The role of mast cells in thioglycollate-induced inflammation

The possible role of mast cells in the initiation of inflammation was studied in genetically mast cell-deficient mice, WBB6F1-W/Wv. Inflammation was induced by i.p. injection of thioglycollate. The influx of neutrophils was markedly delayed in WBB6F1-W/Wv mice as compared to the WBB6F1-+/+, mice (congeneic controls). At the time (14 h) of maximum influx of neutrophils in WBB6F1-+/+ mice, thioglycollate caused a 3-fold increase in the total cell number in the peritoneal lavage fluid, and the neutrophil count was elevated 14-fold. At the same time point in W/Wv mice, the total cell number in the peritoneal lavage fluid was not increased significantly and the neutrophils were increased only three- to four-fold. Not only was the neutrophil influx in WBB6F1-W/Wv mice delayed, but the length of time during which the neutrophil count was elevated in the peritoneal fluid was significantly shortened. Transfer (i.p.) of mast cells cultured from the bone marrow of congeneic controls corrected the delay in the neutrophil influx. The magnitude of the neutrophil influx in WBB6F1-W/Wv mice was equivalent to that of congeneic controls 9 days after mast cell repletion. Histologic studies were performed to follow the migration and differentiation of mast cells after adoptive transfer into WBB6F1-W/Wv mice. No connective tissue mast cells could be identified on day 9 when the inflammatory reaction was restored. Migration of mast cells into the tissue, as studied in the cecum, progressed steadily. On day 9 after adoptive transfer, the mast cell number was 38% of congeneic controls. Therefore, the increase in thioglycollate-induced neutrophil influx in WBB6F1W/Wv mice after mast cell repletion seemed to be correlated, at least to some extent, with the migration of mast cells into tissues and not with differentiation into connective tissue mast cells. However, a certain maturation and differentiation may have occurred. These results suggest that mast cells play an important role, although they do not seem to be the only cell type responsible for the initiation of inflammation.     

Mast cells are critical for the production of leukotrienes responsible for neutrophil recruitment in immune complex-induced peritonitis in mice

Mast cells are secretory cells strategically located in the vicinity of blood vessels where they can readily initiate and modulate various inflammatory processes, including plasma exudation and leukocyte infiltration. We have previously shown that 50% of the neutrophil influx during immune complex peritonitis in mice is due to mast cells. Eicosanoids are important mediators of various inflammatory processes including neutrophil infiltration. The possibility that mast cells are essential for the production of leukotrienes (LT) involved in the elicitation of neutrophils in immune complex peritonitis was investigated in mast cell-deficient, WBB6F1-W/WV, and normal, WBB6F(1-)+/+, mice. The time course and amounts of immunoreactive PGE2, 6-keto-PGF1 alpha, and TX3B2 released into the peritoneal exudates were similar in both sets of mice. LTB4 and LTC4 levels, however, were twofold higher in +/+ than in W/WV mice 2 h after stimulation. HPLC analysis of the peritoneal exudate confirmed the presence of leukotrienes. The 5-lipoxygenase inhibitor A-63162 blocked leukotriene production in a dose-dependent manner in both sets of mice. However, this compound caused a significant reduction (60%) of neutrophil infiltration only in WBB6F(1-)+/+ but not in the mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/WV mice restored the effect of A-63162 on PMN recruitment. These data suggest that mast cells in the vicinity of blood vessels are important for the synthesis of leukotrienes responsible for PMN recruitment.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Hybrid resistance to parental mast cell precursors in the skin of (WB X C57BL/6)F1-W/Wv mice

When bone marrow cells of (WB X C57BL/6)F1-+/+ (WBB6F1-+/+) and WB-+/+ (WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. However, the number of WB bone marrow cells necessary for appearance of mast cell clusters was significantly larger than when bone marrow cells of WBB6F1-+/+ mice were used. When WB bone marrow cells were mixed either with WB thymus cells or with silica particles, the proportion of injection sites at which mast cell clusters appeared increased to the level that was observed after the injection of the same number of WBB6F1-+/+ bone marrow cells. When suckling WBB6F1-W/Wv mice of less than or equal to 18 days of age were used as recipients, bone marrow cells of WBB6F1-+/+ and WB mice produced mast cell clusters with a comparable efficiency. Both syngeneic thymus cells and silica particles are known to abrogate the hybrid resistance that is observed in the spleen against parental hematopoietic stem cells. The hybrid resistance in the spleen is not detectable in suckling mice, either. Thus, the poor growth of mast cell precursors in the skin and the poor growth of hematopoietic stem cells in the spleen seem to be regulated by the same mechanism.     

