Human Mast Cells From Adipose Tissue Target and Induce Apoptosis of Breast Cancer Cells.

Mast cells (MC) are important immune sentinels found in most tissue and widely recognized for their role as mediators of Type I hypersensitivity. However, they also secrete anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The purpose of this study was to investigate adipose tissue as a new source of MC in quantities that could be used to study MC biology focusing on their ability to bind to and kill breast cancer cells. We tested several cell culture media previously demonstrated to induce MC differentiation. We report here the generation of functional human MC from adipose tissue. The adipose-derived mast cells (ADMC) are phenotypically and functionally similar to connective tissue expressing tryptase, chymase, c-kit, and FcεRI and capable of degranulating after cross-linking of FcεRI. The ADMC, sensitized with anti-HER2/neu IgE antibodies with human constant regions (trastuzumab IgE and/or C6MH3-B1 IgE), bound to and released MC mediators when incubated with HER2/neu-positive human breast cancer cells (SK-BR-3 and BT-474). Importantly, the HER2/neu IgE-sensitized ADMC induced breast cancer cell (SK-BR-3) death through apoptosis. Breast cancer cell apoptosis was observed after the addition of cell-free supernatants containing mediators released from FcεRI-challenged ADMC. Apoptosis was significantly reduced when TNF-α blocking antibodies were added to the media. Adipose tissue represents a source MC that could be used for multiple research purposes and potentially as a cell-mediated cancer immunotherapy through the expansion of autologous (or allogeneic) MC that can be targeted to tumors through IgE antibodies recognizing tumor specific antigens.

NF-κB inhibitors that prevent foam cell formation and atherosclerotic plaque accumulation.

The transformation of monocyte-derived macrophages into lipid-laden foam cells is one inflammatory process underlying atherosclerotic disease. Previous studies have demonstrated that fullerene derivatives (FDs) have inflammation-blunting properties. Thus, it was hypothesized that FD could inhibit the transformation process underlying foam cell formation. Fullerene derivatives inhibited the phorbol myristic acid/oxidized low-density lipoprotein-induced differentiation of macrophages into foam cells as determined by lipid staining and morphology.Lipoprotein-induced generation of TNF-α, C5a-induced MC activation, ICAM-1 driven adhesion, and CD36 expression were significantly inhibited in FD treated cells compared to non-treated cells. Inhibition appeared to be mediated through the NF-κB pathway as FD reduced expression of NF-κB and atherosclerosis-associated genes. Compared to controls, FD dramatically inhibited plaque formation in arteries of apolipoprotein E null mice. Thus, FD may be an unrecognized therapy to prevent atherosclerotic lesions via inhibition of foam cell formation and MC stabilization.

Inhibition of inflammatory arthritis using fullerene nanomaterials.

Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

A steroid-mimicking nanomaterial that mediates inhibition of human lung mast cell responses.

Water-soluble fullerenes can be engineered to regulate activation of mast cells (MC) and control MC-driven diseases in vivo. To further understand their anti-inflammatory mechanisms a C70-based fullerene conjugated to four myo-inositol molecules (C70-I) was examined in vitro for its effects on the signaling pathways leading to mediator release from human lung MC. The C70-I fullerene stabilizes MC and acts synergistically with long-acting ß2-adrenergic receptor agonists (LABA) to enhance inhibition of MC mediator release through FcεRI-simulation. The inhibition was paralleled by the upregulation of dual-specificity phosphatase one (DUSP1) gene and protein levels. Concomitantly, increases in MAPK were blunted in C70-I treated cells. The increase in DUSP1 expression was due to the ability of C70-I to prevent the ubiquitination and degradation of DUSP1. These findings identify a mechanism of how fullerenes inhibit inflammatory mediator release from MC and suggest they could potentially be an alternative therapy for steroid resistant asthmatics. FROM THE CLINICAL EDITOR: This study investigates the role and mechanism of action of fullerenes in deactivating mast cell-based inflammation, paving the way to the development of a novel, non-steroid therapy in reactive airway disease.

Human skin mast cells express complement factors C3 and C5.

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1ß, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas ß-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.

Generation of anaphylatoxins by human beta-tryptase from C3, C4, and C5.

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.