Mast Cell Growth Factor Receptor Research Articles

Unusual presentation of telangiectasia macularis eruptiva perstans at the site of healed herpes zoster; Wolf's isotopic response.

There are several reported cases of Wolf's isotopic response, including infections, cancers, inflammatory and immune-related disorders. It is interesting that the majority of these occurred after herpes zoster (HZ) had healed. In this article, we describe an unusual case of adult mastocytosis/telangiectasia macularis eruptiva perstans (TMEP) at the location of recovered HZ. Given that adult mastocytosis is thought to be caused by dysregulation of the mast cell growth factor receptor, the c-Kit proto-oncogene (CD117), and the fact that the varicella zoster virus-infected cutaneous lesions contain CD117-positive mast cells (CD117+MCs), we hypothesize that CD117+ MCs may be in charge of the local immunological response and cytokine release those results in TMEP after HZ.

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived “driver” and “drug-resistant” alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called “liquid biopsy” allows for the dynamic monitoring of the patients’ tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

Physiological Relevance of LL-37 Induced Bladder Inflammation and Mast Cells

We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm(2). Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells.

Lantrunculin B-induced filamentous actin disassembly enables human mast cell degranulation by SCF alone (177.7)

Abstract The release of inflammatory mediators from mast cells following antigen/IgE-mediated FcϵRI aggregation induces allergic reactions including anaphylaxis. Stem cell factor (SCF), the ligand for the mast cell growth factor receptor KIT, potentiates such release. However, despite inducing similar early signaling events as antigen, SCF alone induces minimal, if any, degranulation in the absence of other stimuli. We therefore investigated whether the process underlying these differences may lie in receptor-induced distal events involving the reorganization of the actin cytoskeleton. To explore this issue, we utilized latrunculin B, an inhibitor of actin polymerization which is known to potentiate FcϵRI-mediated mast cell degranulation. As expected, latrunculin B moderately potentiated IgE/antigen-induced human mast cell degranulation, This response was, however, markedly enhanced in the presence of SCF (100 ng/ml), especially at lower antigen concentrations, suggesting that the SCF dependent component was more sensitive to latrunculin B than the antigen component. Furthermore, although SCF alone failed to induce degranulation, in the presence of latrunculin B, it induced degranulation in a concentration-dependent manner to a similar extent as that produced by IgE/antigen. These data thus suggest that SCF provides inadequate signals for the necessary remodeling of the actin filament network for degranulation which is rectified by latrunculin B.

MTORC1 and mTORC2 control normal and neoplastic mast cell homoeostasis through regulation of cell division rather than maintenance of survival (151.20)

Abstract Mature mast cells are terminally differentiated and non-dividing. However, a pathologic increase in mast cells is associated with the myeloproliferative disorder mastocytosis which has been linked to expression of an activating mutation (D816V) in the mast cell growth factor receptor, KIT. We found that in primary human mast cells developed from peripheral blood CD34+ progenitor cells, KIT activation led to activation of mTORC1 and mTORC2 pathways through phosphorylation of mTOR (mammalian target of rapamycin) at S2481 and that these pathways were constitutively activated in both D816V positive (HMC-1.2) and negative (HMC-1.1, LAD2) neoplastic human mast cells. A dual acting inhibitor of the mTORC1 and mTORC2 pathways, Torin1, and a more selective mTORC1 inhibitor, Rapamycin, abolished proliferation of the malignant mast cells regardless of the presence of the D816V mutation. However, little reduction in numbers of the non-dividing primary mast cells was observed. The effects of the inhibitors on cell proliferation correlated to downregulation of mTORC1 and mTORC2 pathways which was verified by shRNA. Furthermore, no evidence of apoptosis in response to the inhibitors was observed in either neoplastic or primary mast cells. Collectively, our data demonstrate that by targeting the mTORC1 and mTORC2 pathways, it may be possible to inhibit the proliferation of rapidly dividing mast cell populations associated with mastocytosis while leaving normal mast cells largely unaffected.

RT‐qPCR Comparison of mast cell populations in whole blood from healthy horses and those with laminitis

Inflammatory damage to the digital laminae, a structure responsible for suspension of the distal skeleton within the hoof capsule, results in a painful and often life-threatening disease in horses called laminitis. There can be many diverse causes of laminitis; however, previous work in the horse has suggested that in each case, the inflammation and resulting tissue damage is consistent with the action of mediators released from mast cells (MC), as well as the downstream consequences of their activation. The recent development of molecular genetics tools to characterize cells based on their transcriptional activity makes a new approach for measuring MCs possible. Healthy thoroughbred horses from a variety of age groups were used to assess the amount of variation in KIT (encoding mast cell growth factor receptor) and TPSB2 (encoding mast cell tryptase beta 2) gene expression present in the population and to establish "normal" values. Horses (n=9) with a wider range of body condition scores (3-8), because of a more lax management setting that could predispose them to laminitis, had significantly higher KIT expression in circulating peripheral blood cells than horses under individualized management conditions (n=10) that produced ideal body condition scores (4-6) (mean 2.573-fold, P<0.0005). Likewise, horses affected with acute laminitis (n=11) had elevated expression of TPSB2 (2.760-fold, P=0.0011) relative to control horses (n=15). These data suggest that investigation of MC-related genes KIT and TPSB2 may be effective to assay MC population and activity. More work is needed to refine the diagnostic criteria to better describe at what point MC activation occurs and illustrate the use of gene expression assays in clinical cases of laminitis. Additionally, MC activation is associated with inflammatory disease in several mammalian species and may prove a valuable therapeutic target in the horse.

