Morphology Of Mast Cells Research Articles

Alterations in Lung Mast Cell Populations in Patients with Chronic Obstructive Pulmonary Disease

Mast cells have important roles in innate immunity and tissue remodeling but have remained poorly studied in inflammatory airway diseases like chronic obstructive pulmonary disease (COPD). To perform a detailed histological characterization of human lung mast cell populations at different severities of COPD, comparing with smoking and never-smoking control subjects. Mast cells were analyzed in lung tissues from patients with mild to very severe COPD, GOLD I-IV (n = 25, 10 of whom were treated with corticosteroids). Never-smokers and smokers served as controls. The density, morphology, and molecular characteristics of mucosal and connective tissue mast cells (MC(T) and MC(TC), respectively) were analyzed in several lung regions. In all compartments of COPD lungs, especially at severe stages, the MC(TC) population increased in density, whereas the MC(T) population decreased. The net result was a reduction in total mast cell density. This phenomenon was paralleled by increased numbers of luminal mast cells, whereas the numbers of terminal transferase dUTP nick end labeling (TUNEL)(+) apoptotic mast cells remained unchanged. In COPD lungs, the MC(T) and MC(TC) populations showed alterations in morphology and expression of CD88 (C5a-R), transforming growth factor (TGF)-beta, and renin. Statistically significant correlations were found between several COPD-related mast cell alterations and lung function parameters. As COPD progresses to its severe stages, the mast cell populations in the lung undergo changes in density, distribution, and molecular expression. In COPD lungs, these novel histopathological features were found to be correlated to lung function and they may thus have clinical consequences.

Novel anti-ulcer α,β-unsaturated lactones inhibit compound 48/80-induced mast cell degranulation

The present study was designed to examine the effects of a sesquiterpene lactone isolated from Artemisia douglasiana Besser (dehydroleucodine), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (xanthatin) and a semisynthetic butenolide (3-benzyloxymethyl-5 H-furan-2-one) on mast cell degranulation induced by compound 48/80. Peritoneal mast cells from male adult Sprague–Dawley rats were purified in Percoll, preincubated in the presence of test lactones (dehydroleucodine, xanthatin or 3-benzyloxymethyl-5 H-furan-2-one) and then challenged with the mast cell activator compound 48/80 (10 µg/ml). Concentration-response and kinetic studies of mast cell serotonin release evoked by compound 48/80, evaluation of mast cell viability and morphology by light and electron microscopy, and comparative studies using ketotifen and sodium chromoglycate were carried out. Serotonin release studies, carried out together with morphological studies, showed the effectiveness of the above lactones to stabilize mast cells. The comparative study with ketotifen and sodium chromoglycate, well known mast cell stabilizers, showed the following order of potency dehydroleucodine = xanthatin > 3-benzyloxymethyl-5 H-furan-2-one ≥ ketotifen/sodium chromoglycate to inhibit mast cell serotonin release induced by compound 48/80. The present study provides the first strong evidence in favour of the hypothesis that dehydroleucodine, xanthatin and 3-benzyloxymethyl-5 H-furan-2-one inhibit compound 48/80-induced serotonin release from peritoneal mast cells, acting thus as mast cell stabilizers. Our findings may provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response.

Utility of the World Heath Organization classification criteria for the diagnosis of systemic mastocytosis in bone marrow

