Presence of a blastocyst and mast cell depletion of the mouse uterus

The mast cell population has been studied during early decidualization of the mouse uterus. The number increases in early pregnancy until the attachment phase when a sharp depletion is noticed. This fall has been correlated with stimuli of maternal origin, but at the same time the presence of a blastocyst at the presumptive implantation sites seems to exert a significant effect on the depletion of mast cells. A relationship between the number of mast cells in the uterus and the physiological state of the organ has definitely been established [Harvey, 1964; Likar and Likar, 1964; Gibbons and Chang, 1972; Brandon and Evans, 1983]. The number of mast cells in the pregnant uterus is known to decrease around the time of implantation in the rat [Shelesnyak, 1960; De Feo, 1967; Brandon and Bibby, 1979]. Several workers believe that the mast cell population and histamine content decrease as a result of a rise in circulating estrogen [Westin, 1955; Gibbons and Chang, 1972; Spaziani, 1975]. In the present communication the possibility of a relationship between the decrease in mast cell population and the presence of a blastocyst, whose estrogenic role has been reported [Dickmann and Dey, 1973; Dickmann et al., 1975; Sengupta et al., 1977], in the uterine horn has been discussed.     

An assessment of mast-cell deficient mice (W/Wv) as a model system to study the role of histamine in implantation and deciduoma formation

The ovaries from mast cell-normal (+/+) and mast cell-deficient (W/Wv) mice were examined with light and electron microscopy. In addition the effect of ovariectomy and subsequent steroid treatment on total uterine histamine content, total mast cell numbers and surface and glandular epithelial cell heights was measured. The ovaries of +/+ mice were normal, displaying various stages of follicular growth and atresia and numerous corpora lutea; the ovaries of W/Wv mice lacked follicles and corpora lutea but contained numerous hyperplastic interstitial cells which contained numerous lipid droplets, vesiculated mitochondria and abundant endoplasmic reticulum suggestive of steroid synthesis. Steroid treatment of ovariectomized +/+ and W/Wv mice caused a significant increase in uterine wet weight and endometrial surface and glandular epithelial cell heights. In +/+ mice, steroid treatment caused a concomitant increase in total mast cells per uterine horn while mast cells were totally absent in W/Wv mice. The increase in uterine histamine in +/+ mice is consistent with the increase in mast cell numbers. Measurable amounts of uterine histamine, which increases slightly after steroid treatment, were demonstrated in W/Wv mice. Since the uteri of +/+ and W/Wv mice respond to steroids in a similar manner with the sole exception being histamine content and mast cell numbers, our results demonstrate the potential of using these animals to investigate the role(s) of uterine mast cells and non-mast cell uterine histamine in the process of implantation and the formation of a decidual cell response.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Effect of histamine receptor agonists and antagonists on the uterine vasculature

Histamine H1 and H2 receptors are known to exist in uterine smooth muscle; however, neither receptor has been clearly identified in the uterine vasculature. In the present study, 12 nonpregnant ewes were chronically instrumented with catheters in the carotid artery, jugular vein, uterine arteries, and electromagnetic flow probes on the uterine arteries for continuous measurement of uterine blood flow. Dose response curves were determined for bolus injections of Histamine (1-10 micrograms), the H1 receptor agonist 2PEA (10-100 micrograms), and the H2 receptor agonist Dimaprit (30-300 micrograms) before H1 receptor blockade with pyrilamine, following H1 receptor blockade, and following H2 receptor blockade with metiamide. Uterine vasodilator responses to histamine and 2PEA were essentially abolished by pyrilamine, while responses to dimaprit were not altered. Following addition of metiamide, responses to histamine were reduced further and responses to dimaprit were abolished. Baseline uterine blood flow was not altered by either H1 or H2 receptor blockade or their combination. Intraarterial bolus injections of the mast cell histamine-releasing compound 48/80 (100-1000 micrograms) had no effect on uterine blood flow. These experiments demonstrate that the uterine vasculature of the ovine contains almost exclusively H1 receptors, does not contain compound 48/80 sensitive mast cells and is not dependent upon endogenous histamine to maintain blood flow.     

