Suppression of muscosal mastocytosis by infection with the intestinal nematode Nematospiroides dubius

Mice exposed to primary infections with the parasite intestinal nematode Nematospiroides dubius failed to show the mucosal mast cell (MMC) response which is characteristic of infections with other species of intestinal nematode and which was readily induced in these mice by infections with Nippostrongylus brasiliensis or Trichinella spiralis. The failure to generate a mucosal mastocytosis was independent of host strain or sex. When infections with N. dubius were established before, or concurrently with, T. spiralis or N. brasiliensis, the MMC response elicited by these species was delayed and/or depressed as was expulsion of the worms themselves. Infection with N. dubius given when a MMC response was already established, by exposure to T. spiralis, had no effect on MMC numbers. The possibility that the effects of N. dubius upon MMC responses reflect a lack of mastocytopoietic potential, rather than an active interference, was excluded by showing that SJL mice, which expel primary infections with N. dubius and express strong immunity to reinfection, developed marked mastocytosis during secondary infections. The depression of MMC responses by N. dubius is discussed in relation to the known immunosuppressive properties of this parasite and in relation to the T cell mediated control of MMC development.     

Reconstitution by bone marrow grafting of the defective protective capacity at the migratory phase but not at the intestinal phase of Nippostrongylus brasiliensis infection in W/Wv mice

After a primary infection by subcutaneous inoculation with the infective larvae (L₃) of Nippostrongylus brasiliensis, the intestinal worm burden was higher and expulsion was slower in W/W^v mice than in ^+/+ mice. When the course of infection was segregated into the migratory and intestinal phases, protection during the migratory phase examined by the larval recovery from the lungs and that during the intestinal phase measured by worm burden after intraduodenal implantation with adult worms were both defective in W/W^v mice. The higher susceptibility of W/W^v mice during the migratory phase was normalized by bone marrow re constitution. On the other hand, higher susceptibility of W/W^v mice during the intestinal phase, which was measured by worm burden 24 h after intraduodenal implantation of the larvae recovered from the lungs of rats, was not normalized by bone marrow grafting. Furthermore, slower expulsion seen in W/W^v mice after intraduodenal implantation with adult worms was not hastened by bone marrow reconstitution. These results indicate that the protective mechanisms against N. brasiliensis operating during the migratory phase and those during the intestinal phase were different in terms of bone marrow dependency and that nonmyeloid cells utilizing c-kit ligand/receptor system to express their functions are involved in the rnucosal defence against N. brasiliensis.     

Nematospiroides dubius in the mouse: evidence that adult worms depress the expression of homologous immunity

Summary Mice immunized by a single infection with irradiated (25 krad) larvae of N. dubius were very resistant to subsequent challenge. However, when normal larvae were administered together with irradiated larvae at immunization, the acquired immunity expressed against a challenge infection was markedly depressed. It was found that as few as 50 normal N. dubius larvae interfered with the immunity that would have otherwise been elicited by the concurrently administered irradiated larvae, but this depressed response was totally alleviated when the normal worms were removed after completing their development in the intestinal mucosa and before they reached adulthood. Adult TV. dubius were transplanted directly into the intestines of mice either 7 days before or after immunization by irradiated larvae; it was shown that the recipient mice were less resistant to challenge than mice which had been sham operated. Transplanted adult worms themselves stimulated very little resistance to challenge in recipient mice. These results established that adult parasites are capable of depressing the expression of homologous immunity in the mouse. The possible mechanisms by which JV. dubius might modulate the host's immunological activity at the intestinal level are discussed and it is proposed that this mechanism is of benefit to the parasite in preventing the host from eliminating the worms during a chronic primary function.     

Transfer of immunity to Nematospiroides dubius: co-operation between lymphoid cells and antibodies in mediating worm expulsion

The effect of transferring immune serum (IS) and immune mesenteric lymph node cells (IMLNC) either alone or in combination was studied in NIH mice infected with partially radiation attenuated (5 krad) N. dubius. It was demonstrated that immunity to N. dubius could be transferred with IS and with IMLNC. However, considerably greater protection was transferred to recipient mice when they received both IS + IMLNC. Animals treated in this way had fewer worms than either of the other groups from as early as day 9 onwards, suggesting that a substantial proportion of the worms in mice given IS + IMLNC was retained in the intestinal tissues. The few surviving worms which completed the tissue phase of their development were then rejected by the fourth week of infection. Dose response data showed that as few as 1 × 10⁷ IMLNC could cause a significant reduction in worm numbers when given in combination with IS. These experiments indicate that both antibodies (IS) and sensitized lymphoid cells (IMLNC) are required for effective resistance to N. dubius.     

