Surface Of Mast Cells Research Articles

Treatment of Allergic Diseases: Application to Clinical Practice of a New Concept of Mutual Substitutions of Antibody Molecules on the Surface of Mast Cells

Treatment of Allergic Diseases: Application to Clinical Practice of a New Concept of Mutual Substitutions of Antibody Molecules on the Surface of Mast Cells

Abnormal Expression of Fcγ Receptors by Human Bone Marrow Mast Cells in Systemic Mastocitosis

RATIONALE: Mast cells from Systemic Mastocytosis (SM) display unique aberrant immunophenotypic characteristics that contribute to the differential diagnosis of this rare disease. Both anaphylaxis and vascular collapse are frequent events in SM and, in a high percentage of cases, neither specific IgE antibodies nor positive skin test can be found. We have analysed the expression of Fcγ receptors (FcγRs) to detect possible differences of expression between normal and pathological bone marrow (BM) mast cells. METHODS: A total of 170 BM samples (109 healthy controls and 61 SM patients) were included in our study. Using multiparametric flow cytometry we have analysed the expression of the FcγRs (CD16 (FcγRIII), CD32 (FcγRII) and CD64 (FcγRI)) on the surface of normal and pathological BM mast cells. RESULTS: Normal and pathological BM mast cells expressed variable levels of FcγRs; however, reactivity for these three antigens was higher among SM BM mast cells than in BM mast cells from control individuals, both when the percentage of positive cases (CD16: 48% vs 14,2%, p<0,0001; CD32: 95% vs 77,9%, p=0,002; CD64: 63,7% vs 20,3%, p<0,0001), and the amount of antigen per cell (CD16, p=0,007; CD32, p<0,0001; CD64, p=0,023) were considered. Furthermore, almost no overlap in the mean fluorescence intensity of CD32 expression on BM mast cells was observed between SM and controls. CONCLUSIONS: BM mast cells in SM overexpress FcγRs that, hypothetically may play a role in mast cell activation in SM. Overexpression of CD32 on pathological BM mast cells is a useful parameter for the differential diagnosis of SM.

IgE Reversibly Enhances FcɛRI levels on the Surface of Human Skin Mast Cells and Increases Sensitivity to Low Levels of FcɛRI Cross-Linking

RATIONALE: In humans, serum IgE levels correlate with FcɛRI levels on short-lived peripheral blood basophils. This study examines IgE-dependent FcɛRI expression on long-lived tissue-dispersed human mature mast cells and the effect on mediator release.

Decoding IgE Fc receptors

Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases by interacting with two membrane receptors: high-affinity FcepsilonRI and low-affinity FcepsilonRII (CD23). Allergeninduced IgE-occupied FcepsilonRI aggregation on the mast cell or basophil cell surface leads to the activation of intracellular signaling events and eventually the release of pre-formed and de novo synthesized inflammatory mediators. The role of FcepsilonRII in allergic diseases has been proposed to include regulation of IgE synthesis, enhanced histamine release from basophils, and a contribution to Ag-IgE complex presentation but the exact function of CD23 remains poorly understood. This review summarizes some new developments in IgE Fc-receptor studies with an emphasis on regulation of FcepsilonRI expression and signal transduction, including monomeric IgE, lipid raft segregation, and some recently identified negative regulators. A better understanding of signaling events following IgE FcR aggregation will shed new light on how allergy patients might be treated more safely and effectively.

Expression of mast cell surface antigen-1 (MASA-1) on bone marrow derived mast cells

Expression of mast cell surface antigen-1 (MASA-1) on bone marrow derived mast cells

Significance of activated mast cells in submucosa and muscularis propria of patients with Crohn's disease detected by a novel antimast cell surface molecule antibody

