Synaptotagmin I expression in mast cells of normal human tissues, systemic mast cell disease, and a human mast cell leukemia cell line.

Synaptotagmin I (STG I) is a Ca(2+) sensor and one of the synaptic vesicle proteins that mediate exocytosis. To determine the mechanism of release of large granules from mast cells, we studied by immunohistochemistry the presence of STG I in mast cells in normal human tissues simultaneously with the mast cell markers mast cell tryptase (tryptase) and c-kit. The tumor cells of systemic mast cell disease (SMCD) and a human mast cell leukemia cell line (HMC-1) were also examined. Human mast cells in normal tissues and the tumor cells of SMCD expressed STG I as well as mast cell tryptase (tryptase) and c-kit. STG I mRNA and its products in HMC-1 were examined by RT-PCR analysis and immunocytochemistry, respectively. STG I expression in HMC-1 cells was compared with that in cells stimulated and non-stimulated by phorbol 12-myristate 13-acetate and also with that in NB-1 and PC12 cells, known to express STG I. STG I mRNA was detected in both non-stimulated and stimulated HMC-1 cells and in NB-1 and PC12 cells. STG I immunoreactivity was weaker than NB-1 or PC12 immunoreactivity. However, it increased in the stimulated HMC-1 cells. Mast cells expressed STG I in various states. STG I may mediate exocytosis of large granules in mast cells.     

Tryptase activates the mitogen-activated protein kinase/activator protein-1 pathway in human peripheral blood eosinophils,causing cytokine production and release

We have previously shown that mast cells enhance eosinophil survival and activation. In this study we further characterized mast cell activity toward eosinophils. Sonicate of both rat peritoneal mast cells and the human mast cell line 1 (HMC-1) induced a concentration-dependent IL-6 and IL-8 release from human peripheral blood eosinophils (ELISA). HMC-1-induced IL-8 release was significantly reduced by the tryptase inhibitors GW-45 and GW-58 (90 and 87%, respectively, at an optimal concentration) but not by anti-stem cell factor, anti-TNF-alpha, or anti-IFN-gamma neutralizing Abs or by the antihistamine drugs pyrilamine and cimetidine. In a manner similar to HMC-1, human recombinant tryptase induced the expression of mRNA for IL-8 (RT-PCR) and caused IL-8 release from the eosinophils. Addition of cycloheximide, actinomycin D, dexamethasone, PD 98059, curcumin, or SB 202190 completely inhibited the tryptase-induced IL-6 and IL-8 release. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release. Tryptase caused phosphorylation of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases 1 and 2, and p38 (Western blot). Tryptase also induced the translocation of c-Jun from the cytosol to the nucleus (confocal microscopy) and enhanced AP-1 binding activity to the DNA (EMSA). Eosinophils were found to express proteinase-activated receptor 2 (FACS). When eosinophils were incubated with tryptase in the presence of anti-proteinase-activated receptor 2 antagonist Abs a significant decrease in the IL-6 and IL-8 release occurred. In summary, we have demonstrated that the preformed mast cell mediator tryptase induces cytokine production and release in human peripheral blood eosinophils by the mitogen-activated protein kinase/AP-1 pathway.     

Bronchial epithelial cells release IL-6, CXCL1 and CXCL8 upon mast cell interaction

Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.     

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.     

Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

STI571 (Glivec) affects histamine release and intracellular pH after alkalinisation in HMC-1560, 816

