Minoo Ghannadan

Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis

OBJECTIVE To analyze the immunophenotype and functional properties of synovial mast cells (SyMC) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS Synovial tissue was obtained from 25 patients with RA and 17 patients with OA. Tissue was dispersed by enzymatic digestion using collagenase. Surface receptor expression on SyMC was analyzed by monoclonal antibodies (MAb) and indirect immunofluorescence staining. Histamine release experiments were performed using the MC agonist recombinant human (rHu) stem cell factor (SCF), the anaphylatoxin rHuC5a, and an anti-IgE antibody. RESULTS In both groups of patients (RA and OA), SyMC were found to react with MAb to IgE, SCF receptor (c-kit, CD117), as well as CD antigens likewise expressed in lung MC (CD9, CD29, CD33, CD43, CD44, CD45). However, a significantly increased proportion of SyMC from RA patients reacted with MAb against C5a receptor (C5aR; CD88), compared with SyMC from OA (mean +/- SD percentage of SyMC reacting with CD88 MAb S5/1 in RA 27.5 +/- 8.6% versus 0.0% in OA, and with CD88 MAb W17/1 in RA 58.3 +/- 15.2% versus 12.5 +/- 15.0% in OA; P < 0.05). Furthermore, in RA, significant histamine release from SyMC above control was induced by rHuC5a, anti-IgE, and rHuSCF, whereas SyMC in OA released histamine after stimulation with anti-IgE and rHuSCF, but not rHuC5a. CONCLUSION SyMC exhibit phenotypic and functional properties similar to MC in other tissues. In patients with RA, but not OA, SyMC express significant amounts of C5aR (CD88) and release histamine in response to rHuC5a. These results indicate a role for SyMC and C5a/C5aR in the pathogenesis of RA.

Increased Sensitivity of Histidinemic Mice to UVB Radiation Suggests a Crucial Role of Endogenous Urocanic Acid in Photoprotection

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Detection of novel CD antigens on the surface of human mast cells and basophils.

Background: Mast cells (MC) and basophils are effector cells of allergic reactions. Growth and function of these cells are regulated by a network of cytokines, other ligands, and respective cell surface membrane receptors. Methods: In the present study, we examined the expression of novel CD antigens on human lung MC, skin MC, blood basophils, the MC line HMC-1, and the basophil cell line KU-812. Expression of surface antigens was analyzed by monoclonal antibodies (mAbs) and indirect immunofluorescence staining techniques. Results: Primary MC were found to react with mAbs against KIT (CD117), the signal regulatory protein SIRP-α (CD172a), and the ectoenzyme E-NPP3 (CD203c). Human basophils were found to express large amounts of E-NPP3 and lower levels of Siglec-5 (CD170), CXCR4 (CD184) and SIRP-α. The HMC-1 cell line was recognized by mAbs against SIRP-α, CXCR4, endothelial protein C receptor (CD201) and E-NPP3. KU-812 cells were found to react with mAbs against E-NPP3, CXCR4 and glycophorin C (CD236R), but did not react with mAb against endothelial protein C receptor. Most of the other CD antigens tested disclosed negative results. Conclusions: In summary, our data provide further evidence that MC and basophils express a unique composition of surface antigens. The use of novel CD antibodies may help to isolate MC and basophils and to study their functional properties.

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Primary sources and immunological prerequisites for sST2 secretion in humans

AIMS Serum levels of the soluble growth stimulation gene-2 (sST2) are elevated in heart and pulmonary diseases. However, the relationship of the sST2/interleukin (IL)-33 axis and its triggers as well as its organ distribution is still not known. This study was thus designed to investigate the cellular origin and regulation of sST2 and IL-33 in vitro and in vivo. METHODS AND RESULTS sST2 and IL-33 gene expression and protein secretion were analysed in pooled organ-specific cDNAs and in primary cell cultures, respectively, by RT-PCR and ELISA technology. The strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines IL-1alpha, IL-1beta, and tumour necrosis factor alpha as well as supernatants of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via an NFkappaB-dependent mechanism. In addition, LPS stimulation in humans in vivo induced a short-term inflammatory response that was followed by a massive enhancement of sST2 secretion. CONCLUSION These results identify the primary sources and inflammatory triggers for the enhancement of sST2 secretion and demonstrate a relationship between inflammation and the secretion of a bioactive member of the IL-1R family, both in vitro and in vivo.

On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts

Several emerging treatment concepts for myeloid neoplasms are based on novel drugs targeting cell surface antigens, signalling pathways, or critical effector molecules. Systemic mastocytosis is a haematopoietic neoplasm that behaves as an indolent myeloproliferative disease in most patients, but can also present as aggressive disease or even as an acute leukaemia. In patients with aggressive disease or mast cell leukaemia, the response to conventional therapy is poor in most cases, and the prognosis is grave. Therefore, a number of attempts have been made to define novel treatment strategies for these patients. One promising approach may be to identify novel targets and to develop targeted drug therapies. In this article, we support the notion that neoplastic mast cells indeed express a number of potential molecular targets including immunoreactive CD antigens, the microphthalmia transcription factor (MITF), and members of the Bcl‐2 family. In addition, the tyrosine kinase receptor KIT and downstream signalling pathways have been proposed as targets of a specific pharmacological intervention. A particular challenge is the disease‐related D816V‐mutated variant of KIT, which is resistant against diverse tyrosine kinase inhibitors including STI571, but may be sensitive to more recently developed targeted compounds. The therapeutic potential of target‐specific approaches in malignant mast cell disorders should be evaluated in forthcoming clinical trials in the near future.

Inactivation of VEGF in mammary gland epithelium severely compromises mammary gland development and function

To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo‐alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up‐regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC‐labeled albumin injected intrave‐nously(i.v.) into lactating mice extravasated rapidlyand accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombi‐nant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium‐derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.— Rossiter, H., Barresi, C., Ghannadan, M., Gruber, F., Mildner, M., Fodinger, D., Tschachler, E. Inactivation of VEGF in mammary gland epithelium severely compromises mammary gland development and function. FASEB J. 21, 3994–4004 (2007)

Stratum corneum‐derived caspase‐14 is catalytically active

Caspase‐14, a cysteine protease with restricted tissue distribution, is highly expressed in differentiated epidermal keratinocytes. Here, we extracted soluble proteins from stratum corneum (SC) of human epidermis and demonstrate that the extract cleaves tetrapeptide caspase substrates. The activity decreased to below 10% when caspase‐14 was removed by immunodepletion showing that caspase‐14 is the predominant caspase in SC. In contrast to normal SC, where caspase‐14 was present exclusively in its processed form, incompletely matured SC of parakeratotic skin from psoriasis and seborrheic dermatitis contained both procaspase‐14 and caspase‐14 subunits. Fractionation of extract from parakeratotic SC revealed that the peak caspase activity coeluted with processed caspase‐14 but not with procaspase‐14. Our results suggest that during regular terminal keratinocyte differentiation, endogenous procaspase‐14 is converted to caspase‐14 subunits that are catalytically active in the outermost layers of normal human skin.

Detection of differentiation- and activation-linked cell surface antigens on cultured mast cell progenitors.

Background:  Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34+ hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation‐ and activation‐linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC.

Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.