SummaryBackgroundDegranulated mast cells may be missed by histochemical and immunohistological techniques, making the histological assessment of activated mast cells difficult.AimTo use a novel mast cell surface antigen 1 (MASA‐1) to detect activated mast cells.MethodsSurgical samples of human intestine were obtained from 15 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 11 controls. Frozen sections were cut and MASA‐1 was detected and quantified by enzyme immunohistology. Immunohistological double staining with anti‐MASA‐1 and either anti‐CD68 or anti‐c‐Kit antibody was also performed.ResultsThe number of MASA‐1‐positive cells was significantly higher in the submucosal layer and muscularis propria in patients with CD than in patients with UC or controls. The number of MASA‐1‐positive cells was significantly higher in the muscularis propria in patients with UC in highly inflamed colon compared with controls and less inflamed colon. Human intestinal MASA‐1‐positive cells are not from the macrophage lineage and showed heterogeneous expression of c‐Kit.ConclusionThe increased number of mast cells in the submucosa and muscularis propria in patients with CD suggests that the involvement of activated mast cells in transmural inflammatory responses possibly including tissue remodelling.

Sensitive Detection of c-kit Point Mutations by D-HPLC in Peripheral Blood and Bone Marrow Samples from Patients with Systemic Mastocytosis.

The majority of patients (pts) with systemic mastocytosis (SM) is characterized by the presence of the transforming mutation D816V of the c-kit gene, resulting in a factor-independent activation of KIT, a receptor tyrosine kinase on the surface of mast cells. The mutation is regarded the causative event for the pathogenesis of the disease and a potential target for therapeutic intervention. Novel tyrosine kinase inhibitors, like dasatinib, nilotinib (AMN107) and midostaurin (PKC412) have inhibitory effects on c-kit D816V mutant cells according to recent in-vitro findings. However, the sensitivity of screening procedures for mutations by direct sequencing may be compromised by a small proportion of malignant cells in the bone marrow (BM) sample. We therefore sought to establish a sensitive strategy for the detection of c-kit mutations in BM and peripheral blood (PB) samples by D-HPLC (denaturing-high performance liquid chromatography) with fragment collection of the mutated proportion combined with direct sequencing. D-HPLC has been established using serial dilutions of c-kit D816V positive HMC-1 cells in a background of NB4 cells harboring wildtype c-kit and pts mRNA in control mRNA. D-HPLC was optimized for the detection of the D816V mutation down to 0.1–0.5% mutant clone (cell line and RNA dilution). In comparison, the detection limit for D816V mutations by conventional sequencing was 10 to 15%. The method was established for c-kit mutations in exon 17, but is applicable for mutations in exons 9, 11 and 13 as well. In case of a positive D-HPLC signal, the presence of the mutation was confirmed by direct sequencing of c-kit exon 17. Secondly, the D-HPLC eluates containing the mutated products were extracted by fragment collection, enriched by PCR and subsequently sequenced. The technique was applied to BM (n=81) and PB (n=38) samples from 95 pts (53 m, 42 f) meeting the WHO criteria for SM. Median age was 53 yrs (range 24–86). At diagnosis, D-HPLC was positive in 89% of the BM samples (72/81), whereas conventional sequencing revealed the D816V mutation in 69% of the pts (56/81), one patient was positive for the variant D816H mutation. However, the combination of fragment collection and direct sequencing improved the D816V mutation detection to 86% of pts (70/81) compared to sequencing only. The analysis of PB samples revealed D-HPLC positivity in 50% of pts (19/38) with a consecutive detection of the D816V mutation by direct sequencing alone in 34% of pts (13/38). Fragment collection followed by direct sequencing improved the D816V mutation detection to 50% (19/38). In conclusion,D-HPLC is a reliable and sensitive method for the screening of c-kit mutations in SM which is superior to conventional direct sequencing. In case of a positive DHPLC signal, sequencing is recommended to confirm and specify the mutation.The method is suitable for the surveillance of pts during therapy with novel tyrosine kinase inhibitors.Employing sensitive screening techniques, the D816V mutation could be detected in BM as well as in PB samples, but the reliability for a positive result was higher using BM.

Phase II Trial of the Tyrosine Kinase Inhibitor PKC412 in Advanced Systemic Mastocytosis: Preliminary Results.

