Mast cells are augmented in deep vein thrombosis and express a profibrinolytic phenotype

A number of recent data suggest that mast cells (MC) and their products are involved in the pathophysiology of thrombosis. In the current study, we have evaluated the number, distribution, and phenotype of MC in patients with deep vein thrombosis of the lower limb (DVT) (n = 15). Contralateral nonthrombosed limb veins served as control (CO). MC were examined by Giemsa staining and by immunohistochemistry using antibodies against tryptase, chymase, tissue-type plasminogen activator (tPA), urokinase (uPA), urokinase receptor (uPAR), and plasminogen activator inhibitors (PAI-1, PAI-2). We found an increase in the number of tryptase-positive MC in DVT compared with CO (DVT: 9.1+/-1.0 v CO: 4.7+/-0.6 MC/mm2, P < .05). Most of these MC appeared to accumulate in the adventitia of the thrombosed veins, in vicinity of the vasa vasorum. In both DVT and CO, MC reacted with monoclonal antibodies to c-kit, tryptase, and chymase. MC also stained positive for tPA and urokinase receptor, but did not express detectable PAI-1 or PAI-2. As compared with CO, a decreased proportion of MC in DVT was found to stain positive for chymase and tPA. Together, our results show that MC increase in number in DVT and express a profibrinolytic phenotype. We hypothesize that MC and MC-derived profibrinolytic molecules play a role in the pathophysiology of DVT.     

Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells

BACKGROUND: During the last years, mast cells have been recognized as a potent cellular source of multiple cytokines. However, little is known about the regulation of cytokine production by mature human mast cells derived from mucosal sites. METHODS: Human mast cells were isolated from intestinal mucosa and cultured for 14 days in the presence of stem cell factor (SCF) alone or in combination with IL-4. Mast cells were then stimulated by IgE receptor cross-linking or bacterial infection and cytokine production was examined by RT-PCR and ELISA. RESULTS: We found that human intestinal mast cells produce proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 without further stimulation. Stimulation of the cells with gram-negative bacteria (Escherichia coli and others) caused an upregulation of TNF-alpha expression. Following IgE receptor cross-linking, we found additional expression of the Th2 cytokines IL-3, IL-5 and IL-13. Interestingly, mRNA for IL-3, IL-5 and IL-13 was also expressed in unstimulated mast cells provided they were cultured in the presence of SCF and IL-4. Moreover, IL-4 rendered mast cells capable of releasing IL-5 in response to bacterial challenge. CONCLUSION: In the presence of the mast cell survival factor SCF, mature human mast cells produce predominantly proinflammatory cytokines, whereas in the presence of SCF and IL-4, mast cells produce not only proinflammatory but also Th2 cytokines.     

Immunosuppression inhibits switch from naive to memory phenotype in human T lymphocytes

Recently, human T cells have been divided into memory (antigen-primed) and naive (unprimed) subsets, which vary in phenotype and function. Immunosuppressive drugs and heparin have been found to inhibit PHA-induced in vitro switching from naive to memory cell phenotype in a process that is at least partly independent of cell proliferation. Furthermore, T cells isolated from immunosuppressed renal allograft recipients were deficient in their in vitro PHA-induced transition from naive to memory phenotype.     

Retrovirally induced switch from production of IL-12 to IL-4 in dendritic cells.

Dendritic cells (DC) in HIV-1 infection show a reduced capacity to stimulate primary T cell proliferation. Exposure of bone marrow-derived DC to Rauscher leukemia virus (RLV) provides a mouse model for studying retrovirally induced reduction in stimulatory capacity for T cells. Treatment with IL-12, a cytokine that promotes the development of Th1 cells, has been postulated as a treatment for AIDS and is effective at restoring cell-mediated immunity in mice infected with mouse AIDS virus or with RLV (see Knight, S. C. and Patterson, S., Annu. Rev. Immunol. 1994. 15: 593-615 for references). Here we studied the direct effect of RLV and of IL-12 on bone marrow-derived DC. Normal DC produced IL-12 and IL-10 and stimulated primary allogeneic T cell proliferation. Exposure of DC to RLV caused reduced production of IL-12, production of IL-4 was seen in DC for the first time and T cell stimulation was inhibited. Addition of IL-12 reinstated and enhanced IL-12 synthesis in RLV-treated DC, abrogated production of IL-10 and IL-4 and restored stimulatory activity. Manipulation of cytokine production in DC could be a stratagem that has evolved in the retrovirus to avoid stimulation of cellular responses.     

