Functional and biochemical characterization of rat bone marrow derived mast cells

The functional and biochemical characterization of rat bone marrow derived mast cells (RBMMC) confirms both species-related differences between rat and mouse bone marrow-derived mast cells (MBMMC) as well as mast cell heterogeneity in a single species. Such RBMMC have the staining characteristics of mucosal mast cells and contain the mucosal mast cell protease. The RBMMC release the preformed granule mediator beta-hexosaminidase both in response to immunologic stimulation with 200 ng Ag (net release 15.8 +/- 3.8%) and in response to 1 microM calcium ionophore A23187 (net release 21.8 +/- 6.8%). However, compound 48/80, substance P, and somatostatin did not induce mast cell degranulation. In experiments with optimal beta-hexosaminidase release, the RBMMC generated similar quantities of the newly formed arachidonic acid metabolites leukotriene C4 and PGD2 when stimulated with either Ag or calcium ionophore A23187. The RBMMC incorporate [35S]sulfate into proteoglycans consisting of 90% chondroitin sulfates and 10% heparin. The chondroitin sulfates were comprised of chondroitin 4 sulfate and chondroitin sulfate diB sulfated disaccharides in a ratio of 4/1. Although we show that RBMMC and MBMMC share a low histamine content, functional IgE receptors and unresponsiveness to cromolyn and selective secretagogues (compound 48/80, substance P, and somatostatin), we also provide evidence that RBMMC differ from MBMMC in their profile of newly generated mediators, preformed granule proteoglycan, and lack of proliferative response to mouse IL-3.     

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.     

Functional compartments in rat mast cells for cAMP and calcium on histamine release

The crosstalk between 3', 5'-cyclic adenosine monophosphate (cAMP), intracellular calcium, and histamine release in rat mast cells using the stimulatory effect of three different drugs, thapsigargin, sodium fluoride (NaF), and compound 48/80 were studied. Each of these drugs induces histamine release by different mechanisms. The transducting pathways modulating cAMP and intracellular calcium levels were modified by using, cholera toxin (CTX) which ADP-rybosylates Gs-protein, pertussis toxin (PTX) which ADP-rybosylates Gi-protein, and okadaic acid (OA) which inhibits phosphatases 1 and 2a. Our results show that CTX increased cAMP levels and inhibited histamine release elicited by thapsigargin and compound 48/80. The inhibitory effect of CTX on histamine release was potentiated by OA in the presence of compound 48/80 but was decreased in the presence of thapsigargin. Calcium uptake was stimulated by NaF and compound 48/80. The previous treatment with OA increased calcium uptake when combined with compound 48/80 but not with NaF. Treatment with NaF highly stimulated calcium uptake and cAMP levels only when combined with OA and CTX. These results suggest that the modulatory effect of intracellular calcium and cAMP on histamine release depend more on the crosstalk of the activated signal transducting pathway than on the final level of calcium or cAMP, further supporting the theory that rat mast cells are divided into functionally distinct compartments.     

Cytofluorometric demonstration of a catecholamine in rat mast cells after the administration of DOPA

Synopsis A catecholamine, probably dopamine, was identified in rat peritoneal mast cells after subcutaneous injection of DOPA. Its identity was established by cytofluorometry of cells treated with hot formaldehyde vapour according to the Falck-Hillarp technique. Injections of 50–200 mg/kgl-DOPA were followed by a dose-dependent increase in fluorescence intensity, measured at the emission maximum for catecholamines. The increase in fluorescence intensity was accompanied by a change in the emission spectrum with displacement of the fluorescence maximum towards a shorter wavelength characteristic for a catecholamine. Recordings of rates of photodecomposition showed a rapid exponential fading of the fluorescence in mast cells of control rats comparable to that of 5-hydroxytryptamine-containing protein droplets, whereas the mast cells of DOPA-treated rats showed a slower fading rate, intermediate between that of dopamine- and 5-hydroxytryptamine-containing models.     

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.     

Fibronectin on the surface of rat mast cells

Rabbit antiserum against rat plasma fibronectin induced histamine release in isolated rat peritoneal mast cells. Immunofluorescence revealed fibronectin on the mast cells in rat mesentery and on the surface of the isolated mast cells. Mast cells adhered to collagen-coated dishes. This cellular adherence was inhibited by the addition of anti-fibronectin. Fibronectin on the surface of mast cells may play a role of attachment of the cells to collagenous connective tissues.     

Development of mucosal mast cells after injection of a single connective tissue-type mast cell in the stomach mucosa of genetically mast cell-deficient W/Wv mice

Mast cells may be classified into at least two phenotypically distinct populations: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). Mast cells in the peritoneal cavity of mice are typical CTMC, whereas mast cells in the mucosa of the stomach show morphologic characteristics of MMC. We investigated whether CTMC may change to MMC. A single peritoneal mast cell of WBB6F1-+/+ mice was identified under the phase-contrast microscope, picked up with the micromanipulator, and injected into the stomach wall of genetically mast cell-deficient WBB6F1-W/Wv mice. The cells with histochemical and electron microscopical features of MMC developed in the mucosa, and those with histochemical features of CTMC in the muscularis propria. This directly demonstrates that a certain proportion of CTMC may function as a bipotent precursor for both MMC and CTMC.     

Effect of exogenous heparin on secretory status of rat mast cells under immobilization stress]

The significant increase of heparin release from mast cells was observed in rats under stress conditions induced by 60 min immobilization. The index of its saturation with heparin became 4 times lower. The highest secretory activity of mast cells was observed during the first 30 min of immobilization. It was shown that at that time the heparin release from mast cells occurred by granulolysis (merocrine type of secretion). In the rats received heparin (15 or 150 u/200 g) during the first 15 min of immobilization the mast cells released heparin with the same intensity as in a 4 control animals. But then in rats with high heparin blood concentration the heparin release from mast cells ceased and mast cells began to accumulate heparin from blood. By the 30th min of immobilization the heparin content in the mast cells has become normal.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.     

Functional status of the rat insular apparatus and adrenal cortex at early periods after irradiation

As early as 0.5-3 h after X-irradiation of rats with a lethal dose of 12 Gy glucocorticoid activity of adrenal cortex was enhanced and 11-oxycorticosteroid level in blood was increased. The increasing of the immunoreactive insulin content of blood was registered at the background of hypercorticoidism and normal function of insular apparatus during the first hours following irradiation. At later times (24-72 h), a stable hypercorticoidism developed, the ability of beta-cells to react adequately to glucose was impaired, and IRI content of blood decreased.     

Functional status of cryopreserved rat liver mitochondria

Mitochondria from the rat liver have been frozen down to -196 degrees C under protection of dimethylsulfoxide, after which they were subjected to periodical increase and decrease in temperature (cycling) in the ranges of -105 degrees C to -196 degrees C or -135 degrees to -196 degrees C. There have been observed some non-lethal damages of mitochondria which were revealed in the decrease in the respiratory control and in the ADP/O ratio after the cycling. The damage rate increased with the increase of the number of temperature decrease-increase cycles. The damages were also greater in the case when the upper limit of cycling temperature was higher than the glass transition temperature of the freezing medium (Tg = -126 degrees C). The non-lethal damages of mitochondria are explained by the influence of electric fields, appearing in the frozen sample during the temperature increase or decrease.     

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.