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X Congreso Nacional de Paleopatología. Univesidad Autónoma de Madrid, septiembre de 2009

P-glycoprotein has been identified in mast cells stabilized in culture as well as in rat peritoneal mast cells, and is primarily concentrated on the granular membrane. This study aimed to define the role of this protein in the transport and accumulation of doxorubicin in mast cell granules and in its histamine releasing effect. The reverting agent verapamil, that is a substrate for P-glycoprotein, inhibited doxorubicin uptake in intact mast cells in a dose and time dependent manner, but had no effect on the exocytotic action of the antineoplastic drug. Doxorubicin was also concentrated in granules with intact membranes and the uptake was dependent on temperature and showed a trend for saturation. Verapamil and vinblastine, another substrate for P-glycoprotein, significantly reduced doxorubicin concentrations in intact granules. Similar results were obtained with the metabolic inhibitors sodium metavanadate, N-ethylmaleimide, and sodium azide, whereas ouabain, an inhibitor of sodium-potassium ATPase, was without effect. Doxorubicin was taken also up in granule remnants, consisting of a proteoglycan matrix without membrane, that are extruded from mast cells upon stimulation. However, the uptake was not dependent on temperature and was not modified by P-glycoprotein substrates or metabolic inhibitors. Rat peritoneal mast cells were examined for the expression of P-glycoprotein at the protein level with C219 monoclonal antibody, using Western blot, confirming that P-glycoprotein was expressed in mast cells. These data suggest the presence of a P-glycoprotein active in the transport of doxorubicin, in mast cell granules.

The process of exocytosis was studied in rat and beige mouse peritoneal mast cells stimulated by adriamycin (ADR) at 37 degrees C and 22 degrees C. ADR induces a non cytotoxic histamine release that is followed by a significant uptake of the drug. Examination was performed by transmission electron microscopy and, at the same time, histamine release and ADR uptake were measured by spectrofluorimetry. ADR accumulation in mast cells was investigated also by fluorescence microscopy. In rat mast cells stimulated at 37 degrees C, the secretory process developed abruptly and was virtually complete after 30 sec. Electron microscopy showed rapid intracytoplasmic channel formation and extrusion of secretory granules; spectrofluorimetry revealed a massive release of histamine and rapid uptake of ADR. In addition, fluorescence microscopy showed mast cells exhibiting an intense orange-yellow fluorescent signal localized at the secretory granules. At 22 degrees C, rat mast cells showed alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane. Histamine release and ADR uptake proceeded less quickly than at 37 degrees C. Quantitative analysis of rat mast cell ultrastructure demonstrated that histamine release induced by ADR stimulation was achieved by sequential exocytosis. This process presents both morphological and biochemical affinities with the exocytosis induced by basic secretagogues such as compound 48/80. In beige mouse mast cells the process of exocytosis progressed more slowly and was completed after 20 min at 37 degrees C. By electron microscopy, the cytoplasm presented a rigid structure due to abundance of actin-like fibrils. Granule fusion was an uncommon feature and exocytosis was mostly the result of single granule opening to the cell exterior without extrusion of granule matrices.

Mast cells stimulated with adriamycin at 4 degrees C underwent a unique exocytotic reaction. Rat peritoneal cells including mast cells were stimulated in vitro with adriamycin (100 micrograms/ml) for 0, 10, 30 or 60 sec and observed by transmission electron microscopy. Early changes could be observed after 10 sec stimulation and consisted in an approximately 5-fold increase (p0.001) of 0.05-0.2 micron diameter cytoplasmic vesicles. The Golgi apparatus showed signs of activation and vacuolization. From 10 to 30 sec, cytoplasmic vesicles fused with the perigranular membranes and with the membranes of developing secretory channels. At 60 sec, the number of vesicles and vacuoles diminished to nearly two-fold starting levels. The exocytotic reaction characteristically resulted in the formation of enormously dilated granular cavities. The secretory process appeared incomplete; after 60 sec, in fact, maximal histamine release was 20% and exocytosis could be found in approximately 30% of mast cells. Pre-incubation with vinblastine followed by adriamycin stimulation at 37 degrees C determined a dose-dependent inhibition of histamine release which was accompanied by the ultrastructural appearance of numerous 0.05-0.5 micron cytoplasmic vesicles and by signs of inhibited exocytosis. Our results support the concept that hyperstability of the cortical cytoskeleton coupled with microtubule perturbation would be responsible for the depressed pattern of mast cell exocytosis observed at 4 degrees C. Although stimulation at 4 degrees C induces a paradoxal secretory process, we believe that this approach may represent a useful model for understanding some basic mechanisms of exocytosis in mast cells.

