy layers was performed on normal-appearing skin of 11 adult patients with atopic dermatitis and high mite-specific IgE antibody scores. Positive skin reactions were observed in 9 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 studies and extraction of messenger RNA (mRNA). Perivascular infiltration of small mononuclear cells began at 2 hours and was followed by eosinophilic infiltration at 6 hours, and the number of eosinophils continued to increase at 24 and 48 hours. In addition to the increased expression of IL-4, IL-5, and tumor necrosis factor-alpha mRNA during the time course detected by reverse-transcription polymerase chain reaction, mRNA of IL-6 and IL-7 was also up-regulated. After the removal of test patches with mite allergen, the number of eosinophils started to decrease in a time-dependent manner. Histopathologic findings at 48 hours after removal showed lymphocyte-dominant infiltration intermingled with occasional eosinophils. These mite allergen patch test reactions may provide a useful model for studying the pathogenesis of atopic eczema, especially with regard to the initiation of eosinophil infiltration and the alternative increase in lymphocytes.

Juvenile temporal arteritis with eosinophilia: a distinct clinicopathological entity.

BACKGROUND: Juvenile temporal arteritis has been proposed as an entity but remains controversial. CASE REPORT: A 39-year-old male, who was otherwise asymptomatic, developed painless bilateral nodules in the temporal areas. His eosinophil blood count was 2,660/mm3 (31%), while the erythrocyte sedimentation rate was normal. Histologic examination of the lesion revealed non-giant-cell granulomatous inflammation with abundant eosinophil infiltration. The clinical and pathologic manifestations in our patient were different from those in classic temporal arteritis, which occurs almost exclusively in individuals over the age of 50 years, allergic granulomatosis and angiitis, and thromboangiitis obliterans. Eight cases of this disease have previously been reported in the literature. CONCLUSION: We consider that 'juvenile temporal arteritis with eosinophilia' is a distinct clinical and pathologic entity. The prognosis of the diseases is considered to be good.

Chronologic analysis of eosinophil granule protein deposition and cell adhesion molecule expression in mite allergen-induced dermatitis in atopic subjects.

To investigate the process of eosinophil recruitment in atopic dermatitis (AD), patch testing with crude dust-mite allergens was performed on normal-appearing skin of 9 adult AD patients with high levels of mite-specific IgE antibodies. Positive reactions were observed in 6 AD subjects, whereas 0 of 7 control subjects showed positive reactions to mite allergens. Positive reaction sites were biopsied chronologically and studied histologically and immunohistochemically. Eosinophils were seen in postcapillary venules in the dermis at 2 h, followed by infiltration of eosinophils at 6 h which peaked at 24 and 48 h. In the epidermis, eosinophilic spongiosis was seen at 48 h. Positive reactions against eosinophil granule proteins were observed in connective tissues as well as on eosinophils. Almost all infiltrating eosinophils were positive for BMK-13 (an antibody against major basic protein); half of them were positive for EG2 (an antibody against eosinophil cationic protein). With regard to adhesion molecules, expression of E-selectin and intercellular adhesion molecule-1 endothelial cells was up-regulated as infiltrating eosinophils increased in number. These findings suggest that eosinophil transmigration from endothelial cells and eosinophil degranulation play important roles in initiating early AD lesions induced by transepidermal mite allergen permeation.

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.

[Chronological analysis of eosinophil infiltration in mite-allergen induced dermatitis in atopic patients].

To elucidate the active process of eosinophil infiltration in atopic dermatitis (AD), we performed patch testing using crude mite allergens on normal-appearing skin in adult AD patients with high levels of mite-specific IgE antibodies and analyzed positive reaction sites chronologically by histological and immunohistological methods. Positive reactions were observed in 6 out of 9 AD patients (66.7%), whereas none of 7 non-AD control subjects showed positive reactions. Skin biopsies were obtained from positive reaction sites at 2,6,12,24 and 48 hours after allergen challenge. Eosinophils were seen within postcapillary venules at 2 hours, followed by increased perivascular eosinophil infiltration at 6 hours which peaked at 24 and 48 hours. Epidermal eosinophilic spongiosis was also observed at 48 hours. The majority of infiltrating eosinophils showed positive reactions for BMK-13 and 15-59% of them were EG2 positive activated eosinophils. BMK-13, EG2 and anti-EP positive reactions were scattered throughout connective tissue and their areas were time-dependently increased. These findings suggest that eosinophil transmigration and activation play an important role in initiating early AD lesions induced by transepidermal mite allergen permeation.