Functional correlations between mucosal mast cell activity and immunity to Trichinella spiralis in high and low responder mice

Levels of intestinal mast cell protease (IMCP) were quantified in serum, gut tissue and in intestinal fluids taken from mice infected with Trichinella spiralis during primary and secondary infections. The ability to generate a mast cell response was dependent on the response phenotype of the mouse strain used. The mast cell response in rapid responder mice (NIH) occurred sooner and was more pronounced than in either intermediate (SWR) and low responder (B1O) mice. This pattern was also reflected in the concentration of IMCP found in various tissues examined. The correlations between IMCP concentrations in blood, and worm expulsion, are discussed.     

The in vivo role of stem cell factor (c-kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice

The role of stem cell factor (SCF) in the generation of intestinal mast cell hyperplasia and host protective immunity following helminth infection was investigated using the Trichinella spiralis/mouse model. In vivo administration of a monoclonal antibody specific for the receptor for SCF (c-kit) was found to completely prevent the generation of intestinal mastocytosis normally observed following T. spiralis infection. This was reflected by markedly reduced intestinal mast cell protease (IMCP) levels in both tissue and serum. Moreover, animals treated with anti-c-kit antibody failed to show any evidence of worm expulsion from the gut. The data demonstrate for the first time, a critical role for the SCF in the generation of mucosal mastocytosis and host protective immunity following an intestinal helminth infection.     

Stem cell factor dependent hyperplasia of mucosal-type mast cells but not eosinophils in Schistosoma mansoni-infected rats

Stem cell factor (SCF) is a growth factor with multiple activities which acts on numerous cell types including primordial germ cells, haemopoietic stem cells, melanocytes and mast cells. SCF is critical for the development of the mast cell hyperplasia associated with infection with the intestinal parasites Nippostrongylus brasiliensis and Trichinella spiralis. In the present study we have assessed the role of SCF in the mast cell and eosinophil responses to Schistosoma mansoni in the rat by blocking its effects in vivo with polyclonal antibody to SCF. Rats treated with sheep anti-SCF antibody on days 21, 24, 27 and 30 of infection with S. mansoni showed a rapid decrease in serum concentrations of the mucosal mast cell-associated protease rat mast cell protease II (RMCP II) by day 24, compared with normal sheep IgG-treated controls. Similarly, the number of mucosal mast cells and RMCP II levels in both small intestine and liver were also significantly reduced by day 32 of infection. In contrast with the depeletion of mast cells and mast cell proteases, eosinophil numbers in liver or intestine did not change significantly after anti-SCF treatment compared with controls. These results confirm that mast cell survival and hyperplasia are dependent on the presence of SCF whilst demonstrating that the eosinophil recruitment to liver and intestine associated with S. mansoni infection is SCF-independent.     

Systemic release of mucosal mast cell protease during infection with the intestinal protozoal parasite, Eimeria nieschulzi. Studies in normal and nude rats

The systemic secretion of rat mucosal mast cell protease (RMCPII), a major product of rat mucosal mast cells (MMC), was examined during primary infections with the protozoan parasite, Eimeria nieschulzi in CFH/B, athymic (rnu/rnu) and euthymic (rnu/+) rats. Release of RMCPII into the blood stream (2.9 micrograms/ml of serum) of normal rats occurred within 1 day after infection. This response developed 3-6 hours after inoculation with oocysts, was dose-dependent, and was found in both naive and immune rats. Maximal release of RMCPII (4.5 micrograms/ml of serum) in naive rats occurs 9 days after primary infection, whereas the numbers of MMC and concentrations of mucosal RMCPII were maximal 14 days after infection, by which time the systemic RMCPII response had begun to decline. The numbers of MMC and concentrations of mucosal RMCPII in uninfected nude rats were similar to those in the heterozygous (rnu/+) litter-mates. After infection, the numbers of MMC and concentrations of mucosal RMCPII increased in the heterozygotes but not in nude rats. Similarly, RMCPII was detected systemically only in the heterozygotes.     

Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N brasiliensis infection

We assessed the effects of the c-kit ligand, stem cell factor (SCF), in the jejunal mucosal mast cell hyperplasia that occurs during infection with the intestinal nematodes, Nippostrongylus brasiliensis or Trichinella spiralis in rats. Compared with vehicle-treated rats, rats treated with SCF (25 micrograms/kg/d, intravenous [i.v.] for 14 days) during N brasiliensis infection exhibited significantly higher levels of the rat mucosal mast cell (MMC)-associated protease, rat mast cell protease II (RMCP II) in the jejunum and serum on day 8 of infection, but not on days 10 or 15 of infection. By contrast, in comparison to rats treated with normal sheep IgG, rats treated with a polyclonal sheep antirat SCF antibody exhibited markedly decreased numbers of jejunal MMCs, levels of jejunal RMCP II, and serum concentrations of RMCP II during infection with either nematode, particularly at the earlier intervals of infection (< or = day 10). Taken together, these findings indicate that SCF importantly contributes to MMC hyperplasia and/or survival during N brasiliensis or T spiralis infection in rats, but that levels of endogenous SCF are adequate to sustain near maximal MMC hyperplasia during infection with these nematodes. Notably, treatment of rats with SCF somewhat increased, and treatment with anti- SCF significantly decreased, parasite egg production during N brasiliensis infection. This finding raises the interesting possibility that certain activities of intestinal MMCs may contribute to parasite fecundity during infection with this nematode.     

Systemic release of a mast cell proteinase following nematode infections in sheep

An enzyme-linked immunosorbent assay (ELISA) for sheep mast cell proteinase (SMCP) has been developed. Concentrations of SMCP in homogenates of abomasal tissue from parasite-immune sheep (341 micrograms SMCP/g tissues) were raised when compared to those in normal (non-infected) abomasa (0.145 micrograms SMCP/g tissue). SMCP was not detected in sera from normal animals challenged with Haemonchus contortus but was present (less than 1.0 ng SMCP/ml) in sera from 8/11 immune sheep 2 h after intra-abomasal challenge with 1 x 10(6) exsheathed Haemonchus larvae. In two further experiments, the SMCP response in gastric lymph was monitored after homologous larval challenge in sheep immune to Ostertagia circumcincta and in normal controls. SMCP (less than 1.4 ng SMCP/ml) was detected in lymph from 2/3 and 4/5 immune animals between 1 and 4 days post-challenge with 50,000 larvae, but not from normal animals. SMCP was not detected in lymph from immune animals following challenge with 1000 Ostertagia larvae. The relatively low concentrations of SMCP in blood and lymph reflect the presence of proteinase inhibitor(s) which interfered with the ELISA.     

Limit dilution analysis of mast cell precursor frequency in the gut epithelium of normal and Trichinella spiralis infected mice

Previous studies have established that the gut nematode Trichinella spiralis induces a dramatic thymus dependent intestinal mastocytosis which peaks within 6 to 12 days after primary oral infection. It is not known, however, if the increase in gut mast cells results from the influx of mast cells or their precursors, or from the expansion and differentiation of mast cell precursors (MCP) that are normally present in the small intestinal epithelium. In the present study, the number of mucosal MCP in the intraepithelial lymphocyte (IEL) population and in bone marrow (BM) cells from normal and 4 day T. spiralis infected mice was compared by culturing the cells at limiting dilutions in medium containing interleukin-3 (IL-3). While the MCP frequency in IEL from infected mice was found to be significantly increased in comparison with that found in normal mice, the numbers of MCP in BM from the two groups were equivalent. Resident intraepithelial mucosal MCP therefore undergo a local expansion before the occurrence of an overt T dependent intestinal mastocytosis. This finding lends support to the view that local mucosal T cells are involved in regulating mast cell numbers in response to intestinal helminth infection.     

