The human cardiac mast cell: localization, isolation, phenotype, and functional characterization

We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.     

Immortalization and characterization of human cell lines with mast cell and monocytic properties

Summary. We have previously derived a cell strain which had both mast cell and monocytic properties from the bone marrow of a child with diffuse cutaneous mastocytosis. This cell strain, termed HBM-M, consisted of two cell populations both of which possessed certain ultrastructural, cytochemi-cal and surface phenotypic features of degranulated mast cells. The cells also displayed cytochemical and surface phenotypic features of monocytes. These cells may represent a common bone marrow derived mast cell/monocyte precursor. Studies of human mast cells have been hindered by the fact that it is difficult to establish such cells in long-term culture. Thus, we sought to immortalize HBM-M cells by introducing Simian virus 40 large T-antigen. Following transfection by the strontium phosphate technique, transformed cells were selected, expanded and passaged until the cells entered a non-proliferative phase termed crisis. Certain clones passed through crisis 3 months later and by this means two immortal cell lines, HBM-MI-1 and HBM-MI-2, were obtained. The criterion for immortality was growth for greater than 100 population doublings. The immortal cell lines retained some, but not all. of the mast cell and monocytic properties of the original HBM-M cell strain. The immortalization of the cell strain HBM-M provides an opportunity to investigate the mast cell and monocytic properties of these cells, and the apparent relationship between mast cells and monocytes.     

Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: Antiproliferative effects of peptide analogues

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4–71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.     

Expression of Bcl-2 by human bone marrow mast cells and its overexpression in mast cell leukemia

Bcl-2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl-2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl-2 was studied using quantitative three-color flow cytometry. We also studied the molecular configuration of the bcl-2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl-2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl-2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl-2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl-2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl-2, which could not be related to molecular rearrangements involving the bcl-2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl-2 by BMMC in MCL may help to explain their resistance to chemotherapy-induced apoptosis.     

Expression of dipeptidylaminopeptidase-IV in some human T and B cell lines

The specificity of the expression of dipeptidylaminopeptidase-IV (DAP-IV) was examined in cells from leukemia patients and in 33 normal and leukemic human cell lines widely used in various studies. There was no correlation between DAP-IV activity and OKT4 positivity or maturation stage in T cells. In addition, DAP-IV was unexpectedly expressed by 4 B cell lines and 1 histiocytic lymphoma cell line, indicating either lack of specificity of DAP-IV or infidelity of gene expression under unnatural culture environment.     

Angiogenesis inhibitor TNP-470: cytotoxic effects on human neoplastic cell lines

Angiostatic substance TNP-470 displayed moderate cytotoxicity towards human leukemia HL-60, HL-60/ADR, HL-60/VCR and myeloma ARH77 cell lines with IC50 in the range 5-10 microM of concentrations and slightly higher IC50 for myeloma cell line U266. IC50 for ovarian CH-1, A2780 and A2780/ADR cell lines was in the range 10-15 microM with the exception of platinum-resistant SKOV3 cell line (more than 40 microM ). The IC50 values for MDA-MB-231 and MCF-7 breast carcinoma cell lines were 15 and 25 microM, respectively. In human hemopoietic neoplastic cell lines examined, TNP-470 induced the appearance of subpopulation with sub-G0 DNA content, suggesting the apoptosis-inducing potential of TNP-470 in these cells. No TNP-470-induced drug uptake modulation in drug-resistant leukemia cell line HL-60/VCR was observed. TNP-470 induced accumulation of cells in G0/G1 phase of cell cycle. There was no TNP-470-induced inhibition of MMP collagenase activity or MMP (MMP2 and MMP9) production in the human fibrosarcoma cells HT 1080 in vitro.     

Evidence of mast cell activation and leukotriene release after mannitol inhalation.

The aim of this study was to investigate if mannitol inhalation, as a model of exercise-induced bronchoconstriction (EIB), causes mast cell activation and release of mediators of bronchoconstriction. Urinary excretion of previously identified mediators of EIB was investigated in association with mannitol-induced bronchoconstriction. Twelve asthmatic and nine nonasthmatic subjects inhaled mannitol and urine was collected 60 min before and for 90 min after challenge. The urinary concentrations of leukotriene (LT)E4, the prostaglandin (PG)D2 metabolite and the mast cell marker 9alpha,11beta-PGF2 were measured by enzyme immunoassay. N(tau)-methylhistamine was measured by radioimmunoassay. In asthmatic subjects, inhalation of a mean+/-SEM dose of 272+/-56 mg mannitol induced a reduction in forced expiratory volume in one second (FEV1) of 34.5+/-2.1%. This was associated with increases in urinary 9alpha,11beta-PGF2 (91.9+/-8.2 versus 66.9+/-6.6 ng x mmol creatinine(-1), peak versus baseline) and LTE4 (51.3+/-7.5 versus 32.9+/-4.7). In nonasthmatic subjects, the reduction in FEV1 was 1.0+/-0.5% after inhaling 635 mg of mannitol. Although smaller than in the asthmatics, significant increases of urinary 9alpha,11beta-PGF2 (68.4+/-6.9 versus 56.0+/-5.8 ng x mmol creatinine(-1)) and LTE4 (58.5+/-5.3 versus 43.0+/-3.3 ng x mmol creatinine(-1)) were observed in the nonasthmatic subjects. There was also a small increase in urinary excretion of N(tau)-methylhistamine in the nonasthmatics, but not in the asthmatics. The increased urinary levels of 9alpha,11beta-prostaglandin F2 support mast cell activation with release of mediators following inhalation of mannitol. Increased bronchial responsiveness to the released mediators could explain the exclusive bronchoconstriction in asthmatic subjects.     