Release of histamine from rat mast cells by the complement peptides C3a and C5a.

Suspensions of rat mast cells were used to study the histamine-releasing actions of anaphylatoxins C3A and C5a in vitro. The peptides, derived from human or porcine complement proteins C3 and C5, were less potent than 48/80 but more potent than bradykinin in stimulating release of histamine from mast cells. The pattern of release resembled that of the anaphylactic release action, e.g. release was limited to less than 30 per cent of the cell histamine, the reaction was calcium-dependent and was potentiated by phosphatidyl serine. When C3a and C5a were added together to mast cell suspensions, the amount of histamine released was additive. Similarly, release by either peptide combined with bradykinin was additive. Histamine-releasing activity (as well as smooth muscle-stimulating activity) was abolished when the peptides were treated with pancreatic carboxy-peptidase B. Active or inactive peptides were bound by mast cells and addition of active C3a in combination with the inactive, des-arginine derivativ, C3ai, resulted in partial inhibition of histamine release.     

Expression of apoptosis-related proteins in Barrett's metaplasia-dysplasia-carcinoma sequence: a switch to a more resistant phenotype

Barrett's esophagus, or columnar-lined esophagus (CLE), is a premalignant disorder in which the stratified squamous epithelium is replaced by metaplastic epithelium. To gain more insight into the process of carcinogenesis in CLE, we studied several factors involved in the apoptotic pathway in biopsies with gastric metaplasia (GM), intestinal metaplasia (IM), dysplasia, and/or adenocarcinoma. Immunohistochemistry was performed for Fas, Bcl-2, Bax, Bcl-xl, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2). Fas staining was positive in the epithelium of all biopsies from patients with CLE but negative in normal gastric mucosa. Fas staining was positive in all tumor cells of the 8 cases containing adenocarcinoma. Bcl-2 was positive in lamina propria immune cells of all specimens. Bax staining was positive in the epithelium of all biopsies, including tumor cells. Bcl-xl was positive in dysplasia and tumor cells, but negative in 8 of 17 biopsies containing IM. iNOS was positive in 20 of 21 biopsies with IM and in 4 of 8 dysplasia biopsies. COX-2 was positive in 7 of 8 adenocarcinomas. We conclude that the apoptotic balance in the transformation from IM to adenocarcinoma switches to an antiapoptotic phenotype because of increased Bcl-xl expression and decreased Bax expression. Fas can be used as a marker for the differentiation of gastric mucosa and metaplasia in the esophagus. iNOS is highly positive in CLE-associated intestinal metaplasia. COX-2 is negative in nonmalignant CLE. Therefore, pharmacologic inhibition of COX-2 activity is unlikely to be effective in the preventing CLE-associated adenocarcinoma. There was no clear correlation between iNOS expression and activation of proapoptotic and antiapoptotic genes.     

Signaling pathway in nerve growth factor induced histamine release from rat mast cells

Objective and Design: We investigated a signal transduction pathway involved in NGF induced histamine secretion from mast cells. We compared this mechanism with the exocytosis induced by basic secretagogue compound 48/80.Materials and Methods: Isolated rat peritoneal mast cells were obtained from male Wistar rats. Histamine release was assayed spectrofl uorometrically.Results: We found that tyrosine kinase inhibitor genistein, phospholipase C (PLC) inhibitor U-73122, phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, but not MAP kinase inhibitors, SB203580 and PD98059, reduce histamine secretion in NGF provoked mast cell degranulation. In compound 48/80 mediated degranulation, we confi rmed only the involvement of tyrosine kinase and PLC, but not PI-3 kinase, PKC and MAP kinases.Conclusions: Our results indicate that release of histamine from mast cells after stimulation with NGF is regulated by tyrosine kinase, PLC, PI-3 kinase and PKC, but not by MAP kinases. This biochemical pathway differs from that provoked by compound 48/80.Received 16 February 2005; accepted by A. Falus 12 May 2005     

The phenotype of mast cells in primary adenoid liver tumours of rats and its relation to tumor cells]

