Lena Lundeberg

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)‐γ] 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‐γ) (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.

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.

Nanovesicles from Malassezia sympodialis and Host Exosomes Induce Cytokine Responses – Novel Mechanisms for Host-Microbe Interactions in Atopic Eczema

Background Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. Objective To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. Methods Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. Results We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-α responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-α responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-α but not IL-4 in undepleted PBMC. Conclusions Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE.

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‐γ (IFN‐γ)], 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 Ni2+. Cytokine production of PBMC stimulated with Ni2+ 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‐γ 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.

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

To cite this article: Ribbing C, Engblom C, Lappalainen J, Lindstedt K, Kovanen PT, Karlsson MA, Lundeberg L, Johansson C, Nilsson G, Lunderius‐Andersson C, Scheynius A. Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin‐1 expression. Allergy 2011; 66: 110–119.

Expression of serotonin receptors in allergic contact eczematous human skin

Abstract. The expression of the serotonin (5-HT) receptors 5-HT1AR, 5-HT2AR and 5-HT3R was investigated in allergic contact eczematous human skin by an indirect fluorescence method. 5-HT1AR expression was found in basal epidermal NK1-beteb-positive cells, which were more elongated and showed longer dendritic processes in contact eczematous skin than in control skin. Immunoreactivity for 5-HT1AR was also found in the upper part of the epidermis, with no difference between eczematous and control skin. 5-HT1AR expression was also found in 33.3±6.5% and 63.7±11.3% of papillary dermal mononuclear cells in inflamed skin and control skin, respectively (P<0.001), as well as in vessel walls. Some of these mononuclear cells were tryptase-positive, and found in both eczematous and control skin. 5-HT2AR-positive cells were found in the upper part of the epidermis in eczematous skin, but were more evenly distributed in the epidermis of control skin. In addition, inflammatory dermal mononuclear cells and vessel walls showed immunoreactivity for this receptor. 5-HT3R expression was found in the basal epidermal layer of eczematous and control skin. These findings indicate a plasticity in the effects of serotonin, especially regarding 5-HT1AR, in allergic contact eczematous skin.

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)-β and interferon (IFN)-γ] 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-3H]thymidine. The production of IL-2, TNF-β, IFN-γ 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-γ and TNF-β 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-β (p < 0.05) and IL-4 (p < 0.01). In the in situ hybridization study, TNF-β 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

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

Abstract 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.

IL-18 skews the invariant NKT-cell population via autoreactive activation in atopic eczema

Atopic eczema (AE) is a chronic relapsing inflammatory skin disease where the commensal yeast Malassezia can act as a microbial trigger factor. Malassezia activates human DC to produce IL‐18, an innate cytokine that is elevated in serum of AE patients; however, the precise role of IL‐18 in human AE etiology is unknown. Herein, we investigated the effect of IL‐18 on the human invariant NKT (iNKT) cell compartment in AE. We found that IL‐18 was a potent activator of human iNKT‐cells and promoted a pro‐inflammatory CD1d‐dependent response, even in the absence of exogenous ligands. Chronic activation via IL‐18 on the other hand was inhibitory and skewed the iNKT‐cell pool by selectively suppressing CD4+ iNKT‐cells. This was mimicked in AE patients where the proportion of CD4+ iNKT‐cells was reduced in peripheral blood and coincided with elevated plasma levels of IL‐18. Furthermore, a reduced CD4+ iNKT‐cell pool was associated with elevated IgE levels in plasma, and the plasma levels of IL‐18 correlated with both total IgE and disease severity in the AE patients. Based on these findings, we propose that IL‐18‐mediated activation and subsequent dysregulation of the CD1d‐restricted iNKT‐cells plays a role in the pathogenesis of human AE.

IgE Sensitization Profiles Differ between Adult Patients with Severe and Moderate Atopic Dermatitis.

Background Atopic dermatitis (AD) is a complex chronic inflammatory disease where allergens can act as specific triggering factors. Aim To characterize the specificities of IgE-reactivity in patients with AD to a broad panel of exogenous allergens including microbial and human antigens. Methodology Adult patients with AD were grouped according to the SCORAD index, into severe (n = 53) and moderate AD (n = 126). As controls 43 patients were included with seborrhoeic eczema and 97 individuals without history of allergy or skin diseases. Specific IgE reactivity was assessed in plasma using Phadiatop®, ImmunoCap™, micro-arrayed allergens, dot-blotted recombinant Malassezia sympodialis allergens, and immune-blotted microbial and human proteins. Results IgE reactivity was detected in 92% of patients with severe and 83% of patients with moderate AD. Sensitization to cat allergens occurred most frequently, followed by sensitization to birch pollen, grass pollen, and to the skin commensal yeast M. sympodialis. Patients with severe AD showed a significantly higher frequency of IgE reactivity to allergens like cat (rFel d 1) and house dust mite (rDer p 4 and 10), to Staphylococcus aureus, M. sympodialis, and to human antigens. In contrast, there were no significant differences in the frequencies of IgE reactivity to the grass pollen allergens rPhl p 1, 2, 5b, and 6 between the two AD groups. Furthermore the IgE reactivity profile of patients with severe AD was more spread towards several different allergen molecules as compared to patients with moderate AD. Conclusion We have revealed a hitherto unknown difference regarding the molecular sensitization profile in patients with severe and moderate AD. Molecular profiling towards allergen components may provide a basis for future investigations aiming to explore the environmental, genetic and epigenetic factors which could be responsible for the different appearance and severity of disease phenotypes in AD.