Suppression of mucosal mastocytosis by Nematospiroides dubius results from an adult worm-mediated effect upon host lymphocytes

Infections with the nematode Nematospiroides dubius fail to elicit mucosal mast cell (MMC) responses in the intestines of host mice, and suppress MMC responses generated by heterologous infection. Larval N. dubius have the capacity to prime for mastocytosis, and to elicit this response in primed mice during a challenge, but only if adult worms are prevented from developing, either by anthelmintic treatment or by irradiation of the larvae themselves. The suppressive effect of the adult stage was confirmed in experiments where such worms were implanted directly into the intestines of mice primed by exposure to irradiated N. dubius larvae or concurrently infected with Trichinella spiralis. Data on the mechanisms underlying this suppressive effect were obtained from experiments involving the adoptive transfer of mastocytosis by mesenteric lymph node cells (MLNC) from T. spiralis infected mice. When MLNC were taken from mice infected concurrently with both T. spiralis and N. dubius no enhanced mastocytosis was seen in recipients after challenge with T. spiralis. Exposure of MLNC from T. spiralis infected donors to the presence of adult N. dubius after transfer did not reduce the adoptively transferred response. The response was also unaffected when MLNC from adult N. dubius infected mice were simultaneously transferred with MLNC from T. spiralis donors. It is concluded that the suppressive effect of adult N. dubius upon the expression of mucosal mastocytosis acts upon the generation of lymphocytes capable of promoting the development of MMC from precursor cells.     

In vitro modelling of rat mucosal mast cell function in Trichinella spiralis infection

Intestinal infection with the parasitic nematode, Trichinella spiralis, provides a robust context for the study of mucosal mast cell function. In rats, mucosal mast cells are exposed to parasites during the earliest stage of infection, affording an opportunity for mast cells to contribute to an innate response to infection. During secondary infection, degranulation of rat mucosal mast cells coincides with expulsion of challenge larvae from the intestine. The goal of this study was to evaluate the rat bone marrow‐derived mast cells (BMMC) and the rat basophilic leukaemia cell line (RBL‐2H3) as models for mucosal mast cells, using parasite glycoproteins and antibody reagents that have been tested extensively in rats in vivo. We found that BMMC displayed a more robust mucosal phenotype. Although T. spiralis glycoproteins bound to mast cell surfaces in the absence of antibodies, they did not stimulate degranulation, nor did they inhibit degranulation triggered by immune complexes. Parasite glycoproteins complexed with specific monoclonal IgGs provoked release of rat mast cell protease II (RMCPII) and β‐hexosaminidase from both cell types in a manner that replicated results observed previously in passively immunized rats. Our results document that RBL‐2H3 cells and BMMC model rat mucosal mast cells in the contexts of innate and adaptive responses to T. spiralis.     

Hepatic recruitment of mast cells occurs in rats but not mice infected with Schistosoma mansoni

The pathogenesis of infection with Schistosoma mansoni in rats is distinct from that in mice. Rats are non-permissive hosts and infection is terminated in the liver before egg laying commences whereas the parasite completes its life cycle in mice. Comparison of the mast cell responses in the two species reveals that a pronounced hepatic mastocytosis occurs in the rat and this is concomitant with the demise of the parasite. The majority of recruited hepatic mast cells contain the highly soluble granule chymase, rat mast cell protease-II, which is released systemically into blood during the period of parasite elimination. In contrast, very few mast cells are found in livers of parasitized mice and none contain the soluble granule chymase mouse mast cell protease-1. However, during egg deposition in the gut, an intraepithelial mastocytosis occurs in parasitized mice. These intraepithelial cells are typical mucosal mast cells as determined by their content of mouse mast cell protease-1. Recruitment of mucosal mast cells occurs in the intestinal lamina propria of infected rats soon after the parasites migrate to the liver. These findings suggest that mast cells of the mucosal phenotype are involved in the pathogenesis of the hepatic response to infection in the rat but that, in the mouse, mucosal mastocytosis is associated with intestinal sensitization by egg antigens.     

