House dust mites as potential carriers for IgE sensitization to bacterial antigens.

BACKGROUND: IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. METHODS: Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. RESULTS: IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. CONCLUSION: House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.

Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression.

BACKGROUND: The disrupted skin barrier of patients with atopic eczema (AE) might facilitate contact between mast cells (MCs) in the skin and environmental triggers of the disease. One such trigger is the skin-colonizing yeast Malassezia sympodialis (M. sympodialis). In this study, we investigated the interaction of MC with M. sympodialis. METHODS: Mast cells were generated from peripheral blood CD34(+) progenitor cells of healthy controls (HC) and M. sympodialis-sensitized AE patients. Biopsy specimens were taken from HC and lesional AE skin for immunohistological stainings. RESULTS: The progenitor-derived MCs expressed the macrophage-inducible C-type lectin receptor Mincle, and exposure of these cells to M. sympodialis induced up-regulation of the mRNA expression of Mincle. Furthermore, we demonstrate that, when compared to HC, the progenitor-derived MCs from AE patients (i) contain more intrinsic granule mediators such as histamine, (ii) exhibit enhanced IL-6 release in response to M. sympodialis exposure, and (iii) have an impaired up-regulation of the fungal recognition receptor Dectin-1. In addition, analysis of skin sections from HC and AE patients revealed MCs as the predominant Dectin-1-expressing cell type in the skin. CONCLUSION: Our data indicate that progenitor-derived MCs from AE patients differ from those from HC. Further investigations with skin-derived MCs are necessary to confirm the observed differences which could provide new insights into the pathogenic mechanisms underlying AE.

Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis.

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.

Nickel, cobalt, chromium, palladium and gold induce a mixed Th1- and Th2-type cytokine response in vitro in subjects with contact allergy to the respective metals.

Nickel (Ni), the main cause of contact allergy to metals, induces in vitro production of both Th1- and Th2-type cytokines in peripheral blood mononuclear cells (PBMC) from allergic subjects. Because the knowledge of the cellular immune response to other metals involved in contact allergy has been limited, we investigated the cytokine profile induced by Ni, cobalt (Co), chromium (Cr), palladium (Pd) and gold (Au) in PBMC from patients with patch test reactivity to the respective metals. PBMC from patients with patch test reactivity to Ni, Co, Cr, Au and/or Pd (n = 31) and non-allergic controls (n = 5) were stimulated in vitro with corresponding metal salts. Th1- [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2- (IL-4 and IL-13) type cytokine responses were measured by enzyme-linked immunospot (ELISpot) and/or enzyme-linked immunosorbent assay (ELISA). All metals induced a mixed Th1- and Th2-type cytokine production in PBMC from individual patients with patch test reactivity to the corresponding metal, but not in control PBMC. Significantly higher responses in the patient versus controls were found for Cr (IL-2 and IL-13), Pd (IL-2 and IL-4), Au (IL-13 and IFN-gamma) (all P < 0.05) and Ni (all four cytokines; P < 0.01) but not Co. Overall, 71% (37/52) and 89% (81/91) of the positive and negative patch test reactivities to metals, respectively, were matched by the in vitro reactivity. In conclusion, our data suggest that sensitization to Co, Cr, Pd and Au results in a cellular immune response of a character similar to the mixed Th1- and Th2-type cytokine profile shown previously to be induced by Ni.

Nickel elicits concomitant and correlated in vitro production of Th1-, Th2-type and regulatory cytokines in subjects with contact allergy to nickel.

Nickel (Ni2+) elicits production of functionally distinct cytokines in vitro, but the relation between the cytokine profile and the degree of the allergic reaction in vivo needs to be better defined in order to improve the understanding of the immunological mechanisms involved in contact allergy and to facilitate development of in vitro diagnostics. The aim of the study was to define Th1-type [interferon-gamma (IFN-gamma)], Th2-type [interleukin-4 (IL-4), IL-5 and IL-13] and regulatory (IL-10) cytokine responses to Ni2+ in peripheral blood mononuclear cells (PBMC) from subjects with varying patch test reactivity to Ni2+. The study included subjects with strong (+3), moderate (+2), weak (+1) or negative (controls) patch test reactivity to Ni2+ (n = 10 per group). All +3 and +2 subjects but only three +1 subjects had a clinical history of contact allergy to Ni(2+). Cytokine production of PBMC stimulated with Ni(2+) was determined by enzyme-linked immunospot and/or enzyme-linked immunosorbent assay. Ni2+ elicited significant production of all cytokines in PBMC from patch-test-positive subjects versus controls with a positive correlation between each cytokine and the patch test reactivity as well as with other cytokines. More subjects responded to Ni2+ above cut-off values with Th2-type cytokines as compared with IFN-gamma or IL-10; 100% of +3, 80% of +2, 50% of +1 and 0% of control subjects displayed reactivity to Ni2+ based on IL-4 and IL-13 assays. Despite the prevailing view of Ni2+ allergy as a type-1-mediated condition, the in vivo reactivity to Ni2+ correlated with a mixed Th1-type, Th2-type and regulatory cytokine response to Ni2+in vitro. The results accentuate the importance of type 2 responses in contact allergy and also demonstrate that IL-4 and IL-13 are reliable markers for Ni2+ allergy.

