R. A. Wilson

Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells

ABSTRACT Ym1 and Fizz1 are secreted proteins that have been identified in a variety of Th2-mediated inflammatory settings. We originally found Ym1 and Fizz1 as highly expressed macrophage genes in a Brugia malayi infection model. Here, we show that their expression is a generalized feature of nematode infection and that they are induced at the site of infection with both the tissue nematode Litomosoides sigmodontis and the gastrointestinal nematode Nippostrongylus brasiliensis. At the sites of infection with N. brasiliensis, we also observed induction of other chitinase and Fizz family members (ChaFFs): acidic mammalian chitinase (AMCase) and Fizz2. The high expression of both Ym1 and AMCase in the lungs of infected mice suggests that abundant chitinase production is an important feature of Th2 immune responses in the lung. In addition to expression of ChaFFs in the tissues, Ym1 and Fizz1 expression was observed in the lymph nodes. Expression both in vitro and in vivo was restricted to antigen-presenting cells, with the highest expression in B cells and macrophages. ChaFFs may therefore be important effector or wound-repair molecules at the site of nematode infection, with potential regulatory roles for Ym1 and Fizz1 in the draining lymph nodes.

Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma receptor gene exposed to the irradiated Schistosoma mansoni vaccine.

A high level of protection against Schistosoma mansoni is elicited in mice by the irradiated cercaria vaccine and interferon‐γ (IFN‐γ) is a key cytokine in the pulmonary effector response. The role of this cytokine has been investigated in mice with a targeted disruption of the IFN‐γ receptor gene (IFN‐γR−/− mice). The level of protection was impaired relative to that elicited in C57BL/6 and 129 wild‐type (WT) animals. These two groups developed compact effector foci, of largely mononuclear cell composition, around individual challenge parasites migrating through the lungs. In contrast the IFN‐γR−/− mice showed a massive and generalized leucocytic infiltration of the airways and interstitium in which eosinophils were a prominent feature. Cultures of airway leucocytes from C57BL/6 mice produced abundant IFN‐γ whilst those from IFN‐γR−/− mice produced interleukin‐4 (IL‐4), IL‐5 and IL‐10, indicating default to the Th2 pathway; the WT animals showed an intermediate response. The pattern of cytokine gene transcripts in whole lung tissue agreed remarkably well with the level of cytokine protein detected in leucocyte cultures, with the exception of substantial IL‐4 mRNA but negligible protein in C57BL/6 mice. The loose but intense infiltrate of leucocytes in the lungs of IFN‐γR−/− mice was clearly ineffective in eliminating challenge parasites, whereas the level of IFN‐γ protein and mRNA in the lungs of C57BL/6 and WT mice correlated with the size and compactness of effector foci. On the basis of these and earlier observations, we suggest that a primary role for IFN‐γ is to promote intercelluar adhesion between the leucocytes in an effector focus, promoting its ability to block parasite migration.

Protein synthesis and release by cultured schistosomula of Schistosoma mansoni

The lung schistosomulum of Schistosoma mansoni is the target of protective immunity in mice singly vaccinated with irradiated cercariae. Since the effector responses are T cell-mediated, their initiation requires the release of antigens from the intact parasite. We have used the technique of biosynthetic labelling with [35S]methionine, before and after transformation of the cercariae, to analyse the kinetics of protein synthesis and release by the schistosomulum. In addition, the proteins present in the soluble fraction of the parasite and those released during in vitro culture have been characterized. During a 7-day culture period schistosomula derived from labelled cercariae lost proteins most rapidly within the first 3 h after transformation. Two proteins of molecular weight 61 and 20 kDa were dominant and may correspond to areas of proteolytic activity. Analysis of the rate of protein synthesis of schistosomula labelled after transformation revealed four different phases, which may relate to the developmental processes occurring in vivo. During the first 24 h, synthesis was very low, increasing to a plateau and then rising to a peak at day 8; therefore the rate declined rapidly. Whilst some stage-specific synthesis of proteins was detected in the soluble fractions of the parasite bodies, the pattern of proteins released by cultured larvae was remarkably uniform. At least 15 proteins were detected by autoradiography with bands at 61, 45 and 20 kDa being particularly prominent. These proteins merit further study as potential mediators of the protective immune response.

