DNA distribution of mast cell populations in growing rats

Summary The proliferation of rat peritoneal mast cells was examined under normal conditions in vivo. DNA content of individual mast cells was measured by cytofluorometry after staining with the bibenzimidazole dye Hoechst 33258. Diploid non mast cells from each rat were used as a biological standard, which resulted in small long-term variations in the method. The proportion of mast cells in the S+G2 region of the DNA distribution was about 4% for young rats (24 days old, body-weights about 60 g). It decreased in relation to body-weight, and was less than 1% for 105-day-old rats weighing 400 g. During the same growth period the total number of mast cells in the peritoneal cavity increased about 8-fold. The total number of proliferating cells, about 30,000, remained constant throughout the observation period. No evidence of polyploidization or accumulation in G2 of mast cell nuclei was found. It is concluded that peritoneal mast cells increase in number by mitotic proliferation of differentiated cells.Supported by grants from the Swedish Medical Research Council, Project No. 2235     

Mast cell dynamics and involvement in the development of peritoneal adhesions in the rat

Mast cells have been implicated in the ethiopathology of post-operative peritoneal adhesions. However an evaluation of their role in this condition is missing. Adhesions were induced in rats using small intestinal scraping. These rats or rats injected ip with either Stem Cell Factor (SCF) or nedocromil sodium or compound 48/80 (day 0-20) were sacrificed for grading of peritoneal adhesions, for evaluating mast cells and inflammatory cells in adhesions and peritoneal lavage (histochemical staining) and for histamine content (peritoneal lavage, radioenzymatic assay) on days 1-21. Mast cell sonicate was added to intestinal fibroblast and their proliferation was assessed (cell counting). All the rats developed adhesions (day 1) and after 3 days the adhesion score remained constant. Early adhesions were avascular and made of fibrinous exudate containing many mast cells. Thereafter adhesions became denser, and the number of stainable mast cells decreased and then stabilized. On the first few days, inflammatory cells in the peritoneal lavage increased while mast cells and histamine content were significantly reduced indicating their activation. Injection of SCF for 1 week slightly increased peritoneal adhesion formation while nedocromil sodium reduced their development. Compound 48/80 had no significant influence. Addition of mast cell sonicate to normal intestine or to peritoneal adhesion fibroblasts resulted in a significant increase of fibroblast proliferation. In conclusion, mast cell presence correlated with the establishment of peritoneal adhesions, and their pharmacological modulation influenced adhesion formation. In vitro mast cell induced fibroplasia. Therefore, mast cells have a profibrogenic role in this model of peritoneal adhesions.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Inhibition of rosette formation of IgE-bearing rat mast cells by disodium chromoglycate.

The interaction of reaginic antibody with specific antigen was studied by the rosette formation of peritoneal rat mast cells. The mast cells were obtained from actively sensitized rats or were passively sensitized in vitro. Rosette formation was of a higher degree with mast cells of actively sensitized rats; in this case 58% of the cells showed a strong rosette-forming effect (blinding more than 5 SRBC). No rosette formation was detected in 18% of the cells. With passively sensitized rat mast cells, rosette formation was 45% and 22%, respectively. Rosette formation of both actively and passively sensitized mast cells could be inhibited by disodium chromoglycate (DSCG); the inhibitory effect of 20 micrograms and 200 micrograms of the drug was the same, and neither dose caused a full inhibition. It is suggested that the linkage of specific antigen to the surface of sensitized mast cells can be inhibited by DSCG in vivo.     

Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.     

Subcellular localization of brain mast cell histamine in developing rat

The intracerebroventricular administration of compound 48/80 or polymixin B to rats 0 to 60 days old, produced a decrease both in the histamine which sediments in the crude nuclear fraction, as well as in the number of mast cells in the brain. In contrast, the histamine-releasers did not affect histamine levels in subcellular fractions where neuronal histamine is found. Once released, histamine disappeared rapidly (t 1/2 = 3.8 min). In untreated animals and in those treated with histamine releasers, the number of mast cells/g in the whole brains of developing rats and in the cerebral regions of adult rats showed a close correlation with the histamine levels in the crude nuclear fraction. The content of histamine per mast cell in adult rat brain was estimated to be about 13 pg/cell. Histologic examination of the subcellular fractions revealed the presence of intact mast cells in the crude nuclear fraction obtained from untreated animals, and of degranulated mast cells in the same fraction obtained from animals treated with histamine releasers. The mast cell contribution to adult rat brain histamine levels was about 22%. Our results strongly support that most of the histamine which sediments in the crude nuclear fraction of the rat brain is located in mast cells. Determination of histamine in the crude nuclear fraction and in the supernatant of this fraction is proposed as an easy way for identifying the cellular pool altered by any treatment affecting brain histamine levels.     