Background: Advanced forms of systemic mastocytosis (aggressive systemic mastocytosis [ASM] and mast cell leukemia [MCL]) are myeloproliferative disorders with few treatment options and a poor prognosis. In the majority of patients (pts), the pathogenesis of ASM/MCL is related to constitutive activation of the receptor tyrosine kinase (TK) KIT, due to an aspartate to valine mutation within protein codon 816 (D816V). D816V KIT is imatinib resistant both in vitro and in vivo. We demonstrated that PKC412 (N-benzoyl-staurosporine), a structurally different KIT TK inhibitor, can block the growth of D816V KIT-transformed cells at a low 50% inhibitory concentration of 30-40 nM (vs. imatinib > 1 uM). In addition, we treated a MCL pt with PKC412 on a compassionate basis, resulting in a good partial response (Gotlib et al, Blood . 2005; 106:2865-70). Therefore, we initiated a phase II study designed to assess PKC412 efficacy and safety in ASM/MCL pts.Methods: PKC412 100 mg bid was administered in 28-day cycles. Pts without a major response (MR) or partial response (PR) by Valent criteria after 2 cycles were discontinued.Results: To date, 11 ASM pts (n=6 male) have been enrolled, 7 with an associated CMML or mixed MDS/MPD. Median age at entry was 62 yrs (range 30–72); the median # of prior therapies was 1 (range 0–3).Efficacy: Currently, 9 pts are evaluable for efficacy. Responses were observed in 6/9 (67%) pts, including 2 pts with a MR (both incomplete remission), and 4 pts with a PR (3 good PR, 1 minor response). Three pts were discontinued after 2 cycles (2 progressive disease, 1 stable disease). The 2 MRs consisted of resolution of hypoalbuminemia and correction of the platelet count to >100,000/mm3 in one pt, and normalization of splenomegaly with a 69% decrease in serum tryptase in the second pt. The 3 good PRs includedalmost complete resolution of large-volume ascites,> 50% reduction in palpable hepatosplenomegaly, and>50% reduction in direct hyperbilirubinemia.Serum tryptase decreased by 60–70% in 2 of these pts. All MRs and good PRs were accompanied by a marked improvement in performance status. Median duration of treatment in responders is 4.5 cycles (2+ - 11+). Bone marrow (BM) mast cell (MC) burden was stable to slightly decreased in responders; in 1 PR pt, the BM MC burden decreased from 50% to 20% with loss of expression of the neoplastic mast cell surface marker CD25.Safety: PKC412 was generally well tolerated. Non-hematologic AEs included grade 1–2 nausea and/or vomiting (N/V), and less commonly diarrhea, fatigue, and headache (HA). Worsening of pre-existing anemia developed in 2 pts (grade 2 and 3, n=1 each). Dose reduction to 50 mg bid was undertaken in 3 pts for grade 3 thrombocytopenia, grade 2 N/V, and grade 2 HA. Two pts discontinued therapy after 4 cycles (1 for grade 3 fatigue and 1 for grade 2 N/V).Conclusion: PKC412 demonstrates a high rate of MRs and PRs in this initial cohort of advanced SM pts, supporting further accrual in this Simon two-stage trial. Ongoing biologic investigations include pharmacokinetics, KIT mutation and phosphorylation status, serum KIT/KIT ligand levels, and ex vivo effects of PKC412 with BM MC.

An Intermediate pH Unfolding Transition Abrogates the Ability of IgE to Interact with Its High Affinity Receptor FcϵRIα

The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcepsilon and IgG1-Fc (Fcgamma). Fcepsilon was found to be significantly less stable than Fcgamma under all pH and NaCl conditions tested. Additionally, the Cepsilon3Cepsilon4 domains of Fcepsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcepsilon and an Fcgamma-FcepsilonRIalpha fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcepsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 10(-12) m). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcepsilon unfolded, and interaction of Fcepsilon with the Fcgamma-FcepsilonRIalpha fusion protein was abrogated. The unusual pH sensitivity of Fcepsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.

