Michael Samoszuk

Eosinophils Are a Major Source of Nitric Oxide-Derived Oxidants in Severe Asthma: Characterization of Pathways Available to Eosinophils for Generating Reactive Nitrogen Species

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 ± 198 μmol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 ± 40.7 μmol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 ± 457 μmol BrY/mol tyrosine; 161 ± 88 μmol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 ± 14.5 μmol BrY/mol tyrosine; 65 ± 69 μmol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO2−, NO3−, or NO2Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO2− or an exogenous NO source. At low, but not high (e.g., >2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

Degranulating mast cells in fibrotic regions of human tumors and evidence that mast cell heparin interferes with the growth of tumor cells through a mechanism involving fibroblasts

BackgroundThe purpose of this study was to test the hypothesis that mast cells that are present in fibrotic regions of cancer can suppress the growth of tumor cells through an indirect mechanism involving peri-tumoral fibroblasts.MethodsWe first immunostained a wide variety of human cancers for the presence of degranulated mast cells. In a subsequent series of controlled in vitro experiments, we then co-cultured UACC-812 human breast cancer cells with normal fibroblasts in the presence or absence of different combinations and doses of mast cell tryptase, mast cell heparin, a lysate of the human mast cell line HMC-1, and fibroblast growth factor-7 (FGF-7), a powerful, heparin-binding growth factor for breast epithelial cells.ResultsDegranulating mast cells were localized predominantly in the fibrous tissue of every case of breast cancer, head and neck cancer, lung cancer, ovarian cancer, non-Hodgkins lymphoma, and Hodgkins disease that we examined. Mast cell tryptase and HMC-1 lysate had no significant effect on the clonogenic growth of cancer cells co-cultured with fibroblasts. By contrast, mast cell heparin at multiple doses significantly reduced the size and number of colonies of tumor cells co-cultured with fibroblasts, especially in the presence of FGF-7. Neither heparin nor FGF-7, individually or in combination, produced any significant effect on the clonogenic growth of breast cancer cells cultured without fibroblasts.ConclusionDegranulating mast cells are restricted to peri-tumoral fibrous tissue, and mast cell heparin is a powerful inhibitor of clonogenic growth of tumor cells co-cultured with fibroblasts. These results may help to explain the well-known ability of heparin to inhibit the growth of primary and metastatic tumors.

Improved Immunohistochemical Method for Detecting Hypoxia Gradients in Mouse Tissues and Tumors

We describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues.

Mast cell inhibitor cromolyn increases blood clotting and hypoxia in murine breast cancer

Human breast cancer is extensively infiltrated by mast cells that contain powerful anticoagulants such as heparin, tryptase and chymase. To determine if human breast cancer is associated with mast cell activation, we measured the levels of mast cell tryptase (an indicator of mast cell activation) in the blood of 20 women with varying stages of breast cancer. The mean level of tryptase in women with breast cancer (10.3 ± 4.2 μg/L) was significantly higher than in 50 normal healthy women without breast cancer (3.0 ± 2.5 μg/L, p < 0.05 by two‐tailed t‐test). To explore the role of mast cells in breast cancer in more detail, we then carried out experiments that were aimed at determining if an inhibitor of mast cell function, sodium cromolyn, could increase blood clotting and hypoxia within subcutaneous implants of the 4T1 mammary adenocarcinoma cell line in mice. We treated tumor‐bearing mice with 5 consecutive daily doses of sodium cromolyn (10 mg/kg, i.p.). An average of 30% of the periphery of the tumors from the 5 drug‐treated mice contained large lakes of clotted blood that were not evident in any of the tumors from the control (untreated) mice. By computerized image analysis of tumors immunostained for a hypoxia marker (pimonidazole), the tumors from the treated mice had significantly more hypoxia (35 ±12 % hypoxic regions, n = 5) than the tumors from untreated (control) mice (16 ± 7%, n = 5). We conclude that sodium cromolyn enhanced peri‐tumoral blood clotting and intratumoral hypoxia. These results suggest that mast cells may play an important role in regulating blood clotting and hypoxia within breast cancer.