Mast cell activation and tissue cell proliferation

Summary The effect of mast cell activation and degranulation on the proliferation in the intact mesentery was studied in Sprague-Dawley rats. Mast cell activation was achieved by a single intraperitoneal injection of Compound 48/80.The proliferation was studied using three independent methods for estimation of cell production and DNA synthesis: 1. the mitotic index, 2. the relative number of cells having a DNA content in the S and G2 regions, by Feulgen photometric measurement in individual cells, and 3. the specific DNA activity, employing a method which combines a liquid scintillation technique after an intravenous injection of ³H-thymidine and Feulgen photometric determination of the DNA content per membrane preparation.It was found that the proliferation of the normal mesenchymal cells adjacent to the activated and degranulated mast cells in the mesentery was significantly increased within 24 and 32 h, the maximum increase being more than 20-fold compared to untreated controls. The results suggest that the common type of mast cell may have a pathophysiological function related to stimulation of local cell proliferation.Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty, University of LinköpingWe thank Brita Söderlund, Margareta Odenö and Iréne Svensson for skilful technical assistance, and Erik Leander, Ph. D., for help with statistical methodsPart of this work was presented at the 7th Meeting of the European Study Group for Cell Proliferation, 5–9 May 1975, in Amsterdam, The Netherlands     

Cultures of mast cell-like (MCL) cells from human pleural exudate cells

Under special culture conditions, rat peritoneal macrophages have previously been shown to transform into mast cells. This method has been adapted here to the human species. Adherent large mononuclear cells from human pleural exudates were cultured in a medium supplemented with horse serum (30%) and fibroblast supernatants (30%). Metachromatic staining (toluidine blue, pH 3.6) of cytoplasmic granules appeared first in a small percentage of cells by days 5-6 of culture and reached a high intensity in 50% of the cells between days 12-22. Histamine levels within the cells increased by a factor of 7 during this same time period and the cell size by a factor of 3. Cultures could be maintained for about three weeks, since viability and total cell number decreased on extended culture. The data suggest that mononuclear cells in inflammatory exudates can transform into mast cell-like cells under the influence of high levels of specific conditioning factors in their microenvironment.     

Murine mast cell colony formation supported by IL-3, IL-4, and recombinant rat stem cell factor, ligand for c-kit.

Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c-kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin-3 and interleukin-4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue-type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin- and berberine sulfate-positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow-derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin-3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC.     

Lymphocytes bearing Fc receptors for IgE. II. Induction of Fcepsilon-receptor bearing rat lymphocytes by IgE.

The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.     

The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity

Background

The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated.

Methodology

Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water.

Results

ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells.

Conclusions

The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.     

High rate of IgE-mediated histamine release from rat mesenteric mast cells.

IgE-dependent histamine release from rat mesenteric mast cells was investigated. Excised mesenterium was cut into pieces and incubated with IgE overnight at 4 degrees C for sensitization. Over 10 pieces of mesenterium specimen could be prepared from a rat. Antigen-induced histamine release from mesenterium specimen was initiated rapidly and reached a plateau in 5 min. In an optimal condition, over 50% of total histamine was released. In contrast, unpurified and purified peritoneal mast cells released only 22.5% and 5.3% of total histamine, respectively, upon IgE stimulation. Tranilast, a mast cell stabilizer, inhibited the histamine release from mesenteric mast cells significantly. The mesenterium might be useful material for studying tissue-associated mast cell activation.     

Age-dependent mitogenesis in normal connective tissue cells

We have previously described ways to use the mesentery for studies of proliferation in intact tissue. Here we have studied a weak and a strong mitogenic response in the mesentery of rats aged 6--42 weeks, induced by a single intraperitoneal injection of saline or the mast-cell-degranulating drug 48/80. Proliferation was measured by the specific DNA activity, the fraction of fibroblast- and mesothelial-like cells in the S+G2 cell-cycle-phases, and the mitotic index. We also counted the relative number of mast cells in the tissue (normalized to 5,000 fibroblast-like and mesothelial-like cells), since this might influence 48/80-induced mast-cell-mediated proliferation. In old animals there was a decline in the proliferative response and the time required to initiate DNA synthesis was prolonged. This appears to be the first report of such an age-dependent proliferation characteristic in the mesentery, and probably in any connective tissue. The normalized number of mast cells in the mesentery also declined with increasing age.     

BN大鼠和豚鼠对卵清白蛋白致主动过敏反应的免疫学特性比较

目的 比较BN大鼠和豚鼠对卵清白蛋白(OVA)致敏前后机体免疫学特性的变化.方法 BN大鼠和豚鼠分别用OVA(每只1 mg)隔日致敏(i.p.),共5次;于末次致敏第10天以OVA(每只2 mg)激发致敏(i.v.);分别设正常对照组和OVA致敏组.于激发致敏后1h处死动物,分离腹腔肥大细胞、脾脏和骨髓,并制备脾脏和骨髓淋巴细胞.以annexin-V作为标志检测肥大细胞活性,同时以Fluo-3/AM标记胞内钙离子,检测钙离子水平;以PHA和LPS作为有丝分裂原,分别检测脾脏和骨髓T、B淋巴细胞活性.结果 ①致敏BN大鼠和豚鼠脾脏及骨髓T、B淋巴细胞活性均升高,其中骨髓淋巴细胞活性BN大鼠显著高于豚鼠,脾脏淋巴细胞活性两种属间差异无显著性;②致敏后,腹腔肥大细胞活性两种属间差异无显著性,但BN大鼠致敏后是致敏前的6倍,豚鼠是3倍;③肥大细胞内钙离子水平两种属致敏后均升高,豚鼠致敏前后钙离子水平具有统计学意义.结论 OVA致敏后,BN大鼠骨髓淋巴细胞活性明显高于豚鼠,豚鼠肥大细胞内钙离子较BN大鼠升高明显,肥大细胞活性两者无明显差异.因此,在实验中可以根据两种属在过敏反应中的特点以及具体的实验要求选择动物模型.     