The role of pulmonary cellular reactions in the resistance of vaccinated mice to Schistosoma mansoni

Summary A histopathological and ultrastructural study was made of schistosomula and associated inflammatory reactions in the lungs of normal mice, and mice previously vaccinated with irradiated cercariae. In normal mice at day 7 postinfection all schistosomula were located in blood vessels. From day 11 onwards an increasing proportion of schistosomula were intra‐alveolar (80% from day 20). No cellular reactions were evident around intravascular parasites in normal mice but at later sampling times large compact foci were associated with alveolar parasites. Initial reactions, probably in response to non‐specific tissue damage, were approximately 50% polymorphonuclear, and 50% mononuclear. Mononuclear cells predominated at later times. In spite of inflammation, no damage to the schistosomula was observed. There was no evidence for re‐entry of schistosomula into blood vessels, and it was assumed entry into alveoli occurred accidentally as parasites attempted to traverse pulmonary blood vessels. The pattern of localization of schistosomula in vaccinated mice was similar to that in normal mice, the proportion in alveoli increasing with time (64% from day 20). The most significant difference was that intravascular schistosomula attracted foci of host leucocytes which were always 85% or more mononuclear, containing both lymphocytes and macrophages. The infiltrating cells enlarged the intersititium, separating the vascular endothelium from the alveolar epithelium. Fibrous protein was also deposited in the interstitial region. In some instances the complete blood‐air barrier was destroyed by the infiltrates. Unusual paracrystalline inclusions were observed in alveolar macrophages and giant cells. The differences in cellular responses in vaccinated and normal mice suggest that challenge schistosomula stimulated an anamnestic immune response. The resulting inflammation, by impeding movement through the vasculature, terminated migration in the lungs, and accounted for the observed resistance to reinfection. The reactions in vaccinated mice have many of the features of a delayed hypersensitivity response implying that lung phase resistance in vaccinated mice may be T‐cell rather than antibody‐mediated.

The profile of IgG1 and IgG2a antibody responses in mice exposed to Schistosoma mansoni

The segregation of IgG2a and IgG1 immunoglobulin isotypes as markers for Th1 and Th2 lymphocytes respectively, was investigated in mice exposed to normal or optimally‐irradiated S. mansoni cercariae. Using a panel of ELISAs, soluble antigens from lung‐stage schistosomula, adult worms, or eggs, were probed with serum samples collected at biweekly intervals. Infected mice developed increased IgG1 responsiveness to all three antigens, especially between weeks five and seven, whereas IgG2a responses were lower, particularly to egg antigens. This confirms that Th2 responses are dominant after the onset of patency in infected mice. In comparison, vaccinated mice developed lower levels of IgG1, and higher levels of IgG2a to larval and worm antigens. Thus, they had balanced expression of IgG1 and IgG2a, despite having a dominant Th1 lymphocyte population. An elevated IgG1 response to egg antigens in vaccinated mice challenged with normal parasites, occurred two weeks later than in normal mice. Mice exposed to male‐only cercariae developed IgG1 and IgG2a antibodies to larval and worm antigens. However, they also had elevated IgG1 to egg antigens from week five, despite a total absence of eggs. Therefore, adult worm antigens may cross react with the egg and stimulate the switch to Th2 dominated responsiveness.

Protective immunity to Schistosoma mansoni induced in the olive baboon Papio anubis by the irradiated cercaria vaccine.

The radiation-attenuated schistosome vaccine induces a high level of protective immunity in rodents. In order to assess its potential relevance to man, we have tested its efficacy in the non-human primate Papio anubis. A vaccination regime consisting of 3 exposures of approximately 9000 cercariae irradiated with 30 or 60 krad. of gamma radiation induced > 50% protection to a challenge with normal larvae. A lower attenuating dose of 20 krad., optimal for vaccination of mice, was less effective. All vaccination regimes elicited a population of PBMC which proliferated in vitro in response to antigen. These responses peaked after the third exposure but were significantly lower after challenge. They revealed relatively little cross-reactivity with adult Schistosoma haematobium antigens and provided some evidence for stage-specific antigens. Circulating IgM reactive with adult S. mansoni antigen was detected after the second vaccination but levels remained low throughout. In contrast, IgG levels were boosted by successive vaccinations, although they showed a tendency to decline from 14 days after each exposure. There also appeared to be a lag of about 14 days after challenge before levels began to rise. Thus, both proliferation and antibody data suggest a lower responsiveness after challenge which may reflect either the reduced antigenic load or immunogenicity of normal, compared to vaccinating larvae. The data indicate that the attenuated schistosome vaccine is capable of inducing protection in a highly permissive primate host, with the implication that the mechanisms involved may also be relevant to man.

Schistosoma mansoni: dynamics of migration through the vascular system of the mouse.