IgE, Allergic Diseases, and Omalizumab

Immunoglobulin E (IgE) plays a central role in the development of allergic diseases. In sensitized individuals, IgE antibodies bind to receptors on mast cell and basophil surfaces, releasing preformed and newly generated mediators that initiate an immunologic cascade and inflammatory symptoms. Omalizumab (Xolair) is a humanized monoclonal antibody designed to bind specifically to IgE. It was approved by the United States Food and Drug Administration in 2003 for the treatment of patients with moderate-to-severe persistent asthma that is inadequately controlled with inhaled corticosteroids (ICS) and who have a positive skin test or in vitro reactivity to a perennial aeroallergen. In clinical trials in such patients, omalizumab reduced the incidence of asthma exacerbations, severe exacerbations, the use of rescue medication, and improved both symptoms and quality of life (QOL).

This Month in Gastroenterology

This Month in Gastroenterology

Urticaria pigmentosa and mastocytosis: The role of immunophenotyping in diagnosis and determining response to treatment

Recent advances in our understanding of mast cell biology and disease resulted in identification of important differences in expression of mast cell surface antigens in normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts leads to the inclusion of this immunophenotypic abnormality in the World Health Organization's diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients with limited disease that lacks histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. Flow cytometric analysis of bone marrow mast cells is therefore a sensitive method of diagnosis of mast cell disease and is expected to find increasing use in determining response to emerging mast cell cytoreductive therapies.

Significance of activated mast cells in submucosa and muscularis propria of patients with Crohn's disease detected by a novel antimast cell surface molecule antibody

SummaryBackgroundDegranulated mast cells may be missed by histochemical and immunohistological techniques, making the histological assessment of activated mast cells difficult.AimTo use a novel mast cell surface antigen 1 (MASA‐1) to detect activated mast cells.MethodsSurgical samples of human intestine were obtained from 15 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 11 controls. Frozen sections were cut and MASA‐1 was detected and quantified by enzyme immunohistology. Immunohistological double staining with anti‐MASA‐1 and either anti‐CD68 or anti‐c‐Kit antibody was also performed.ResultsThe number of MASA‐1‐positive cells was significantly higher in the submucosal layer and muscularis propria in patients with CD than in patients with UC or controls. The number of MASA‐1‐positive cells was significantly higher in the muscularis propria in patients with UC in highly inflamed colon compared with controls and less inflamed colon. Human intestinal MASA‐1‐positive cells are not from the macrophage lineage and showed heterogeneous expression of c‐Kit.ConclusionThe increased number of mast cells in the submucosa and muscularis propria in patients with CD suggests that the involvement of activated mast cells in transmural inflammatory responses possibly including tissue remodelling.

Regulation of Microtubule Formation in Activated Mast Cells by Complexes of γ-Tubulin with Fyn and Syk Kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcɛRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523 ng/ml with a range of 425–82,610 ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls ( P < 0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly ( P < 0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.

Immunogenic and allergenic potentials of natural and recombinant innocuous proteins

A new aspect of protein immunogenic and allergenic properties has become important recently, when there is a higher chance that our immune system will be exposed to novel protein antigens and/or familiar protein antigens with an unprecedented high frequency and large amount. These proteins are innocuous, nontoxic, and noninvasive by themselves, and include various natural proteins from the environment and recombinant proteins from industry. The technical term allergenic has been used for such proteins and their abilities to induce specific IgE production and to cross-link IgE/Fc epsilonRI on the surface of mast cells and basophiles have been recognized. As for the environmental proteins, some physicochemical properties (solubility, stability, and permeability across a mucosal epithelium) of the proteins indirectly play important roles in their allergenic potential because they do not originate from invasive pathogens as vehicles. Indeed, several lines of experimental evidences have been accumulated indicating that all proteins are absorbed across mucosal epithelia by transcellular transport and/or through interstitial spaces among the epithelial cells but not at equal levels. Some animal models have been established for natural sensitization to some allergenic proteins by feeding or intragastric administration without an adjuvant and, in a few cases, some symptoms resembling human allergy and even anaphylaxis have been induced by oral challenge with the proteins. Sometimes, even to self-proteins, the immunogenic or allergenic potential is given by post-translational modifications and possibly by unknown structural/conformational alterations, when they are exogenous self-proteins, such as recombinant human proteins for drug use. Despite the accumulation of knowledge and the progress in analytical technology on protein allergenicity, it is still crucial to predict the allergenic potential of novel and unused proteins. However, some animal models are applicable for assessing the relative allergenic potential of processed proteins in comparison with that of native proteins in preclinical studies.

