Karin Kissel

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88–97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI‐TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N‐linked carbohydrates. Following N‐terminal amino acid sequencing and NB1‐specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614‐bp cDNA for NB1. COS‐7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311‐bp sequence was identified to be the entire coding region. The 5′ and 3prime; untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N‐terminal extracellular protein with two cysteine‐rich domains, three N‐linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl‐phosphatidylinositol attachment (o) site. Database searches revealed homology to Ly‐6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly‐6 snake toxin superfamily.

Human platelets target dendritic cell differentiation and production of proinflammatory cytokines

BACKGROUND:  Dendritic cells (DCs) are the most potent antigen‐presenting cells that initiate and regulate immune responses. They are unique in their feature to produce bioactive interleukin (IL)‐12, a major proinflammatory cytokine connecting innate and adaptive immunity. Platelets (PLTs) are highly reactive components of the circulatory system with fundamental importance in hemostasis and innate immunity. Recently, immunomodulatory capacities of single specific human PLT‐derived products on DC effector functions were identified. To improve the understanding of PLT‐DC interactions, this study investigates the influence of intact resting and activated PLTs on DC phenotype and key functions.

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency ≥0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency ≥0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.

Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells

Carriage of the TNF -308 A allele (rs1800629 A) has been associated with increased serum TNF-alpha levels, the development of sepsis syndrome, and fatal outcome, in severely traumatized patients (Menges et al., 2008 [1]). Herein, we analysed the putative allelic imbalance of TNF-alpha release from myeloid cells. Circulating peripheral blood cells from healthy human blood donors (n=104) and monocyte-derived macrophages (n=158) were analysed for their ex vivo capacity of TNF-alpha expression. Our findings indicate that carriage of the TNF -308 A allele is not associated with high TNF-alpha expression in circulating human leucocytes and monocyte-derived macrophages. Other cellular sources, e.g. tissue-resident cells like mast cells and/or tissue specific macrophages might be the cellular source of high TNF-alpha serum levels shortly after trauma.