No effect of anti-interleukin-5 therapy (mepolizumab) on the atopy patch test in atopic dermatitis patients.

BACKGROUND: The atopy patch test (APT) is an in vivo model to study the induction of eczema by inhalant allergens in atopic dermatitis (AD) patients. Mepolizumab is a monoclonal antibody to interleukin-5, which reduces peripheral blood eosinophils. Previously, we reported that mepolizumab treatment did not result in clinical improvement in AD. The current study investigates the effect of mepolizumab therapy on the APT in the same patients. METHODS: Mepolizumab treatment was given at days 0 and 7 in a double-blind placebo-controlled design. The APT was applied at days -2, 0, 14 and 28. Clinical evaluation of each APT was conducted 48 h after application at days 0, 2, 16 and 30. Skin biopsies were taken at days 0, 2 and 16 for eosinophil counts. RESULTS: The mepolizumab-treated group showed no significant reduction in macroscopic outcome of the APT. Tissue eosinophils were reduced in the mepolizumab-treated group at day 16 compared with placebo; however, this was not significant. CONCLUSION: Mepolizumab therapy cannot prevent the eczematous reaction induced by the APT. Furthermore, the influx of tissue eosinophil numbers in the APT is not significantly inhibited after mepolizumab treatment compared with placebo, despite a significant reduction in peripheral blood eosinophils.

Salivary gland and peripheral blood T helper 1 and 2 cell activity in Sjögren's syndrome compared with non-Sjögren's sicca syndrome.

OBJECTIVES: To investigate whether differences in T helper (Th) 1 and Th2 cell activity in salivary glands ("local") or ("peripheral") blood can discriminate between Sjögren's syndrome (SS) and non-Sjögren's sicca syndrome (nSS-sicca). Additionally, to study relationships of local and peripheral Th cell activities with each other and with disease activity measures. METHODS: 62 sicca patients (32 with SS, 30 with nSS-sicca) were studied. Local Th1 (interferon gamma (IFNgamma)) and Th2 (interleukin (IL) 4) activity were determined using immunohistochemistry. T cell production of IFNgamma and IL4 in peripheral blood (PB) was determined by ELISA. Erythrocyte sedimentation rate (ESR) and serum IgG were considered disease activity measures. RESULTS: ESR and serum IgG were higher in patients with SS than in patients with nSS-sicca. Local Th1 cell activity was higher and PB Th1 activity lower in patients with SS than in those with nSS-sicca. Th2 cell activity did not differ significantly between the patient groups. The ratio IFNgamma/IL4 was higher in salivary glands and lower in PB in patients with SS than in patients with nSS-sicca. Local and peripheral Th1 and Th2 cell activities correlated with ESR and serum IgG levels. ESR, serum IgG, and local or peripheral Th1 or Th2 cell activity did not discriminate between patients with SS and nSS-sicca. CONCLUSIONS: An imbalance between Th1 and Th2 activity in sicca patients is clearly related to the severity of disease, but cannot be used to distinguish between patients with SS and those with nSS-sicca.

Atopy patch test in patients with atopic eczema/dermatitis syndrome: comparison of petrolatum and aqueous solution as a vehicle.

BACKGROUND: The atopy patch test (APT) is an in vivo model to study the induction of eczema by inhalant allergens. This study was designed to compare two commonly used APT methods. METHODS: In the first method, the allergen is dissolved in aqueous solution, which is applied on tape-stripped skin. In the second method, the allergen is dissolved in petrolatum and applied without tape stripping. Thirteen patients with atopic dermatitis sensitized to inhalant allergens were patch tested using both methods. Reactions were evaluated macroscopically and microscopically after 48 h. RESULTS: Nine out of 13 patients displayed a positive reaction for both methods. One patient had a positive APT for the aqueous method alone and three for the petrolatum method alone. Reactions were significantly stronger when using the petrolatum method. Histological evaluation of the nine patients positive for both methods showed no significant differences in number of eosinophils, T-cells and neutrophils. CONCLUSION: The APT using the petrolatum vehicle induces a higher number of positive reactions and is significantly stronger relative to the APT using allergen in aqueous vehicle. The cellular influx in both test methods is comparable. Both methods can be used to study the mechanisms in the induction of eczema by inhalant allergens.