Mast cells in adenoid liver tumours of 32 rats induced with nitrosomorpholine were observed ultrastructurally, and among them, some were studied immunocytochemically via immunogold techniques. Data indicated that mast cells which located in tumour tissues presented positive expression of rat mast cell protein (RMCP) II, indicating origination from the mucosal mast cells, while those in the connective tissues around tumours were largely stained negatively with either RMCP II or RMCP I antisera, with the exception of only a few cells showing positive RMCP II staining. Ultrastructural observation showed that mast cells in tumors contacted closely with the tumor cells. Membranes of the intracellular granules in these mast cells were fusing together. The content inside the granules were discharged and spread along the intercellular space between the tumor cells. There was not any lesion obtained ultrastructurally at the contacting point between the tumor cells and the mast cells. The significance of mucosal mast cells in adenoid liver tumors is briefly discussed.     

Inhibition of protein kinase C results in a switch from a non-motile to a motile phenotype in diverse human lymphocyte populations.

Circulating lymphocytes are rounded, non-motile cells which on contact with cytokines, specialized or activated endothelium, acquire a constantly shape-changing, polarized morphology which enables migration into appropriate sites. The biochemical mechanisms which regulate this switch are not understood but the various stimuli may have a common final pathway. In this study we show that protein kinase C (PKC) inhibitors of the bisindolylmaleimide type (GF 109203X, Ro 31-8220, CGP 41,251) induce resting, spherical lymphocytes to change rapidly (< 30 min) into polarized, locomotory cells. This phenomenon was seen with diverse populations of blood T lymphocytes, tonsillar B cells and Jurkat and Molt4 T-cell lines. Consistent with this, down-regulation of PKC by chronic treatment (44 hr) with bryostatin also induced the polarized phenotype in blood lymphocytes and non-motile Molt4 cells. Conversely, treatment of a spontaneously motile subline of Molt4 cells with various PKC activators caused a reversion to the non-motile phenotype within minutes. PKC activation must be sufficient to overcome the effects of a constitutively active phosphatase because bisindolylmaleimide induction of motility could be prevented by pretreatment of the cells with a phosphatase inhibitor, calyculin A. It is concluded that, in resting lymphocytes, chronic activation of a PKC offsets the action of a constitutively active phosphatase and the net result is maintenance of the non-motile state. Agents which alter the kinase/phosphatase balance in favour of dephosphorylation result in induction of the locomotory phenotype.     

Human eosinophils induce histamine release from antigen-activated rat peritoneal mast cells: a possible role for mast cells in late-phase allergic reactions

BACKGROUND: Mast cells and eosinophils are believed to interact during the late and the chronic stages of allergic inflammation. OBJECTIVE: In this study we investigated whether eosinophils can cause activation and consequent histamine release of already challenged mast cells, a situation likely to take place during the allergic late-phase reaction. METHODS: Rat peritoneal mast cells presensitized with IgE anti-dinitrophenol-human serum albumin and challenged by dinitrophenol-human serum albumin or compound 48/80 were incubated with either eosinophil sonicate or major basic protein (MBP). Eosinophils were purified from the peripheral (>98%) blood of mildly allergic patients. Heparin and pertussis toxin and different extracellular Ca(2+) concentrations were used to modulate mast cell reactivation by MBP. Histamine release was assessed as a marker of mast cell activation. RESULTS: IgE-challenged mast cells were sensitive to reactivation induced by eosinophil sonicate and MBP. Reactivation was not cytotoxic for the mast cells. Mast cells previously challenged with compound 48/80 did not respond to subsequent MBP activation. Furthermore, heparin and pertussis toxin both inhibited mast cell reactivation induced by MBP. The ability of eosinophil sonicate and MBP to activate mast cells was not significantly affected at the different Ca(2+) concentrations. CONCLUSIONS: In summary, we have shown a direct activating activity of eosinophils, partially due to MBP, toward IgE-challenged and immunologically desensitized mast cells. This suggests that in vivo mast cells can be reactivated during a late-phase reaction to release histamine by a non-IgE-dependent mechanism.     

