Maria Tengvall Linder

Atopic Eczema/Dermatitis Syndrome and Malassezia

Atopic dermatitis is a chronic multifactorial inflammatory skin disease, which has had a marked increase in prevalence during the last decades. Recently, a new nomenclature was recommended where the term ‘atopic eczema/dermatitis syndrome’ (AEDS) should be used to reflect the heterogeneity in this group of patients and where those patients without measurable IgE reactivity should be classified as either ‘nonallergic AEDS’ or ‘non-IgE-associated allergic AEDS’. For nearly 20 years it has been discussed whether the opportunistic yeast Malassezia, previously designated Pityrosporum, is a contributing factor to AEDS. Today there are several reports that demonstrate specific serum IgE or positive skin prick test and/or atopy patch test reactions to Malassezia in patients with AEDS. Several IgE-binding components have been identified in extracts of Malassezia ranging in molecular mass between 10 and 100 kD. The genes for nine Malassezia allergens with molecular weights ranging from 14 to 36 kD have hitherto been identified and cloned. Six of them are now produced by recombinant techniques and used in diagnostic tests. At present the genus Malassezia is subdivided into seven different species, which all have been isolated from human skin. The respective contribution of different Malassezia spp. to AEDS and in what proportion they share allergens remains to be clarified. We summarize here data that Malassezia can play a role in eliciting and maintaining eczema in patients with AEDS.

The prevalence of Malassezia yeasts in patients with atopic dermatitis, seborrhoeic dermatitis and healthy controls

Cultures for Malassezia yeasts were taken from both normal-looking skin and lesional skin in 124 patients with atopic dermatitis, 16 patients with seborrhoeic dermatitis and from normal skin of 31 healthy controls. Positive Malassezia growth was found in fewer patients with atopic dermatitis (56%) than in patients with seborrhoeic dermatitis (88%) or in healthy controls (84%, p<0.01). In the patients with atopic dermatitis, fewer positive cultures were found in lesional (28%) than in non-lesional skin (44%, p<0.05), while positive cultures were found in 75% of both lesional and non-lesional skin of patients with seborrhoeic dermatitis (not significant). M. sympodialis dominated in patients with atopic dermatitis (46%) and in healthy controls (69%). In patients with seborrhoeic dermatitis both M. sympodialis and M. obtusa were cultured in 43%. A Malassezia species extract mixture would increase the possibility of detecting IgE sensitization to Malassezia in patients with atopic dermatitis.

Itch and inflammation induced by intradermally injected interleukin-2 in atopic dermatitis patients and healthy subjects

To explore the pruritogenic and inflammatory effects of cytokines, a single dose of 20 μg recombinant human interleukin-2 was injected intradermally into eight patients with atopic dermatitis and eight healthy controls. The study was double-blind and randomized with glucose as a negative control. The effects were evaluated by recording local itch and erythema over 72 h and by examining skin biopsies taken at 24 h and 72 h. In patients and controls, interleukin-2 provoked a low-intensity local itch with maximal intensity between 6 h and 48 h and erythema with maximal extension between 12 h and 72 h. In the atopic dermatitis patients, these reactions tended to appear earlier and were less pronounced than in the healthy controls. Interleukin-2 induced dermal mononuclear cell infiltrates consisting mainly of CD3+ cells. A majority of the T cells were CD4+. The number of dermal CD25+, HLA-DR+ and ICAM-1+ cells was also increased at the interleukin-2 injection sites. In the epidermis, interleukin-2 induced spongiosis and exocytosis as well as HLA-DR+ and ICAM-1+ keratinocytes. The microscopic findings tended to be more prominent at 72 h than at 24 h in both groups, but with a somewhat slower onset in the atopic dermatitis patients. In conclusion, a single intradermal injection of interleukin-2 induced local itch, erythema, dermal T-cell infiltrates, spongiosis, exocytosis and activation of keratinocytes both in atopic dermatits patients and in healthy controls.

Not only Th2 cells but also Th1 and Th0 cells express CD30 after activation.