Heterogeneity within tissue-specific macrophage and dendritic cell populations during cutaneous inflammation in atopic dermatitis.

BACKGROUND: Macrophages and dendritic cells may play a role in chronicity of atopic dermatitis (AD); however, so far only limited data are documented on the distribution of these cells in the skin during cutaneous inflammation. OBJECTIVES: To gain better insight into the presence and distribution of macrophage and dendritic cell (sub)populations in acutely and chronically inflamed skin of AD patients. METHODS: Chronic inflammatory reactions were studied in lesional AD skin biopsies; the atopy patch test was used as a model for the initiation of AD lesions, representing acute inflammation. To determine the number and phenotype of different dermal macrophage and dendritic cell populations immunohistochemistry and digital imaging were used. RESULTS: There was an increase in macrophage numbers in acutely and chronically inflamed AD skin, whereas absolute dendritic cell numbers were unchanged, compared with non-lesional AD skin. Furthermore, phenotypically heterogeneous and overlapping macrophage and dendritic cell populations were present in inflamed AD skin. The classic macrophage marker CD68 and prototypic dendritic cell marker CD1a could bind to the same cell subpopulation in the dermis of inflamed AD skin. Mannose receptors were expressed mainly by macrophages in inflamed AD skin. CONCLUSIONS: In this study we observed changes in macrophage number and phenotype during cutaneous inflammation in AD. Dendritic cell numbers did not change; however, phenotypically dendritic cell and macrophage subpopulations showed increasing overlap during inflammation in AD skin. We show for the first time that within tissue-specific macrophage populations further subpopulations are present, and that monocyte-derived cells may express markers for both dendritic cells and macrophages. Our results point to the existence of a heterogeneous pool of macrophage/dendritic cell-like cells, from which subpopulations of dermal macrophages and dendritic cells arise.

Interleukin 4 and interferon-gamma expression of the dermal infiltrate in patients with erythroderma and mycosis fungoides. An immuno-histochemical study.

BACKGROUND: Erythroderma, or generalized erythema of the skin, may result from different causes. At present it is unclear whether the underlying patho-mechanisms that lead to erythroderma are identical or different depending on the original disease. The aim of this study was to investigate the dermal cytokine profile in different types of erythroderma and mycosis fungoides. METHODS: Snap-frozen skin biopsy specimens from 33 patients with erythroderma were studied. Thirteen had idiopathic erythroderma, 7 erythrodermic atopic dermatitis, 5 Sézary syndrome and 8 had erythroderma from miscellaneous causes. We also studied 6 patients with mycosis fungoides (5 plaques and 1 tumor) and 5 healthy non-atopic volunteers. The biopsies were immunohistochemically stained for interleukin 4 (IL-4) and interferon gamma (IFN-gamma). All positive cells for IL-4 and IFN-gamma in the dermis were counted and the number of positive cells was calculated per mm2. IL-4/IFN-gamma ratio was calculated for each biopsy. RESULTS: The patients with idiopathic erythroderma, atopic dermatitis and miscellaneous erythroderma, all showed more IFN-gamma-positive cells than IL-4-positive cells in the dermis. The median IL-4/ IFN-gamma ratio for these three groups was 0.6, 0.9 and 0.45, respectively. These differences were not statistically significant. All patients with Sézary syndrome however, showed more IL-4-positive cells than IFN-gamma-positive cells. The median IL-4/IFN-gamma ratio was 1.8, which is significantly higher than in the other groups p<0.05). In mycosis fungoides roughly the same number of cells expressed IL-4 and IFN-gamma. The median IL-4/IFN-gamma ratio was 1.0, which is significantly lower than in Sézary syndrome (p<0.05). CONCLUSIONS: The dermal infiltrate in patients with Sezary syndrome mainly shows a T-helper 2 (Th2) cytokine profile, this in contrast to T-helper 1 (Th1) cytokine profile in benign reactive erythroderma. This indicates that although a relative uniform clinical picture of erythroderma is obvious, a different patho-mechanisms may be underlying.

Expression of VCAM-1, ICAM-1, E-selectin, and P-selectin on endothelium in situ in patients with erythroderma, mycosis fungoides and atopic dermatitis.

