Coagulation/fibrinolysis and inflammation markers are associated with disease activity in patients with chronic urticaria.

BACKGROUND: The evaluation of disease severity and activity of chronic urticaria (CU) is essential for the adequate treatment of patients. However, there is no reliable biomarker for such evaluations. Recently, markers of blood coagulation and fibrinolysis have been revealed to be elevated in severe cases of CU. In this article, we studied the coagulation/fibrinolysis and inflammation markers and their relationship to disease activity in patients with CU. METHODS: Plasma fibrin degradation products (FDP), d-dimer and serum C-reactive protein (CRP) were measured with the assessment of disease severity and skin reaction to autologous serum in 82 patients with CU and 37 patients with acute urticaria, idiopathic angioedema (AE) or inducible types of urticaria (IU). RESULTS: The levels of FDP in patients with CU were significantly higher than those in patients with IU, but no other differences in FDP, d-dimer and CRP were observed among patients with different types of urticaria. These markers of patients with CU were well correlated with each other and significantly associated with disease severity of CU, but not with skin reactions to autologous serum. In 37 patients with CU, levels of all these parameters reduced as their disease condition improved, while they increased when the disease became aggravated. Regarding FDP, this relationship was observed even if FDP concentrations were within normal range throughout the study. CONCLUSIONS: The measurement of plasma FDP, d-dimer and serum CRP may be useful for the assessment of disease activity of CU.

Sweat antigen induces histamine release from basophils of patients with cholinergic urticaria associated with atopic diathesis.

BACKGROUND: We previously demonstrated that the semipurified human sweat antigen causes skin reactions and histamine release from basophils via specific IgE in patients with atopic dermatitis (AD). Patients with cholinergic urticaria (ChU) also develop skin reactions and histamine release of basophils in response to autologous sweat. OBJECTIVES: To study whether or not patients with ChU share sensitivity for the sweat antigen with patients with AD and to study the clinical characteristics among patients with ChU and the relationship with histamine-release activity of basophils. METHODS: The sweat antigen that induces histamine release from basophils of patients with AD was prepared by Con-A, anion-exchange and reverse-phase chromatography. Relationships between histamine-release activity against the sweat antigen and clinical features of patients with ChU were analysed. RESULTS: Twenty-three of 35 patients with ChU showed > 5% net histamine release in response to the semipurified sweat antigen, whereas none of healthy controls did so. In patients with ChU, histamine release in response to semipurified sweat antigen significantly correlated with the level of serum IgE and eosinophil numbers in peripheral blood. Incidence of each atopic disease in patients with ChU tended to be higher than in the general Japanese population. When the patients were categorized according to their responses in the histamine release test, the positive group tended to show a higher incidence of AD and bronchial asthma compared with the negative group. CONCLUSIONS: ChU and AD may share hypersensitivity to common antigens in sweat. The sweat allergy and atopic diathesis are associated with each other.

Semi-purification of the immunoglobulin E-sweat antigen acting on mast cells and basophils in atopic dermatitis.

BACKGROUND: Sweating aggravates the symptoms of atopic dermatitis (AD). We have recently reported positive skin reactions and histamine release from basophils in response to autologous sweat in patients with AD. OBJECTIVE: To characterize the biochemical and immunological properties of the substance in sweat that evokes histamine release and to study the usability of the basophil-histamine release test with the sweat antigen for AD. METHODS: Sweat collected from healthy volunteers was purified using chromatographies. Serum immunoglobulin (Ig)E of four patients with AD were purified using an affinity-chromatography column with anti-IgE antibodies. The amount of semi-purified sweat antigen (138 ng protein/ml) that induced a half-maximum reaction of basophils of a patient with AD was utilized for the basophil histamine release test. The involvement of specific IgE and high-affinity IgE receptor (FcepsilonRI) in the reactions was examined using basophils of healthy volunteers, a human mast cell line (LAD2), and a rat basophilic leukemia cell line transfected with human alpha-subunit of FcepsilonRI (RBL-48). RESULTS: The semi-purified sweat antigen induced histamine release from the basophils of 47 of 61 (74.6%) patients with AD and four of 46 (8.7%) healthy controls. Both basophils and mast cells sensitized with the patient-derived IgE showed degranulation upon stimulation with the sweat antigen. However, no reaction was observed when cells were sensitized with myeloma IgE or the antigen was treated with proteases. CONCLUSION: The semi-purified standardized sweat antigen consists of a protein that induces degranulation of basophils and mast cells via antigen-specific IgE and FcepsilonRI in patients with AD.