The tyrosine kinase network regulating mast cell activation

Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (Fc epsilon RI) on the mast cell surface, a response which can be further enhanced following stem cell factor-induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both Fc epsilon RI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, Fc epsilon RI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor-proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton's tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and Fc epsilon RI-mediated mast cell activation.

Combined Mastocytoma-Junctional Nevus

A unique combined mastocytoma-junctional nevus presented as a 4-mm dark brown macule in the axilla of a 57-year-old white female. Histopathologic examination revealed a proliferation of mast cells partially or completely filling the dermal papillae, hyperpigmentation of the basal keratinocytes and mildly increased basal melanocytes. Overlying the mast cell proliferation, pigmented junctional nevus nests were present. The mast cells were strongly positive with Giemsa stain and mast cell tryptase immunohistochemical stain; nevomelanocytic cells were negative. Nevomelanocytes were strongly immunoreactive for S100, HMB-45, Mart-1, and tyrosinase; mast cells were negative. The clinicopathologic features suggested a synchronous proliferation of 2 cell types in the same small cutaneous field rather than a collision tumor. While the cutaneous mast cells probably originated as a disseminated clone, it is postulated that local mast cell growth factor induced nevomelanocytic proliferation and modulated mast cell growth. In fact, the tumor exhibited strong immunoreactivity for the mast cell growth factor receptor (CD117) in mast cells, basal melanocytes, and nevus nests. The incidence of dual mast cell-melanocytic tumors appears to be very low, as only 3 total cases have now been reported. However, since in patients with multiple mastocytomas only a small fraction of lesions are biopsied, the true incidence may be higher than supposed.

Urticaria. New developments and perspectives

Although the etiology of urticaria is mostly unclear, there are a number of recent new insights into the underlying pathomechanisms and causes. Dermal mast cell numbers and endothelial cell adhesion molecule and cytokine expression are also increased in uninvolved skin, while serum P-selectin levels are elevated, suggesting a systemic activation of the cutaneous inflammatory system in urticaria. In all adults, but not children with indolent mastocytosis, we found activating point mutations of c-kit, the mast cell growth factor receptor, in lesional cutaneous mast cells. In acute urticaria, IgE-dependent mechanisms are only rarely involved (0.9%), in contrast to generally held beliefs, whereas acute upper respiratory infections (39.3%) and drug intolerance (9.2%) are more frequent. In chronic urticaria, pseudoallergies to food (73%) and more rarely chronic inflammatory gastrointestinal diseases (11%) play a major role, with avoidance or elimination of the eliciting factors leading to long term remission. On the basis of recent findings, autoantibodies must be viewed mostly as secondary rather than causative in chronic urticaria.

Close association between sequence polymorphism in the KIT gene and the roan coat color in horses.

The roan coat color in horses is controlled by a dominant allele that is lethal in the homozygous condition. Phenotypic similarities to some pigmentation disorders in human and mouse, combined with comparative mapping data, identified KIT, encoding the mast cell growth factor receptor, as a major candidate gene for the roan locus (Rn). Rn has previously been mapped to equine linkage group (LG) II. In this study, LGII was expanded with KIT and PDGFRA (platelet-derived growth factor receptor alpha) by use of RFLP and linkage analysis. Moreover, highly significant linkage disequilibrium between Rn and a KIT TaqI RFLP, representing a synonymous substitution in exon 19, was revealed. There was a strong KIT-Rn association in most breeds. Almost the complete KIT-encoding sequence was determined by sequence analysis of RT-PCR products. Comparison of horse KIT cDNA sequences, representing three different alleles (two different rn and one Rn), revealed five sequence polymorphisms and several mRNA splice variants, but none of these proved to be specifically associated with Rn. An insertion of a partial (79 bp) LINE1-element between exons 1 and 2, leading to a frameshift, represented about 30% of KIT transcripts in the Belgian roan horse used for the sequence analysis. However, an association between this L1 splice insertion and the roan phenotype was not verified when testing additional unrelated roan and non-roan horses from different breeds. The study strengthens the hypothesis that the roan coat color is controlled by KIT, but further analyses are needed to reveal the causative mutation(s).

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.

