CD4 T-cell hyporesponsiveness induced by schistosome larvae is not dependent upon eosinophils but may involve connective tissue mast cells.

In areas endemic for schistosomiasis, people can often be in contact with contaminated water resulting in repeated exposures to infective Schistosoma mansoni cercariae. Using a murine model, repeated infections result in IL-10-dependent CD4(+) T-cell hyporesponsiveness in the skin-draining lymph nodes (sdLN), which could be caused by an abundance of eosinophils and connective tissue mast cells at the skin infection site. Here, we show that whilst the absence of eosinophils did not have a significant effect on cytokine production, MHC-II(+) cells were more numerous in the dermal cell exudate population. Nevertheless, the absence of dermal eosinophils did not lead to an increase in the responsiveness of CD4(+) T cells in the sdLN, revealing that eosinophils in repeatedly exposed skin did not impact on the development of CD4(+) T-cell hyporesponsiveness. On the other hand, the absence of connective tissue mast cells led to a reduction in dermal IL-10 and to an increase in the number of MHC-II(+) cells infiltrating the skin. There was also a small but significant alleviation of hyporesponsiveness in the sdLN, suggesting that mast cells may have a role in regulating immune responses after repeated exposures of the skin to S. mansoni cercariae.

'Seeing is Believing'; the use of novel imaging approaches towards creating a greater understanding of parasite: host interactions.

This editorial highlights some of the key points made in the six invited reviews in this special issue of Parasite Immunology on the use of contemporary imaging technologies to investigate the parasite: host interface. Three of the reviews deal with the protozoa Trypanosoma, Leishmania, and Plasmodium, whilst the remainder focus on helminth parasites particularly Schistosoma. The reviews cover aspects related to how the development of transgenic parasites has vastly advanced our understanding of how parasites interact with host cells, particularly as a cause of pathology. Imaging technologies have also been exploited in revealing parasite localisation within host tissues and identifying novel therapeutic targets. Combined the reviews show how 'state of the art' imaging technologies have resulted in a seismic advance in our understanding of parasite biology and how this has the potential to develop new, and improved, strategies to combat disease caused by parasite infections.

Schistosome infection is associated with enhanced whole-blood IL-10 secretion in response to cercarial excretory/secretory products.

Infection of the human host by schistosome parasites follows exposure of skin to free-swimming cercariae and is aided by the release of excretory/secretory (E/S) material, which is rich in proteases and glycoconjugates. This material provides the initial stimulus to cells of the innate immune system. The study presented here is the first to examine human innate/early immune responsiveness to cercarial E/S in subjects from an area co-endemic for Schistosoma mansoni and S. haematobium. We report that in infected participants, stimulation of whole-blood cultures with cercarial E/S material (termed 0-3 hRP) caused the early (within 24 h) release of greater quantities of regulatory IL-10, compared with uninfected controls. Elevated levels of IL-10 but not pro-inflammatory TNFα or IL-8 were most evident in participants co-infected with S. mansoni and S. haematobium and were accompanied by a higher 0-3 h RP-specific IL-10: TNFα ratio. We also report that glycosylated components within 0-3 h RP appear to be important factors in the stimulation of IL-8, TNFα and IL-10 production by whole-blood cells.

Interleukin-10 secretion from CD14+ peripheral blood mononuclear cells is downregulated in patients with acne vulgaris.

BACKGROUND: Acne is a common chronic inflammatory dermatosis of the pilosebaceous unit. It is characterized by seborrhoea, comedone formation and an inflammatory response consistent with defective cellular immunity to Propionibacterium acnes. OBJECTIVES: The objective of this study was to investigate the immune reactivity of patients with acne compared with healthy controls by examining the response of peripheral blood mononuclear cells (PBMCs) to stimulation with P. acnes. Particular focus was placed upon measuring the production of interleukin (IL)-10, which has an established immunoregulatory role. PATIENTS AND METHODS: Venous blood was collected from 47 patients with acne and 40 age- and sex-matched healthy controls with no prior history of acne. PBMCs were cultured and their cytokine response to P. acnes investigated. RESULTS: Proinflammatory IL-8 and tumour necrosis factor (TNF)-alpha secretion from PBMCs was higher in patients with acne when stimulated with P. acnes. In contrast, a statistically significant reduction in PBMC secretion of anti-inflammatory IL-10 in patients with acne was identified. The impaired production of IL-10 by PBMCs from patients with acne was confined to CD14+ cells presumed to be monocytes. The ability of CD14 cells from patients with acne to phagocytose P. acnes bacteria was also observed to be defective but the addition of exogenous IL-10 to PBMC cultures restored phagocytic activity. CONCLUSIONS: These data suggest that patients with acne have a proinflammatory cytokine milieu and crucially are unable to contain early inflammatory changes due to a specific defect in immunosurveillance, namely low monocyte IL-10 production. Our observations raise the possibility that acne therapeutics might profitably target IL-10 both as a regulator of proinflammatory cytokines and in augmenting the CD14+ cell phagocytic response.