There is much evidence that rheumatoid arthritis is closely linked to angiogenesis. Important angiogenic mediators have been demonstrated in synovium and tenosynovium of rheumatoid joints. VEGF (Vascular Endothelial Growth Factor), expressed in response to soluble mediators such as cytokines and growth factors and its receptors are the best characterized system in the angiogenesis regulation of rheumatoid joints. Moreover, other angiogenic mediators such as platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor beta (TGF-ß), tumor necrosis factor alpha (TNF- a), interleukin-1 (IL-1), IL-6, IL-8, IL-13, IL-15, IL-18, angiogenin, platelet activating factor (PAF), angiopoietin, soluble adhesion molecules, endothelial mediator (endoglin) play an important role in angiogenesis in rheumatoid arthritis. On the other hand, endostatin, thrombospondin-1 and -2 are angiogenic inhibitors in rheumatoid arthritis. The persistence of inflammation in rheumatoid joints is a consequence of an imbalance between these inducers and inhibitors of angiogenesis.

In adult life, the architecture of the intestinal villus is maintained by a complex series of epithelialstromal interactions that involve different types of fixed and mobile cells located in the intestinal mucosa. Mast cells (MC) are normal constituents of the small bowel mucosa where they reside in the villous and pericryptal lamina propria as well as within the columnar epithelial cell layer. Besides being involved in numerous immune and inflammatory reactions in the context of both innate and acquired host defence, MC are known to exert important non-immunological functions like wound repair, extracellular matrix remodelling, angiogenesis and neurotrophism as well as modulation of fibroblast, epithelial cell and smooth muscle cell activity. These pleiotropic functions put MC in a central, strategic position to organize tissue defence, restore tissue damage and maintain tissue homeostasis. This review summarizes the most recent advances concerning the functional anatomy of the crypt-villus unit and discusses the way intestinal MC might become part of the instructive circuits that ultimately lead to the maintenance of a proper villous shape.

There is currently great excitement and expectation in the stem cell community following the discovery that multipotent stem cells can be cultured from human fetal tissue and retain their ability to give rise to a variety of differentiated cell types found in all three embryonic germ layers. Although the earliest sites of hematopoietic cell and endothelial cell differentiation in the yolk sac blood islands were identified about 100 years ago, cells with hemangioblast properties have not yet been identified in vivo. Endothelial cells differentiate from angioblasts in the embryo and from endothelial progenitor cells, mesoangioblasts and multipotent adult progenitor cells in the adult bone marrow. Circulating endothelial progenitor cells (EPC) have been detected in the circulation after vascular injury and during tumor growth. The molecular and cellular mechanisms underlying EPC recruitment and differentiation are not yet understood, and remain as one of the central issues in stem cell biology. For many years, the prevailing dogma stated that the vessels in the embryo develop from endothelial progenitors, whereas sprouting of vessels in the adult results only from division of differentiated endothelial cells. Recent evidence, however, indicates that EPC contribute to vessel growth in the embryo and in ischemic, malignant or inflammed tissues in the adult, and can even be therapeutically used to stimulate vessel growth in ischemic tissues.

It has recently been shown that adriamycin is accumulated in mast cells by an active transport system, which closely resembles the outward transport system of multidrug resistant (MDR) cells. The present study was undertaken in order to test the effect of substances which are known to limit or reverse resistance in MDR cells on adriamycin uptake and histamine release in rat peritoneal mast cells. The lysosomotropic amines chloroquine, nicotine, propranolol, atropine, methylamine, ammonium chloride and quinacrine were only slightly effective at very high concentrations; no effect could be observed with the lysosomotropic amine amantadine. The carboxylic ionophores monensin and nigericin were, on the contrary, extremely efficacious; in particular, the effect of monensin was more evident when mast cells were preincubated with the ionophore for 10 or 30 minutes while only a slight inhibition was evident when the two substances were added simultaneously. Removal of monensin from the incubation medium before challenge with adriamycin did not abolish the inhibitory action of the ionophore. Among the tested membrane active agents, Tween 80 and Triton WR-1339 were able to limit adriamycin uptake and histamine release. The microscopical examination showed that in mast cells treated with adriamycin, an intense fluorescence was present in cytoplasmic granules; on the contrary, mast cells preincubated with monensin showed hardly any fluorescence.