To investigate whether the CD30 molecule, expressed only by a minority of T and B cells, defines a subtype of T helper cells, Pityrosporum orbiculare‐specific CD4+ T cell clones were assessed for CD30 protein and gene expression. The clones were defined as Th1, Th0, and Th2 according to their cytokine mRNA profile detected by reverse transcription PCR (RT‐PCR). The kinetics of CD30 expression after OKT3 (anti‐CD3) stimulation was analyzed by flow cytometry, immunocytochemistry, and RT‐PCR. OKT3 activation induced a high expression of CD30 in cells of both Th1 and Th0 as well as Th2 type after 1‐3 days. A difference between the clones was noted in that the Th2 clones remained highly positive in CD30 expression, whereas expression in the other clones started to decline from day 3. These data indicate that CD30 is expressed in activated CD4+ T cells of all three subtypes, and that the expression is sustained in Th2 cells.

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.

Elevated Levels of IgG and IgG4 to Malassezia Allergens in Atopic Eczema Patients with IgE Reactivity to Malassezia

Background: The opportunistic yeast Malassezia is considered to be one of the factors that can contribute to atopic eczema (AE). Elevated serum IgE levels, T-cell proliferation and positive skin prick test (SPT) and atopy patch test (APT) reactions to Malassezia are found among AE patients. Methods: Sera from 127 AE patients, 14 patients with seborrheic dermatitis (SD) and 33 healthy controls were investigated for IgE and IgG4 to M. sympodialis extract and four recombinant Malassezia allergens; rMala s 1, rMala s 5, rMala s 6, and rMala s 9. In addition, IgG to the recombinant allergens was analyzed. The IgG and IgG4 levels were compared to IgE levels and in vivo reactions (SPT and APT) to Malassezia. Results: AE patients with serum IgE levels >0.35 kU/l to M. sympodialis extract had significantly higher IgG4 levels to M. sympodialis extract than AE patients without detectable serum IgE to M. sympodialis extract, SD patients and healthy controls. Among the AE patients with and without detectable serum IgE to M. sympodialis extract, respectively, there were no differences in IgG4 levels between patients with positive or negative in vivo reactions to M. sympodialis extract. IgG4 to the rMala s allergens was almost exclusively found among patients with IgE to the same allergen. Within the four tested rMala s allergens, most IgG4 reactions were found to rMala s 6, an allergen with homology to cyclophilin. Conclusions: Elevated serum IgG4 to M. sympodialis extract accompanies elevated serum IgE to the extract. This is further confirmed by the association between IgG/IgG4 and IgE to recombinant Malassezia allergens.

Elevated Peripheral Allergen-Specific T Cell Response Is Crucial for a Positive Atopy Patch Test Reaction

Background: Atopic eczema is a chronic inflammatory skin disease in which several subgroups of cases can be identified. Atopy patch testing (APT) reveals allergen sensitization also in atopic eczema patients devoid of detectable allergen-specific IgE, suggesting the importance of factors other than IgE in the reaction. Here we investigate the relationship between APT reactions and allergen-specific peripheral IgE and T cell reactivity in atopic eczema patients. Methods: Adult patients with atopic eczema (n = 64) and healthy controls (n = 24) were analyzed for reactivity to Malassezia sympodialis extract by APT, measurement of specific plasma IgE and in vitro determination of the frequency of allergen-reactive peripheral blood mononuclear cells producing interleukin-4 and interleukin-5 using the ELISpot method. Results: When combining the results of the APT, IgE measurements and the ELISpot analyses, reactivity to M. sympodialis was found in a majority of the atopic eczema patients (69%), whereas the healthy controls were negative throughout. T cell reactivity to M. sympodialis, manifested by production of both interleukins 4 and 5, was highly predictive for a positive APT reaction and displayed a strongly positive correlation with the APT score. In contrast, the allergen-specific IgE levels did not predict the APT outcome, and no correlation could be found between the IgE levels and the APT score. Conclusion: Peripheral allergen-specific T helper 2 cell-mediated reactivity appears to be required for a positive APT reaction to M. sympodialis. The diagnostic potential of measuring peripheral allergen-specific T cell responses should be considered in atopic eczema.