BACKGROUND: Erythroderma may result from different causes. At present it is unclear whether the patho-mechanisms that lead to these different types of erythroderma are identical or different. Adhesion molecules and their ligands play a major role in endothelial-leukocyte interactions, which affect the binding, transmigration and infiltration of lymphocytes and mononuclear cells during inflammation, injury, or immunological stimulation. The aim of this study was to investigate the adhesion molecule expression on endothelial cells in erythroderma in situ. METHODS: Snap-frozen skin biopsy specimens from 23 patients with erythroderma were studied. Eight had idiopathic erythroderma, 5 erythrodermic atopic dermatitis, 4 Sézary syndrome and 6 had erythroderma from miscellaneous causes. As a control we studied skin specimens from 10 patients with mycosis fungoides, 5 patients with atopic dermatitis and 5 healthy non-atopic volunteers. To determine adhesion molecule expression on endothelial cells in situ, sections were immuno-histochemically double stained with biotinylated Ulex Europaeus agglutinin 1 as a pan-endothelial cell marker, and for the adhesion molecules VCAM-1, ICAM-1, E-, and P-selectin. All double- and single-stained blood vessels in the dermis were counted. RESULTS: Mean endothelial expression in erythroderma was as follows: VCAM-1 51.4%, ICAM-1 70.1%, E-selectin 43.5%, and P-selectin 52.6%. There was no statistical difference between different groups of erythroderma. Mean expression of all adhesion molecules tested, was in Sézary syndrome higher than in mycosis fungoides albeit not significant. In erythrodermic atopic dermatitis only VCAM-1 expression was significantly higher than in lesional skin of atopic dermatitis. No differences were observed in expression of the other three adhesion molecules. CONCLUSIONS: There is no difference regarding adhesion molecule expression on endothelial cells between different types of erythroderma.

Modulation of the atopy patch test reaction by topical corticosteroids and tar.

BACKGROUND: Pharmacologic studies in atopic eczema (AE) are difficult to standardize. Patients with AE differ in the stage of their skin disease (acute, subacute, chronic). OBJECTIVE: This study was designed to assess macroscopic and microscopic effects of pretreatment with topical glucocortico-steroids (GCSs) and tar on the atopy patch test (APT) reaction in patients with atopic eczema. METHODS: Nonlesional skin of the back of patients with AE (n = 6) was treated for 3 weeks at 3 different sites with triamcin-olonacetonide 0.1% in cetamacrogol ointment (GCSs), pix liquida 10% in cetamacrogol ointment (tar), and cetamacrogol ointment (vehicle), respectively. APTs were performed, and biopsy specimens were taken from all these sites (time = 0 and 24 hours) for immunohistochemical analysis. RESULTS: Treatment with both GCSs and tar was able to reduce the macroscopic outcome of the APT reaction. Furthermore, both treatment modalities had an almost equally inhibiting effect on the influx of T cells, eosinophils, and CD1(+), RFD1(+), IFN-gamma(+), and IL-4(+) cells, as well as on the percentage of vessels expressing the adhesion molecules vascular cell adhesion molecule 1 and E-selectin in response to epicutaneous aeroallergen challenge. CONCLUSION: Although both treatments significantly reduced the various cellular constituents of allergic inflammation, all cell types remained present. In addition, this study shows that the APT can be used to evaluate the effect of topical anti-inflammatory treatments on allergic inflammation in patients with AE.

Expression of Fc receptors for IgG during acute and chronic cutaneous inflammation in atopic dermatitis.