In the World Health Organization classification, one major and four minor criteria are specified for the diagnosis of systemic mastocytosis. We report our experience using these criteria to diagnose systemic mastocytosis involving bone marrow. A total of 59 patients with clinically suspected systemic mastocytosis underwent comprehensive bone marrow examination, including immunophenotyping by immunohistochemistry and/or flow cytometry and molecular studies for KIT exon 17 mutations. Serum tryptase levels were also assessed. Of these 59, 53 (90%) patients met the diagnostic criteria for systemic mastocytosis. In these patients, multifocal dense infiltrates of mast cells, the major criterion, was observed in 36 (68%) patients. Atypical mast cell morphology was observed in 53 (100%), an aberrant immunophenotype was identified in 50 of 52 (96%), KIT mutation was present in 33 of 44 (75%), and an elevated serum tryptase (>20 ng/ml) was detected in 44 of 52 (85%). In the six patients in which bone marrow examination could not confirm systemic mastocytosis, one had systemic mastocytosis involving spleen, one patient had chronic idiopathic myelofibrosis, and four had no specific diagnosis, but systemic mastocytosis was still considered most likely. Of these six patients, atypical mast cell morphology was identified in five, aberrant immunophenotype in five, KIT mutation in two, and elevated serum tryptase in two. None of these cases met the major criteria. We conclude that the World Health Organization criteria are useful for the diagnosis of systemic mastocytosis in bone marrow specimens. The results also show the relative values of traditional morphologic criteria (ie, major criterion) and the results of ancillary testing (ie, minor criteria). However, as illustrated by the case of splenic systemic mastocytosis as well as the patient with chronic idiopathic myelofibrosis, the current World Health Organization system is neither completely sensitive nor specific for systemic mastocytosis.

Inhibitory Activity of α,β-Unsaturated Lactones on Histamine Release from Rat Peritoneal Mast Cells

The present study was designed to examine the effects of a sesquiterpene lactone isolated from Artemisia douglasiana Besser (dehydroleucodine, DhL), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (xanthatin, Xt) and a semisynthetic butenolide (3-benzyloxymethyl-5 H-furan-2-one, But) on mast cell histamine release induced by compound 48/80. Peritoneal mast cells from male adult Sprague-Dawley rats were purified in Percoll, preincubated in the presence of test lactones (DhL, Xt or But) and then challenged with the mast cell activator compound 48/80. Concentration-response studies of mast cell histamine release evoked by compound 48/80, evaluation of mast cell viability and morphology studies by light microscopy were carried out. Biochemical and morphological studies showed the effectiveness of the above lactones to inhibit secretory responses of rat peritoneal mast cells. The present study provides evidence in favor of the hypothesis that DhL, Xt and But inhibit compound 48/80-induced histamine release from rat peritoneal mast cells. Our findings may provide an insight into the design of novel pharmacological agents that may be used to regulate the mast cell response.

An RGS for PI3K

Immunoglobulin E (IgE) triggers the activation of the FcεRI immune receptor, which signals through immunoreceptor-based tyrosine activation motifs, in response to allergens. FcεRI activation on mast cells produces an increase in intracellular calcium concentration, which causes degranulation and the release of inflammatory mediators such as histamine. The signaling pathway from the FcεRI to calcium involves the tyrosine kinases Lyn and Syk, the adaptor proteins Gab2 and Grb2, phosphoinositide 3-kinase (PI3K), and phospholipase C-mediated production of inositol 1,4,5-trisphosphate (IP 3 ). G protein-coupled receptors, such as adenosine or prostaglandin receptors, may amplify IgE-mediated mast cell activation. Bansal et al . found that RGS13, a member of the R4 subfamily of regulator of G protein signaling (RGS) proteins, provided an inhibitory input into IgE-mediated mast cell degranulation through a mechanism involving the p85 subunit of PI3K instead of a G protein. RGS proteins are best known for attenuating G protein signaling through their actions as GTPase-activating proteins (GAPs) for specific Gα subunits and for interfering with the interactions between Gα subunits and downstream effectors. Bansal et al . studied bone marrow-derived mast cells (BMMCs) from Rgs13 knockout ( Rgs13 -KO) mice and found that antigen-elicited degranulation was greater and inhibition of G proteins with pertussis toxin did not alter this enhanced response, suggesting that G proteins were not mediating the effect. Furthermore, reconstitution of the Rgs13 -deficient BMMCs with mutant forms of RGS13 that could not interact with G proteins prevented the enhanced degranulation response. The Rgs13 -KO mice also exhibited elevated cutaneous allergic response compared with wild-type mice, yet did not show altered mast cell numbers or morphology. Using glutathione S -transferase fused to RGS13, the p85 subunit of PI3K was identified from lysates of BMMCs as a tyrosine-phosphorylated protein that interacted with RGS13. The interaction between RGS13 and p85 was direct and occurred with recombinant proteins, although it did require p85 tyrosine phosphorylation (achieved by incubation with Lyn). Rgs13 -deficient BMMCs showed increased production of IP 3 , phosphorylation of Akt, and increased intracellular calcium concentrations in response to antigen. Transduction of wild-type BMMCs with a cell-permeable RGS13 construct inhibited antigen-stimulated calcium flux and PI3K activity. Immunoprecipitation experiments with BMMCS transduced with the cell-permeable RGS13 indicated that RGS13 interfered with the recruitment of p85 to Gab2 of the FcεRI signaling complex. G. Bansal, Z. Xie, S. Rao, K. H. Nocka, K. M. Druey, Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13. Nat. Immunol. 9 , 73-80 (2008). [PubMed]