Degranulating Eosinophils in Human Endometriosis

Degranulating eosinophils have been described in most endometrial cancers. We hypothesized that endometriosis (ectopic, nonneoplastic endometrial tissue) would be an appropriate model system for determining whether eosinophil degranulation is part of a specific immune response to endometrial cancer or if it is related to the more general phenomenon of tissue remodeling (wound healing) that is common to both disorders. To test this hypothesis, we performed immunohistochemistry and Western blotting to evaluate the presence of eosinophil peroxidase (a marker of eosinophil degranulation) in normal endometrium (n = 20) and endometriosis samples (n = 24) and to define the coexpression of three eosinophil chemoattractants: interleukin-5 (IL-5), eotaxin, and regulated on activator-normal T cell expressed and secreted (RANTES). There was focally intense deposition of eosinophil peroxidase in the fibrotic connective tissue and blood vessels of 21 of 24 human endometriosis specimens; two samples showed weak staining, and only one tissue was negative for eosinophil degranulation. None of the 10 normal proliferative endometrial specimens had evidence of eosinophil degranulation, and four of 10 secretory tissues stained only weakly for eosinophil peroxidase. The presence of degranulating eosinophils was also associated with the presence of eotaxin and IL-5 in some samples and with RANTES in others. We conclude that the abundant presence of degranulating eosinophils in the fibrous regions of endometriosis supports the interpretation that eosinophils are involved in general tissue remodeling and wound healing rather than a tissue-directed immune response.

Hyperplasia of mantle/marginal zone B cells with clear cytoplasm in peripheral lymph nodes. A clinicopathologic study of 35 cases.

We describe 35 peripheral lymph nodes classified as mantle cell/marginal zone B-cell hyperplasia with clear cells using morphologic and immunologic findings. For the purpose of this study, we obtained clinical follow-up information and performed immunoglobulin gene rearrangement studies on paraffin sections by polymerase chain reaction. Architecturally, the nodes were suggestive of a benign process: no pericapsular infiltration, sinuses readily identified, scattered reactive follicles present, and paracortical nodular hyperplasia present. No monocytoid B cells were present. Focally, small lymphoid cells with round nuclei and clear cytoplasm (clear cells) formed monomorphic nodular, inverse follicular, and/or marginal zone patterns. Flow cytometry and immunohistochemical analysis revealed neither light chain restriction nor an aberrant B-cell phenotype. Immunoglobulin gene rearrangement studies showed a clonal band in 1 of 26 cases in which DNA was amplified. To ascertain the clinical relevance of this positive case, follow-up information was obtained 30 months after the initial biopsy; the 83-year-old woman was alive without treatment but had splenomegaly and bone marrow involvement by marginal zone B-cell lymphoma. The morphologic and immunologic criteria used for diagnosis of mantle cell/marginal zone B-cell hyperplasia with clear cytoplasm are valid; however, to rule out the possibility of occult lymphoma, immunoglobulin gene rearrangement studies and clinical follow-up are necessary.

Disseminated persistent lymphoid hyperplasia containing Epstein-Barr virus and clonal rearrangements of DNA.

We describe the pathologic, molecular, and clinical features of a 52-year-old man who had a 7-year history of widely disseminated, persistent lymphoid hyperplasia. Exuberant follicular and interfollicular lymphoid hyperplasia with some histologic features of Castlemans disease were present at various times in the submental and cervical lymph nodes, lacrimal and parotid glands, right and left orbits, mediastinum, and hard palate of this patient. Flow cytometric and immunoperoxidase studies of two of the specimens indicated a slight predominance of cells expressing-immunoglobulin light chain. In one specimen, there were clonal rearrangements of DNA coding for immunoglobulin heavy chain and for the T-cell (3-receptor. When DNA from this specimen was also examined by the polymerase chain reaction technique. Epstein-Barr viral DNA was detected. This case suggests that Epstein-Barr virus may be associated with an unusual form of aggressive and persistent lymphoid hyperplasia that contains clonal rearrangements of DNA.