Expulsion of Hymenolepis nana from mice with congenital deficiencies of IgE production or of mast cell development

The roles of IgE and mast cells on expulsion of adult Hymenolepis nana from the intestine were examined in mice. IgE-dependency was determined by comparing congenitally IgE-deficient SJA/9 and IgE-producing SJL/J mice infected with 50 H. nana eggs. Anti-H. nana IgE antibody was detected at three weeks post infection (p.i.) in SJL but not in SJA mice. The number of adult worms in the intestines of SJA and of SJL mice were similar at two weeks, but significantly more were found in SJA mice at three weeks p.i. Treatment of mice with anti-ɛ antibody also resulted in an increased worm burden at three weeks, suggesting participation of IgE in expulsion of H. nana. Intestinal mastocytosis was induced by infection regardless of the IgE status of the mice. Mast cell-dependency was tested in mast cell-deficient W/W^u and in normal littermate +/+ mice infected with 100 H. nana eggs. Anti-H. nana antibody was detected in both groups of mice at three weeks p.i. Worm expulsion seemed to be mast cell dependent because expulsion was less complete in W/W^u mice at three weeks p.i. Peripheral blood eosinophilia was comparable at three weeks p.i. in both IgE and mast cell sufficient and deficient mice. These results suggest that IgE and mast cells participate in the expulsion of H. nana adults from intestine in mice.     

Mouse mast cell protease-1 is required for the enteropathy induced by gastrointestinal helminth infection in the mouse

BACKGROUND & AIMS: The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. Mast cell hyperplasia is a hallmark of infection with gastrointestinal nematodes, in particular Trichinella spiralis. Although the precise mechanism by which mast cells induce expulsion of these parasites has not been elucidated, it has been proposed that mast cell mediators, including cytokines and granule chymases, act to create an environment inhospitable to the parasite, part of this being the induction of intestinal inflammation. Therefore, the aims of this study were to dissect the role of mast cells and mast cell proteases in the induction of parasite-induced enteropathy. METHODS: Mast cell-deficient W/Wv and mouse mast cell protease-1 (mMCP-1)-deficient mice were infected with T. spiralis, and parasite expulsion, enteropathy, and Th2 responses were determined. RESULTS: Expulsion of the parasite was delayed in both strains of mice compared with wild-type controls; additionally, in both cases, the enteropathy was significantly ameliorated. Although Th2 responses were significantly reduced in mast cell-deficient W/Wv mice, those from mMCP-1-deficient mice were similar to wild-type mice. Additionally, levels of TNF-alpha and nitric oxide were significantly reduced in both W/Wv and mMCP-1 deficient mice. CONCLUSIONS: These results imply that mast cells may contribute to the induction of protective Th2 responses and, importantly, that the intestinal inflammation associated with gastrointestinal helminths is partly mediated by mMCP-1.     

Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell deficient W/Wv mice

Mucosal mast-cell hyperplasia is frequently observed in intestinal nematode infections and it has been suggested that mast-cell responses to parasite antigens are involved in worm expulsion (self cure). To evaluate the importance of this mechanism, the course of infection and expulsion of Nippostrongylus brasiliensis was compared in mast-cell deficient W/WV and normal (+/+) mice. Initial infectivity rates were similar, but the subsequent kinetics of expulsion of adult worms differed principally in that the onset of expulsion in mast-cell deficient mice appeared to occur 24-36 h later than that in normal mice. Expulsion was complete by the 14th day post infection in both W/WV and normal mice. Worm fertility (as estimated by faecal egg output) also differed in W/WV and normal mice, with maximal egg output in W/WV mice occurring 24 h later than that in normal mice. Although a few mast cells were present in the intestinal mucosa and tongue of W/WV mice, their numbers did not change during the course of infection with N. brasiliensis. In contrast, worm expulsion in normal mice was associated with a moderate increase in numbers of intestinal mast cells, commencing at the onset of expulsion and peaking several days after expulsion was completed.     