Antibody responses induced by Trichobilharzia regenti antigens in murine and human hosts exhibiting cercarial dermatitis.

Cercariae of bird schistosomes (genus Trichobilharzia) are able to penetrate the skin of mammals (noncompatible hosts), including humans, and cause a Th2-associated inflammatory cutaneous reaction termed cercarial dermatitis. The present study measured the antibody reactivity and antigen specificity of sera obtained after experimental infection of mice and natural infection of humans. Sera from mice re-infected with T. regenti showed a bias towards the development of antigen-specific IgM and IgG1 antibodies and elevated levels of total serum IgE, indicative of a Th2 polarized immune response. We also demonstrate that cercariae are a source of antigens triggering IL-4 release from basophils collected from healthy human volunteers. Analysis of sera from patients with a history of cercarial dermatitis revealed elevated levels of cercarial-specific IgG, particularly for samples collected from adults (> 14 years old) compared with children (8-14 years old), although elevated levels of antigen-specific IgE were not detected. In terms of antigen recognition, IgG and IgE antibodies in the sera of both mice and humans preferentially bound an antigen of 34 kDa. The 34 kDa molecule was present in both homogenate of cercariae, as well as cercarial excretory/secretory products, and we speculate it may represent a major immunogen initiating the Th2-immune response associated with cercarial dermatitis.

Comparison of cysteine peptidase activities in Trichobilharzia regenti and Schistosoma mansoni cercariae.

Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

Modulation of the host's immune response by schistosome larvae.

Schistosomes appear to have evolved several strategies to down-regulate the host's immune response in order to promote their own survival. For the host, down-regulation is also beneficial as it can limit the extent of pathology. It is widely accepted that schistosomes modulate the immune response during the chronic phase of infection after egg deposition has started. However, there is increasing evidence that modulation of the immune response can occur much earlier at the time infective cercariae penetrate the host skin. In this review, we explore the various lines of evidence that excretory/secretory (ES) molecules from cercariae down-regulate the host's immune response. We highlight the immunological factors that are produced and may be involved in regulating the immune system (e.g. IL-10, and eicosanoids), as well as speculating on possible mechanisms of immune modulation (e.g. mast-cell activation, T-cell apoptosis, and/or the skewed activation of antigen-presenting cells [APCs]). Finally, we draw attention to several molecules of schistosome origin that have the potential to stimulate the regulatory response (e.g. glycans) and link these to potential host receptors (e.g. TLRs and C-type lectins).

Immunity induced by the radiation-attenuated schistosome vaccine.

As a paradigm for the development of a vaccine against human schistosomiasis, the radiation-attenuated (RA) vaccine has enabled the dissection of different immune responses as putative effector mechanisms. This review considers advances made in the past, and updates our knowledge with reference to recent studies that have provided new information relevant particularly to the early innate events after vaccination, and to the nature of the protective effector mechanism. Priming of a protective response by RA larvae is a highly co-ordinated series of events starting in the skin, draining lymph nodes and lungs, leading to the development of various effector responses, ranging from Th1-associated cell-mediated activity, to anti-parasitic antibodies, all of which contribute to the elimination of challenge larvae to varying extents. In this respect, the RA vaccine elicits a multifaceted immune response, from which we can derive valuable insights relevant to the future design of novel delivery systems and adjuvants for recombinant and subunit vaccines.

Signaling via interleukin-4 receptor alpha chain is required for successful vaccination against schistosomiasis in BALB/c mice.