Antiproliferative effects of natural human tumor necrosis factor-alpha, interferon-alpha, and interferon-gamma on human pancreatic carcinoma cell lines

The antiproliferative effects of natural human tumor necrosis factor-alpha (nHuTNF-alpha), natural human interferon-alpha (nHuIFN-alpha), and natural human interferon-gamma (nHuIFN-gamma) were investigated in human pancreatic carcinoma cell lines. HuP-T3, HuP-T4, and BxPC-3 carcinoma cells exhibited mild growth inhibition after treatment with nHuTNF-alpha. HuP-T3, HuP-T4, MIA-PaCa-2, and BxPC-3 cells exhibited mild or marked growth inhibition after treatment with nHuIFN-alpha. Incubation with nHuIFN-gamma caused marked growth inhibition of HuP-T4 and BxPC-3 cells, whereas HuP-T1, HuP-T3, and MIA-PaCa-2 cells showed only mild growth inhibition with the same dose of nHuIFN-gamma. Combined nHuTNF-alpha and nHuIFN-alpha (1:1) demonstrated marked synergism in comparison with their effects as single agents on HuP-T1, HuP-T3, and MIA-PaCa-2 cells. The combination of nHuTNF-alpha and nHuIFN-gamma (100:1) also demonstrated a marked synergistic effect in comparison with these cytokines alone in four out of five pancreatic carcinoma cell lines (HuP-T1, HuP-T3, HuP-T4, and MIA-PaCa-2 cell lines). The marked increase in efficacy brought about by using combinations of cytokines may enable some improvement in the treatment of pancreatic carcinoma patients in the future.     

Dickkopf1在人脑胶质瘤细胞株中的表达及意义

目的探讨Wnt通路抑制因子Dickkopf-1（DKK1）在人胶质瘤发生、发展中的作用。方法采用ELISA、RT-PCR法检测人胶质瘤细胞株U251和T98G中DKK1蛋白及DKK1 mRNA表达水平。结果DKK1蛋白在U251和T98G中均呈高表达;DKK1mRNA在胶质瘤细胞株U251和T98G中高表达,但在正常脑组织中表达不明显。结论DKK1在人胶质瘤细胞株中高表达;其可能在胶质瘤发生发展中起重要作用。     

Expression and function of beta-adrenergic receptors in human hematopoietic cell lines.

We investigated the expression and functional characteristics of beta-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [125I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of beta-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimal or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of beta-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32P-labelled beta 2-adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their beta-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage.     

Expression, regulation, and chromosomal localization of the Max gene.

The Max gene encodes a protein that interacts specifically with the Myc protein to form a heterodimer with high affinity for the specific cognate DNA binding site of Myc. Here we examine the expression of Max RNA in comparison to Myc RNA during cell growth and differentiation. Two species of RNA, a major 2.0- and a minor 1.7-kilobase species, hybridized specifically to a Max cDNA probe in all human and murine cell lines that were tested. Unlike Myc, the steady-state level of Max RNA is not significantly modulated with respect to proliferation or differentiation. Max RNA is expressed in quiescent BALB/c 3T3 cells and is modestly increased 3 h after addition of serum to the quiescent cells. In contrast to Myc RNA, Max RNA does not decline immediately upon induction of differentiation of HL60 cells by dimethyl sulfoxide, and only a modest decrease of Max RNA was observed 72 h after induction of differentiation. Unlike Myc RNA, Max RNA is relatively stable with a half-life of greater than 3 h and, therefore, does not exhibit the characteristic short half-life of RNAs encoded by most immediate early genes. The human Max gene was localized to chromosome 14, band q23. With respect to the recurring abnormalities in human tumors, this region of chromosome 14 is involved in deletions in B-cell chronic lymphocytic leukemia and malignant lymphomas and in the 12;14 translocation in uterine leiomyomas.