Mast cells (MCs) arise in situ from circulating stem cell factor (SCF)-dependent committed progenitors (PrMCs) and accumulate at sites of allergic mucosal inflammation. We hypothesized that human (h)PrMCs and their mature counterparts might share overlapping patterns of chemokine and cytokine receptor utilization with eosinophils, basophils, and T helper type 2 (Th2) lymphocytes for their homing and allergy-associated hyperplasia. We have characterized committed hPrMCs and fully mature hMCs derived in vitro from cord blood for their functional responses to chemokine and cytokine agonists germane to allergic inflammation and for their maturation-related expression of the corresponding receptors. After 4 wk of culture in the presence of recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-10, the cells were characterized as hPrMCs based upon their uniform surface expression of c-kit and CD13, low-level expression of Fc∈RIα, absence of CD14 and CD16 expression, and immunoreactivity for MC chymase in >80%, and about half were immunoreactive for tryptase and metachromatic with toluidine blue. By week 9, the cells had matured into hMCs, identified by higher levels of c-kit, continued expression of CD13 and low-level Fc∈RIα, uniform toluidine blue metachromasia, and uniform immunoreactivity for both tryptase and chymase. The 4-wk-old hPrMCs expressed four chemokine receptors (CXCR2, CCR3, CXCR4, and CCR5). Each receptor mediated transient rapid calcium fluxes in response to its respective ligand. Both recombinant human eotaxin and stromal cell–derived factor 1α elicited chemotaxis of hPrMCs. Only CCR3 was retained on the mature 9-wk-old hMCs from among these chemokine receptors, and hMCs responded to eotaxin with a sustained calcium flux but without chemotaxis. The Th2 cytokines IL-3, IL-5, IL-6, IL-9, and granulocyte/macrophage colony-stimulating factor each augmented the SCF-dependent proliferation of hPrMCs and hMCs. In contrast, the prototypical Th1 cytokine, interferon γ, suppressed SCF-driven proliferation of both hPrMCs and hMCs. Thus, throughout their development in vitro, hMCs obey SCF-dependent, cytokine-driven mitogenic responses that reflect a Th2-type polarization characteristic of allergy and asthma. Furthermore, committed hPrMCs have a unique profile of chemokine receptor expression from among reported hematopoietic cells, including CCR3, which is shared with the other cells central to allergic inflammation (eosinophils, basophils, and Th2 lymphocytes).
Endothelial dysfunction, or activation, elicited by oxidized low density lipoprotein (Ox-LDL) and its lipid constituents has been shown to play a key role in the pathogenesis of atherosclerosis. We recently have identified a novel receptor for Ox-LDL-designated lectin-like Ox-LDL receptor (LOX-1) in vascular endothelial cells. To examine ligand specificity of LOX-1, we established CHO cell lines stably expressing both human and bovine LOX-1 (LOX-1-CHO). LOX-1-CHO bound and degraded 125I-labeled Ox-LDL but did not significantly degrade 125I-labeled acetylated LDL (Ac-LDL). Fucoidin and maleylated BSA (M-BSA), which inhibit 125I-Ox-LDL binding to class A scavenger receptors, did not inhibit 125I-Ox-LDL binding or degradation in LOX-1-CHO. Polyinosinic acid and carrageenan, in contrast, significantly reduced 125I-Ox-LDL binding to LOX-1-CHO by 62% and 60%, respectively. Delipidated and untreated 125I-Ox-LDL were bound and degraded equally in LOX-1-CHO; furthermore, excess amounts of unlabeled, delipidated Ox-LDL inhibited binding and degradation of untreated 125I-Ox-LDL. Taken together, LOX-1 is a receptor for Ox-LDL but not for Ac-LDL. LOX-1 recognizes protein moiety of Ox-LDL, and its ligand specificity is distinct from other receptors for Ox-LDL, including class A and B scavenger receptors.
Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro . Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcɛRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-α, IL-5, IL-13, macrophage inflammatory protein 1α, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcɛRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcɛRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.
Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit؉ cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.
Bone density of lumbar vertebrae (L2 to L4) and the whole body in 29 patients with anorexia nervosa were measured by dual photon absorptiometry, and the results were compared with those of 10 age-matched normal controls. The patients had significantly lower bone mineral density (BMD) in L3 and L2-4 than controls. However, there was no difference in whole-body BMD. L3 and L2-4 BMD was positively correlated with body weight and was negatively correlated with duration of illness and amenorrhea. Patients who had been more active 6 months before the time of the study had significantly higher L3 BMD than the less active patients. Most patients had an abnormally low serum estrogen level, whereas the mean serum levels of thyroid hormone (T3, T4), cortisol, calcitonin, parathyroid hormone and vitamin D were within the normal range. No correlation was found between L3 or L2-4 BMD and the levels of these hormones. These results suggest that severe weight loss, low physical activity, longer duration of amenorrhea and deficiency of estrogen contribute to bone loss in patients with anorexia nervosa, whereas calcium-regulating hormones such as parathyroid hormone, calcitonin and vitamin D are unlikely to be a primary contributor to bone loss.
Polyphenols, retained in black tea wastes following the commercial production of tea beverages, represent an underutilized resource. The purpose of this study was to investigate the potential use of hot-compressed water (HCW) for the extraction of pancreatic lipase-inhibiting polyphenols from black tea residues. Black tea residues were treated with HCW at 10 °C intervals, from 100 to 200 °C. The resulting extracts were analyzed using high-performance liquid chromatography-mass spectrometry and assayed to determine their inhibitory effect on pancreatic lipase activity in vitro. Four theaflavins (TF), 5 catechins, 2 quercetin glycosides, quinic acid, gallic acid, and caffeine were identified. The total polyphenol content of extracts increased with increasing temperature but lipase inhibitors (TF, theaflavin 3-O-gallate, theaflavin 3'-O-gallate, theaflavin 3,3'-O-gallate, epigallocatechin gallate, and epicatechin gallate) decreased over 150 °C. All extracts inhibited pancreatic lipase but extracts obtained at 100 to 140 °C showed the greatest lipase inhibition (IC(50) s of 0.9 to 1.3 μg/mL), consistent with the optimal extraction of TFs and catechins except catechin by HCW between 130 and 150 °C. HCW can be used to extract pancreatic lipase-inhibiting polyphenols from black tea waste. These extracts have potential uses, as dietary supplements and medications, for the prevention and treatment of obesity.
Accumulation of substantial numbers of activated T lymphocytes, as well as monocyte/macrophages, in focal areas of arterial intima appears to be a hallmark of atherogenesis. Our previous report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate the expression of heparin-binding epidermal growth factor-like growth factor and the interleukin (IL)-2 receptor in cultured human peripheral T lymphocytes. In this study, we show that lyso-PC can also enhance interferon gamma (IFN-gamma) secretion and gene expression in human T lymphocytes. Lyso-PC-induced upregulation of IFN-gamma depended on the presence of IL-2, IL-12, or phytohemagglutinin in culture media and was similarly observed in both CD4+ and CD8+ subsets. Actinomycin D chase by Northern blotting showed that lyso-PC significantly prolonged IFN-gamma mRNA half-lives in human T cells. Transient transfection of IFN-gamma promoter-reporter gene construct in the human T-cell line Jurkat cells demonstrated that lyso-PC stimulated the transcription of IFN-gamma promoter-driven luciferase gene. Analyses of serial deletion mutations of IFN-gamma promoter revealed that the lyso-PC-responsive element is located between base pairs - 102 and -78 of the transcription initiation site of the IFN-gamma gene. Enhanced expression of IFN-gamma in T lymphocytes by lyso-PC may play a crucial role in atherogenesis.
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