Immune-mediated damage is not essential for the expulsion of Nippostrongylus brasiliensis adult worms from the small intestine of mice

Nippostrongylus brasiliensis adult worms are expelled from the rat small intestine during a primary infection by two steps. First, host immune responses cause damage to the worms, and then a nonspecific inflammatory response initiates expulsion. We have tested the two-step expulsion hypothesis in mice infected with N. brasiliensis. After a primary infection in C57BL/6 mice, adult worms started to lay eggs on day 5 postinfection (p.i.) and were expelled around day 9-10 p.i. According to the rat system, 5 day- and 8-day-old worms were assumed to be 'normal' and 'damaged', respectively. When 5 day- and 8 day-old worms obtained from C57BL/6 mice were transferred surgically into the small intestine of naive C57BL/6 mice, both 5 day- and 8 day-old worms were almost simultaneously expelled by day 6 postworm implantation (p.w.i.). In contrast, when 5 day- and 8 day-old worms of mouse origin were implanted into naive Wistar rats, 8 day-old worms were expelled by day 5 p.w.i., while 5 day-old worms were expelled by day 8 p.w.i. Similar results were obtained when BALB/c mice were used. Therefore, mice can expel N. brasiliensis adult worms as rapidly as rats expel 'damaged' worms, regardless of the status of the worms ('normal' or 'damaged'). Stat6-deficient mice were unable to expel implanted 5 day-old worms up to day 10 p.w.i., suggesting that cellular mechanisms depending on Stat6-signalling system are necessary for the expulsion. When N. brasiliensis adult worms obtained from Stat6-deficient mice 5 and 15 days after a primary infection were implanted into Wistar rats, the former established in the recipient rats for approximately 1 week and were then expelled by day 10 p.w.i., whereas the latter were expelled by day 4 p.w.i. These results suggest that immune-mediated damage of N. brasiliensis adult worms (first step) is not a prerequisite for expulsion from the small intestine of mice, although adult worms are actually damaged by Stat6-independent immune mechanisms.     

Expulsion of Nematospiroides dubius from the intestine of mice treated with immune serum

This paper describes experiments which demonstrated that the survival of Nematospiroides dubius was severely impaired in mice treated with immune serum. CFLP donor mice were given a series of infections ranging from 25 to 200 infective larvae, at weekly intervals for 6 weeks. The mice were treated with anthelmintic on day 21 and/or day 28 to prevent the accumulation of lethal numbers of parasites in the intestine, and were bled between day 42 and day 49. Female NIH recipient mice were given a total of 2.0-2.5 ml of immune serum i/p., in several separate smaller doses at various times in relation to the day of infection. Between the administration of immune serum begun during the first 4 days of infection and the animals being killed within the next 3 weeks, the mice harboured fewer worms than control animals, the worms were stunted and their fecundity was greatly reduced. Furthermore, these worms were subsequently lost from the intestines of treated mice, during and after the fourth week of infecton. These effects on N. dubius were not observed when mice were given normal serum nor when immune serum was administered after day 6 of the infection. The delayed rejection of adult worms from mice treated with immune serum is of particular significance and suggests that immune serum contained factors which facilitated the expression of a second component in worm expulsion not nornally effective in a primary infection. The possible immunologcal mechanisms underlying these findings are discussed and related to the immunosuppression which N. dubius is known to induce in the host.     

Different susceptibility to the IL-3 induced-protective effects between Strongyloides ratti and Nippostrongylus brasiliensis in C57BL/6 mice

Repetitive administration of recombinant IL-3 induced protection against Strongyloides ratti but not against Nippostrongylus brasiliensis in C57BL/6 mice. Numbers of S. ratti were negligible from day 4 to day 6 post-infection in mice injected with IL-3, whereas N. brasiliensis burdens were almost equal from day 4 to day 6 between mice injected with IL-3 or with medium. Mice treated with IL-3 and then concurrently infected with S. ratti and N. brasiliensis were protected from intestinal S. ratti but not from N. brasiliensis. The numbers of intestinal mucosal mast cells were increased by the repetitive IL-3 treatment on one day after the final injection and was augmented by subsequent infection with both nematodes.     

Variability of the intestinal immunoglobulin E response of rats to infection with Trichinella spiralis, Heligmosomoides polygyrus or Nippostrongylus brasiliensis

Total intestinal IgE level increased in rats infected with Trichinella spiralis or Heligmosomoides polygyrus (peak levels of 2.6 microg and 3.7 microg, respectively), but not in rats infected with Nippostrongylus brasiliensis. Intestinal implantation of young adult N. brasiliensis did not stimulate an intestinal immunoglobulin (Ig)E response, suggesting that mucosal penetration may be required for local intestinal IgE responses in rats. During a T. spiralis infection, total IgE levels in the intestinal lumen were consistently higher in LEWIS and LOU rats (rat strains that eliminate T. spiralis worms earlier in the infection) than in PVG, AO and WKA/H strain rats. There was no correlation in either the total level of serum IgE and IgA, or of intestinal IgA with differences between strains in the rate of worm elimination from the gut. Furthermore, the intestinal IgE immunoprecipitated from LEWIS rats 12 days after infection reacted with T. spiralis adult worm metabolic antigens, while intestinal IgE from PVG rats only became reactive with adult worm metabolic antigens from 14 days after infection. These data emphasize the significance of the intestinal IgE response and its unique features by comparison with serum IgE and IgA or intestinal IgA.     