Effects of fluoxetine on mast cell morphology and protease-1 expression in gastric antrum in a rat model of depression

To investigate the effects of fluoxetine on depression-induced changes of mast cell morphology and protease-1 (rMCP-1) expression in rats. A Sprague-Dawley rat model of chronic stress-induced depression was established. Fifty experimental rats were randomly divided into the following groups: normal control group, fluoxetine+normal control group, depressed model group, saline+depressed model group, and fluoxetine+depressed model group. Laser scanning confocal microscopy (LSCM) immunofluorecence and RT-PCR techniques were used to investigate rMCP-1 expression in gastric antrum. Mast cell morphology was observed under transmission electron microscopy. ANOVA was used for statistical analysis among groups. Morphologic observation indicated that depression induced mast cell proliferation, activation, and granule hyperplasia. Compared with the normal control group, the average immunofluorescence intensity of gastric antrum rMCP-1 significantly increased in depressed model group (37.4+/-7.7 vs 24.5+/-5.6, P<0.01) or saline+depressed model group (39.9+/-5.0 vs 24.5+/-5.6, P<0.01), while there was no significant difference between fluoxetine + normal control group (23.1+/-3.4) or fluoxetine+depressed model group (26.1+/-3.6) and normal control group. The average level of rMCP-1mRNA of gastric antrum significantly increased in depressed model group (0.759+/-0.357 vs 0.476+/-0.029, P<0.01) or saline+depressed model group (0.781+/-0.451 vs 0.476+/-0.029, P<0.01), while no significant difference was found between fluoxetine+normal control group (0.460+/-0.027) or fluoxetine+depressed model group (0.488+/-0.030) and normal control group. Fluoxetine showed partial inhibitive effects on mast cell ultrastructural alterations and de-regulated rMCP-1 expression in gastric antrum of the depressed rat model. Chronic stress can induce mast cell proliferation, activation, and granule hyperplasia in gastric antrum. Fluoxetine counteracts such changes in the depressed rat model.

Effect of cold‐induced stress on rat bladder tissue contractility and histomorphology

To investigate the effects of cold-induced stress on bladder tissue histomorphology and contractility in a rat model. Eighteen male Sprague-Dawley rats were divided into three groups: Control group (Group 1), acute cold-stress group (Group 2, kept at +4 degrees C for 8 hr), and chronic cold-stress group (Group 3, kept at +4 degrees C for 4 hr/day for 21 days). At the end of protocols, histological examination of general bladder tissue morphology and determination of mast cells was performed. Organ bath studies were conducted at basal tone where contractile responses to 120 mM potassium, electrical field stimulation (EFS), and carbachol (10(-9)-10(-4) M) were assessed. Relaxation responses to EFS, isoproterenol (10(-9)-10(-4) M), papaverine plus sodium nitroprusside (10(-4) M each) were recorded in carbachol pre-contracted strips. All data were compared by one-way ANOVA test. Group 1 revealed regular bladder mucosa with intact urothelium. Groups 2 and 3 showed degeneration of urothelium with accumulation of neutrophils and significantly increased number of mast cells in both mucosa and muscularis. Mast cell counts were significantly higher in Group 3 compared to Group 2. Contractile responses to 120 mM potassium and EFS were significantly greater in the control group compared to other groups. Carbachol caused dose-dependent contractions that were significantly higher in the control group (at 10(-5), 3 x 10(-5), and 10(-4) M doses). There was no statistical difference between the groups in terms of relaxation responses. In vivo cold exposure induces significant bladder injury and decreased tissue contractility. Mechanistic pathways involved in the response of the urinary bladder to cold-induced stress need further investigation.