Paget's cells. New evidence linking mammary and extramammary Paget cells to a common cell phenotype

Three mouse monoclonal anti-human cytokeratin antibodies were made against human sole epidermis. One of these (KA4) was shown to react with a variety of human simple epithelium, including eccrine and apocrine sweat glands and the luminal cells of the breast ducts and lobules, but failed to decorate interfollicular stratified squamous epithelium. This antibody reacted by the immunoblot technic with cytokeratins of Mr values 54, 46, and 40 kdaltons. KA4 reacted strongly with clear cells found in 11% of breast epithelium in clinically uninvolved nipples and with all Paget's cells in four cases of mammary and five cases of extramammary Paget's disease. These findings suggest a common cellular phenotype for Paget's cells and relates them to a population of cells found in breast epithelium.     

Histamine release from isolated rat mast cells induced by opiates: effect of sterical configuration and calcium

The stereospecificity of the action of opiates on rat mast cells was investigated by means of thel-andd-isomers levorphanol and dextrorphan. The dose-response curves for histamine release induced by the 2 drugs were of a similar shape with a maximum at 2×10^–3 M and a pronounced minimum at 5×10^–3 M. At concentrations below 5×10^–3 M the effect of both drugs resembled that of morphine, i.e. the release occurred rapidly and inhibition was observed with naloxone, codeine, and antimycin A. Levorphanol, dextrorphan, and the antagonist levallorphan at concentrations above 5×10^–3 M seemed to be toxic to mast cells.The uptake of⁴⁵Ca in connection with histamine release induced by pethidine, levorphanol, and dextrorphan was lower than that of control cells, whereas the uptake induced by morphine did not differ from that of controls. The inhibition of compound 48/80-induced histamine release by morphine was paralleled by a reduced⁴⁵Ca uptake.The time course for the inhibitory effect of preincubation with morphine was similar for the histamine release induced by morphine and by compound 48/80. Washing of the cells after preincubation with morphine was without effect on the inhibition of morphine-induced histamine release, whereas the inhibition of compound 48/80. was reduced.The present observations with morphine and compound 48/80 support our previous impression of similarities in their mode of action, but a mechanism implying an interference by morphine with the disposition of calcium could also account for the findings. The observed antagonism between morphine and calcium resembles that of opiate receptors, but histamine release induced by opiates does not involve sterospecific opiate receptors.     

The mechanism of histamine release induced by pilocarpine from different tissues: Studies on rat peritoneal mast cells

Pilocarpine releases histamine from mast cells of cat submandibular gland and rat liver. In the salivary gland, histamine is released into the saliva and venous outflow. Atropine blocks the salivation, but not histamine release from the submandibular gland into the blood. Histamine release from the gland could be due to a direct action of pilocarpine on tissue mast cells or to an indirect action of mediators (acetylcholine and peptides). These hypotheses were further investigated in our present studies on rat peritoneal mast cells. Our results show: (1) histamine release from rat peritoneal mast cells induced by pilocarpine (ED₅₀=1.7×10⁻² mol/l) occurs at 1000-fold higher concentrations than by substance P (ED₅₀=1.7×10⁻⁵ mol/l) and in 6.5-fold higher concentrations than by atropine (ED₅₀=2.6×10⁻³ mol/l), (2) pilocarpine injected directly into the rat peritoneal cavity causes histamine release from peritoneal mast cells in 1.8-fold higher concentrations than from isolated rat peritoneal mast cells. These results would support the hypothesis that histamine release from cat submandibular gland is caused by peptidergic co-transmission during the stimulation of the organ.

     

Human placental mast cells: a role in pre-eclampsia?

The human placenta contains a significant amount of histamine, a potent vasoconstrictor of the placental vasculature, shown by the author to be stored within the tissues' mast cells. The proposed hypothesis suggests that placental mast cells may have an important role in normal and, or pathological processes during pregnancy. This suggestion will be applied to the confounding problem of pre-eclampsia, a complication of pregnancy characterised by hypertension, oedema and proteinuria, which is associated with increased morbidity and mortality of mother and baby. It is postulated that pre-eclampsia reflects an inflammatory-type reaction, in which mast cell-mediated events play a significant role. The mediators released upon mast cell activation, such as histamine and prostaglandins, may be involved in the vasospasm that characterises pre-eclampsia; while processes such as uptake and clearance of vasoactive mediators by mast cells may be important in normotensive pregnancies and upset in those women who develop pre-eclampsia.