Oral administration of pulverized Konjac glucomannan prevents the increase of plasma immunoglobulin E and immunoglobulin G levels induced by the injection of syngeneic keratinocyte extracts in BALB/c mice.

BACKGROUND: Immunoglobulin (Ig) E plays a key role in the pathogenesis of atopic diseases, such as asthma, atopic dermatitis and allergic rhinitis. Oral administration of pulverized Konjac glucomannan (PKGM) has recently been demonstrated to prevent both plasma IgE elevation and developing dermatitis in NC/Nga mice, a model of atopic dermatitis. OBJECTIVE: To clarify the direct effect of PKGM on the increase of plasma IgE, we employed the system of BALB/c mouse that increases IgE level without developing dermatitis in response to continuous injection of the extract of syngeneic keratinocytes, PAM 212 cells (PAM extract). METHODS: Three weeks after the start of feeding with either control or PKGM diet, mice were injected subcutaneously with PAM extract bi-weekly for 10 weeks. The levels of plasma Igs were measured by enzyme-linked immunosorbent assay every 2 weeks after the injection. The levels of epsilon germline transcription and the amounts of mRNA for IL-4, IFN-gamma, GATA-3 and T bet gene in the spleen were evaluated by real-time RT-PCR at the end of the experiment. RESULTS: On the one hand, PKGM prevented the increase of plasma IgE and IgG (IgG1, IgG2b) induced by PAM extract, and on the other hand, it enhanced the levels of plasma IgG3. However, it did not affect the level of plasma IgM. PKGM also reduced the levels of plasma ovalbumin (OVA)-specific IgE in OVA-sensitized mice. Moreover, PKGM attenuated the induction of epsilon germline transcription and expression levels of mRNA for IL-4, IFN-gamma and GATA-3 in the spleen of PAM extract-injected mice. PKGM also attenuated the induction of epsilon germline transcription and mRNA for IFN-gamma and T bet in the spleen of phosphate-buffered saline-injected control mice. CONCLUSIONS: These results suggested that oral administration of PKGM prevents the elevation of plasma IgE by suppressing IgE class switching in B cells and/or the commitment development of naive lymphocytes to both T-helper type 1 (Th1) and Th2.

The IL-6 family cytokines, interleukin-6, interleukin-11, oncostatin M, and leukemia inhibitory factor, enhance mast cell growth through fibroblast-dependent pathway in mice.

Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the mechanisms involved in the pathogenesis of these conditions remains largely uninvestigated, it is speculated that mediators produced in the lesional skin provide a favorable microenvironment for mast cell growth. Among the proinflammatory mediators, leukemia inhibitory factor (LIF), which shares a receptor component (gp130 subunit) with interleukin-6 (IL-6), has been identified as a mast cell growth-enhancing factor produced by cells of the keratinocyte-derived cell line (KCMH-1). In this study, we investigated the effect of four IL-6 family cytokines, IL-6, IL-11, oncostatin M (OSM) and LIF on mast cell growth in a mast cell/fibroblast co-culture system. When mouse bone marrow-derived cultured mast cells (BMMC) were maintained on a NIH/3T3 fibroblast monolayer, these cytokines induced proliferation of the mast cells, but none of the cytokines had any effect on mast cell proliferation in the absence of fibroblasts. mRNA for gp130 and receptors for the four IL-6 family cytokines were detected in NIH/3T3 fibroblasts by reverse transcriptase-mediated polymerase chain reaction. In contrast, only mRNA for the IL-11 receptor and gp130 were detected in BMMC. Tyrosine phosphorylation of gp130 was observed in NIH/3T3 fibroblasts after stimulation with all the cytokines. Some IL-6 family cytokines enhanced the production of stem cell factor (SCF), a potent mast cell growth factor, from NIH/3T3 fibroblasts, but the amount of SCF produced by NIH/3T3 fibroblasts was not paralleled by the mast cell growth-enhancement induced by the IL-6 family cytokines. When anti-SCF antibody was added with the IL-6 family cytokines in the BMMC/fibroblast coculture system, a significant effect of these cytokines remained, although the growth-enhancing activity was markedly reduced. A similar result was obtained when BMMC were prepared from W/W(V)-mice, which lack functional c-kit, in the BMMC/ fibroblast coculture system. These results suggest that IL-6 family cytokines stimulate mast cell growth by a fibroblast-dependent mechanism, and also suggest the existence of another pathway between BMMC and NIH/3T3 fibroblasts cooperating with the SCF/c-kit pathway. IL-6 family cytokines may thus contribute to mast cell hyperplasia in skin diseases.

Food-dependent exercise-induced anaphylaxis: a report of two cases and determination of wheat-gamma-gliadin as the presumptive allergen.

Water/salt-insoluble wheat proteins have been identified as the most frequent allergenic foodstuffs in patients with food-dependent exercise-induced anaphylaxis (FDEIA) in Japan. However, the specific allergenic proteins in wheat-dependent exercise-induced anaphylaxis have not been well defined. Challenge testing, skin testing and a fluoroenzyme immunoassay were used for diagnosis in two patients suspected by history of having wheat-dependent exercise-induced anaphylaxis. Gel chromatography and IgE immunoblotting followed by N-terminal amino acid sequencing were used to identify the allergenic wheat protein. The challenge test revealed that both patients had FDEIA. The skin tests and the immunoassay results suggested that wheat gluten was the allergen in both patients. Gel chromatography of wheat gluten revealed that the antigens had molecular weights ranging from 40 to 250 kDa. IgE immunoblotting and subsequent N-terminal amino acid sequencing revealed that wheat-gamma-gliadin was the antigen predominantly bound by IgE in the two patients.

Interleukin-1 alpha enhances mast cell growth by a fibroblast-dependent mechanism.

Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the pathogenesis of these conditions remains largely uninvestigated, it has been speculated that lesional mediators provide a favorable microenvironment for mast cell growth. We investigated the effect of an inflammatory cytokine, IL-1 alpha, on mast cell growth in a mast cell/fibroblast coculture system. When mouse bone marrow-derived cultured mast cells (BMMC) were cultured on a NIH/3T3 fibroblast monolayer, IL-1 alpha stimulated mast cell proliferation. However, IL-1 alpha did not stimulate 3H-thymidine incorporation in BMMC in the absence of fibroblasts. Separation of BMMC from fibroblasts by a permeable micropore membrane reduced the effect of IL-1 alpha. When BMMC were prepared from W/Wv mice, which lack a functional c-kit, or when NIH/3T3 fibroblasts were substituted with Sl/Sld-derived fibroblasts, which lack membrane-bound stem cell factor (SCF), a lower, but significant, effect of IL-1 alpha was observed. Flow cytometric analysis revealed no enhancement of SCF expression on fibroblasts following stimulation with IL-1 alpha. Neutralizing antibodies against IL-3, IL-4, IL-10, and nerve growth factor (NGF) showed no inhibition. On the other hand, indomethacin inhibited the effect of IL-1 alpha, and prostaglandin E2 induced mast cell growth in the co-cultures. These results indicate that IL-1 alpha stimulates mast cell growth by a fibroblast-dependent mechanism, in which SCF/c-kit interaction may participate in a major way. The mast cell growth activity induced by this cytokine can, at least in part, be attributed to prostaglandins. Inflammatory cytokines may thus contribute to mast cell hyperplasia in skin diseases.