Differentiation and maintenance of mast cells from CD34 + human cord blood cells

Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells. Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy. CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro. Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.

Evaluation of neoplastic human mast cells by tryptase‐immunoelectron microscopy

To analyse and characterize the ultrastructural morphology of normal tissue mast cells (MC) and neoplastic bone marrow MC. We have examined the ultrastructure and cytomorphological features of MC derived from cord blood cells, neoplastic bone marrow MC in patients with systemic mastocytosis (SM, n = 4), myelomastocytic leukaemia (MML, n = 2), mast cell leukaemia (MCL, n = 2) and tryptase-positive acute myeloid leukaemia (AML, n = 4). Based on their ultrastructure and morphology, four distinct cell types could be delineated: (i) mature well-granulated tissue MC exhibiting a round central nucleus; (ii) atypical MC type I with oval nuclei, hypogranulated cytoplasm, and prominent surface projections; (iii) immature atypical MC with bi- or polylobed nuclei (atypical MC type II = promastocytes); and (iv) metachromatic blasts. Type I atypical MC were detected in a patient with indolent SM, whereas type II MC and metachromatic blasts were primarily found in MML, MCL and tryptase-positive AML. In all samples examined, the identity of MC could be reconfirmed by immunoelectron microscopy, irrespective of the stage of cell maturation or the disease variant, all types of MC contained tryptase in their cytoplasmic granules. Immunoelectron microscopy may be a helpful approach in confirming the identity of neoplastic MC in myeloid neoplasms.

Characteristic alterations of cutaneous neurogenic factors in photoaged skin

Although it has been recognized that photoageing and chronological ageing differ in various morphological and biological aspects, the characteristic alterations of cutaneous neurogenic factors in photoaged skin are poorly characterized. To characterize cutaneous neurogenic factors, including innervation, neuropeptides, nerve growth factor and interactions of mast cells, in photoaged skin. Paired biopsy specimens were obtained from sun-exposed volar forearm skin and from sun-protected dorsal upper arm skin of 20 elderly subjects. Various cutaneous neurogenic factors, including innervation, neuropeptides, neurokinin receptor, nerve growth factor, neurogenic inflammation and morphology of mast cells, were compared in sun-exposed vs. sun-protected skin quantitatively and qualitatively. Cutaneous neurogenic factors associated with photoageing were characterized by a significant increase in the densities of dermal and intraepidermal nerve fibres, a correlation between epidermal innervation and the severity of photodamage, increases in the number of neuropeptidergic sensory nerve fibres in the dermis and in tissue levels of sensory neuropeptides, increases in the content of nerve growth factor, reduced expression of neurokinin receptor 1 by epidermal keratinocytes and by vascular endothelial cells and a tachykinin-specific reduction of cutaneous neurogenic inflammation. Mast cells in photodamaged skin showed several characteristic morphological features, including various degrees of activation and an intimate association with fibroblasts, which were distinct from those in sun-protected skin. Furthermore, mast cells in photodamaged skin possessed larger amounts of substance P within their granules than did those in sun-protected skin. These findings document for the first time characteristic alterations of cutaneous neurogenic factors in photodamaged skin and suggest that the cutaneous nervous system may be involved in photoageing processes.

Bullous delayed pressure urticaria: pathogenic role for eosinophilic granulocytes?

Bullous delayed pressure urticaria (DPU) is a rare variant of DPU. Treatment of DPU is difficult and the underlying pathogenic mechanism of DPU remains elusive. We report a 72-year-old man with DPU and associated chronic urticaria as well as delayed urticarial dermographism. Pressure challenge gave rise to a deep weal covered by multiple vesicles and bullae after 24 h. Histological examination of a skin biopsy specimen obtained 24 h after pressure challenge demonstrated intraepidermal bullae filled with eosinophils accompanied by a dense, predominantly eosinophilic infiltrate in the dermis. Whereas the numbers and morphology of mast cells were unaltered, the extracellular deposition of eosinophil cationic protein revealed evidence for eosinophil activation. Concomitantly, both CD4+ and CD8+ T lymphocytes were present in the infiltrate and expressed interleukin 5. As bullous DPU may represent the maximal variant of DPU, the investigation of the cellular infiltrate and the chemokines/cytokines released may reveal potential pathogenic mechanisms. A possible effector role of eosinophilic granulocytes, T-cell subsets and mast cells is discussed.