The athymic nude rat. IV. Immunocytochemical study to detect T-cells, and immunological and histopathological reactions against Trichinella spiralis

The anti-parasite response was investigated after oral infection of athymic nude (rnu/rnu) rats and heterozygous (+/rnu) littermates with 1000 muscle larvae of Trichinella spiralis. No IgM, IgG and IgE antibodies were detected in serum of rnu/rnu rats. Expulsion of adult worms from the small intestine was prolonged (worms were nearly all expelled at days 14 and 91 in +/rnu and rnu/rnu rats respectively). The yield of muscle larvae in the carcasses of nude rats at day 91 was 33 times higher than in +/rnu rats. In contrast to the strong inflammatory reaction in the parasitized tongue of +/rnu rats, no infiltration was observed in rnu/rnu rats. Using an immunoperoxide method with monoclonal anti-rat T-cell antibody, no T cells were identified in spleen, mesenteric lymph node and Peyer's patches. These data support earlier studies that the nude rat lacks functional T cells. As the counts of connective tissue mast cells (CTMC), intestinal mast cells (IMC) and globule leucocytes (GL) in small intestine of uninfected rnu/rnu rats were equal or higher than in +/rnu rats, it is concluded that the origin of these cells is thymus-independent. In contrast to +/rnu rats, infection of rnu/rnu rats induced no increase of CTMC, IMC or GL. Thus, these cells depend on T cells to undergo proliferation. Finally, results of this study were inconclusive whether IMC are precursors for GL, or that they represent independent cell populations.     

Proteinase phenotypes and fixation properties of rat mast cells in parasitic lesions caused by Mesocestoides corti: selective and site-specific recruitment of mast cell subsets

The distribution, fixation properties, and protease phenotypes of mast cells populating lesions caused by the metacestode stage of the cestode Mesocestoides corti in the rat were characterized. Intraperitoneal infection with M. corti induced severe granulomatous types of reactions around the pancreas and further lesions in the liver. These sites were infiltrated with mast cells which contained either rat mast cell protease I or II derived respectively from connective tissue (CTMC) or mucosal mast cells (MMC). A proportion of cells in pancreatic granulomas had staining and fixation properties identical to those of intestinal mucosal mast cells; others were typical connective tissue mast cells. Subcutaneous inoculation of parasites was associated with nodular dermal reactions, and all of the infiltrating mast cells had the fixation and staining properties of CTMC and contained RMCPI uniquely. Increased numbers of RMCPII-containing mast cells were present in the intestines of rats infected intraperitoneally. Significant quantities of RMCPII were present in homogenates of pancreatic granulomas and in livers of rats harbouring intraperitoneal infections but none was detected in skin. These findings suggest that mast cells of different phenotypes are selectively recruited to some, but not all, lesions.     

Clonogenic mast cell progenitors and their excess numbers in chimeric BALB/c mice with inactivated GATA-1

In agar cultures of marrow cells from adult female BALB/c chimeric GATA-1(Plt13/+) mice, a high frequency of unusual dispersed colonies was noted. Analysis showed that these were colonies of mast cells and that mast cell colony-forming cells (progenitors) could be detected in clonal cultures of adult marrow, neonatal marrow, or fetal liver if the combined stimulus of stem cell factor and interleukin-3 was used. Mast cell progenitors were in active cell cycle and showed an extensive capacity for self-generation. Mast cell colonies both from control GATA-1(+/+) mice and GATA-1(Plt13/+) mice could generate growth factor-dependent cloned cell lines that grew for >18 months. Surprisingly, the majority of the excessive numbers of mast cell progenitors in chimeric GATA-1(Plt13/+) mice were transcribing the inactive Plt13 allele of GATA-1, suggesting that GATA-1 normally acts to restrict the emergence of committed mast cell progenitors. In sharp contrast, all eosinophil progenitors in these mice were transcribing the normal GATA-1 allele. No excess tissue mast cells were observed in GATA-1(Plt13/+) mice, suggesting that the excess mast cell progenitors in these mice might be generating mast cells with a defective in vivo proliferative or tissue homing capacity.     