Acceleration of tumor growth and peri-tumoral blood clotting by imatinib mesylate (Gleevec™)

Imatinib mesylate (Gleevec™) inhibits the BCR‐ABL tyrosine kinase in chronic granulocytic leukemia. Previous studies have demonstrated that imatinib mesylate also inhibits the survival and functions of normal mast cells by interfering with the receptor tyrosine kinase for stem cell factor (SCF), c‐kit, which is expressed by mast cells. Because mast cells extensively surround many types of cancer and contain powerful anticoagulants such as heparin, we investigated the effects of imatinib mesylate on blood clotting and tumor growth within subcutaneous implants of a mammary adenocarcinoma cell line (4T1) in BALB/c mice. After 5 days of oral treatment with 10 mg/kg of the drug, the average mass of the tumors in treated mice (198 ± 42 mg, n = 5) was significantly (p < 0.05) greater than the average mass of the tumors from untreated (control) mice (60 ± 23 mg, n = 5). Moreover, the tumors in the treated mice were frequently surrounded by large lakes of clotted blood that were not evident in tumors from the control mice. Accelerated growth and blood clotting were also observed in tumor‐bearing mice treated with heparinase I enzyme to destroy endogenous mast cell heparin and in NDST‐2 knockout mice in which there is a targeted disruption in the gene coding for mast cell heparin synthesis. We conclude that imatinib mesylate accelerated the growth and peri‐tumoral blood clotting of implants of mammary adenocarcinoma in mice. These results suggest that imatinib mesylate may have significant effects on mast cells infiltrating tumors, in addition to its other biologic activities. Our results also indicate that the mechanism of this effect may be related to the anticoagulant properties of mast cell heparin.

Association Between Nitrotyrosine Levels and Microvascular Density in Human Breast Cancer

Nitrotyrosine (NO2Y) is a global marker of protein modification by reactive nitrogen species such as peroxynitrite derived from nitric oxide (NO). Because NO and its derivatives are postulated to enhance carcinogenesis, we used stable isotope dilution mass spectrometry to measure the levels of NO2Y in 30 samples of human breast cancer of varying pathologic types. In the samples tested, the NO2Y levels were generally low (average of 14.1 ± 9.2 μmol NO2Y per mole of tyrosine). Breast cancers with a high microvascular density, however, had a significantly higher average level of NO2Y than tumors with a low microvascular density (20 v.s. 10 μmol NO2Y per mole of tyrosine, p = 0.007 by two-tailed t-test, assuming unequal variances of two samples). There was no apparent association between NO2Y levels and the differentiation of the tumors, tumor aneuploidy, estrogen receptor status, HER-2 expression, lymph node status, or infiltration of the tumors by neutrophils or eosinophils. When the tissues were stained by immunohistochemistry for NO2Y, the NO2Y was localized predominantly within inflammatory cells located immediately adjacent to blood vessels at the edges of the tumors. NO2Y was generally not evident within the tumor cells or inflammatory cells in the stroma. We conclude that low levels of reactive nitrogen species are located predominantly within inflammatory cells near blood vessels of breast cancer and that higher NO2Y levels are associated with an increased density of blood vessels. Our findings, therefore, support a possible association between inflammatory cells and reactive nitrogen species in modulating microvascular density at the edges of breast cancer.

Selective Thrombosis of Tumor Blood Vessels in Mammary Adenocarcinoma Implants in Rats

Adenocarcinomas in rats and humans frequently contain perivascular, degranulating mast cells that release heparin. Protamine is a low-molecular weight, cationic polypeptide that binds avidly to heparin and neutralizes its anticoagulant properties. We hypothesized that mast-cell heparin functions as a localized anticoagulant that modulates hemostasis and blood perfusion in tumors. Consequently, systemically administered protamine should be able to neutralize the endogenous heparin within tumors, thereby inducing selective thrombosis of blood vessels within tumors. Here we demonstrate with magnetic resonance imaging (MRI) that an intravenous dose of protamine labeled with gadolinium accumulated within the parenchyma of subcutaneous implants of a mammary adenocarcinoma in Fischer 344 rats. Moreover, we show with dynamic contrast enhanced MRI that sequential intravenous doses of protamine in 12 tumor-bearing rats resulted in significantly decreased signal enhancement kinetics (blood perfusion) of the tumor. This functional impairment of MRI signal enhancement was accompanied by histological evidence of thrombosis in the blood vessels within the tumor. There was no histological evidence of thrombosis within normal liver, kidney, lung, spleen, or adjacent muscle of tumor-bearing animals that received protamine treatment or in the tumors of animals that had not been pretreated with protamine. On the basis of these results, we conclude that protamine accumulates within adenocarcinoma implants and induces selective thrombosis of blood vessels within the tumor, probably by neutralizing the endogenous heparin within tumors.