Atopic dermatitis is an allergic skin disease characterized by elevated total and antigen-specific serum IgE and IgG4 levels. In acute and chronic cutaneous inflammation, large cellular infiltrates including T cells, dendritic cells and macrophages are found, especially in the dermis. These cells play an important part in the regulation of local inflammatory reactions. Receptors binding IgG (FcgammaR) are involved in dendritic cell and macrophage function. In this study, we examined the in vivo distribution and cellular expression of the three classes of leucocyte FcgammaR in human skin during acute and chronic cutaneous inflammation in atopic dermatitis. Atopy patch test skin was used as a model for acute inflammation in atopic dermatitis, while chronic lesional skin was used to investigate FcgammaR expression in chronically inflamed skin. In atopy patch test sites no increase in the number of CD1a+ dendritic cells and a slight increase in macrophages compared with non-lesional skin was observed. Our results showed increased expression of FcgammaRI (CD64) and FcgammaRIII (CD16) in acutely inflamed skin as well as in chronically inflamed lesional skin, compared with healthy and non-lesional atopic dermatitis skin. FcgammaRI was expressed by RFD1+, RFD7+ and CD68+, but not by CD1a+ dermal dendritic cells. RFD1+ dendritic cells and CD68+ macrophages were the main FcgammaRIII-expressing cells during the acute inflammatory reaction. The significant increase in expression of FcgammaRIII (CD16) and FcgammaRI (CD64) probably results from upregulation of the receptors on resident cells. Insight into the presence of FcgammaR+ cells in human skin during inflammation is important both for our understanding of skin immune reactions and the development of new therapeutic concepts.

Clinical and immunologic variables in skin of patients with atopic eczema and either positive or negative atopy patch test reactions.

BACKGROUND: Epicutaneous application of aeroallergens induces a positive atopy patch test (APT) response in about 50% of patients with atopic eczema (AE) and sensitization for these allergens. OBJECTIVE: To elucidate the mechanisms determining the outcome of the APT, the following questions were addressed. Are there differences in clinical features between patients with AE who have positive versus negative APT responses? Is a macroscopically negative APT response also histologically negative, and if so, are there differences in clinically noninvolved skin between the two groups regarding (1) the sensitivity toward an irritant, (2) the composition of cellular infiltrate, (3) the presence of aeroallergen-specific T cells, and (4) the number of IgE(+) cells? METHODS: Punch biopsy specimens from both house dust mite patch tested and the clinically noninvolved skin of patients with AE who have positive APT responses (n = 10) and negative APT responses (n = 10) and those from the normal skin of atopic individuals without AE (n = 10) and nonatopic volunteers (n = 10) were analyzed by using immunohistochemistry with mAbs against eosinophil cationic protein, IgE, the high-affinity receptor for IgE, and CD3 and CD25 mAbs. Furthermore, T-cell lines were propagated from noninvolved skin of all patient and control groups. The T-cell lines were tested for house dust mite specificity. RESULTS: Negative APT sites were immunohistochemically similar to clinically noninvolved AE skin. There were no significant differences between patients with AE who had positive and negative APT results regarding either clinical features, the composition of cellular infiltrate, or the presence of allergen-specific T cells in clinically noninvolved skin. However, differences were observed regarding the presence of IgE on epidermal CD1a(+) cells. CONCLUSION: Our results indicate that a positive APT reaction requires the presence of epidermal IgE(+) CD1a(+) cells in clinically noninvolved skin, but that also other, as yet unknown, discriminatory factors are involved.

Adhesion molecule expression on skin endothelia in atopic dermatitis: effects of TNF-alpha and IL-4.

BACKGROUND: Atopic dermatitis (AD) is characterized by skin infiltrates of leukocytes, such as lymphocytes and eosinophils. OBJECTIVE: To describe the mechanisms determining this inflammatory process, we have analyzed expression of adhesion molecules and their regulation on skin endothelial cells (ECs). METHODS: Expression of adhesion molecules on ECs was analyzed by immunohistochemistry by using Ulex europaeus agglutin 1 as a pan-endothelial marker. RESULTS: Vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin were not found in skin of nonatopic individuals, whereas expression of these surface molecules was observed in nonlesional skin of patients with AD and was even more pronounced in lesional skin or after epicutaneous application of aeroallergen. Induction of adhesion molecule expression was examined on both macrovascular ECs from human umbilical cord vein (HUVECs) and human microvascular ECs (HMEC-1) from skin. TNF-alpha very potently upregulated adhesion molecule expression in vitro on both EC cell types. To verify the in vivo relevance of TNF-alpha, we performed TNF-alpha staining in the skin. TNF-alpha was observed in the dermis of nonatopic skin, both in chymase-containing mast cells and CD68+ macrophages. The increase in the number of TNF-alpha-containing cells was concomitant with the increase in adhesion molecule expression in the skin of patients with AD. IL-4 is supposed to be important in atopic diseases because of its IgE- and VCAM-1-inducing properties. However, IL-4 addition failed to induce VCAM-1 expression on HMEC-1, although in the same set of experiments, a clear induction of VCAM-1 expression by IL-4 on HUVECs was demonstrated. Flow cytometry revealed the absence of 11-4 receptor alpha-chains on HMEC-1 and their presence on HUVECs. Immunohistochemistry examination on skin sections showed no binding of the IL-4R alpha-chain antibodies to ECs. CONCLUSION: We conclude that adhesion molecule expression is increased in the skin of patients with AD. Most probably, this increased expression is not a (direct) effect of IL-4 on skin endothelium, but other cytokines, such as TNF-alpha, might be responsible for this increased adhesion molecule expression. Continuous adhesion molecule expression may facilitate T-cell extravasation in a nonantigen-specific manner, thus explaining the presence of increased T-cell numbers in nonlesional skin of patients with AD.