The human mast cell line (HMC-1(560, 816)) was used to study the effect of the tyrosine kinase inhibitor STI571 (Glivec) on exocytosis, intracellular Ca(2+) and pH changes, because STI571 inhibits the proliferation of HMC-1(560) and induces its apoptosis. This drug does not have these effects on HMC-1(560, 816). Exocytosis in HMC-1(560, 816) cells can be stimulated by alkalinisation with NH(4)Cl as well as with ionomycin. Surprisingly 24-h pre-incubation with STI571 decreases spontaneous histamine release of HMC-1(560, 816) cells, but increases the histamine response after alkalinisation and not after ionomycin-stimulation. After addition of NH(4)Cl, pH(i) has a higher increase in STI571 pre-incubated cells, without changing intracellular Ca(2+) concentration. Activation of PKC in combination with tyrosine kinase inhibition increases also histamine release in HMC-1(560, 816) cells. Strangely, STI571 pre-incubated cells with PKC inhibited by rottlerin show the same effects. In these cells, cytosolic pH increases more than in control cells. This is the first report of STI571 effect in HMC-1(560, 816) cells. It seems that different pathways modulate signals for proliferation and exocytosis. STI571 does not only inhibit KIT TyrK, but may also influence cytosolic pH after alkalinisation in both cell lines, HMC-1(560) and HMC-1(560, 816), and this ends in induced histamine release. This work is important since HMC-1(560, 816) cells are reported in 80% of aggressive systemic mastocytosis cases and the understanding of some signalling pathways involved in mast cell response could facilitate drug targeting.     

Mast cell exosomes promote lung adenocarcinoma cell proliferation – role of KIT-stem cell factor signaling

Background

Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.

Methods and results

Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.

Conclusions

Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

     

Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates

Herein we investigate the ability of live promastigotes and total lysate of Leishmania (Viannia) braziliensis, derived from parasites in the logarithmic (L-Lb) or stationary phase (S-Lb), to induce human mast cell line (HMC-1) activation. In comparison with medium-treated cells, a significant histamine release was observed in HMC-1 cultures stimulated with S-Lb. Lipophosphoglycan also induced histamine release by HMC-1 cells. In immunocytochemical assays, we found a marked staining for tryptase in medium-treated HMC-1 cells, however, stimulation with L-Lb or S-Lb caused a marked decrease in the color reaction as well as in the number of tryptase-positive cells. L-Lb and S-Lb induced an evident decrease in the intracellular expression of IL-4 but not IL-12. Live stationary promastigotes were able to induce high levels of IL-4 release in HMC-1 cultures. Furthermore, these cells released significant amounts of IL-12 when incubated with both types of live promastigotes. These results indicate that L. (V.) braziliensis promastigotes differ in their ability to induce direct human mast cells activation, according to the growth phase of the parasite. Furthermore, the release of pro-inflammatory mediators and cytokines could represent an important phenomenon that might favor the initial establishment of the infection.     

Galectin-9 Enhances Cytokine Secretion,but Suppresses Survival and Degranulation,in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.     

Dengue virus infection of mast cells triggers endothelial cell activation

Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection.     

LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells.

BACKGROUND: Mast cells are resident tissue cells that induce anaphylactic reactions by rapidly releasing mediators after antigen-mediated cross-linking of immunoglobulin E receptors. In the similarly active peripheral blood basophilic leukocyte, lysosome-associated membrane protein 3 (LAMP-3; CD63) has been described as an activation marker, but LAMPs have not been investigated in normal tissue mast cells. METHODS: Intra- and extracellular expressions of LAMP-1 (CD107a), LAMP-2 (CD107b), and LAMP-3 (CD63) were analysed by flow cytometry, immunocytochemistry, and functional assays in unstimulated and stimulated leukemic human mast cell line 1 (HMC-1) and skin mast cells. RESULTS: On flow cytometry, all mast cells expressed LAMP-3 at their cell membranes, whereas LAMP-1 and LAMP-2 were barely detectable (HMC-1 cells) or expressed at low levels (<10% of skin mast cells). After fixation and permeabilisation, high intracellular levels of all three LAMPs were noted in both cell types. After stimulation, a rapid translocation of intracellular LAMPs to the cell membrane, with an associated release of histamine, leukotriene C(4) and prostaglandin D(2), was ascertained in skin mast cells only. CONCLUSION: These results show that LAMP-1 and LAMP-2 are activation markers for normal mast cells. The lack of LAMP translocation after activation of leukemic mast cells may be related to maturation or malignancy-associated defects of these cells.     