Köpek derisindeki mast hücreleri: dağılım,yoğunluk, heterojenite ve tespit tekniklerinin etkisi

This study was conducted with the aim of examining the location, the morphology, and numerical distribution of mast cells in the different areas of the healthy adult dog skin. Skin samples taken from the cheek, pinna, thorax and thigh areas of 10 healthy dogs were used as the study material. The results show the existence of mast cell heterogeneity in dog skin with respect to staining with alcian blue / safranin O and formaldehyde fixation sensitivity. Mast cells characterized by four combination of these parameters were observed: Mast cells containing only alcian blue-positive granules which were formalin-sensitive, mast cells containing only alcian blue-positive granules which were formalin-resistant, mast cells containing only safranin O-positive granules which were formalin resistant and mast cells containing a mixture of alcian blue-positive and safranin O-positive granules which were formalin resistant. With toluidine blue staining method, it was founded that IFAA fixed tissues were contained more mast cells than those tissues fixed with the 10% formaldehyde in all four regions. The difference in average number of mast cells per mm² between the IFAA and the 10% formaldehyde fixed tissues was statistically significant (p0.05), in the other three areas, statistically significant differences (p

Mast cells in the dog skin: distribution, density, heterogeneity and influence of fixation techniques

Bu araştırma sağlıklı erişkin köpeklerde farklı deri bölgelerindeki mast hücrelerinin yerleşim yerlerini, morfolojilerini ve sayısal dağılımlarını incelemek amacıyla yapıldı. Çalışmada materyal olarak 10 adet sağlıklı köpeklerin yanak, kulak kepçesi, kaburga ve but bölgelerinden alınan deri örnekleri kullanıldı. Bulgularımız, alcian blue / safranin O ile boyanmasına ve formaldehit tespitine duyarlılığına göre köpek derisinde mast hücre heterojenitesini gösterdi. Mast hücreleri bu parametrelerin dört kombinasyonu ile tanımlandı: Formole duyarlı sadece alcian blue-pozitif granüller içeren mast hücreleri, formole dirençli sadece alcian blue-pozitif granüller içeren mast hücreleri, formole dirençli sadece safranin O-pozitif granüller içeren mast hücreleri ve formole dirençli hem safranin O-pozitif hem de alcian blue-pozitif mikst granüller içeren mast hücreleri. Toluidin blue ile boyanmış kesitlerde dört bölgede de IFAA tespitli dokuların % 10 formolle tespit edilenlere göre daha fazla mast hücresi içerdiği dikkati çekti. Bu iki tespit arasında mm²’de mast hücre sayı ortalamalarındaki farklılığın istatistiksel olarak önemli (p0.05) iken, diğer üç bölgede istatistiksel fark önemli (p

243 REPROGRAMMING OF FIBROBLASTS TO EXPRESS MALE GERM CELL OR MAST CELL FUNCTIONS USING TESTIS OR MAST CELL-DERIVED CELL EXTRACTS