Increased numbers of CD4+ and CD8+ T cells in lesional skin of cats with allergic dermatitis.

The aim of this study was to characterize T cells in the skin of cats with an allergic dermatitis histologically compatible with atopic dermatitis, since T cells play an important role in the pathogenesis of atopic dermatitis in humans. We observed a significantly greater number of T cells in lesional skin of domestic short-haired cats with allergic dermatitis (n = 10; median age 5.8 years) than in the skin of healthy control animals (n = 10; median age 5.0 years). In the skin of the healthy control animals, one or two CD4+ cells and no CD8+ cells were found. A predominant increase of CD4+ T cells and a CD4+/CD8+ ratio (mean +/- SD: 3.9 +/- 2.0) was found in the lesional skin of 10 cats with allergic dermatitis. The CD4+/CD8+ cell ratio in the skin of healthy control animals could not be determined because of the absence of CD8+ cells. The CD4+/CD8+ cell ratio in the peripheral blood of 10 cats with allergic dermatitis (mean +/- SD: 1.9 +/- 0.4) did not differ significantly from that in 10 healthy control animals (2.2 +/- 0.4). The CD4+/CD8+ cell ratio and predominance of CD4+ T cells in the lesional skin of cats with allergic dermatitis is comparable to that found in atopic dermatitis in humans. In addition, the observed increase of CD4+ T cells in the nonlesional skin of cats with allergic dermatitis compared to the skin of healthy cats is similar to what is seen in humans. Cytokines produced by T cells and antigen-specific T cells are important mediators in the inflammatory cascade resulting in atopic dermatitis in humans. This study is a first step to investigate their role in feline allergic dermatitis.

Nonspecific T-cell homing during inflammation in atopic dermatitis: expression of cutaneous lymphocyte-associated antigen and integrin alphaE beta7 on skin-infiltrating T cells.

Atopic dermatitis (AD) is a chronic skin disorder, characterized by infiltration of activated memory CD4+ T cells into skin. A model to study the onset of allergic inflammation in a patient with AD is the atopy patch test (APT), in which, by epicutaneous application of aeroallergen, an eczematous reaction is induced in 50% of sensitized patients with AD. Extravasation of T cells into skin is thought to be critically dependent on expression of the surface molecule cutaneous lymphocyte-associated antigen (CLA), which recognizes and binds its ligand E-selectin on endothelium. We studied the dynamics of expression of CLA and the gut homing receptor alphaE beta7 (HML-1) on T cells in the skin of patients with AD and in APT reactions and nickel and sodium lauryl sulfate patch test reactions by means of immunohistochemical double staining of skin biopsy specimens. The results show an increase in the number of CD3+ T cells in the lesional skin of patients with AD, APT reactions, and nickel and sodium lauryl sulfate patch test reactions as compared with nonlesional skin of the same patients and nonatopic individuals. In contrast, the percentages of CLA+ T cells in the lesional skin of patients with AD, in the APT reactions, and in sodium lauryl sulfate and nickel patch test reactions were decreased. In addition, we found a marked expression of alphaE beta7 by T cells present in skin, indicating a nonspecific influx of T cells during allergic skin inflammation. We propose that during allergic skin inflammation CLA expression is not a prerequisite for cutaneous T-cell infiltration. CLA expression may be important for T cells to extravasate from blood into skin during immune surveillance or for retention of allergen-specific T cells in skin.