Although protective immunity in C57BL/6 mice induced by a single dose of the radiation-attenuated schistosome vaccine is believed to be mediated by Th1-type immune responses, we here report that in BALB/c mice protection can also depend upon signaling via the interleukin-4 (IL-4) receptor which conventionally governs the development of Th2-type immune responses. We show that in BALB/c mice deficient for the IL-4 receptor alpha chain (IL-4Ralpha(-/-)), which are unresponsive to IL-4 and IL-13, vaccine-induced protection is abrogated compared with that in wild-type (WT) mice. In vaccinated IL-4Ralpha(-/-) mice, IL-12p40 production by cells from the skin exposure site was elevated, although gamma interferon (IFN-gamma) production in draining lymphoid tissues was similar in WT and IL-4Ralpha(-/-) mice. Nevertheless, the effector response in IL-4Ralpha(-/-) mice was Th1 biased with elevated IFN-gamma in the lungs and higher immunoglobulin G2a (IgG2a) and IgG2b titers but negligible quantities of Th2-associated IgG1 and IgE. Interestingly, levels of IL-4 were equivalent in WT and IL-4Ralpha(-/-) mice, indicating that Th2 responses were not dependent upon signaling by IL-4 or IL-13. No differences in the phenotype and composition of the pulmonary effector mechanism that might explain the failure to induce protection in IL-4Ralpha(-/-) mice were detected. However, passive transfer of partial protection to naive IL-4Ralpha(-/-) mice, using serum from vaccinated WT mice, indicates that Th2-associated antibodies such as IgG1 have a role in parasite elimination in BALB/c strain mice and that signaling via IL-4R can be an important factor in the generation of protection.

Characterization, cloning and immunogenicity of antigens released by transforming cercariae of Schistosoma mansoni.

A schistosome infection is initiated when the parasite penetrates the skin of a susceptible host. Relatively large quantities of protein are released by transforming cercariae compared to later larval stages. This represents the first parasite material to which the host's immune system is exposed, yet little is known about the proteins which are released during the first few hours post-transformation. We have shown that antiserum raised against such molecules was capable of imparting protection against a schistosome challenge infection upon passive transfer to naïve mice. By screening a cercarial cDNA library with this serum, 38 positive clones were identified. Sequence analysis showed these to represent 8 different molecules which included Schistosoma mansoni 21-7 kDa antigen, calcium-binding-protein and the vaccine candidate glutathione S-transferase (Sm28GST). In addition, 5 clones were isolated, 1 of which had significant homology to many cytochrome C proteins, another with leukocyte elastase inhibitors and 3 which represented novel molecules. Four clones were expressed in a prokaryotic high-level expression vector, sera produced against each purified recombinant protein and used subsequently to probe Western blots and parasite sections. The leukocyte elastase inhibitor homologue and 2 unknowns induced significant proliferation by lymph node cells recovered from mice vaccinated with irradiated cercariae. More strikingly, the 2 novel proteins stimulated very high levels of interferon gamma (IFNgamma) secretion both by lymph node cells and those recovered by broncho-alveolar lavage from the lungs of vaccinated mice. Such results will be discussed in the context of vaccine development.

IL-18 potentiates the adjuvant properties of IL-12 in the induction of a strong Th1 type immune response against a recombinant antigen.

Due to the synergistic effects of IL-12 and IL-18, and to their importance in establishing a Th1 type immune response, we investigated the potential of a combined administration of both cytokines as an adjuvant for recombinant antigens. As a model system, we used a schistosome T cell antigen recently identified in our group. By co-adsorption of this antigen on alum in the presence of IL-12 and IL-18, we demonstrate that IL-18 enhances the effects of IL-12 in inducing an antigen-specific Th1 type CD4(+) T cell response as well as high titres of IgG1, IgG2a, and IgG2b antibodies.

Interleukin-12 can directly induce T-helper 1 responses in interferon-gamma (IFN-gamma) receptor-deficient mice, but requires IFN-gamma signalling to downregulate T-helper 2 responses.

An in vivo model of pulmonary granuloma formation around embolized schistosome eggs was investigated as an environment in which to analyse a role for interleukin-12 (IL-12) in the differentiation of T-helper 1 (Th1) and Th2 subsets. Specifically, mice deficient for the interferon-gamma receptor (IFN-gammaR-/-) were used to determine the role for IL-12 in the absence of IFN-gamma-mediated signalling. We show that recombinant IL-12 administered to IFN-gammaR-/- mice caused the up-regulation of mRNA for IFN-gamma in lung tissue, and the secretion of abundant IFN-gamma by in vitro-cultured lymph node cells in response to egg antigens. This indicates that IL-12 can act independently of IFN-gamma to induce the development of Th1 cells. Administration of rIL-12 to wild-type mice markedly reduced the secretion of Th2-associated cytokines, IL-4 and IL-5. However, these cytokines were not dramatically reduced in IFN-gammaR-/- mice treated with IL-12. We conclude that inhibition of these cytokines by IL-12 is primarily dependent upon effective IFN-gamma signalling, although abrogation of T-cell derived IL-10 appeared to be dependent upon IL-12. We also show that increases in mRNA for the beta2 subunit of the IL-12 receptor and the p40 subunit of IL-12 after rIL-12 treatment were lower in IFN-gammaR-/- mice, compared to wild-type mice, indicating that their expression was primarily dependent upon IFN-gamma with only a minor role for IL-12.

