Theo Thepen

A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis.

Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intensely pruritic subacute and chronic eczematous plaques, the pathogenesis of which appears to involve a complex interplay of genetic, pharmacological, environmental and psychological factors. Here, Markus Grewe and colleagues propose that the development of skin lesions in AD patients results from sequential activation of T helper 2 (Th2)- and Th1-type cells.

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.

Evaluation of variables influencing the outcome of the atopy patch test

BACKGROUND The number of positive atopy patch test (APT) results in patients with atopic eczema (AE) varies in different studies, probably because of different test techniques. Variables that may influence the outcome of the APT were evaluated. METHODS APTs were performed in 84 patients with AE, 30 control patients with atopic disease, and 85 healthy volunteers, with house dust mite and grass pollen allergens in concentrations of 100, 1000, 10,000, and 100,000 allergenic units/ml. The influence of 0, 10, or 20 tape strippings was investigated. The tests were performed on the back and/or the antecubital fossa and evaluated after 20 minutes and 24, 48, and 72 hours. In all patients the total and specific serum IgE levels were measured. RESULTS The maximal number of positive APT results were obtained under the following conditions: an allergen concentration equal to 10,000 allergenic units/ml, 10 tape strippings and readings at 24 and 48 hours. Positive APT results were observed in five of 30 control patients with atopic disease and in none of 85 healthy volunteers. Statistically significantly higher total and allergen-specific serum IgE levels were found in the group of patients with AE with positive APT results. CONCLUSIONS We recommend the previously described conditions to get an optimal method for APT. The correlation between the APT and the total and specific serum IgE suggests an important role for IgE in the reaction mechanism behind the APT.

Regulation of IgE production in pre‐sensitized animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen*

Intratracheal inoculation of dichloromethylene diphosphonate encapsulated in liposomes leads to the rapid accumulation of this drug in alveolar macrophage (AM) phagolysosomes, and the death of the majority of these cells over the ensuing 24–48 hr. The technique is highly selective for phagocytes and has no detectable side‐effects on other cells in the lung. The present experiments demonstrate that following AM depletion, pre‐sensitized animals respond to aerosol challenge via secondary serum IgE (but not IgG) responses, and the accumulation of large numbers of allergen‐specific and non‐specific antibody forming cells in respiratory tract regional lymph nodes and in lung and airway tissues; the latter comprise both IgE and IgG plasma cells, which were detected in the approximate ratio of 2.5:1. Moreover, aerosol challenged AM‐depleted animals develop large mononuclear cell infiltrates in the lung and airways, which includes a substantial CD4+ T‐cell component. These results suggest a major role for AM in regulating the magnitude of secondary IgE responses to inhaled allergen.

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.

Adhesion molecule expression on skin endothelia in atopic dermatitis: Effects of TNF-α and IL-4

Abstract 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-α very potently upregulated adhesion molecule expression in vitro on both EC cell types. To verify the in vivo relevance of TNF-α, we performed TNF-α staining in the skin. TNF-α was observed in the dermis of nonatopic skin, both in chymase-containing mast cells and CD68 + macrophages. The increase in the number of TNF-α–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 IL-4 receptor α-chains on HMEC-1 and their presence on HUVECs. Immunohistochemistry examination on skin sections showed no binding of the IL-4R α-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-α, 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. (J Allergy Clin Immunol 1998;102:461-8.)

Nonspecific T-cell homing during inflammation in atopic dermatitis: Expression of cutaneous lymphocyte-associated antigen and integrin αEβ7 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.

Increased specific IgE production in lungs after the induction of acute stress in rats

Under normal conditions, immune responses in the lung are suppressed, resulting in low antigen-specific serum antibody titers after intratracheal immunization of rodents. We attempted to determine whether mild inescapable electric foot-shock stress, immediately followed by intratracheal instillation of trinitrophenyl-conjugated keyhole limpet hemocyanin, could enhance the primary and secondary humoral immune responses in male Wistar rats. This acute emotional stress paradigm and subsequent intratracheal administration of trinitrophenyl-conjugated keyhole limpet hemocyanin resulted in a dramatic increment of trinitrophenyl-specific immunoglobulin isotype concentrations in serum, including IgE. Analysis of paratracheal lymph node and spleen tissue for trinitrophenyl-specific antibody-forming cells revealed that the increased specific antibody concentrations resulted from a local production in the paratracheal lymph nodes. Because the presence of aeroantigen-specific IgE is associated with diseases such as allergic asthma, we advocate the view that acute emotional stress can contribute to the onset or severity of allergic asthma by lowering the threshold of induction of aeroantigen-specific IgE production in the lungs.

Migration of Alveolar Macrophages from Alveolar Space to Paracortical T Cell Area of the Draining Lymph Node

In this report we studied the translocation of fluorescent particulate antigens to the draining lymph node, and the migration of fluorescent labeled alveolar macrophages (AM) and peritoneal macrophages (PM) in mice. The results show that intratracheally (IT) instilled particulate antigens translocate to the paracortical T cell area of the draining lymph node. When labeled AM were injected IT, they were found to migrate from the alveolar space into the paracortical T cell area of the draining lymph node. An identical localisation was found after IT injection of labeled PM. When either labeled AM, or PM were injected into the peritoneal cavity, a different migration pattern was observed. Via this route the labeled macrophages migrated to the subcapsular sinus and medulla of the draining lymph nodes. It is shown that the migrated cells are not dendritic cells (DC) present in the cell preparations. A possible role for the micro-environment of the injection site, and the significance of the specific migration pattern of AM is discussed.

Peritoneal Cell Labelling: A Study on the Migration of Macrophages and Dendritic Cells Towards the GUT

In this study the migration of peritoneal cells was investigated by a fluorescence labelling technique. We found that peritoneal cells migrate to the subcapsular sinus and medulla of the parathymic lymph node (PTLN) and paratracheal lymph node (PTrLN). It was also observed that fluorescence labelled cells possibly granulocytes, macrophages and dendritic cells were found in the B cell follicles of Peyer patches and the dome area after intraperitoneal (ip) labelling. The implication of the migration of antigen presenting cells to the gut on the mucosal immune response is discussed.