Description and Characterization of a Novel Human Mast Cell Line for Scientific Study.

BACKGROUND: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. METHODS: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (ß-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. RESULTS: LADR cells were larger and more granulated as seen with Wright-Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. CONCLUSIONS: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.

Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis.

Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in HPS1 which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the HPS1 mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients' serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with HPS1 rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by HPS1 transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.

IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3.

BACKGROUND: IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES: We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS: HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS: IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION: IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Helicobacter pylori: A significant and treatable cause of chronic urticaria and angioedema.

Two outpatient medical offices evaluated 204 patients with chronic urticaria during 2012. This article presents a retrospective study showing that 10% of patients with chronic urticaria may be infected with H. pylori. Furthermore, eradication of infection can be followed by remission of urticaria, reduced morbidity from gastric ulcers, and cancer.

Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells.

Human mast cells (HuMCs) are derived from CD34(+) pluripotent hematopoietic cells which are KIT (CD117)(+) and FcεRI(-), and lack lineage-specific surface markers. Bone marrow and peripheral blood are the two readily available sources for obtaining CD34(+) cells from which HuMCs can be cultured. CD34(+) cells are isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34(+) cells may be immediately cultured in serum-free culture media containing recombinant human (rh) stem cell factor (SCF), rhIL-6, and rhIL-3 (added only during the first week). Weekly hemidepletions and removal of adherent cells and/or debris enables the investigator to obtain HuMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 weeks.

A ten-year retrospective analysis of the distribution, use and phenotypic characteristics of the LAD2 human mast cell line.

BACKGROUND: In 2003, this laboratory published an account of the human mast cell line LAD2 (Laboratory of Allergic Diseases 2) that expressed FcεRI, responded to recombinant human stem cell factor (rhSCF) and resembled CD34+-derived human mast cells. LAD2 cells have now been distributed worldwide. To study the impact of this transfer, we analyzed the number of investigators receiving LAD2 cells and resulting publications. METHODS: Records maintained in our laboratory, the Technology Transfer and Intellectual Property Office and Office of Technology Transfer, were reviewed for material transfer agreements (MTAs) and licensing agreements (LAs). Journals and impact factors were obtained from PubMed.gov by cross-referencing LAD2 and human mast cells from 2003 through November 2013. RESULTS: Over 300 MTAs and 40 LAs were approved. LAD2 cells were shipped to over 30 countries. More than 80 papers have been published in journals with impact factors from 1.31 to 13.21. Intended uses include the study of receptors, degranulation, and cell signaling. LAD2 cells continue to express described markers and have consistent FcεR1-mediated degranulation. CONCLUSIONS: Success of the LAD2 line reflects the demand for a human mast cell line in research, the uniqueness of this cell line, and that it continues to exhibit minimal variation from its original description. We hope that the awareness of the impact of this cell line on mast cell research will encourage others to develop and distribute other similar cell lines with additional characteristics so as to address the limitations of depending on the study of cultured human mast cells from tissues.

Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype.

BACKGROUND: Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. OBJECTIVES: We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. METHODS: Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. RESULTS: KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. CONCLUSION: Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing.

Glycomic analysis of human mast cells, eosinophils and basophils.

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

IgE-FcepsilonRI interactions determine HIV coreceptor usage and susceptibility to infection during ontogeny of mast cells.

Progenitor mast cells (prMCs), derived from CD34(+) precursors are CD4(+)/CCR5(+)/CXCR4(+) and susceptible to CCR5(R5)-tropic virus but only marginally susceptible to CXCR4(X4)-tropic HIV. As infected prMCs mature within extravascular compartments, they become both latently infected and HIV-infection resistant, and thus capable of establishing an inducible reservoir of CCR5-tropic infectious clones. In this report we provide the first evidence that IgE-FcepsilonRI interactions, occurring during a unique period of mast cell (MC) ontogeny, enhance prMC susceptibility to X4 and R5X4 virus. IgE-FcepsilonRI interactions significantly increased expression of CXCR4 mRNA ( approximately 400- to 1800-fold), enhanced prMC susceptibility to X4 and R5X4 virus ( approximately 3000- to 16,000-fold), but had no significant effect on CD4, CCR3, or CCR5 expression, susceptibility to R5 virus, or degranulation. Enhanced susceptibility to infection with X4 virus occurred during the first 3-5 wk of MC ontogeny and was completely inhibited by CXCR4-specific peptide antagonists and omalizumab, a drug that inhibits IgE-FcepsilonRI interactions. IgE-FcepsilonRI coaggregation mediated by HIVgp120 or Schistosoma mansoni soluble egg Ag accelerated maximal CXCR4 expression and susceptibility to X4 virus by prMCs. Our findings suggest that for HIV-positive individuals with atopic or helminthic diseases, elevated IgE levels could potentially influence the composition of CXCR4-tropic and R5X4-tropic variants archived within the long-lived tissue MC reservoir created during infection.