Histamine is involved in gastric vasodilation during acid back diffusion via activation of sensory neurons

Protective vasodilation during acid back diffusion into the rat gastric mucosa depends on activation of sensory neurons and mast cell degranulation with histamine release. We hypothesized that these two mediator systems interact and that histamine partly exerts its effect via sensory nerves. Gastric blood flow (GBF) and luminal histamine were measured in chambered stomachs, and mast cell numbers were assessed by morphometry. Ablation of sensory neurons and depletion of mast cells were produced by pretreatment with capsaicin or dexamethasone, respectively. Mucosal exposure to 1.5 M NaCl and then to pH 1.0 saline in ablated and control rats caused increased luminal histamine and reduced numbers of mast cells. Enterochromaffin-like cell marker pancreastatin remained unchanged. Only control rats responded with an increase in GBF. Capsaicin stimulation (640 microM) of the undamaged mucosa induced identical increase in GBF and unchanged mast cell mass in normal and dexamethasone-treated rats. Increase in GBF after topical exposure to histamine (30 mM) in rats pretreated with capsaicin or a calcitonin gene-related peptide (CGRP)(1) antagonist human CGRP(8-37) or exposed to the calcium pore blocker ruthenium red was less than one-half of that in control rats. These data suggest that mast cell-derived histamine is involved in gastric vasodilatation during acid back diffusion partly via sensory neurons.     

Polyamines affect histamine synthesis during early stages of IL‐3‐induced bone marrow cell differentiation

Mast cells synthesize and store histamine, a key immunomodulatory mediator. Polyamines are essential for every living cell. Previously, we detected an antagonistic relationship between the metabolisms of these amines in established mast cell and basophilic cell lines. Here, we used the IL‐3‐driven mouse bone marrow‐derived mast cell (BMMC) culture system to further investigate this antagonism in a mast cell model of deeper physiological significance. Polyamines and histamine levels followed opposite profiles along the bone marrow cell cultures leading to BMMCs. α‐Difluoromethylornithine (DFMO)‐induced polyamine depletion resulted in an upregulation of histidine decarboxylase (HDC, the histamine‐synthesizing enzyme) expression and activity, accompanied by increased histamine levels, specifically during early stages of these cell cultures, where an active histamine synthesis process occurs. In contrast, DFMO did not induce any effect in either HDC activity or histamine levels of differentiated BMMCs or C57.1 mast cells, that exhibit a nearly inactive histamine synthesis rate. Sequence‐specific DNA methylation analysis revealed that the DFMO‐induced HDC mRNA upregulation observed in early bone marrow cell cultures is not attributable to a demethylation of the gene promoter caused by the pharmacological polyamine depletion. Taken together, the results support an inverse relationship between histamine and polyamine metabolisms during the bone marrow cell cultures leading to BMMCs and, moreover, suggest that the regulation of the histamine synthesis occurring during the early stages of these cultures depends on the concentrations of polyamines. J. Cell. Biochem. 108: 261–271, 2009. © 2009 Wiley‐Liss, Inc.     

天花粉对小鼠子宫肥大细胞超微结构的影响

本研究用雌性中国１号小鼠１２只,分实验组和对照组,实验组分别腹腔注射天花粉后处死,取子宫组织作超薄切片,电镜观察。在天花粉作用下,常见子宫肥大细胞脱颗粒,呈功能活化状态,并与子宫平滑肌细胞紧密相邻。提示肥大细胞可能通过脱颗粒释放组胺促进子宫平滑肌的收缩。同时,也见肥大细胞与子宫结缔组织中的成纤维细胞,淋巴细胞等密切接触,提示成纤维细胞和淋巴细胞与天花粉作用下子宫肥大细胞的数量增多有关。     

Uterine histamine content in mast-cell deficient (W/WV) mice during experimentally induced deciduoma formation

This study examines the changes in uterine histamine content in mast-cell normal and deficient mice during deciduoma formation. Surgical trauma (e.g., sutures) and intraluminal injection of oil produce deciduoma formation in ovariectomized mast-cell normal (+/+) and mast-cell deficient (W/W^V) pseudopregnant mice that had previously received a single control ovary transplanted under the kidney capsule. Mast-cell normal (+/+) mice exhibit significant elevation of total uterine histamine content during both trauma and oil induced deciduoma formation. Mast-cell deficient (W/W^V) mice under conditions of similar deciduoma formation did not show any change in total uterine histamine content. These results indicate that the change in histamine observed in mast-cell normal mice during deciduoma formation is most likely of mast cell origin. The lack of any measurable change in uterine histamine in mast-cell deficient mice indicates that an increase of non-mast cell histamine is not a prerequisite for deciduoma formation.     