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): downregulation of specific cytokine secretion (IL-9 and IL-10) correlates with poor mastocytosis and chronic survival of adult worms

Mice were infected either with Trichinella spiralis (day 0). Heligmosomoides polygyrus (day – 14) or concurrently with both species and were killed in groups, together with naïve control mice, on 2 occasions (day 8 and 15 post infection with T. spiralis, corresponding to days 22 and 29 p.i. with H. polygyrus). The expulsion of T. spiralis was slowed significantly in concurrently infected mice and this was associated with a reduced mastocytosis and lower serum mucosal mast cell protease levels. Mesenteric lymph node (MLN) lymphocytes from all three experimental groups secreted IL-3 and IL-4 in copious amounts when stimulated in vitro by Concanavalin A (Con-A), but the secretion of high levels of IL-9 and IL-10 was essentially confined to mice infected with T. spiralis alone. It is suggested that adult H. polygyrus selectively modulate cytokine secretion by Th2 cells within the MLN during infection and that this is brought about as a direct consequence of the mechanism employed by H. polygyrus to depress mucosal inflammatory responses in order to facilitate its own survival.     

Temporal distribution of distinct mast cell phenotypes during intestinal schistosomiasis in mice

Mastocytosis is a common feature of helminth infection in most host species. We examined the temporal distribution and phenotype of mast cells during intestinal schistosomiasis in mice, using antibodies directed against histamine, a general mast cell marker, against mouse mast cell protease-1 (MMCP-1), a mucosal mast cell (MMC) marker, and against tryptase, a predominantly connective tissue mast cell (CTMC) marker. Ileal paraffin and/or cryosections of control, 8- and 15-week-infected mice were quantitatively analysed. In the intestinal wall of non- and unisexual infected mice, a few dispersed mast cells were detected. In infected mice, a transient increase of mast cells in the mucosa and a gradual increase in the outer muscle layer were observed. MMCP-1 expressing MMCs were predominantly present in the mucosa during the acute phase [8 weeks postinfection (p.i.)], while tryptase and histamine immunoreactivity demonstrated that two subsets of CTMCs were predominantly present in the outer muscle layer at 15 weeks p.i. (chronic phase). In conclusion, these results reveal that, in mice, both MMCs and CTMCs are involved in the inflammatory response during schistosomiasis. The recruitment of each mast cell population is time-dependent and occurs at different locations. These data suggest that mastocytosis is associated with motility-related gastrointestinal symptoms and egg excretion.     

The influence of challenge dose, duration of immunity, or steroid treatment on mucosal mast cells and on the distribution of sheep mast cell proteinase in Haemonchus-infected sheep

Summary The distribution of granule-specific sheep mast cell proteinase (SMCP). was assayed by immunocytochemistry and quantified by immunoassay in sheep immune to Haemonchus contortus. Repeated infection with Haemonchus larvae over 10–12 weeks induced a pronounced mucosal mastocytosis, including intraepithelial globule leukocytes (GL), which. 7 days after ceasing this dosing regime, was associated with the inability of incoming larvae to establish within the abomasal mucosa. Loss of this resistance, due to the cessation of stimulation with Haemonchus larvae 84 days previously or to treatment of sheep with cortico-steroid. was associated with a marked decline in mast cell density and concentrations of SMCP in abomasal mucosal tissues. Nevertheless, larvae also failed to establish in immune sheep rested from challenge 42 days previously and in which mast cell counts were not significantly different from those of control sheep. A small, but significant, release of SMCP was demonstrated in gastric mucus from immune sheep following larval challenge, whereas little or no SMCP was detected in mucus from naïve animals.     

Immunoglobulin E and mast cell responses are related to worm biomass but not expulsion of Hymenolepis diminuta during low dose infection in rats

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.