Autoradiography of compressed mouse tissues has been used to estimate the numbers of 75Se-labelled schistosomula present in different mouse organs. Day 7 parasites extracted from the lungs of donor mice were delivered by injection to the lungs, systemic organs and liver of recipient mice as a discrete pulse. The numbers detected in various locations with time post-injection were then used to analyse the dynamics of intravascular migration. Approximately 98% of cercaria-associated label was lost during the first 14 days of parasite life, two-thirds of this in the first 7 h post-infection. Nevertheless, 99-113% of schistosomula could be detected 30 min post-injection into the locations chosen. The efficiency of the parasite delivery system was 95%. The time required for the number of foci in the lungs to decline to 50%, after injection of parasites via the femoral vein, was 55 h. Adjustment of this data to allow for parasites returning to the lungs after passage round the systemic vasculature gave a value of 30-35 h for the true mean time of lung transit. The distribution of parasites to systemic organs after their exit from the lungs was proportional to the fractional distribution of cardiac output. The probability (P) of a schistosomulum being distributed to splanchnic beds was estimated at 0.32 and its P of being trapped in the hepatic portal distributaries within the liver as 0.72-0.86. On this basis, the entire hepatic portal population of adult schistosomes would be recruited during 2-3 circuits of parasites around the pulmonary--systemic vasculature. The mean transit time of schistosomula through intestinal capillaries was 6.5 h whilst that through other systemic organs combined (muscles, kidneys, brain, etc) was 16 h, considerably more rapid than lung transit. The time taken for schistosomula to pass between organs, in arterial and venous blood, was shown to be less than 30 min in both cases (probably much less).

Antigen localization and the induction of resistance in mice vaccinated with irradiated cercariae of Schistosoma mansoni

The fate of 75Se-labelled parasites and their released pre-synthesized macromolecules has been followed in three murine infection models. Parasite numbers in specific tissues were determined by autoradiography, and released material was estimated by gamma-counting of tissues, with adjustment for the presence of parasite-associated radiolabel. Marked differences were found between the three models. The pattern of migration of normal schistosomula was similar to that previously reported. In addition we have described the transit of parasites through the lymph nodes draining the infection site. Significant quantities of released material were detected in the skin, draining lymph nodes, bloodstream and liver. The circulating material was of parasite origin, macromolecular, and hence potentially antigenic. In comparison to the normal infection, radiation-attenuated parasites (inducing a high level of resistance to challenge) persisted in the skin, draining lymph nodes and lungs, releasing a proportionally greater amount of material in the nodes. In mice exposed to attenuated parasites and treated with the compound RO11-3128 at 24 h (inducing a low level of resistance) there was an early death and rapid clearance of the parasites whilst still in the skin. This situation resulted in the highest levels of released material in the skin, bloodstream and liver, but negligible levels in the draining lymph nodes. We suggest that the persistence of radiation-attenuated parasites in the skin and draining lymph nodes, together with the prolonged release of antigen in the latter site, compared to the normal situation, are major factors in the induction of resistance.

Antibodies elicited by the secretions from schistosome cercariae and eggs are predominantly against glycan epitopes.

The glycoproteins secreted by Schistosoma mansoni cercariae and eggs play a key role in parasite transmission to and from the mammalian host. We used secreted preparations from these two life cycle stages to characterize the reactivity of sera from baboons exposed to normal and/or attenuated cercariae, in comparison with somatic antigen preparations and defined glycan epitopes. Periodate treatment of native antigens revealed that responses to the two secreted preparations were overwhelmingly directed against glycans rather than peptides. Considerable immunological cross‐reactivity between glycans in the two preparations was inferred from a comparison of sera from infected‐only and vaccinated‐only animals, predominantly exposed to egg and cercarial secretions, respectively. In contrast, when somatic antigen preparations derived from adult worms or eggs were used to probe sera, a stronger antipeptide response was seen that accounted for up to 66% of maximum reactivity. Probing of sera with defined glycan structures confirmed the time course of responses and the presence of cross‐reactive epitopes. In spite of the intense antiglycan response elicited in mice by administration of live eggs, no protection against a cercarial challenge was observed. Our data further support the hypothesis that antiglycan responses are a smokescreen with negligible protective potential.

Apoptosis: a mechanism of immunoregulation during human schistosomiasis mansoni

People infected with schistosomes may present with a variety of clinical manifestations ranging from the relatively asymptomatic intestinal (INT) form to the hepatointestinal (HI) or hepatosplenic (HS) forms characterized by hepatomegaly and hepatosplenomegaly with severe portal hypertension, respectively. Flow cytometry analyses were used to evaluate the contribution of apoptosis in specific cell populations from schistosomiasis patients to the development of the different clinical forms of the disease. The results showed that cell death induced by combinations of specific antigen and cytokines corresponds with specific clinical presentations. It was shown that soluble egg antigen (SEA) increased the level of apoptosis only in T cells from INT patients. Stimulation with soluble lung worm antigen preparation (SLAP) did not induce significant differences in the levels of apoptosis in T cells from the patients with the different clinical forms of schistosomiasis. These results suggest for the first time that apoptosis plays an important role in the modulation of the anti‐SEA response in INT patients.