Maya Zimmermann

Interleukins, from 1 to 37, and interferon-γ: Receptors, functions, and roles in diseases

Advancing our understanding of mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections could lead to effective and targeted therapies. Subsets of immune and inflammatory cells interact via ILs and IFNs; reciprocal regulation and counter balance among T(h) and regulatory T cells, as well as subsets of B cells, offer opportunities for immune interventions. Here, we review current knowledge about ILs 1 to 37 and IFN-γ. Our understanding of the effects of ILs has greatly increased since the discoveries of monocyte IL (called IL-1) and lymphocyte IL (called IL-2); more than 40 cytokines are now designated as ILs. Studies of transgenic or knockout mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided important information about IL and IFN functions. We discuss their signaling pathways, cellular sources, targets, roles in immune regulation and cellular networks, roles in allergy and asthma, and roles in defense against infections.

Distinct regulation of IgE, IgG4 and IgA by T regulatory cells and toll-like receptors

Background:  Allergic diseases are characterized by the activation of the immune system and formation of immunoglobulin (Ig)E antibodies against normally innocuous environmental antigens, whereas IgG4 and IgA represent noninflammatory and blocking antibody isotypes. The T helper 2 (Th2) cells induce and T regulatory (Treg) cells suppress several features of allergic inflammation. Our aim was to investigate the role of allergen‐specific T regulatory type 1 (Tr1) cells and CD4+CD25+ Treg cells and toll‐like receptors (TLRs) on IgE, IgG4 and IgA production.

IL-32 is expressed by human primary keratinocytes and modulates keratinocyte apoptosis in atopic dermatitis

BACKGROUND Keratinocyte (KC) apoptosis is an important mechanism of eczema and spongiosis in patients with atopic dermatitis (AD) and is mediated by IFN-gamma, which is secreted by T(H)1 cells. IL-32 is a proinflammatory cytokine that is involved in the inflammatory processes of rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn disease. Recently, it was shown that upregulation of IL-32 induces apoptosis. OBJECTIVE The aim of the study was to investigate the expression and function of IL-32 in patients with AD. METHODS The expression of IL-32 in KCs was analyzed by means of RT-PCR, ELISA, and flow cytometry. Transfections of small interfering RNA were performed in primary KCs, and apoptosis was analyzed by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, annexin-V, and 7-amino actinomycin D stainings. Immunofluorescence stainings were used to detect IL-32 in skin biopsy specimens, and serum levels of IL-32 were analyzed by means of ELISA. RESULTS We report that IL-32 is expressed in human primary KCs on stimulation with IFN-gamma, TNF-alpha, and T(H)1 cells in contrast to T(H)2, regulatory T (Treg), or T(H)17 cells, which showed no effect. Transfection of primary KCs and artificial skin equivalents with small interfering RNA to IL-32, which resulted in a clear decrease in IL-32 expression, significantly reduced KC apoptosis. Immunofluorescence staining demonstrated that IL-32 was expressed in AD lesional skin, whereas it was present in neither skin biopsy specimens from healthy donors nor in lesional skin from patients with psoriasis. Serum levels of IL-32 from patients with AD correlated with disease severity, but increased serum levels of IL-32 were also detected in asthmatic patients. CONCLUSION The present study demonstrates KCs as a source of IL-32, which modulates KC apoptosis and contributes to the pathophysiology of AD.

Identification of germline susceptibility loci in ETV6-RUNX1-rearranged childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant disease of the white blood cells. The etiology of ALL is believed to be multifactorial and likely to involve an interplay of environmental and genetic variables. We performed a genome-wide association study of 355 750 single-nucleotide polymorphisms (SNPs) in 474 controls and 419 childhood ALL cases characterized by a t(12;21)(p13;q22) — the most common chromosomal translocation observed in childhood ALL — which leads to an ETV6–RUNX1 gene fusion. The eight most strongly associated SNPs were followed-up in 951 ETV6-RUNX1-positive cases and 3061 controls from Germany/Austria and Italy, respectively. We identified a novel, genome-wide significant risk locus at 3q28 (TP63, rs17505102, PCMH=8.94 × 10−9, OR=0.65). The separate analysis of the combined German/Austrian sample only, revealed additional genome-wide significant associations at 11q11 (OR8U8, rs1945213, P=9.14 × 10−11, OR=0.69) and 8p21.3 (near INTS10, rs920590, P=6.12 × 10−9, OR=1.36). These associations and another association at 11p11.2 (PTPRJ, rs3942852, P=4.95 × 10−7, OR=0.72) remained significant in the German/Austrian replication panel after correction for multiple testing. Our findings demonstrate that germline genetic variation can specifically contribute to the risk of ETV6–RUNX1-positive childhood ALL. The identification of TP63 and PTPRJ as susceptibility genes emphasize the role of the TP53 gene family and the importance of proteins regulating cellular processes in connection with tumorigenesis.