ICAM-3 (CD50) is expressed by human mast cells: induction of homotypic mast cell aggregation via ICAM-3

Intercellular adhesion molecule-3 (ICAM-3, CD50), an adhesion receptor of the immunoglobulin superfamily, is suggested to play a key role in adhesive cellular interactions during the initial phase of an immune response. We here provide evidence that ICAM-3 is abundantly expressed by cells of the human mast cell line HMC-1 and, to a lower degree, by purified skin mast cells, as demonstrated by flow-cytometry, ELISA and RT-PCR. ICAM-3 immunoprecipitated from surface biotinylated HMC-1 cells migrates as a broad band of Mr 124,000 by Western blot analysis. We also demonstrate that monoclonal antibodies directed against ICAM-3 are capable of inducing rapid HMC-1 cell aggregation, the extent of which strongly depends on the epitope recognized by the mAb applied. Interestingly, although inhibitable by two of six mAbs against LFA-1, HMC-1 aggregation induced via ICAM-3 appears to be mediated by an adhesive pathway independent of LFA-1. Dermal mast cells are also aggregated with anti-ICAM-3 mAbs, a phenomenon which has not been described before for isolated tissue mast cells. However, this process displays slower kinetics, as compared to HMC-1 cells. That anti-ICAM-3 mAbs are able to mediate biological effects is further illustrated by their capability to increase stimulation-dependent release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 from HMC-1 cells. Taken together, these results indicate that ICAM-3 is not only expressed by immature and mature human mast cells, but also possesses functional relevance and may therefore play a significant role in mast cell associated processes.     

IL-4 regulates c-kit proto-oncogene product expression in human mast and myeloid progenitor cells.

The c-kit proto-oncogene encodes the receptor for a novel hemopoietic cytokine, termed stem cell factor (SCF) or mast cell growth factor (MGF) according to its stimulating spectrum. The human receptor for SCF/MGF is expressed in a subset of normal bone marrow progenitor cells, in leukemic myeloid cells, and in mast cells. In the present study, the effects of recombinant human growth regulators (IL-1 through -9, granulocyte-macrophage/granulocyte/macrophage-CSF, IFN, and TNF) on c-kit proto-oncogene product expression were analyzed by indirect immunofluorescence, by using the anti-SCF/MGFR mAb YB5.B8, and Northern blot analyses, by using a c-kit oligonucleotide probe. Of all cytokines tested, IL-4 was found to down-regulate expression of YB5.B8 Ag in the human mast cell line HMC-1 (maximum inhibition, 51.05 +/- 16.36% mean fluorescence intensity of control; p less than 0.02), as well as in primary leukemic myeloid cells. IL-4 was also found to down-regulate expression of YB5.B8 Ag in normal enriched bone marrow progenitor cells. The effects of IL-4 on expression of YB8.B8 Ag in myeloid/mast cell progenitors was dose and time dependent (maximum effects observed on days 2 and/or 4, by using 50 U/ml of rIL-4) and could be neutralized by using anti-IL-4 mAb. Moreover, IL-4 was found to down-regulate expression of c-kit mRNA in leukemic myeloid cells as well as in HMC-1 cells. Together, these observations identify IL-4 as a regulator of c-kit proto-oncogene product expression in the human system. The effects of IL-4 on human hemopoietic progenitor cells and mast cells may be mediated in part through regulation of SCF/MGFR expression.     

5-Lipoxygenase upregulation by dexamethasone in human mast cells

In spite of intensive research, our understanding of the regulation of expression of 5-LO (the key enzyme in the leukotriene metabolism) remains fragmentary. We investigated the effects of dexamethasone on the expression of this gene in a binary model consisting of two clones of the human mast cell line HMC-1, one with a 5-LO-negative and the other with a 5-LO-positive phenotype, respectively. When dexamethasone was included in the culture medium at a physiologically relevant concentration, biosynthesis of 5-LO derivatives increased considerably not only in the 5-LO-negative HMC-1 cells (approx 10-fold) but also in the 5-LO-positive cells, characterized by an already substantial enzyme activity. Consistently, Northern blot analysis revealed that a dramatic increase in the abundance of 5-LO mRNA occurred when the cells were exposed to dexamethasone. Likewise, a significant increase in the immunoreactive 5-LO protein was detected by Western blotting. In contrast, dexamethasone seemed to have no effect on the expression of two other genes of pivotal importance in leukotriene biosynthesis, viz. FLAP and LTC(4) synthase. We conclude that in human mast cells glucocorticoids effectively and selectively upregulate the expression of 5-LO.     