Effect of lipopolysaccharide (LPS) and peptidoglycan (PGN) on human mast cell numbers, cytokine production, and protease composition.

BACKGROUND: Human mast cell (HuMC) maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. It is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS) or peptidoglycan (PGN), influences HuMC biology. RESULTS: Over 6 wks of culture, LPS had minimal effect on HuMC numbers but increased CD117, tryptase and chymase expression. PGN inhibited HuMC development. For mature mast cells, LPS in the presence of rhSCF (10 ng/ml) increased CD117, tryptase, chymase and carboxypeptidase expression, primarily in CD117low HuMC. LPS decreased FcepsilonRI expression and beta-hexosaminidase release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1beta and IL-6 (in addition to IL-8 and IL-12) were detected in short-term culture supernatants of LPS treated cells, and reproduced the increases in CD117, tryptase, chymase, and carboxypeptidase expression observed in the presence of LPS. Comparative studies with mouse bone marrow-derived mast cells from wild type, but not TLR4 knockout mice, showed increases in mRNA of mouse mast cell chymases MMCP-1, MMCP-2 and MMCP-4. CONCLUSION: PGN inhibits HuMC growth, while LPS exerts its primary effects on mature HuMC by altering cytokine production and protease composition, particularly at low concentrations of SCF. These data demonstrate the ability of bacterial products to alter HuMC mediator production, granular content, and number which may be particularly relevant at mucosal sites where HuMC are exposed to these products.

Human tissue mast cells are an inducible reservoir of persistent HIV infection.

We have proposed that, unlike other HIV-vulnerable cell lineages, progenitor mast cells (prMCs), cultured in vitro from undifferentiated bone marrow-derived CD34(+) pluripotent progenitors (PPPs), are susceptible to infection during a limited period of their ontogeny. As infected prMCs mature in culture, they lose expression of viral chemokine coreceptors necessary for viral entry and develop into long-lived, latently infected mature tissue mast cells (MCs), resistant to new infection. In vivo recruitment of prMCs to different tissue compartments occurs in response to tissue injury, growth, and remodeling or allergic inflammation, allowing populations of circulating and potentially HIV-susceptible prMCs to spread persistent infection to diverse tissue compartments. In this report, we provide in vivo evidence to confirm this model by demonstrating that HIV-infected women have both circulating prMCs and placental tissue MCs (PLMCs) that harbor inducible infectious HIV even after highly active antiretroviral therapy (HAART) during pregnancy. Furthermore, infectious virus, capable of infecting alloactivated fetal cord blood mononuclear cells (CBMCs), could be induced in isolated latently infected PLMCs after weeks in culture in vitro. These data provide the first in vivo evidence that tissue MCs, developed from infected circulating prMCs, comprise a long-lived inducible reservoir of persistent HIV in infected persons during HAART.

Growth of human mast cells from bone marrow and peripheral blood-derived CD34+ pluripotent progenitor cells.

Human mast cells (HMCs) are derived from a CD34+ pluripotent progenitor cell that is Kit (CD117+), CD13+, Fc epsilonRI- and lacks lineage-specific surface markers. Bone marrow and peripheral blood are two tissue sources available for obtaining CD34+ progenitor cells from which to culture HMCs. CD34+ cells can be isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34+ cells may be immediately cultured in serum-free culture media containing recombinant human stem cell factor (rhSCF), rhIL-6, and rhIL-3 (first week only). Weekly hemidepletions and the removal of adherent cells and/or debris enables the investigator to obtain HMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 wk.