Altered in vitro apoptosis of cultured mast cells prepared from an inbred strain of mice, NC/Kuj.

BACKGROUND: An inbred strain of mice, NC, develops dermatitis associated with highly elevated serum IgE and dermal mast cell hyperplasia. OBJECTIVES AND METHODS: To clarify the mechanisms for the dermal mast cell hyperplasia in NC, we prepared bone marrow-derived mast cells (BMMCs) from three strains of mice, NC/Kuj, C57BL/6 and BALB/c, and compare histamine contents, histamine release abilities, adhesive properties and apoptosis of the BMMCs. RESULTS: Compared with BMMCs obtained from C57BL/6 and BALB/c, NC/Kuj BMMC possessed higher histamine content and higher adhesive ability to plastic plates, although histamine release from BMMCs was found to be similar in the three strains. The most intriguing finding is the lack of apoptosis in the BMMCs from NC/Kuj upon growth factor deprivation as determined by DNA ladder formation and loss of membrane phospholipid asymmetry. CONCLUSION: The altered in vitro properties of mast cells in NC/Kuj partially account for an increase of dermal mast cells, which might be involved in the development of skin lesions in NC.

Leukemia inhibitory factor enhances mast cell growth in a mast cell/fibroblast co-culture system through stat3 signaling pathway of fibroblasts.

Leukemia inhibitory factor (LIF) enhanced mast cell growth in a mast cell/3T3 fibroblast co-culture system, however the precise mechanisms have not been defined. Western blot analysis showed that bone marrow-derived mast cells failed to express both LIF receptor (LIFR) and gp130, whereas 3T3 fibroblasts expressed both LIFR and gp130. This result indicates that the activity of LIF for mast cell growth is mediated by 3T3 fibroblasts. Signal transducer and activator of transcription (Stat) 3-transfected 3T3 fibroblasts enhanced mast cell growth. In addition, dominant-negative Stat3-transfected fibroblasts blocked LIF-mediated mast cell growth in the co-culture system. In conclusion, LIF-induced mast cell growth in the co-culture system is mediated by an indirect pathway via 3T3 fibroblasts through activating Stat3 signaling pathway in 3T3 fibroblasts.

Histamine enhances UVB-induced IL-6 production by human keratinocytes.

Histamine, an important mediator in immediate-type hypersensitivity, is elevated in the skin of patients with atopic dermatitis and is considered to play a pathogenic role in atopic dermatitis. In this study, to elucidate the mechanism of sun exposure-induced exacerbation of skin lesions in atopic dermatitis, we examined the effect of histamine on proinflammatory cytokine production of keratinocytes induced by ultraviolet (UV) B irradiation. Cultured human keratinocytes were irradiated with 30 mJ/cm2 of UVB and incubated with histamine over the concentration range 10(-7) to 10(-4) M, and the IL-1alpha and IL-6 released into the medium were measured using an ELISA. Histamine weakly stimulated IL-6 production by itself. However, together with UVB, it synergistically enhanced IL-6 production and the amount of IL-6 mRNA as estimated by reverse-transcription polymerase chain reaction (RT-PCR). Histamine had a dose-dependent effect which was maximal at a concentration of 10(-5) M, and had no effect on the kinetics of IL-6 production. In contrast, histamine had no effect on IL-1alpha production by keratinocytes. The effect of histamine was completely blocked by pyrilamine, an H1 receptor antagonist, and mimicked by the H1 receptor agonist, 2-methylhistamine. Whereas the H2 receptor antagonist, cimetidine, slightly inhibited the effect of histamine and the effect of the H2 receptor agonist, 4-methylhistamine, was minute. These results show that histamine augments UVB-induced IL-6 production by keratinocytes predominantly via the H1 receptor at the level of transcription. This suggests a contributory role for histamine in the exacerbation of atopic dermatitis induced by sun exposure.

