Jürgen Grabbe

Human skin mast cells rapidly release preformed and newly generated TNF‐α and IL‐8 following stimulation with anti‐IgE and other secretagogues

Abstract: Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF‐α and IL‐8 as well as the pro‐allergic cytokines IL‐4, IL‐5 and IL‐13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE‐dependent or IgE‐independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF‐α and IL‐8 following activation with either anti‐IgE, SCF, substance P, compound 48/80 or A23187. This release was dose‐ and time‐dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF‐α and IL‐8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL‐4, IL‐5 or IL‐13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro‐inflammatory cytokines like TNF‐α and IL‐8 following IgE‐dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL‐4, IL‐5 or IL‐13 in these settings.

Treatment of severe pemphigus with protein A immunoadsorption, rituximab and intravenous immunoglobulins.

Backgroundu2002 Pemphigus is a life‐threatening autoimmune blistering disease usually treated with high‐dose corticosteroids and other immunosuppressants. However, this regimen may prove inadequate in severe cases and cause dangerous side‐effects. While protein A immunoadsorption (PAIA) induces a rapid remission in severe pemphigus, the disease usually recurs once the treatment is stopped. In contrast, anti‐CD20 antibody rituximab has a delayed onset of action but may lead to a long‐term remission of pemphigus.

Cutaneous manifestations in patients with mastocytosis: Consensus report of the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology.

Cutaneous lesions in patients with mastocytosis are highly heterogeneous and encompass localized and disseminated forms. Although a classification and criteria for cutaneous mastocytosis (CM) have been proposed, there remains a need to better define subforms of cutaneous manifestations in patients with mastocytosis. To address this unmet need, an international task force involving experts from different organizations (including the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology) met several times between 2010 and 2014 to discuss the classification and criteria for diagnosis of cutaneous manifestations in patients with mastocytosis. This article provides the major outcomes of these meetings and a proposal for a revised definition and criteria. In particular, we recommend that the typical maculopapular cutaneous lesions (urticaria pigmentosa) should be subdivided into 2 variants, namely a monomorphic variant with small maculopapular lesions, which is typically seen in adult patients, and a polymorphic variant with larger lesions of variable size and shape, which is typically seen in pediatric patients. Clinical observations suggest that the monomorphic variant, if it develops in children, often persists into adulthood, whereas the polymorphic variant may resolve around puberty. This delineation might have important prognostic implications, and its implementation in diagnostic algorithms and future mastocytosis classifications is recommended. Refinements are also suggested for the diagnostic criteria of CM, removal of telangiectasia macularis eruptiva perstans from the current classification of CM, and removal of the adjunct solitary from the term solitary mastocytoma.

Regulation of mediator secretion in human basophils by p38 mitogen-activated protein kinase: phosphorylation is sensitive to the effects of phosphatidylinositol 3-kinase inhibitors and calcium mobilization

Although human basophils modulate allergic diseases by secreting histamine, leukotriene C4, interleukin (IL)‐4, and IL‐13, the intermediary signals controlling the release of these mediators are poorly understood. Here, we show that p38 mitogen‐activated protein kinase (MAPK) crucially affects basophil activation following stimulation with various secretagogues. Phosphorylation of p38 MAPK occurred within 5 min following anti‐immunoglobulin (Ig)E stimulation, but was more rapidly activated in basophils stimulated with formyl‐Met‐Leu‐Phe or A23187. Additionally, activation of p38 MAPK to the above stimuli was dependent on extracellular influx and intracellular mobilization of calcium. SB 203580, a specific p38 MAPK inhibitor, blocked anti‐IgE‐induced secretion of all basophil mediators and reduced not only p38 MAPK, but also extracellular signal‐regulated kinases 1 and 2 activity, whereas the MAPK antagonist, PD 098059, did not affect p38 MAPK. IgE‐dependent activation of p38 MAPK and MKK3/6 was affected by LY 294002 and wortmannin, suggesting that these kinases are targets for phosphatidylinositol 3 kinase (PI 3‐K). We conclude that p38 MAPK is a pivotal regulator of basophil function downstream of PI 3‐K activation and calcium mobilization.

Inhibitors of PI 3-kinase and MEK kinase differentially affect mediator secretion from immunologically activated human basophils.

Effects of inhibitors of PI 3‐kinase and MEK kinases were investigated on histamine, leukotriene C4 (LTC4), and cytokine release from human basophils stimulated with anti‐IgE. The PI 3‐kinase antagonists wortmannin (> 10 nM) and LY 294002 (> 1 μM) strongly inhibited anti‐IgE‐induced release of all mediators by 40–100%. This was contrasted by the effects of the MEK kinase inhibitor PD 098059, which weakly inhibited histamine, interleukin (IL)‐4, and IL‐13 release but was substantially more efficacious at blocking LTC4 production (> 70% at 10 μM). Previous studies have shown that arachidonic acid synthesis is controlled by MEK kinases. We observed that wortmannin, LY 294002, and PD 098059 reduce basophil ERK‐1,2 activation, thus implying that, with regard to arachidonic acid metabolism, MEK kinases are a downstream target for PI‐3‐kinase. Our results demonstrate a universal regulatory role played by PI 3‐kinases in basophil mediator production and release, whereas MEK kinase signaling is largely limited to controlling arachidonic acid metabolism. J. Leukoc. Biol. 65: 883–890; 1999.