The recovery of the neurally evoked secretory response of rat colonic mucosa after irradiation is independent of mast cells

The ability of the enteric submucosal plexus to influence the transport of water and electrolytes in the colon was investigated in rats for 1 week after acute whole-body gamma irradiation. The involvement of neuroimmune links in the epithelial responses to nerve stimulation was confirmed by the sensitivity of the tissue to tetrodotoxin, mepyramine and doxantrazole. At 1 and 3 days after irradiation, colon tissues were hyporesponsive to nerve stimulation. This was associated with a drastic diminution of mucosal mast cell numbers, tissue histamine levels, and rat mast cell protease II (RMCP II) levels, and by a decreased maximal epithelial response to exogenously added histamine. The responses to electric-field stimulation were insensitive to both mepyramine and doxantrazole. At 7 days, neurally evoked responses recovered, despite the virtual absence of mast cells, tissue histamine and RMCP II, and the continuing decreased response to histamine. The responses were insensitive to doxantrazole but were decreased by mepyramine. This study showed that the establishment of a normal epithelial response to neural stimulation can occur despite the radiation-induced depletion of mucosal mast cells. The recovery of the epithelial response, which was sensitive to mepyramine, may be ascribed to the reappearance of an unknown histaminergic pathway, which probably has indirect effects on epithelial transport but is independent of nerve-mast cell connections.     

Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells

Human cultured mast cells (HCMC) release histamine and sulfidoleukotrienes (LTs) upon IgE-FcepsilonRI-mediated mast cell activation. We analyzed the Ca2+ and PKC signaling in HCMC and compared it to that in rodent mast cells. In HCMC, after IgE-mediated stimulation, an elevation of [Ca2+]i and PKC translocation to the membrane fraction was observed. As concerns Ca2+ signaling, 1) IgE-mediated histamine and LTs release was abolished after Ca2+ depletion, and the reconstitution of Ca2+ recovered the release of histamine and LTs. As regards PKC signaling, 1) staurosporine inhibited IgE-mediated mediator release. 2) PKC-downregulated mast cells did not release histamine and LTs. A23187 and PMA synergistically potentiated the activation of extracellular-regulated kinase and synergistically induced histamine and LTs release. These results demonstrated that HCMC might be useful for analysis of the signal transduction pathway for mediator release, such as histamine and LTs.     

Histamine content and mast cells distribution in mouse uterus: the effect of sexual hormones, gestation and labor

The histamine content of uteri from mice was analyzed in terms of both concentration and total amount per uterine horn a) at two stages of the estrous cycle (estrous and diestrous), b) under sex hormone treatment, c) during pregnancy and after delivery. Histamine concentration and mast cell density were greater during diestrous and in mice treated with progesterone (p less than 0.001). This effect was attributed to a reduction in uterine mass weight, since the amount of histamine per uterine horn remained constant throughout the estrous cycle. During pregnancy, both concentration and amount of histamine per uterine horn were increased, values were significantly higher than in estrous (p less than 0.001) from day 14-17 until day 21 when labor occurred. After six to eight hours post-partum an abrupt reduction on histamine content was observed. Mast cells were more abundant in myometrium than in endometrium, their density followed the same pattern as histamine concentration throughout the estrous cycle.     