A fibrogenic cytokine, platelet-derived growth factor (PDGF), enhances mast cell growth indirectly via a SCF- and fibroblast-dependent pathway.

Mast cell hyperplasia is often observed in dermatoses characterized by fibrosis. Evidence has accumulated showing that a potent fibrogenic cytokine, platelet-derived growth factor (PDGF), plays a pathogenic role in dermal fibrosis. To clarify the mechanism of mast cell hyperplasia associated with fibrosis, we investigated the effect of PDGF on mast cell proliferation and the expression of stem cell factor (SCF), a potent growth factor for mast cells, in fibroblasts. When mouse bone marrow-derived mast cells (BMMC) were cultured on a NIH/3T3 fibroblast monolayer, mast cell proliferation was stimulated in both cell number and total histamine content by all isoforms of PDGF (-AA, -AB, and -BB); however, none of the isoforms had any effect on [3H] thymidine incorporation in BMMC in the absence of fibroblasts. The effect of PDGF-AB and -BB were abrogated either by the addition of anti-PDGF-AB antibody or by the separation of mast cells and fibroblasts by a permeable membrane filter with a pore size of 0.2 microm. Immunoblotting of the NIH/3T3 fibroblasts treated with PDGF revealed an enhanced expression of SCF in the membrane fraction and the effect of PDGF was neutralized by the addition of antibody against SCF. Moreover, no effect of PDGF was observed when BMMC were prepared from W/W(v) mice that lack functional c-kit as the SCF receptor or when 3T3 fibroblasts were prepared from Sl/Sl(d) mice that lack membrane-bound SCF. These results suggest that the fibrogenic cytokine PDGF stimulates mast cell hyperplasia via the expression of membrane-bound SCF by fibroblasts in association with fibrosis of the skin.

A novel point mutation in the steroid sulfatase gene in X-linked ichthyosis.

We analyzed the steroid sulfatase (STS) gene in nine Japanese patients with X-linked ichthyosis (XLI) by a polymerase chain reaction technique and subsequent DNA sequencing. Eight of nine patients showed complete deletion of the STS gene. In a patient of XLI exhibiting a normal amplifying pattern with predicted sizes of the STS gene, a novel mutation was found resulting in the appearance of a stop codon in exon 7 of the STS gene. This suggests that exon 7 or an area in its downstream region is important for STS activity.

Human eosinophils lack human leukocyte elastase.

Recently, human leukocyte elastase has been detected in human eosinophils. Reinvestigating these findings, 2.5 pg active human leukocyte elastase (E.C. 3.4.21.37) were found per neutrophil isolated from peripheral blood, whereas the elastase activity of eosinophil preparations was linearly correlated with the content of contaminating neutrophils. Also spontaneous or stimulated release of active elastase was absent in eosinophils. By immunohistochemistry no elastase immunoreactivity could be demonstrated in human eosinophils. Therefore, we conclude that human eosinophils do not contain considerable amounts of human leukocyte elastase.

Detection of the chemokine RANTES in cytokine-stimulated human dermal fibroblasts.

A novel family of structurally and functionally related polypeptides has recently been detected that are now referred to as chemokines. Within this family, a peptide with the acronym RANTES was shown to be chemotactic for memory T cells, monocytes, and eosinophilic and basophilic granulocytes, thus suggesting it plays an important role in chronic inflammatory and allergic diseases. Murine monoclonal antibodies as well as cDNA probes specific for human RANTES were raised and extensively characterized. With these antibodies, stimulated human dermal fibroblasts were shown to express intracellular RANTES peptide by immunocytochemistry. Furthermore, similar kinetics could be demonstrated in fibroblasts for both RANTES mRNA expression and secretion of RANTES peptide using Northern blot hybridization and sandwich-enzyme-linked immunosorbent assay, respectively. RANTES expression was induced upon stimulation with tumor necrosis factor-alpha as well as with interleukin-1 alpha and -beta in a concentration- and time-dependent manner. These results reinforce the role of both resident and circulating cells in the production and release of RANTES and their participation in inflammatory processes.