Alterations in mast cell function and survival following in vitro infection with human immunodeficiency viruses-1 through CXCR4

HIV-1 infection leads to a disease that attacks the central regulatory mechanisms of the immune response. As mucosal tissue is one of the primary sites infected with HIV in vivo, we examined the effects of HIV exposure on human mast cells, important components of mucosal defense. Using the human mast cell line, HMC-1, which expresses CXCR4 but not CCR5 on the cell surface, we found that several HIV-1 X4 tropic lab (IIIB, RF) and primary isolates but not R5 (BAL, ADA) isolates productively infected these cells. Furthermore, stem cell factor-dependent mast cells derived from primary fetal liver or cord blood cultures were also productively infected with both X4 and R5 HIV-1 strains. Infection was blocked at the level of viral entry using monoclonal antibodies to CXCR4 and CD4. Treatment of HMC-1 with TNF-alpha and TGF-beta stimulated cell surface expression of CCR5 and up-regulated expression of both CCR5 and CXCR4 on primary mast cells, leading to increased susceptibility to both X4 and R5 viral isolates. HIV-1 infection also resulted in histamine release from these mast cells, most due in part to HIV-mediated cell death. These results demonstrate that X4 viruses can use CD4 and the CXCR4 receptor to infect mast cells, suggesting that mast cell-T cell interactions may contribute to HIV mediated immune dysfunction in the mucosa.     

Influence of the tyrosine kinase inhibitors STI571 (Glivec), lavendustin A and genistein on human mast cell line (HMC-1(560)) activation

The human mast cell line (HMC-1(560)) was used to study the effects of tyrosine kinase (TyrK) inhibition on histamine release in consequence of intracellular Ca2+ or pH changes. This is important since the TyrK inhibitor STI571 (Glivec) inhibits proliferation and induces apoptosis in HMC-1(560). HMC-1(560) cells have a mutation in c-kit, which leads to a permanent phosphorylation of the KIT protein and their ligand-independent proliferation. The TyrK inhibitors STI571, lavendustin A and genistein decrease spontaneous histamine release in 24-h pre-incubated cells. Results are compared with those of the mast cell stabiliser cromoglycic acid, which also drops spontaneous histamine release. When exocytosis is stimulated by alkalinisation, STI571 pre-incubated cells release more histamine than non-pre-incubated cells. Alkalinisation-induced histamine release reaches still higher levels in STI571 cells with activated protein kinase C (PKC) by PMA. We do not observe modifications on histamine release in cells, treated with PKC inhibitors (rottlerin, Gf109203 or G?6976). Lavendustin A- and genistein 24-h incubated cells behave similar to STI571 cells, whereas cromoglycic acid does not show effects after stimulation with alkalinisation. Stimulation of exocytosis with the Ca2+ ionophore ionomycin does not modify histamine response in TyrK inhibited cells. Ca2+ and pH changes are observed after long-time incubation with STI571. Results show that pH is still higher in STI571 pre-incubated cells after alkalinisation with NH4Cl, whereas intracellular Ca2+ concentration remains stable. This work further strength the importance of pHi as a cell signal and suggest that STI571 has transduction pathways in common with other TyrKs.     

Thymic stromal lymphopoietin is regulated by the intracellular calcium

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic diseases such as atopic dermatitis. However, it has not been clarified that TSLP would be regulated by intracellular calcium in mast cells yet. To determine it, we blocked intracellular calcium by treatment with calcium chelator, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) in human mast cell line (HMC-1) cells. BAPTA-AM inhibited the production and mRNA expression of TSLP in phorbol myristate acetate plus A23187- stimulated HMC-1 cells. BAPTA-AM also inhibited the nuclear factor-κB activation, IκBα phosphorylation, receptor interacting protein2 (RIP2) expression, and caspase-1 activation in HMC-1 cells. These results provide evidence that calcium regulates the level of TSLP through RIP2/caspase-1/NF-κB/IκBα signal.