Participation of mast cell 5-hydroxytryptamine in the vasoconstrictor effect of neurotensin in the rat perfused hindquarter

Neurotensin (NT) (1 X 10(-8) - 1.5 X 10(-6) g ml-1) caused a transient, dose-dependent increase in perfusion pressure in the rat perfused hindquarter. The vasoconstrictor effect of NT was associated with a short-lived, dose-dependent release of histamine and 5-hydroxytryptamine (5-HT) in the hindquarter effluent. Compound 48/80, a classical mast cell secretagogue, also elicited a vasoconstrictor effect in, and release of histamine from, the rat hindquarter. The vasoconstrictor effect and the release of histamine and 5-HT evoked by NT were much smaller in hindquarters derived from rats pretreated with compound 48/80 for 4 days to cause mast cell depletion than in hindquarters derived from control rats. The mast cell inhibitor cromoglycate (4 mg ml-1) inhibited by about 50% the histamine releasing effect and vasoconstriction produced by the lowest concentrations of NT utilized. The histamine releasing effect of compound 48/80 was more sensitive to blockade by cromoglycate than that of NT. The steroidal antiinflammatory and antiallergic drug dexamethasone did not affect the histamine and 5-HT releasing effect of NT. The vasoconstrictor effects of NT, compound 48/80 and 5-HT were markedly reduced by the 5-HT receptor antagonist methysergide (1 X 10(-7) g ml-1). Histamine (1 X 10(-6) - 10(-4) g ml-1) evoked a decrease in perfusion pressure in hindquarters pre-exposed to noradrenaline. The results suggest the participation of mast cell 5-HT in the vasoconstrictor effect of NT in the rat perfused hindquarter.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.     

Inhibition of compound 48--80 induced histamine liberation from mast cells by triton X-100]

Triton X-100 at concentrations preceding those which liberated histamine, produced dose-dependent inhibition of compound 48/80-induced histamine release from rat mast cells. Triton X-100 (0.00002 1/1) depleted ATP content in the mast cells and blocked compound 48/80-induced histamine release. The inhibition of compound 48/80-induced histamine release and depletion of the ATP content in the mast cells was reversed by glucose (10 mmole). It is concluded that inhibition by Triton X-100 of histamine release induced by compound 48/80 is dependent on inhibition of energy production.     

IgE immunotoxins. Effect of an IgE-ricin A chain conjugate on rat skin histamine content

Immunotoxins--toxins covalently conjugated to specific antibodies--have been studied as possible agents in the treatment of cancer. The avid binding of IgE antibodies to FcR on mast cells and basophils suggested the possible use of an IgE-immunotoxin in the treatment of malignant mastocytosis or as a method to generate mast cell-depleted animals for study. To this end, the effect of a covalent conjugate of rat myeloma IgE and ricin A chain on rat cutaneous mast cells was examined in vivo. IgE-ricin A chain was capable of binding to and sensitizing cutaneous mast cells in vivo as indicated by a bluing response to intracutaneous anti-ricin A chain. IgE-ricin A chain, given either as a single dose or, even more effectively, as two split doses, significantly reduced cutaneous histamine content for 6 to 8 days. Neither a mixture of IgE and ricin A chain that were not conjugated nor the induction of cutaneous mast cell degranulation with anti-IgE affected cutaneous histamine levels. Therefore, IgE-ricin A chain produces a prolonged depletion of cutaneous histamine levels.     

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Localization of histamine in uterine structures

By means of luminescent-histochemical method of Cross, Even, Rost histamine is revealed in all uterine structures. Visual and fluorometric data demonstrate uneven distribution of histamine in the organ's structures. A high content of histamine is specific for macrophages and mast cells, less high--in tegmental epithelium and endometrial glands. A low level of histamine have endometrial stroma, smooth myocytes, cells of the serous membrane and vessels. Basing on the literature data, concerning various sensitivity of the uterine tissues to estrogens and regarding effect of the estrogens upon histamine metabolism in the uterine and regarding interconnection of the histamine receptors in the uterus and the estrogens, a suggestion is made that various contents of histamine in the uterine structures depend on various amount of the histamine receptors in them and on different abilities of the uterine tissues to inactivate histamine. The ability of macrophages to accept free forms of bioamines, as it is described in the literature, evidently can be spread to the uterine macrophages, where a high content of histamine is revealed.     

Ketotifen, a selective histamine liberator]

Ketotifen-induced histamine release from isolated rat mast cells is inhibited under conditions of ATP content depletion in the mast cells. The histamine release is a temperature-dependent process and is blocked at a low temperature. It is concluded that ketotifen is a selective histamine liberator. Time course of ketotifen-induced histamine release corresponds with the process of partial cellular uptake of histamine released.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.