Recovery of rat mast cells after secretion: a morphometric study

Granule reconstitution in rat peritoneal mast cells following massive secretion was studied by morphometric techniques. Immediately following secretion, the earliest identifiable mast cells showed a substantial decrease in cell volume associated with granule loss. Cell volume then increased almost to the original level over a period of a month. The size of the Golgi apparatus increased markedly in the week following secretion and then returned to its original size. The total volume of granules increased slowly after the secretory depletion and by 34 days had not returned to the original value although the number of granules had recovered fully. The reconstitution of mast cells after secretion is a prolonged process with several phases resulting in mast cells of varying appearance and content. This heterogeneity generated by reconstitution post secretion must be considered in studies of populations of mast cells in vivo.     

Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE

Anti-IgE-dependent activation of rat and human mast cells resulted in the preferential generation of the cyclooxygenase products prostaglandin D2 (PGD2) and prostaglandin I2 (PGI2) in the rat and PGD2 in the human. The average net generation of PGD2, determined by gas chromatography-mass spectrometry, was 13.1 ng/10(6) purified rat mast cells and 39.5 ng/10(6) dispersed, enriched human mast cells. After IgE-dependent activation, there was a linear relationship between the net quantities of PGD2 generated and of histamine secreted from dispersed human pulmonary cells when the number of mast cells was varied but the total number of cells was held constant, indicating that it is the number of mast cells participating in IgE-dependent activation, rather than total mast cell number, that determines PGD2 generation. A linear relationship was also shown between PGD2 generation, determined by radioimmunoassay, and the release of the granule marker beta-hexosaminidase from purified rat mast cells on the dose-response portion of the plot of their response to anti-IgE challenge. With higher concentrations of anti-IgE, PGD2 generation from rat mast cells plateaued, whereas net percent beta-hexosaminidase release increased further. In kinetic studies of rat mast cells activated with anti-IgE, the onset (1 to 2 min) and time of maximum generation (5 to 10 min) for PGD2 were delayed relative to the onset (15 to 30 sec) and completion (1 to 2 min) of beta-hexosaminidase release. Thus, the extracellular appearance of PGD2 during IgE-dependent mast cell activation represents a response additional to the secretion of granule-associated mediators.     

The number of the rat peritoneal cells in normal condition, blood loss, and under colchicine injection

The modification of peritoneal cells derivation method proposed for the peritoneum rat, and the absolute amount of peritoneal lymphocytes, monocytes, macrophages, stem and polymorphonuclear neutrophyles, mat cells was determined. The procedure of acute 2% blood loss was found to reduce the total amount of the peritoneal cells due to depletion of lymphocytes accompanied by an increase in amount and proliferative activity of monocyte peritoneal cells, stem neutrophiles appearance in the peritoneal cavity. The subcutaneous injection of colchicine solution reduces the number of the peritoneal macrophages in normal rats, decreases the number of the monocytes, polymorphonuclear neutrophiles and led to disappearance of the stem neutrophiles on the second day of blood loss.     

Stimulation of mouse connective tissue-type mast cells by hemopoietic stem cell factor, a ligand for the c-kit receptor.

The proliferative capacity of mouse connective tissue-type mast cells (CTMC) was analyzed by using a newly discovered c-kit ligand, termed stem cell factor (SCF). More than 90% of CTMC in the peritoneal cavity responded to recombinant rat SCF (rrSCF) and were able to give rise to pure mast cell colonies in methylcellulose culture. Serial observation (mapping) of growth of individual CTMC in culture containing rrSCF confirmed their striking proliferative ability. No serum but accessory cells (non-CTMC cells) in the peritoneal population were required for the clonal growth of CTMC induced by rrSCF in our methylcellulose culture of whole peritoneal cells. The rrSCF-induced mast cell colony formation from peritoneal CTMC was completely inhibited by the addition of anti-c-kit antibody, which can block the binding of SCF to c-kit, to the culture. When IL-3 was combined with rrSCF, mast cell colonies dramatically increased in size. Mapping studies revealed that the combination of the two factors augmented the proliferative rate of CTMC. Approximately 60% of the constituent cells of the mast cell colonies which were formed from peritoneal CTMC in the culture containing rrSCF alone were stained with berberine sulfate, which is a characteristic of CTMC. However, most mast cells which were induced by rrSCF+IL-3 from peritoneal CTMC contained berberine(-)-safranin(-)-Alcian blue(+) granules. Although IL-4 exhibited little synergism with rrSCF in the induction of CTMC proliferation, the addition of IL-4 to the culture containing rrSCF+IL-3 resulted in an increase in mast cells which retained CTMC characteristics.     

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.