Mechanisms of IFN-γ–induced apoptosis of human skin keratinocytes in patients with atopic dermatitis

BACKGROUND Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). OBJECTIVE The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. METHODS Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. RESULTS We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. CONCLUSION Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.

TNF-like weak inducer of apoptosis (TWEAK) and TNF-α cooperate in the induction of keratinocyte apoptosis

BACKGROUND Activation of skin keratinocytes followed by their apoptotic death leads to eczema and spongiosis formations in patients with atopic dermatitis (AD). TNF-like weak inducer of apoptosis (TWEAK) binds to its receptor, fibroblast growth factor-inducible 14 (Fn14), and controls many cellular activities, including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. OBJECTIVE The aim of the study was to investigate the role of TWEAK and Fn14 in the formation of eczema in patients with AD. METHODS Primary keratinocytes were isolated from nonlesional skin from patients with AD and psoriasis and from normal skin of healthy donors. Apoptosis analysis was performed by using annexin V/7-aminoactinomycin D and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. The expression and regulation of TWEAK, TNF-α, Fn14, TNF receptor (TNFR) 1, and TNFR2 were measured by means of RT-PCR, flow cytometric analysis, and ELISA. TWEAK and Fn14 expression of lesional AD and psoriatic skin and normal control skin was analyzed by using immunohistochemistry and immunofluorescence. RESULTS TWEAK and TNF-α cooperate in the induction of apoptosis in primary keratinocytes obtained from patients with AD, patients with psoriasis, and healthy subjects and in artificial skin equivalents. TNFR1 and Fn14 were the main receptors involved. TWEAK upregulates TNF-α expression in primary keratinocytes, whereas TNF-α did not affect the expression of TWEAK and its receptors. High TWEAK expression was observed in AD lesions but not in psoriatic lesions or normal skin. Fn14 was highly expressed in the lesional skin of patients with AD and patients with psoriasis and in healthy control skin. CONCLUSION The high expression of TWEAK in lesional AD skin contributes to the difference in keratinocyte apoptosis and lesional formation between AD and psoriasis.

Dual nature of T cell―epithelium interaction in chronic rhinosinusitis

BACKGROUND T-cell infiltration of submucosa, release of proinflammatory cytokines leading to epithelial activation, and contributions to inflammation are observed in chronic rhinosinusitis (CRS). OBJECTIVES Molecular mechanisms and kinetics of T-cell interaction with sinus epithelium leading to activation followed by subsequent apoptosis of epithelial cells were the focus of the current study. METHODS Primary human sinus epithelial cells and T cells generated from sinus tissues of healthy individuals and patients with CRS with or without allergy and sinus tissue biopsies were characterized in terms of activation (surface marker expression, cytokine production via real-time PCR, confocal microscopy, ELISA) and apoptosis (annexin V/7-amino-actinomycin D staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, receptor expression by flow cytometry, confocal microscopy) of epithelial cells. RESULTS Primary human sinus epithelial cells isolated from patients with CRS were at an activated state with upregulated expression of HLA-DR, IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and TNF-related apoptosis-inducing ligand (TRAIL) compared with healthy individuals. The expressions of these chemokines, HLA-DR, TRAIL, and TNF receptor 2 were significantly induced by IFN-gamma, whereas TRAIL receptor 4 was downregulated. Epithelial cells started to undergo apoptosis 48 hours after IFN-gamma stimulation when the transcription of proinflammatory cytokines and chemokines decreased to initial levels. The essential factors for sinus epithelial apoptosis were T(H)1 cells and IFN-gamma. Epithelial apoptosis was enhanced by Fas-Fas-ligand and TRAIL-TRAIL receptor 2 interactions. Remarkable apoptosis of epithelial cells and shedding was observed in CRS in situ. CONCLUSION Epithelial cell interaction with activated T cells is a biphasic phenomenon in CRS. Initially activated T cells lead to activation and induction of proinflammatory functions of epithelial cells, and thereafter their apoptotic death, resulting in no more contribution to inflammation, takes place.

Annexin V/7-AAD staining in keratinocytes.

Annexin V/7-amino-actinomycin staining is a convenient way to discriminate early apoptosis from late apoptosis and necrosis. Early apoptotic cells express phosphatidylserines (PS) on the outer leaflet of the plasma membrane. PS can be stained by labeled annexin V. Late apoptotic cells and necrotic cells lose their cell membrane integrity and are permeable to vital dyes such as 7-AAD (DNA intercalator).