Human mastocytosis is characterized by increased mast cells. It usually occurs as a sporadic disease that is often transient and limited in children and persistent or progressive in adults. The c-KIT protooncogene encodes KIT, a tyrosine kinase that is the receptor for mast cell growth factor. Because mutated KIT can transform cells, we examined c-KIT in skin lesions of 22 patients with sporadic mastocytosis and 3 patients with familial mastocytosis. All patients with adult sporadic mastocytosis had somatic c-KIT mutations in codon 816 causing substitution of valine for aspartate and spontaneous activation of mast cell growth factor receptor (P = 0.0001). A subset of four pediatric onset cases with clinically unusual disease also had codon 816 activating mutations substituting valine, tyrosine, or phenylalanine for aspartate. Typical pediatric patients lacked 816 mutations, but limited sequencing showed three of six had a novel dominant inactivating mutation substituting lysine for glutamic acid in position 839, the site of a potential salt bridge that is highly conserved in receptor tyrosine kinases. No c-KIT mutations were found in the entire coding region of three patients with familial mastocytosis. We conclude that c-KIT somatic mutations substituting valine in position 816 of KIT are characteristic of sporadic adult mastocytosis and may cause this disease. Similar mutations causing activation of the mast cell growth factor receptor are found in children apparently at risk for extensive or persistent disease. In contrast, typical pediatric mastocytosis patients lack these mutations and may express inactivating c-KIT mutations. Familial mastocytosis, however, may occur in the absence of c-KIT coding mutations.

Altered Expression of Bladder Mast Cell Growth Factor Receptor (c-kit) in Interstitial Cystitis

Objectives. To investigate the presence of mast cell growth factor receptors (c-kit) on bladder mast cells in interstitial cystitis (IC), a bladder condition occurring primarily in women. IC is characterized by pain, urgency, frequency, and mucosal microhemorrhages discernible with cystoscopy under general anesthesia. One of the prevailing theories to explain IC pathophysiology is the increased number of bladder mast cells, many of which are activated in at least a subgroup of IC patients. Stem cell factor (SCF), also known as c-kit ligand, is now recognized as the key molecule responsible for mast cell proliferation and is known to exert its action through specific surface receptors. Methods. Bladder specimens from patients with IC, identified by the criteria established by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and control patients were obtained during diagnostic cystoscopy and were immediately fixed in 4% paraformaldehyde. They were then examined by immunohistochemistry for the unique proteolytic mast cell enzyme tryptase or the presence of c-kit, or both. Results. Bladders of IC patients contained a higher number of mast cells than control patients. However, mast cells in IC patients expressed fewer c-kit on their surface than those in control patients. These results could be explained if the c-kit were occupied by endogenous SCF or were downregulated, possibly by internalization after ligand-receptor interactions, making them inaccessible to immunocytochemical detection. Conclusions. Bladder mastocytosis and/or activation of mast cells, in at least a subpopulation of IC patients, may be explained by increased stimulation of mast cells by SCF. These results could be explained either by a mutation leading to constitutive activation of c-kit or overproduction of c-kit ligand leading to bladder mast cell proliferation in IC.

Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase.

Mast cell growth factor (MGF) (also called stem cell factor) synergizes with several lymphokines, including interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), to promote proliferation and differentiation of certain hemopoietic progenitor cells. Although similar patterns of tyrosine-phosphorylated proteins characterize cells stimulated by MGF, IL-3, and GM-CSF, only the MGF receptor is a tyrosine kinase, and the heterodimeric receptors for IL-3 and GM-CSF share a common beta subunit that is devoid of enzymatic activity. Here we show that signaling pathways utilized by all three cytokines include the cytoplasmic tyrosine kinase JAK2. Analysis of several factor-dependent myeloid cell lines indicated that JAK2 is physically associated with the common beta subunit and with MGF receptor (c-Kit) even prior to ligand binding. However, each of the ligands induced elevated tyrosine phosphorylation of JAK2 and a consequent increase in its catalytic activity. These results demonstrate for the first time the convergence within the same myeloid cells of signaling pathways originating in two distinct lymphokine receptors and a tyrosine kinase receptor on activation of a cytoplasmic tyrosine kinase.

Differential expression of mast cell growth factor receptor (c-kit) by peritoneal connective tissue-type mast cells and tissue culture-derived mast cells.

The peritoneal cavity of the mouse is a major source of connective tissue-type mast cells (CTMCs). Flow cytometric analysis using biotinylated recombinant murine mast cell growth factor (rMuMGF) showed that 1 to 3% of the cells in the peritoneal cavity exhibited MGF receptor (MGFR) (or c-kit). CTMCs were the only cell types expressing MGFR in the peritoneal cavity, and every one of them expressed MGFR. More than half the peritoneal CTMCs retained the potential to proliferate in the presence of recombinant murine interleukin 3 (rMuIL-3), rMuIL-4, and rMuMGF and gave rise to pure, alcian blue-positive "mast cells," which actively expressed c-kit transcripts and MGFR. Flow cytometric analysis and receptor assay carried out at 4 degrees C showed that the number of MGFRs on culture-derived mast cells (CDMCs) was one-third that of peritoneal CTMCs (6 x 10(4) vs. 1.8 x 10(5) MGFR/cell). At 37 degrees C, the total number of membrane MGFRs detected in CDMC was two to three times more than that detected at 4 degrees C, indicating that nearly 70% of total MGFR in CDMCs, compared with only 40% in peritoneal CTMCs, existed as "cryptic sites" unable to interact with exogenous ligand at 4 degrees C. Thus, diminished expression of MGFR is one of the phenotypic characteristics associated with CDMCs.