The development of resistance in different inbred strains of mice to infection with Nematospiroides dubius

Infection by the intestinal nematode parasite Nematospiroides dubius was studied in seven different inbred mouse strains. Although there was some minor variation in the susceptibility of the different strains to a primary infection there were marked differences in their ability to develop resistance to infection following repeated exposure to infective larvae. The strains of mice which developed the best resistance also expelled adult worms arising from the previous infections. The adult worms resulting from a primary infection were slowly eliminated in two inbred strains studied whereas no loss occurred from outbred LACA mice. Although males and females of two strains, C3H/HeJ and CBA/H were equally susceptible to a primary infection, the females developed better resistance than the male mice following two oral administrations of third stage larvae. Infected mice of every strain and both sexes contained high levels of IgG1 in the serum.     

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.     

Immunological consequences of intestinal helminth infections. Humoral responses to ovalbumin

Humoral responses to ovalbumin (OA) administered i.p. with Al(OH)3 were depressed in C57BL mice immunized 5-13 days after infection with N. dubius, but not N. brasiliensis or T. muris. These two parasites induced elevated IgM responses, possibly as a result of systemic contact with parasite larvae or debris since i.v. administered N. dubius also increased IgM titres to OA. Depression of anti-OA IgG titres by N. dubius was also observed in infected BALB/c and CBA mice given OA-Al(OH)3 (i.p.), but not in C57BL mice given OA-Al(OH)3 (s.c.), OA-FCA (i.p. or s.c.), or OA-B, pertussis (i.p.). These findings suggest that local effects of N. dubius within the peritoneum reduce the adjuvanticity of Al(OH)3, resulting in depressed responses to OA-Al(OH)3.     

Genetic control of immunity to Nematospiroides dubius: a 9-day anthelmintic abbreviated immunizing regime which separates weak and strong responder strains of mice

Experiments were designed to re-examine the variables which influence the ability of single primary infections to elicit acquired immunity to Nematospiroides dubius, in particular the importance of the presence or absence of adult worms, as these are known to exert immunomodulatory effects. Briefly, anthelmintic abbreviated infections were considerably more effective at eliciting acquired immunity than longer infections in which adult worms were allowed to reside in the intestine. A 9-day anthelmintic abbreviated infection was extremely effective at stimulation of acquired immunity in NIH mice and very few immunising infection larvae were required. Immunity to subsequent reinfection developed rapidly after the primary infection worms had been eliminated; by day 21 post-infection, the mice were almost totally immune. Abbreviated infections were used to examine the capacity of a number of mouse strains to develop immunity to reinfection. Strains of mice were chosen to allow the effects of MHC linked and non-MHC linked (background) genes to be identified. CBA and C3H strains (both H-2k) were found to be weak responders to N. dubius. B10G (H-2q) mice responded better than C57Bl/10 (H-2b), although these strains have identical background genes. DBA/2 mice were stronger responders compared to BALB/c mice, both strains sharing a common MHC haplotype (H-2d). (NIH X B10G) F1 mice (H-2q) were better responders than either of the parental strains. Several mouse strains all sharing the H-2q haplotype were particularly effective at developing immunity to N. dubius, as were also SJL mice which were the sole representatives of the H-2s haplotype, in the present study. The results established that the response phenotype is influenced by both background and MHC genes and demonstrated gene complementation in the capacity of mice to acquire immunity to N. dubius.     

Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.     

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.     

Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice

Mechanism of spontaneous cure was studied in mice infected with mouse-nonadaptive Nippostrongylus brasiliensis. Adult BALB/c mice were cured spontaneously of infection with this strain of N. brasiliensis by Day 7 post-infection. Expulsion of intestinal worms was delayed dose-dependently by a treatment with anti-CD4 antibody. However, the treatment had no significant effect on larval recovery from the lungs. Treatment of mice with anti-IL-5 antibody suppressed intestinal tissue eosinophilia induced by the infection, but did not affect intestinal worm recovery. Antigen specific IgE antibody was not detected in the sera obtained from Days 5 to 15. Therefore, IL-5 and specific IgE antibody are probably not important in the spontaneous cure. Treatment of mice with anti-CD4 antibody had no significant effect on number of intestinal goblet cells or on expression of terminal sugars of goblet cell mucins. However, histological and quantitative analyses revealed that significantly less intestinal mucus was released in anti-CD4 antibody treated mice than in control mice. These results suggest that CD4+ lymphocytes control the amount of intestinal mucus and consequently the reduced mucus interferes with the spontaneous cure. Quantity of mucus released in the intestinal lumen may have an essential role in the spontaneous cure ofN. brasiliensis-in/fcfio/i of mice.     