The concept of epigenetic reprogramming of a somatic nucleus was supported by the birth of cloned animals and the derivation of embryonic stem cells after nuclear transplantation into oocytes. Moreover, recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. In this study, we were able to modulate the cell fate of fibroblasts by introducing cell extracts derived from a mast cell line, RBL-2H3. NIH-3T3 cells were treated with streptolysin O (SLO; 230 ng/mL), which reversibly permeablizes plasma membrane, and incubated for 30 min with the mast cell- derived cell extracts (4 mg/mL). After resealing the membrane of the cells, we incubated the cells for 3 weeks and then analyzed them for the expression of mast cell-specific genes such as MAFA (mast cell function-associated antigen) and FceRI (high-affinity IgE receptor). Our results showed that the cell extracts can activate the expression of mast cell-specific genes, implying that cell extracts can provide regulatory components required for reprogramming the cell fate to initiate a transcriptional program specific for the cell type. Moreover, mast cell-specific degranulation and cell morphology changes were observed in cultured mouse fibroblasts. We could detect mast cell-specific functions even after 15 days of incubation. Next, to induce porcine fibroblasts to take on testis sertoli cell-specific properties, we reversibly permeablized porcine primary fibroblasts with SLO (230 ng/mL) and incubated the cells with porcine testis extracts (4 mg/mL). As expected, in the reprogrammed primary porcine fibroblasts, the porcine testis extracts activated the expression of porcine testis sertoli cell-specific genes including protamine 1 and 2, SOX9, MIS (mullerian inhibitory substance), preproacrosine (ACR), phosphoglycerate kinase-2 (PGK-2), protein C, and c-kit ligand. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Taken together, our data suggest that testis- or mast cell-derived cell extracts can reprogram fibroblasts to express male germ cell or mast cell functions, respectively, supporting the concept that cell extracts can reprogram the genome activity to activate cell-specific gene expression. This work was supported by ARPC (Grant no. 204117-03-1-HD110) in Korea, and by Biogreen 21 program (Grant no. 20050401034658).

De Novo ABL Kinase Domain Mutations in Murine Bone Marrow Cells Retrovirally Transduced with Wild-Type p210 BCR-ABL and Amplified in Culture with Imatinib Mesylate.

Imatinib mesylate (IM) is a tyrosine kinase inhibitor which is highly efficient in chronic myelogenous leukemia (CML), especially in the firts chronic phase of the disease. Recent data showed, however, that resistance to IM can develop in patients in more aggressive phases of their disease, which mainly occurs through mutations within the ABL kinase domain that interfere with IM binding, leading to IM-resistant relapses. The mechanisms of the occurrence of ABL kinase domain mutations in patients on IM therapy are not well understood, and in some of them, a mutation pre-existing to the introduction of IM was described, suggesting the possibility of a clonal selection under IM therapy.To determine if ABL kinase domain mutations could be induced de novo in primary marrow cells, we used an ecotropic BCR-ABL retrovirus (MIGR-p210 vector, 5.105 viral particles / ml) and infected 5-FU-treated bone marrow cells from C57BL/6 mice. Retrovirally transduced cells (30% GFP+) were transplanted in lethally irradiated animals in which they induced lethal leukemia in 3 weeks. Both BCR-ABL-transduced and control C57BL/6 bone marrow cells were seeded in liquid cultures (104 cells/well) in the presence of 0.25 μM IM with weekly half medium changes during which the concentration of IM was increased gradually from 0.25 to 2 μM over 2 months. In these conditions, no growth could be obtained from normal bone marrow cells (0/192 wells) whereas in 10/192 wells containing BCR-ABL-transduced cells, we observed significant growth on IM. These cells were then amplified in the presence of murine stromal MS-5 cells and 2 μM IM for over 6 months and 2 clones (C3 and C10) exhibiting persistent growth were further characterized. At cytological analysis both cell lines had a typical mast cell morphology. Flow cytomery analyses demonsrated the presence of CD41 marker on both cell lines, with absence of myeloid (Gr1), erythroid (Ter119) and B-cell (B220) markers. Cells were not polyploid and interestingly, they exhibited higher growth rates in the presence of IM, with reduced growth upon IM deprivation. Both cell lines had evidence of BCR-ABL vector integration by PCR analysis and were highly GFP+. To explore the mechanisms of IM-resistance in these cells, we extracted high molecular weight genomic DNA and amplified a BCR-ABL fragment of 1236 bp encompassing the ABL kinase domain of the integrated construct. We then sequenced the ABL kinase domain using internal primers in both 5′-3′ directions. Plasmid DNA from the original MIGR-p210 BCR-ABL vector served as control. In both clones (C3 and C10) ABL kinase point mutations were readily detectable which were not found in the BCR-ABL retroviral vector. C3 carried two mutations interesting the C helix (E300K) and the SH2 contact region (E371K) of ABL kinase domain, whereas C10 carried a single mutation in the C helix (D295N). These mutations were previously detected in a random in vitro mutagenesis assay of BCR-ABL in bacterial systems. Thus, our model is the first demonstration of the occurrence of ABL kinase domain mutations and the concomittant generation of an IM-resistant phenotype in primary marrow stem cells transduced with BCR-ABL vector DNA and selected in the presence of IM. The mechanisms of the occurrence of these mutations in vitro are currently under study but these results suggest that de novo ABL kinase mutations could also occur in vivo in CML patients treated with IM.