Studies of the role of mast cells in contact sensitivity responses. Passive transfer of the reaction into mast cell-deficient mice locally reconstituted with cultured mast cells: effect of reserpine on transfer of the reaction with DNP-specific cloned T cells

The role of mast cells in the elicitation of contact sensitivity (CS) responses was evaluated by transferring different aliquots of the same preparations of immune lymph node cells (I-LNC) into naive, genetically mast cell-deficient (WBB6F1-W/Wv or WCB6F1-S1/S1d) mice and the corresponding congenic normal (+/+) mice. We found that the 24-hr CS responses elicited in the recipient mast cell-deficient mice were statistically indistinguishable from those in the congenic +/+ mice according to four different criteria: micrometer measurements of ear swelling, ratios of the weight or [125I]iododeoxyuridine-labeled leukocyte infiltration-associated cpm in challenged and contralateral control ears, and amount of 125I-fibrin deposition. We also transferred I-LNC into WBB6F1-W/Wv mice which, 5 months earlier, had undergone local repair of their mast cell deficiency by the intradermal injection (into the left ear only) of growth factor-dependent cultured mast cells derived from congenic +/+ mice. When 24-hr CS responses were elicited in both ears of these mice, the reactions in the mast cell-reconstituted left ears were similar to those in the mast cell-deficient right ears. We also found that treatment of antigen-specific cloned T cells with reserpine in vitro markedly impaired their ability to transfer reactivity for CS, providing further evidence that reserpine can interfere with the expression of T-cell-mediated responses through effects independent of its action on mast cells.     

Opposing pharmacological actions of cepharanthin on lipopolysaccharide-induced histidine decarboxylase activity in mice spleens

A biscoclaurin alkaloid preparation, cepharanthin (Ceph), is reported to have opposing pharmacological effects, enhancement or depression, on several cells and tissues, although detailed mechanisms remain unclear. Previously, we reported that Ceph enhanced lipopolysaccharide (LPS)-induced histidine decarboxylase (HDC) activity in mice spleens by consecutive pre-administration. In this study, we examined the pharmacological effects on HDC activity of a single Ceph pre-administration to test the influence of the administration method. Consequently, HDC activities were decreased by a single administration 15 minutes before LPS challenge in ddY and ICR mice spleens. Moreover, to further examine this suppressing effect, we employed genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice to avoid the influence of mast cells. In W/Wv mice, HDC activity was enhanced, but not in the congenic WBB6F1 +/+ mice. These findings suggest that mast cells influence the depressant effect on HDC activity by a Ceph single administration in mast cell sufficient mice.     

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.     

Inability of genetically mast cell-deficient W/Wv mice to acquire resistance against larval Haemaphysalis longicornis ticks

Genetically mast cell-deficient (WB X C57BL/6)F1-W/Wv mice were used to investigate the role of mast cells for the acquisition of resistance against larval Haemaphysalis longicornis ticks. Resistance against ticks was evaluated by reduction in both number and weight of engorged ticks. Although (WB X C57BL/6)F1-+/+ mice with a normal number of mast cells acquired resistance after repeated infestation of ticks, the congenic W/Wv mice did not acquire it. Bone marrow transplantation from the +/+ mice were grafted onto the back of the W/Wv mice, resistance against the ticks was detectable in the grafted skin. In contrast, resistance was not detectable in the skin of the W/Wv mice which had been grafted onto the back of the syngenic W/Wv mice. Thus, we consider that the failure of the W/Wv mice to manifest resistance is attributable to the mast cell depletion.     

Substance P induces granulocyte infiltration through degranulation of mast cells

Substance P, a potent vasodilatory neuropeptide, is released from peripheral nerve endings of sensory neurons by various stimuli. Although in vitro incubation of rat and human mast cells with substance P causes their degranulation, it is not known whether inflammatory changes induced by substance P are mediated by degranulation of mast cells. We investigated this point by using genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-Sl/Sld mice. The s.c. injection of substance P induced degranulation of mast cells in the skin of WBB6F1-+/+ mice, and then a marked eosinophil infiltration around the degranulated mast cells. However, WBB6F1-W/Wv and WCB6F1-Sl/Sld mice showed little or no eosinophil infiltration in the skin after the injection of substance P. When the mast cell deficiency of WBB6F1-W/Wv mice was rescued either systemically by bone marrow transplantation or locally by injection of cultured mast cells, injection of substance P induced the infiltration of eosinophils, suggesting that substance P-induced eosinophil infiltration was mediated through degranulation of mast cells.     

