Arnold S. Kirshenbaum

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 FcεRI or FcγRI

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.

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.

Expression of a Functional High-Affinity IgG Receptor, FcγRI, on Human Mast Cells: Up-Regulation by IFN-γ

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor FcεRI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor FcγRI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for FcγRI. We next found that IFN-γ up-regulated the expression of FcγRI. This was confirmed by flow cytometry, where FcγRI expression on human mast cells was increased from ∼2 to 44% by IFN-γ exposure. FcεRI, FcγRII, and FcγRIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4–5 × 108 M−1) increased from ∼2,400 to 12,100–17,300 per cell. Aggregation of FcγRI on human mast cells, and only after IFN-γ exposure, led to significant degranulation as evidenced by histamine release (24.5 ± 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-α, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis

OBJECTIVE The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. MATERIALS AND METHODS The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. RESULTS The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. CONCLUSION These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact.

Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC‐1 human mast cells or primary bone marrow‐derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and β‐hexosaminidase and produced tumor necrosis factor α (TNF‐α), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF‐α production. Addition of the PI3‐kinase inhibitor, wortmannin, inhibited the heterotypic adhesion‐associated degranulation but not TNF‐α production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule‐associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells. J. Leukoc. Biol. 63: 337–341; 1998.

Treatment of three patients with systemic mastocytosis with interferon alpha-2b.

It has been reported that the administration of interferon alpha-2b is of potential benefit in the treatment of mastocytosis based on a single patient study (NEJM, Feb 27, 1992, 326(9):619-623). Following this report, we administered interferon alpha-2b at a dose of 4 to 5 million units per square meter of body surface area for at least 12 months to one patient with mastocytosis with an associated hematologic disorder (patient 1), one patient with aggressive systemic mastocytosis (patient 2), and one patient with indolent mastocytosis (patient 3). Patients were monitored with the following clinical and laboratory parameters: serial bone marrow biopsies and aspirates, patient log of histamine release attacks, medication dependency, plasma tryptase levels, serum lactate dehydrogenase (LDH) levels, white blood cell counts and differentials, extent of urticaria pigmentosa lesions, bony involvement, and extent of gastrointestinal involvement and hepatomegaly. We also examined the ability of interferon alpha-2b to inhibit recombinant human stem cell factor (rhSCF)-dependent mast cell proliferation from CD34+ bone marrow-derived cells. All patients demonstrated continued progression of disease in one or more clinical criteria at one year of therapy. Similarly, interferon alpha-2b did not inhibit the culture of mast cells from CD34+ bone marrow-derived cells in the presence of SCF. Thus, in our study of three patients with systemic mastocytosis, treatment with interferon alpha-2b was found to be ineffective in controlling progression of disease.

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.

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

BackgroundHuman 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.ResultsOver 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 FcεRI expression and β-hexosaminidase release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1β 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.ConclusionPGN 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.

Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+/CD117+/CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+/CD117+/CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3′-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

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.