Immunophenotyping of skin-infiltrating T-cell subsets in dogs with atopic dermatitis.

Atopic dermatitis in dogs has many clinical features that are identical to those of the same disorder in man. To investigate the pathogenesis of this disease in dogs and the possibility of similarities to the pathogenesis in humans we compared the presence and ratio of CD4+ and CD8+ T-cells in the cutaneous infiltrate of lesional and non-lesional skin of atopic dogs with that in the skin of healthy dogs. In ten dogs with atopic dermatitis and ten healthy dogs the skin was biopsied at the predilection sites for atopic dermatitis and histological sections were immunohistochemically stained for CD4 and CD8. The staining showed an increase in CD4+ and CD8+ T-cells in canine lesional atopic skin, with a predominance of CD4+ T-cells in the epidermis. In non-lesional atopic skin there was also an infiltration with CD4+ and CD8+ T-cells, but without predominance of CD4+ T-cells. The results in the separate predilection sites did not differ substantially from the mean results. These observations indicate further similarities in the immunopathogenesis of atopic dermatitis in dogs and humans, which may have consequences for the control of atopic dermatitis in dogs and contributes to a possible role of the dog as a model for human atopic dermatitis.

Evaluation of the atopy patch test and the cutaneous late-phase reaction as relevant models for the study of allergic inflammation in patients with atopic eczema.

OBJECTIVE: The study was designed to evaluate the atopy patch test (APT) and the late-phase reaction (LPR) after intracutaneous allergen injection as models for the study of allergic inflammation in atopic eczema. METHODS: Immunocytochemistry was used to analyze skin biopsy specimens from sites of APTs and LPRs at 2 and 24 hours and to compare these with lesional and nonlesional skin of patients with atopic eczema. RESULTS: A lack of neutrophil infiltration in specimens from both the APT and lesional skin sites was observed, whereas neutrophils were abundantly present in the specimens from LPR sites. With double-staining techniques it was demonstrated that the few neutrophils present in specimens from APT sites and in lesional skin were mostly located in intravascular areas, whereas in the LPR specimens they were located predominantly in extravascular areas. Eosinophils infiltrated at an earlier time point in the LPR as compared with the APT. Furthermore, there was a decrease of intact mast cells in the LPR site compared with the APT sites and lesional skin. No significant difference in T-cell number was observed between the two tests. Upregulation of E-selectin expression on endothelial cells occurred at an earlier time point in the LPR as compared with the APT. CONCLUSION: There are important differences in cellular infiltrate between the APT and the LPR. The close macroscopic and microscopic similarities between the specimens from APT sites and lesional skin of patients with atopic eczema support the argument that the APT is a more valid in vivo model with which to study allergic inflammation in atopic eczema than the LPR.

Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial TH2 response to a TH1 response in situ: an immunocytochemical study.

In the pathogenesis of atopic dermatitis (AD), IgE plays an important role; and TH2 cells, producing IL-4, have been ascribed a key role in allergic diseases such as AD. To investigate the role of TH subpopulations in the onset and continuation of AD, we performed atopy patch tests (APTs) with house dust mite allergen in patients with AD. Punch biopsy specimens were taken from the APT site, and sections were immunocytochemically double-stained for IL-4 and interferon-gamma together with different membrane markers. This provides a unique model for studying the kinetics of the TH0, TH1, and TH2 responses in situ. The results show that in lesional skin interferon-gamma-positive cells predominate over IL-4-positive cells. The interferon-gamma-positive cells are mainly CD3+ and, in particular, CD4+ cells; the remainder are CD8+, RFD-1+, and RFD-7+ cells. The IL-4-positive cells are exclusively CD4+ T cells; no eosinophils or mast cells were found to stain for IL-4. With regard to the TH cell response, a clear dichotomy of the eczematous response to allergen in skin was observed. In the initiation phase IL-4 production by TH2 and TH0 cells is predominant over interferon-gamma production by TH1 and TH0 cells. In the late and chronic phases the situation is reversed and interferon-gamma production by TH1 and TH0 cells predominates over IL-4 production by TH2 and TH0 cells. Understanding the relationship between the observed biphasic response and clinical manifestation of AD is important for the development of therapeutic strategies.