Expression of SCF splice variants in human melanocytes and melanoma cell lines : potential prognostic implications

Stem cell factor (SCF), the ligand for c-Kit, is known to regulate developmental and functional processes of haematopoietic stem cells, mast cells and melanocytes. Two different splice variants form predominantly soluble (sSCF or SCF-1) and in addition some membrane-bound SCF (mSCF or SCF-2). In order to explore the prognostic significance of these molecules in melanoma, total SCF, SCF splice variants and c-Kit expression were studied in normal skin melanocytes and in 11 different melanoma cell lines, using reverse transcription polymerase chain reaction, immunocytochemistry and enzyme-linked immunosorbent assay. Nine of the 11 melanoma cell lines expressed SCF-1 mRNA, only two of them SCF-2, and these two also SCF-1. Coexpression of both SCF-1 and c-Kit was noted in five cell lines, and only one cell line as well as normal melanocytes expressed both SCF-1 and SCF-2 as well as c-Kit. Corresponding results were obtained on immunocytochemical staining. Of three exemplary melanoma cell lines studied, two expressing SCF mRNA also released SCF spontaneously and on stimulation, whereas the line lacking SCF and c-kit mRNA (SK-Mel-23) failed to do so. These data demonstrate thus that melanoma cell lines, particularly those known to metastasize in vivo, lose the ability to express SCF-2 mRNA, suggesting that this molecule may serve, next to c-Kit, as a prognostic marker for malignant melanoma.

Urticaria: Its History-Based Diagnosis and Etiologically Oriented Treatment

INTRODUCTIONnThe term urticaria refers to any of a group of distinct skin conditions that are characterized by itchy, wheal-and-flare skin reactions (hives). In spontaneous urticaria, the most common type, the hives seem to arise without provocation.nnnMETHODSnSelective review of the literature, including current guidelines.nnnRESULTSnSpontaneous urticaria is divided into acute (lasting less than six weeks) and chronic types. The pathognomonic itching, hives, and angioedema arise by the same mechanism--cutaneous mast cell activation and release of histamine and other mediators of inflammation--in both acute and chronic urticaria, but these two disorders have different etiological profiles. The underlying cause of acute urticaria cannot be identified in about half of all cases. Chronic urticaria, which is much rarer, is usually caused by autoreactivity, chronic infection, or intolerance to food additives. If the condition persists after the underlying cause has been treated or eliminated, non-sedating antihistamines are the agents of first choice for symptomatic treatment.nnnDISCUSSIONnUnlike acute urticaria, which is self-limited and should be treated symptomatically, chronic urticaria should be treated by the identification and elimination of underlying causes, which is usually curative.

Calcineurin Antagonists Differentially Affect Mediator Secretion, p38 Mitogen Activated Protein Kinase and Extracellular Signal Regulated Kinases from Immunologically Activated Human Basophils.

Background Basophils participate in allergic diseases by invading affected tissues and secreting histamine, leukotriene (LT)C4, IL‐4 and IL‐13 following FcεRI cross‐linking. A reduction of basophil mediator production is therefore of considerable therapeutical interest. Macrolactam derivatives, which inhibit calcineurin activation, may be candidates for antiallergic therapy as they reduce both symptoms of inflammatory skin disease in animal models and mast cell degranulation.

Early IgE-dependent release of IL-4 and IL-13 from leukocytes is restricted to basophils: a comparison with other granulocytes and mononuclear cells

We have recently shown that human basophils rapidly release IL-4 (within 10 min–4 h) as well as IL-13 (within 2–16 h) following IgE-dependent activation [1]. However, these cytokines, which play a crucial role in the expression of adhesion molecules, Th2 cell development as well as IgE synthesis, may also be produced by a variety of other leukocytes. Activated Th2 cells are known to produce both IL-4 and IL-13, whereas eosinophils have been reported to secrete IL-4 following IgE-dependent antigen stimulation within a similar time scale to basophils [2]. Since activated eosinophils and Th2 cells may also express IgE receptors the early release of IL-4 and IL-13 may indeed not be as exclusive to basophils as previously thought. We therefore compared the release of IL-4 and IL-13 from basophil depleted granulocytes and mononuclear cells (MNCs) to highly purified basophils following IgE-dependent activation.

Differential role for mitogen-activated protein kinases in IgE-dependent signaling in human peripheral blood basophils: in contrast to p38 MAPK, c-Jun N-terminal kinase is poorly expressed and does not appear to control mediator release.

Background: Exposure of human basophils to allergens results in a rapid secretion of histamine, LTC4, IL-4 and IL-13, which dominate both the symptomology of allergic diseases and support the underlying Th2/IgE predominance associated with these reactions. The IgE-dependent release of these mediators in basophils crucially involves PI 3-kinase and the subsequent activation of p38 MAPK and ERK1&2. Here, we investigated the role of the third major member of the mitogen activated kinase family, namely the c-Jun amino terminal kinase (JNK), which is rapidly activated following IgE receptor cross-linking in murine mast cells. Methods: Human basophils were highly purified by magnetic cell sorting. The activities of various intracellular signaling components, in basophils that had been stimulated under various conditions, were assessed by Western blotting. Mediator secretions were also determined using either spectrofluorometric analysis (histamine) or ELISA (LTC4, IL-4 and IL-13). Results: Our results show that while JNK is moderately expressed in human basophils, it is not consistently phosphorylated upon anti-IgE stimulation. Phosphorylation of the transcription factor c-Jun, a downstream target of JNK, was also undetected in contrast to p38 MAPK and ERK1&2, which were clearly activated following anti-IgE stimulation of the cells. Additionally, inhibitors of the JNK pathway failed to prevent basophil mediator release and had no effect on the phosphorylation of p38 MAPK or ERK1&2 at concentrations which were specific for JNK blockade. Conclusions: These data suggest major differences in utilizing various members of the mitogen-activated kinase family in the signal transduction cascade of IgE-receptor-bearing cells.