Decrease in Skin Test Wheal and Flare Response after Omalizumab Therapy

RATIONALE: Omalizumab binds free IgE and prevents its binding to the surface of mast cells and basophils leading to decreased expression of FcεRI. The onset of associated decreases in skin test reactivity to aeroallergens needs to be further defined.

Expression and Inhibitory Function of Siglecs on Culture-Derived Human Mast Cells (MC)

RATIONALE: Siglecs (sialic acid-binding immunoglobulin-like lectins) are inhibitory receptors. Their expression and function on human MC is unknown. METHODS: Human MC were cultured from CD34+ precursors. mRNA was assessed using gene arrays. Positive results were validated using flow cytometry and Western blotting. Histamine and PDG2 were quantified using standard assays. RESULTS: Among all 11 known siglecs, MC expressed mRNA for Siglec-2, Siglec-3, Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for Siglec-2 and Siglec-10, where levels were low or undetectable. However, Western blotting detected expression Siglec-2 and Siglec-10 in MC lysates. Kinetic analysis of surface phenotype showed CD34+ precursor cells constitutively expressed surface Siglec-3, Siglec-5 and Siglec-10. As they matured into MC, their levels of Siglec-3 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 increased, in parallel with accumulation of intracellular histamine and MC surface markers (e.g., FceRIa and CD51). In functional studies, pre-crosslinking with Siglec-8 antibody, but not a CD51 control antibody, significantly (p<0.05) inhibited FceRI-dependent histamine and PGD2 release. For example, with 1 mg/ml anti-FceRIa antibody CRA-1, histamine and PGD2 release for no pre-crosslinking antibody versus CD51 control pre-crosslinking versus Siglec-8 pre-crosslinking were 28±4, 27±5, and 13±2 net % HR and 59±10, 61±13 and 25±8 net ng PGD2/106 cells, respectively (mean ± SEM, n=10). CONCLUSIONS: Human MC express many Siglecs. Antibody pre-crosslinking of Siglec-8 inhibits FceRI-dependent mediator release. Together with prior data showing eosinophil apoptosis, Siglec-8 and perhaps others are interesting targets for inhibition of allergic inflammation.

Asthma, Allergy and Chemokines

In the human, chemokines represent a structurally related family of more than forty cytokines which act on distinct subsets of leukocytes via specific G protein-coupled receptors expressed on the cell surface. The induction of select repertoires of chemokines following exposure to allergen provides a biological basis for the selective leukocyte recruitment, observed both clinically and experimentally. The chemokine receptor CCR3 is expressed on the cell surface of eosinophils, Th2 lymphocytes, basophils and mast cells and binds the Eotaxin family of chemokines (CCL11, CCL24 and CCL26), whose production is upregulated following allergen challenge. Once recruited, eosinophils are a source of growth factors associated with tissue repair and remodelling and also have the ability to induce tissue damage, a capacity that extends from their traditional role in protecting the host against parasitic worms. Thus, impairment of their recruitment by selective blockade of the CCR3:eotaxin axis represents an attractive target for the therapeutic treatment of asthma. In this review, we will examine recent developments in the field and highlight the roles of other chemokine:chemokine receptor axes implicated in leukocyte recruitment during allergic inflammation.

Measurement of rat mast cell surface antigen-1 (MASA-1) mRNA in a rat model of type I allergy

Measurement of rat mast cell surface antigen-1 (MASA-1) mRNA in a rat model of type I allergy