Estrogen-induced increase in eosinophil number and peroxidase activity in uterus of genetically mast cell-deficient W/Wv mice

The role of mast cells in induction of uterine eosinophilia was investigated by using genetically mast cell-deficient (WB X C57BL/6)F1-W/Wv (hereafter called WBB6F1-W/Wv) mice. The injection of estradiol-17 beta (0.16 micrograms/g body weight) increased the peroxidase activity and eosinophil number in the uteri of castrated WBB6F1-W/Wv and WBB6F1-+/+ mice. Since no significant differences were detectable between these two type of mice, mast cells did not seem to be essential for the estrogen-induced uterine eosinophilia, at least in mice.     

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.     

An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model

Immunization of BALB/c mice with alum-adsorbed OVA, followed by three bronchoprovocations with aerosolized OVA, resulted in the development of airway hyperresponsiveness (AHR) and allergic inflammation in the lung accompanied by severe infiltration of eosinophils into airways. In this murine asthma model, administration of monoclonal anti-IL-5 Ab before each Ag challenge markedly inhibited airway eosinophilia, but the treatment did not affect the development of AHR. Immunization and aerosol challenges with OVA following the same protocol failed to induce AHR in the mast cell-deficient W/Wv mice, but induced AHR in their congenic littermates, i.e., WBB6F1 (+/+) mice. No significant difference was found between the W/Wv mice and +/+ mice with respect to the IgE and IgG1 anti-OVA Ab responses and to the airway eosinophilia after Ag provocations. It was also found that reconstitution of W/Wv mice with bone marrow-derived mast cells cultured from normal littermates restored the capacity of developing Ag-induced AHR, indicating that lack of mast cells was responsible for the failure of W/Wv mice to develop Ag-induced AHR under the experimental conditions. However, the OVA-immunized W/Wv mice developed AHR by increasing the frequency and Ag dose of bronchoprovocations. The results suggested that AHR could be developed by two distinct cellular mechanisms. One would go through mast cell activation and the other is IgE/mast cell independent but an eosinophil/IL-5-dependent mechanism.     

Proliferation and differentiation in culture of mast cell progenitors derived from mast cell-deficient mice of genotype W/Wv

Mice of genotype W/Wv have less than 1% of normal mast cells in the skin, stomach, and cecum. In order to further clarify the mechanism of this deficiency, we studied committed mast cell progenitors and multipotent progenitors, which are capable of mast cell differentiation in clonal culture. The relative concentration of mast cell progenitors in the bone marrow, spleen, and peripheral blood of W/Wv mice was similar to that of +/+ mice. However, the cellularity of the marrows of W/Wv mice was 54% of that of their normal littermates. Identification of mast cells was established by metachromatic staining with toluidine blue, transmission electron microscopy, and demonstration of membrane receptors for immunoglobulin E. The time course of colony formation and the morphology of W/Wv mast cell colonies in culture was identical to that of normal littermates. The percentages of mast cells in individual multi-lineage colonies were extremely variable. The histamine content of mast cells derived from W/Wv mice was similar to that of mast cells from +/+ mice. These studies demonstrated the normal capacity for differentiation and proliferation in culture of mast cell progenitors from W/Wv mice.     

Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate

Mast cells clearly are critical for the expression of some IgE-dependent responses, but their roles in other forms of inflammation are uncertain. We previously described a new model system for defining the unique contribution of mast cells to biologic responses in vivo, genetically mast cell-deficient WBB6F1-W/Wv mice that have undergone selective local repair of their mast cell deficiency by the injection of IL-3-dependent cultured mast cells derived from the congenic normal (WBB6F1-+/+) mice. Using this approach, we analyzed the contribution of mast cells to the acute inflammation induced by the epicutaneous application of PMA. Even though PMA can activate a wide variety of cell types that may contribute to acute inflammation, we found that mast cells were required for the full expression of the tissue swelling and leukocyte infiltration associated with the response to the agent in vivo. Thus, in WBB6F1-W/Wv mice selectively reconstituted with dermal mast cells by intradermal injection of cultured WBB6F1-+/+ mast cells into the left ear only, PMA induced approximately twice the tissue swelling and neutrophil infiltration in the mast cell-reconstituted left ears as in the contralateral control ears. This represents the first use of W/Wv mice locally reconstituted with mast cells to confirm the hypothesis that mast cells can represent an important amplification mechanism in acute inflammatory responses of nonimmunologic origin. It also defines a model system that may be generally useful for investigating mast cell-dependent and -independent aspects of acute inflammatory responses.     

Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.     