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.

The chymase, mouse mast cell protease 4, constitutes the major chymotrypsin-like activity in peritoneum and ear tissue. A role for mouse mast cell protease 4 in thrombin regulation and fibronectin turnover.

To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 null cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 null peritoneal cells to process endogenous fibronectin.

New functions of histamine found in histidine decarboxylase gene knockout mice

Gene targeting techniques have revolutionized the investigation of the effects of bioactive substances in pathological and physiological conditions. Histamine synthesis is uniquely catalyzed by l-histidine decarboxylase. The knockout mice of this gene express no histamine-producing activity and lack histamine. These mice have been used to examine the mechanisms of histamine in several known phenotypes, e.g., gastric acid secretion, contraction of smooth muscles, vascular permeability, and awakening, and have also been used to explore unreported effects of histamine in the whole body. First, we will review the former mechanisms and then move to the latter, new effects. Especially, in the latter mechanisms, we focus on several important roles of histamine in angiogenesis, neutrophil and eosinophil recruitment, bacterial infection, and systemic anaphylaxis in this review. Moreover, to our surprise, the morphology of mast cells in the knockout mice was severely affected by the absence of histamine in terms of their granules.

Systemic mastocytosis with associated clonal hematological non-mast-cell lineage disease: analysis of clinicopathologic features and activating c-kit mutations.

The majority of patients with systemic mastocytosis with associated clonal, hematological non-mast cell lineage disease (SM-AHNMD) have a myeloid stem cell malignancy including myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative disorders, acute myeloid leukemia (AML), or chronic myeloproliferative disease. The clinicopathologic features of SM-AHNMD have not been fully characterized. We describe seven cases of this entity: 3 with MDS, 3 with AML, and 1 with chronic myelomonocytic leukemia. In the majority of cases, SM was diagnosed concurrently with the myeloid malignancy and aberrant mast cell morphology was observed. The commonly described c-kit enzymatic site mutation Asp816Val was detected only in 2 cases, while 3 patients carried the Asp816His mutation. Among the 3 cases with AML, 2 patients carried the translocation t(8;21). On the basis of our results and other reported cases, there appears to be a specific association between SM and AML with t(8;21). Concurrent occurrence of SM may define a subset of patients with de novo AML and other myeloid malignancies who have an adverse prognosis. As clinically effective tyrosine kinase inhibitors that inhibit enzymatic-type c-kit mutations are being developed, detection of mast cell proliferation associated with myeloid malignancy may have important therapeutic implications.

Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.

Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7-10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro. (1) To determine whether mouse bone marrow-derived mast cells (mBMMC) grown in the presence of transforming growth factor (TGF)-beta1 could develop over the same time frame (7-10 days) as MMC in parasitized mice. (2) To compare the early expression of surface receptors (integrins alphaE and beta7, c-kit and FcepsilonR) with that of the MMC-specific granule chymase mouse mast cell protease-1 (mMCP-1). Mouse bone marrow cells were cultured in the presence of IL-9, IL-3 and Stem Cell Factor (SCF) with or without TGF-beta1. mBMMC were quantified after toluidine blue or Leishmans' staining. Expression of MMC-specific mouse mast cell proteases was analysed by ELISA, immunohistochemistry and RT-PCR. Surface antigen expression was characterized by flow cytometry and confocal microscopy. TGF-beta1 promotes the development of abundant MMC-like mBMMC from bone marrow progenitor cells with kinetics, which closely parallel that seen in vivo. mRNA transcripts encoding mMCP-1 and -2 are readily detectable by day 4 ex vivo in cultures grown in the presence of TGF-beta1. Between 30 and 40% and 75-90% of the cells in these cultures on days 4 and 7, respectively, have typical mast cell morphology, are c-kit+, FcepsilonR+, integrin alphaEbeta7+, and express and secrete abundant mMCP-1. The integrin alphaE subunit is coexpressed with mMCP-1. The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.