Study of innervation, sensory neuropeptides, and serotonin in murine contact allergic skin.

Density of nerve fibers, axonal growth, calcitonin gene-related peptide (CGRP), and substance P, and serotonin immunoreactivity as well as concentration were all determined in a murine model of contact allergy. Female Balb/c mice were sensitized on the back with oxazolone and 6 days later challenged with the same antigen on the dorsal surface of the ears, while control mice received the vehicle only. Then, 24 hr postchallenge, one ear was processed for immunohistochemical staining, while the other was frozen and processed for gas chromatography-mass spectrometry or radioimmunoassay (RIA). Protein gene product 9.5 (PGP 9.5) positive nerve fibers showed a tendency to increase in inflamed ears versus control ears in epidermis as well as the dermis. Growth-associated protein-43 (GAP-43) positive fibers in the epidermis were increased (p < .01) in inflamed ears, compared with control ears, as was the case for the dermal fibers, indicating increased axonal growth. Total (epidermis and dermis) numbers of CGRP and substance P positive nerve fibers tended to increase in the inflamed skin in contrast to control skin. In contrast, RIA demonstrated a lower (p < .05) concentration of CGRP in the inflamed ears compared with controls and a tendency for substance P to decrease in concentration in eczematous ears versus controls. There was no difference in serotonin concentration, or in the number of serotonin positive mast cells, between the inflamed and control skin, whereas semiquantification of serotonin positive platelets showed an increase in the inflamed (+/+) compared with control ears (+). Our results indicate that 24 hr after being challenged with the antigen, at the peak of murine skin inflammation, axonal growth, sensory neuropeptides, as well as serotonin may be involved.

Impaired responses of peripheral blood mononuclear cells to nickel in patients with nickel-allergic contact dermatitis and concomitant atopic dermatitis.

BACKGROUND: Allergic contact dermatitis (ACD) is pathogenetically dependent on cell-mediated immune responses mediated by type 1 T lymphocytes. Atopic dermatitis (AD), in contrast, occurs as a result of sustained activation of type 2 subsets of T cells. Although atopic patients may become sensitized to various contact allergens, little is known about the influence of atopy on delayed-type hypersensitivity. OBJECTIVES: To investigate the in vitro responses of peripheral blood mononuclear cells (PBMC) to nickel stimulation in groups of atopic and nonatopic patients with patch test-verified nickel ACD. METHODS: Ten nonatopic patients with nickel ACD, 10 patients with nickel ACD and concomitant AD, 10 patients with AD but with no contact allergy, and 10 healthy persons participated in the study. PBMC were cultured in the presence or absence of nickel sulphate, phytohaemagglutinin (PHA) or tetanus toxoid (TT). [(3)H]thymidine incorporation was used to measure the rate of antigen-induced DNA synthesis and enzyme-linked immunosorbent assay was used to measure the production of interleukin (IL)-2 (type 1 cytokine) and IL-5 (type 2 cytokine). RESULTS: Nickel-stimulated PBMC of nickel-allergic patients with AD proliferated significantly less and secreted significantly lower amounts of IL-2 than cells of nonatopic nickel-allergic patients. IL-5 production was also lower in the former group, although the difference was nonsignificant. Moreover, neither the nickel-specific DNA synthesis nor the cytokine production by PBMC of atopic nickel-allergic patients differed significantly from those of healthy control persons and AD patients without contact allergy. Proliferative and secretory responses of PBMC to PHA or TT stimulation differed nonsignificantly between the groups. Nickel-induced IL-2 production correlated well with IL-5 production in nickel-allergic patients regardless of their atopic status. CONCLUSIONS: Our results indicate that PBMC of nickel-allergic patients with concomitant AD are characterized by impaired in vitro proliferative and secretory responses to the contact allergen nickel but not to the mitogen PHA or the recall antigen TT. The type 2 cytokine IL-5 may play a role in the development of ACD.

Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis.

BACKGROUND: Patients with suspected allergic contact dermatitis still have to undergo patch testing for a correct diagnosis. As this has several disadvantages there is a need for additional methods, preferentially those that can be performed in vitro. Objectives To investigate the possibility of diagnosing contact allergy to nickel (Ni2+) using the enzyme-linked immunospot (ELISpot) assay that allows the analysis of cytokines at a single-cell level in ex vivo activated peripheral blood mononuclear cells (PBMC). METHODS: Eleven female patients and nine age- and sex-matched healthy volunteers participated in the study. All patients had a history of nickel allergy and a positive patch test reaction to NiSO4, while the controls' test was negative. PBMC were cultured in the presence or absence of NiCl2. Cell proliferation was measured with [3H]thymidine incorporation, and the number of cytokine-producing cells analysed with the ELISpot assay. RESULTS: The proliferative response of PBMC to Ni2+, expressed as stimulation index, was significantly higher in the nickel-allergic patients than in the control group. Using the ELISpot assay, we found that PBMC from nickel-allergic individuals responded to Ni2+ with significantly greater production of interleukin (IL)-4, IL-5, IL-13 and interferon-gamma, but not IL-12, compared with the healthy controls. The number of IL-4- and IL-5-producing cells correlated with the number of IL-13-producing cells in the nickel-allergic patients, but Ni2+-induced PBMC proliferation did not correlate with the number of cytokine-producing cells for any of the cytokines tested. CONCLUSIONS: Our results indicate that the ELISpot assay could be a tool in the discrimination between nickel-allergic and non-allergic individuals.

Early DNA synthesis and cytokine expression in the nickel activation of peripheral blood mononuclear cells in nickel-allergic subjects.

BACKGROUND AND OBJECTIVE: Nickel sulphate stimulates the proliferation of lymphocytes in nickel-allergic subjects. However, nickel-induced stimulation of lymphocytes from control persons without clinical symptoms of nickel allergy has also been reported. The aim of the present study was to correlate T cell activity, measured by DNA synthesis and the expression of Th1 [interleukin (IL)-2, tumour necrosis factor (TNF)-beta and interferon (IFN)-gamma] and Th2 (IL-4) cytokines, in short-term (up to 72 h) culture of nickel-stimulated peripheral blood mononuclear cells (PBMC) from nickel-allergic patients compared to control subjects. METHODS: DNA synthesis was measured by the incorporation of [methyl-(3)H]thymidine. The production of IL-2, TNF-beta, IFN-gamma and IL-4 was measured in the supernatants of the cultures by ELISA. In situ hybridization for mRNA was performed using oligonucleotide probes for IL-4, IFN-gamma and TNF-beta in cell smears. RESULTS: Already after 24 h and proceeding through the remaining culture period, there was a statistically significant (p<0.001) difference in the concentrations of IL-2 between patients and controls. There was a significant (p<0.01) difference in DNA synthesis (stimulation index) between the patients and control subjects at 72 h, and also at the same time a difference in the concentrations of TNF-beta (p<0.05) and IL-4 (p<0.01). In the in situ hybridization study, TNF-beta was found to be the only one of the studied cytokines that differed between the nickel-allergic and control subjects, this difference being most evident at 72 h (p<0.01). CONCLUSIONS: Our results indicate a difference between nickel-allergic and non-allergic subjects in the synthesis of DNA and production of cytokines when PBMC are stimulated by nickel sulphate, and IL-2 may be regarded as a critical and early-occurring cytokine.

Inhibitory effect of vasoactive intestinal peptide on the challenge phase of allergic contact dermatitis in humans.