Dural Mast Cells: Source of Contaminating Histamine in Analyses of Mouse Brain Histamine Levels

Abstract: Histamine levels were determined in mouse brains from WBB6F₁- +/+ (mast cell normal) and WBB6F₁- W/W^v (mast cell-deficient) mice whose brains were dissected immediately after decapitation or after freezing the severed heads in liquid nitrogen for 10 s. In WBB6F₁-+/+ mice, brains obtained from frozen heads contained significantly higher levels of histamine than those obtained from unfrozen heads. The converse was found in brains obtained from the WBB6F₁- W/W^v mice. When CF-1 mice (which also contain brain-associated mast cells) were treated as described above, results very similar to those found with the WBB6F₁- +/+ mice were obtained. Further, the high levels of histamine found in CF-1 mice whose brains had been frozen in situ were accompanied by an extensive degranulation of mast cells in the dura mater of these mice. Because of this degranulation of mast cells, and the fact that increased levels of brain histamine were not found in mast cell-deficient mice, it is concluded that dural mast cells are the likely source of the artifactually higher levels of histamine seen in brains frozen in situ.     

Protective roles of mast cells and mast cell-derived TNF in murine malaria

TNF plays important roles in the protection and onset of malaria. Although mast cells are known as a source of TNF, little is known about the relationship between mast cells and pathogenesis of malaria. In this study, mast cell-deficient WBB6F1-W/W(v) (W/W(v)) and the control littermate WBB6F1+/+ (+/+) mice were infected with 1 x 10(5) of Plasmodium berghei ANKA. +/+ mice had lower parasitemia with higher TNF levels, as compared with W/W(v) mice. Diminished resistance in W/W(v) mice was considered to be due to mast cells and TNF. This fact was confirmed by experiments in W/W(v) mice reconstituted with bone marrow-derived mast cells (BMMCs) of +/+ mice or of TNF-/- mice. W/W(v) mice with BMMCs of +/+ mice exhibit lower parasitemia and mortality accompanying significantly higher TNF levels than those of W/W(v) mice. Parasitemia in W/W(v) mice with BMMCs of TNF-/- mice was higher than that in +/+ mice. Activation of mast cells by anti-IgE or compound 48/80 resulted in release of TNF and decrease of parasitemia. In addition, splenic hypertrophy and increased number of mast cells in the spleen were observed after infection in +/+ mice and W/W(v) mice reconstituted with BMMCs of +/+ mice as compared with W/W(v) mice. These findings propose a novel mechanism that mast cells and mast cell-derived TNF play protective roles in malaria.     

Mast cells enhance the antibody-mediated injury of skin basement membrane in mice.

Immunologic basement membrane injury occurs in certain human diseases. We investigated the role of mast cells in the initiation of inflammation induced by selective deposition of antibody on the basement membrane in the skin. Intradermal injection of the antibody into mast cell-deficient WBB6F1-W/Wv mice and their congenic controls, WBB6F1-+/+, caused C (C3) deposition and tissue damage preferentially at the dermo-epidermal junction (basement membrane). Damage occurred earlier and was more extensive in normal than in WBB6F1-W/Wv mice. Hemorrhage in WBB6F1-W/Wv was reduced by 50%. In both groups of mice, a dose- and time-dependent neutrophil infiltration reached maximum at 8 h. At the peak, neutrophil accumulation in WBB6F1-W/Wv was only 50% of that in normal mice. Mast cell reconstitution of WBB6F1-W/Wv mice normalized the inflammatory response. Pretreatment with a 5-lipoxygenase inhibitor, A-63162, reduced neutrophil infiltration by 60% in normal but not in WBB6F1-W/Wv mice. Mast cell repletion restored the effect of A-63162. The results indicate that mast cells are important for the initiation of inflammation induced by the deposition of antibody on the basement membrane and the production of leukotrienes participating in neutrophil elicitation.     

Changing processes from bone marrow-derived cultured mast cells to connective tissue-type mast cells in the peritoneal cavity of mast cell-deficient w/wv mice: association of proliferation arrest and differentiation

Connective tissue-type mast cells (CTMC) and mast cells grown in vitro exhibit many differences in morphology, biochemistry, and function. When cultured mast cells of WBB6F1-+/+ mouse origin were injected into the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice, however, the cultured mast cells acquired characteristics similar to CTMC. In this study, we analyzed the changing process. When the density of the cultured mast cells was measured by Percoll density gradient centrifugation, the proportion of dense mast cells increased after injection into the peritoneal cavity. Because the increase in proportion of dense mast cells paralleled the increase in proportion of heparin-containing mast cells, both parameters may be used as an index for differentiation activity of cultured mast cells into CTMC. When proliferation activity of mast cells was estimated by the incorporation of bromodeoxyuridine, the proliferation activity decreased after the i.p. transfer. Moreover, when cultured mast cells were recovered 10 wk after the i.p. transfer, the mast cells almost lost proliferation activity in the same culture condition that had been used for establishment of cultured mast cells from the bone marrow of WBB6F1-+/+ mice. These results demonstrate that the proliferation arrest and the acquisition of CTMC-like characters are associated after i.p. transfer of cultured mast cells.