Hypersensitivity reactions in small intestine. I Thymus dependence of experimental 'partial villous atrophy'.

Rats infected with the intestinal nematode Nippostrongylus brasiliensis have crypt hyperplasia with villous atrophy in affected areas of the small intestine. In thymus-deprived (B) rats the course of infection is prolonged but, despite the presence of many worms in the intestinal lumen, villi and crypts appear largely normal. This suggests that the tissue damaged associated with N. brasilliensis infection is caused, not by the worms, but by a local thymus-dependent immune reaction. There is some evidence to implicate lymphocytes rather than antibodies in this reaction. It is already know that T-cell-associated damage to the small intestine, such as occurs in allograft rejection, produces subtotal villous atrophy. The present findings suggest that when T cell react locally with helminth antigens a similar type of damage occurs. The presence of a local cell-mediated immune reaction may be the common factor which causes villous atrophy and crypt hyperplasia in many small intestinal diseases, eg, viral enteritis, giardiasis, cow's milk allergy, and coeliac disease.     

Histochemical characteristics of the goblet cell mucins and their role in defence mechanisms against Nippostrongylus brasiliensis infection in the small intestine of mice

The histochemical characteristics of the small intestinal goblet cell mucins of mice and their protective role during the expulsion of Nippostrongylus brasiliensis adult worms were examined. Lectin histochemistry revealed that, before infection, strongly positive stainability of Helix pomatia agglutinin (HPA) and Limax flavus agglutinin (LFA) was observed in all of the small intestinal goblet cell mucins of mice. When ‘normal’or ‘damaged’N. brasiliensis adult worms were implanted separately into the small intestine of euthymic mice, 50% of them were established in the intestine on day one after implantation, but were subsequently expelled within one week in association with HPA and LFA positive goblet cell hyperplasia. On the other hand, in dexamethasone treated euthymic mice, 70% of implanted ‘normal’worms were expelled by day 7 and 80% of implanted ‘damaged’worms were expelled by day 4 in association with HPA and LFA positive goblet cell mucins but no goblet cell hyperplasia. This is consistent with the fact that, in spite of no increase in goblet cell number in hypothymic nu/nu mice, all of the implanted ‘damaged’worms were expelled within day 5 and 80% of implanted ‘normal’worms were expelled by day 14 post implantation, possibly associated with strngly expressed terminal GalNAc and sialic acid residues of the small intestinal goblet cell mucins prior to infection.     

Survival to patency of low level infections with Trichuris muris in mice concurrently infected with Nematospiroides dubius

Large, single-pulse laboratory infections with Trichuris muris are rejected by mice before patency, but low-level infections of fewer than 20 worms survive for long periods. Data are presented to show that the threshold at which an effective immune response takes place is significantly higher in mice concurrently infected with Nematospiroides dubius. In control CFLP mice trickle infections did not survive to maturity but in the slower responder C57 Bl10 mice egg production began on Day 35 and continued for a further seven weeks, with some mature worms present at autopsy. Concurrent infection with N. dubius resulted in trickle infections, T. muris surviving much better than in control mice, although these still showed some resistance to T. muris. It is suggested that the results support the hypothesis that T. muris elicits concomitant immunity in the host. Thus, the first worms to establish survive to patency at which time they can no longer be removed by the host, but once the immunological threshold has been exceeded incoming larvae are rejected by the host. Such a survival strategy would be very useful to T. muris in the wild.     

The effect of Trypanosoma brucei infection on local and systemic antibody responses of rats to Nippostrongylus brasiliensis

Adult Hooded Lister rats were given 5000 Nippostrongylus brasiliensis larvae on day 3 or 7 after infection with Trypanosoma brucei and a second dose of 5000 nematode larvae 28 days later. A similar number of rats was infected only with N. brasiliensis larvae. Comparison of antibody levels in serum and the respiratory and alimentary tracts showed that T. brucei infection influenced both systemic and local antibody responses of rats to N. brasiliensis antigens. After primary infection systemic antibody responses were mainly impaired, the level of suppression depending upon the interval between trypanosome and nematode infections. Anamnestic responses were diminished in both antibody systems. The number of worms reaching the small intestine of T. brucei parasitised rats after primary infection was twice- and after reinfection three-times higher than in rats subjected to nematode infections alone. However, adult nematode expulsion was not delayed. The results suggest that N. brasiliensis infection causes a multiantigenic stimulation of both systemic and local humoral responses of the host. Furthermore, they indicate that depression of systemic antibody responses may enhance worm establishment.     

Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation

Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.