There is increasing evidence that the nervous system has influence on the immune response. The effect of vasoactive intestinal peptide (VIP) and of serotonin and its antagonists on the challenge phase of allergic contact dermatitis in humans were tested. The substances were injected intracutaneously shortly before and 6 h after application of patch tests with nickel sulphate in nickel-allergic patients and the test areas were measured after a further 18 h. Biopsy specimens were also taken for immunohistochemistry. The diameter of the nickel sulphate-induced test reaction was significantly reduced after injection of VIP at 10(-6)-10(-5) mol/l, but was not affected by serotonin or ketanserin. Also tested was the influence of the substances on the response of peripheral blood mononuclear cells from nickel-allergic subjects to nickel sulphate, when added at the same time as the antigen. No effect on the cell proliferative rate was seen, except for an inhibitory effect of serotonin and its antagonists at 10(-5)-10(-4) mol/l. VIP, at 10(-5) mol/l and serotonin at 10(-4) mol/l stimulated the secretion of interferon gamma. The interleukin-2 soluble receptor secretion was slightly stimulated by 5-HT at 10(-4) mol/l and by ketanserin at 10(-6) mol/l. In conclusion, our results show that when injected intracutaneously in the challenge phase of allergic contact dermatitis, VIP has an inhibitory effect, which might be explained by enhanced leukocyte production of interferon gamma.

Serotonin in human allergic contact dermatitis. An immunohistochemical and high-performance liquid chromatographic study.

Allergic contact dermatitis (ACD) is a common clinical condition leading to considerable morbidity. We have recently demonstrated that ketanserin, a serotonin antagonist, significantly inhibits nickel sulphate-induced ACD. Furthermore, serotonin-immunoreactive (IR) cells have previously been demonstrated in normal human cutaneous melanocytes. To further elucidate the role of serotonin in cutaneous contact hypersensitivity, we compared ACD involved skin and uninvolved skin from nickel-allergic patients, and normal skin from healthy volunteers, for the presence of serotonin-like immunoreactive cells using immunohistochemistry. In addition, serotonin concentrations in ACD involved and uninvolved skin were compared by high-performance liquid chromatography (HPLC). In the skin of normal healthy volunteers, the serotonin-IR cells were situated in the basal layer of the epidermis. In uninvolved skin the cells were also situated in the basal layer, but they were more numerous and the immunofluorescence intensity was greater. In involved skin, the IR cells were fewer and they were found higher up in the epidermis. Also, the configuration of these cells was different: they showed enlarged and elongated dendrites as well as dendritic spines. The serotonin antiserum-labelled cells in ACD involved skin were also NKI-beteb positive (the latter is known as a reliable marker of melanocytes). The concentration of serotonin in involved skin was significantly higher than that in uninvolved skin in ACD patients (P < 0.05). Taken together, our previous and present results indicate that serotonin plays an important role in ACD. The basal epidermal serotonin-IR cells are more dendritic in ACD, and are found more superficial in the epidermis, where they might release their content of serotonin, thereby influencing the inflammatory process.

Vasoactive intestinal polypeptide in allergic contact dermatitis: an immunohistochemical and radioimmunoassay study.

Vasoactive intestinal polypeptide (VIP) is a neuropeptide with immunomodulatory properties. To elucidate the possible role of VIP in the pathophysiology of cutaneous contact hypersensitivity, we compared involved with uninvolved skin of allergic contact dermatitis (ACD) from nickel-allergic patients. Assays included quantification of VIP-immunoreactive (VIP-IR) nerve fibres and cells bearing immunoreactivity for VIP1 and VIP2 receptors in skin biopsy specimens, and of the concentration of VIP-like immunoreactivity (VIP-LI) in extracts of biopsy specimens. VIP-IR nerve fibres were found in the deeper part of the dermis close to sweat glands and hair follicles. No difference in the presence of VIP-IR nerve fibres was found between involved and uninvolved skin of ACD. VIP1 and VIP2 receptor immunoreactivity was seen on keratinocytes in the basal layer of the epidermis, with no difference between involved and uninvolved skin. Staining was also seen on vessel walls and mononuclear cells in the dermis. The highest staining intensity in the mononuclear cells was noted with the antibodies against the VIP2 receptor. While most of the mononuclear cells were stained in uninvolved skin, a minority of the cells showed a positive signal in involved skin. The concentration of VIP-LI in uninvolved skin was 1.53+/-0.790 pmol/g and in involved skin 1.41+/-0.735 pmol/g. It is concluded that there is no significant difference in either the distribution of VIP-IR fibres or the concentration of VIP-LI between involved and uninvolved skin of ACD. However, the number of dermal mononuclear cells showing VIP2 receptor immunoreactivity in skin of ACD was reduced.