Thrombopoietin alone or in the presence of stem cell factor supports the growth of KIT(CD117)low/ MPL(CD110)+ human mast cells from hematopoietic progenitor cells.

OBJECTIVES: Thrombopoietin (TPO) is known to promote platelet number, have growth-promoting potential for human megakaryocytes (HuMKs), and increase erythrocyte, monocyte, mast cell, and granulocyte numbers in the presence of additional growth factors. We explored the ability of TPO alone or in the presence of stem cell factor (SCF) to support human mast cells (HuMCs). METHODS: CD34+ pluripotent and CD34+/CD117+/CD13+ HuMC progenitor cells were cultured in rhTPO and examined for HuMCs. Similarly, we added rhTPO to CD34(+) cells cultured in stem cell factor (SCF), which promotes HuMC development. RESULTS: When CD34+ cells were cultured in 10 ng/mL rhTPO and 10 ng/mL rhSCF, TPO enhanced HuMC numbers compared to rhSCF alone. Higher concentrations of rhTPO (50 ng/mL) in the presence of 100 ng/mL rhSCF inhibited the rhSCF-dependent subpopulation of CD117high HuMCs, while promoting CD117low HuMCs. Human CD34+/CD117+/CD13+ cells cultured in rhTPO alone for 1 to 2 weeks differentiated into CD41+/CD110+ HuMKs (85-90%) and FcepsilonRI+/CD117low/CD13+ HuMCs (5-10%). RhTPO-induced HuMCs expressed the TPO (CD110) receptor, tryptase, and chymase and survived when recultured in rhSCF. CONCLUSION: The effect of TPO on HuMCs in the presence of rhSCF varies, depending on the relative concentration of each growth factor, while TPO alone or in combination with rhSCF supports a unique population of CD117low/CD110+ HuMCs.

Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated c-kit.

OBJECTIVE: STI571 is a tyrosine kinase inhibitor which inhibits the kinase activity of kit, the receptor for stem cell factor (SCF). Because activating mutations of c-kit affecting codon 816 are associated with human mast cell neoplasms, we determined whether STI571 exerted a similar cytotoxic effect on neoplastic and normal human mast cells. METHODS: We investigated the effect of addition of STI571 in increasing concentrations (0.01 to 10 micromolar) to two HMC-1 human mast cell leukemia cell lines carrying two different activating c-kit mutations in codons 816 or 560, as well as the effect of the drug on short-term bone marrow cultures obtained from patients who carry a mutated codon 816 or wild-type c-kit. RESULTS: STI571 failed to inhibit the growth of HMC-1(560,816) cells bearing a codon 816 mutation but effectively suppressed the proliferation of HMC-1(560) carrying c-kit with the wild-type codon 816. STI571 did not induce preferential killing of neoplastic bone marrow mast cells in short-term cultures from patients bearing a codon 816 c-kit mutation. In contrast, STI571 caused a dramatic reduction in mast cells in patients without codon 816 c-kit mutations. CONCLUSION: These results suggest that STI571, while effectively killing mast cells with wild-type c-kit, did not show preferential cytotoxicity to neoplastic human mast cells and thus may not be effective in the treatment of human systemic mastocytosis associated with codon 816 c-kit mutations.

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI.

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

An immunohistochemical study of the bone marrow lesions of systemic mastocytosis: expression of stem cell factor by lesional mast cells.

The bone marrow biopsy in mastocytosis often reveals characteristic mast cell collections associated with lymphoid cell aggregates. There is limited information on the composition of these aggregates and whether they contain cytokines important to mast cell growth and development. We thus performed an immunohistochemical characterization of bone marrow lesions in 7 patients with systemic indolent mastocytosis. In nodular lesions, the collections of mast cells were surrounded by lymphoid aggregates; these aggregates consisted of a mixture of B and T cells. Immunohistochemical staining for stem cell factor (SCF) revealed the presence of this cytokine in the lesional mast cells with a granular staining pattern. These results show that the characteristic nodular lesions observed in bone marrow biopsy specimens of patients with mastocytosis bearfeatures of benign lymphoid aggregates and that SCF is present in these lesions, creating a potential autocrine or paracrine growth and differentiation loop for mast cells and lymphoid progenitors.