Isolation of T-cell antigens by using a recombinant protein library and its application to the identification of novel vaccine candidates against schistosomiasis.

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterial EcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins, and (iii) a procedure of simultaneous purification of recombinant proteins from large numbers of isolated clones (representing the protein library) in a single step from pools consisting of up to 24 individual clones. The feasibility of the screening system was confirmed by constructing a protein library of the human parasite Schistosoma mansoni. The recombinant antigens of this library were used to stimulate CD4(+) T cells derived from the axillary lymph nodes of mice vaccinated with irradiated cercariae. In initial screening experiments, we detected parasite-specific proliferation and gamma interferon (IFN-gamma) secretion in response to several pools of cDNA clones. Further analysis of one particular pool revealed that only one of its constituents stimulated considerable IFN-gamma secretion by CD4(+) T cells and that the expressed antigen is identical to a small fragment of myosin heavy chain.

The radiation-attenuated schistosome vaccine induces high levels of protective immunity in the absence of B cells.

Radiation-attenuated cercariae of Schistosoma mansoni elicit consistently high levels of protective immunity in mice. The cell-mediated pulmonary effector mechanisms have been well characterized but the role of B cells and antibodies remains ill defined. We have compared the immune responses of B-cell-deficient (muMT) mice and their wild-type (WT) counterparts following exposure to the attenuated vaccine. Both groups mounted a T helper type 1 (Th1)-biased response in the skin-draining lymph nodes after vaccination. Interferon-gamma was the dominant cytokine secreted by airway leucocytes after challenge in both muMT and WT mice, but there was a somewhat greater Th2 component in the former animals. The cellular infiltrates observed in the airways, and the pulmonary effector foci, were of similar composition in the two groups although some large foci were present in the muMT mice. There was a marked dichotomy in the protection induced in muMT animals by a single vaccination, with two-thirds showing levels similar to their WT counterparts, demonstrating that cell-mediated mechanisms alone can provide adequate protection. The remaining muMT mice had a mean worm burden identical to that of their challenge controls. A possible explanation is that a proportion of the muMT animals have a genetic defect closely associated with the mu-heavy-chain locus on chromosome 12, which affects their ability to mount a protective cell-mediated response. Three vaccinations enhanced the immunity of WT animals, most likely by augmenting antibody-mediated mechanisms. In contrast, no enhancement was seen in muMT mice, suggesting that the cell-mediated response is not boosted by multiple exposures to attenuated larvae.

Immune responses to the radiation-attenuated schistosome vaccine: what can we learn from knock-out mice?

The goal of an effective schistosomiasis vaccine has proved elusive but the protective immunity induced in mice by radiation-attenuated cercaria larvae provides an appropriate model from which such a vaccine might be developed. Using gene-disrupted mice, we have analysed the process of immune priming by attenuated larvae of Schistosoma mansoni and the nature of the pulmonary effector response directed against a challenge infection. Vaccination stimulates expansion of IFNgamma-producing T-helper cells in the skin-draining lymph nodes. IL-12 is crucial in determining the Thl direction of this initial response but the cells of origin and the parasite components which stimulate its production are unknown. In the effector response, focal aggregates comprising mainly mononuclear cells accumulate around challenge larvae in the lungs, a process orchestrated by IFNgamma. This cytokine up-regulates nitric oxide synthase activity but we were unable to implicate nitric oxide as a cytotoxic agent causing challenge parasite elimination. An alternative action for IFNgamma may be to up-regulate adhesion molecule expression, increasing the cohesiveness of effector foci the better to block parasite migration, but the adhesive interactions so far examined do not appear relevant. In contrast, TNF induction is essential to protection, and we are currently testing the hypothesis that it determines the speed of the effector response following arrival of challenge larvae in the lungs.

Formation of multinucleated giant cells in the mouse lung is promoted in the absence of interleukin-12.

The formation of multinucleated giant cells (MGCs) in an in vivo model of pulmonary inflammation was investigated to determine whether these cells are the result of a dominant T helper (Th) 1 or Th2 cytokine environment. We report that knockout (KO) mice with a disrupted interleukin (IL)-12 p40 gene exposed to the helminth Schistosoma mansoni had abundant and very large MGCs (> 50 microm) in their lungs concurrent with extensive eosinophilia and a population of large macrophages. Many of the MGCs and macrophages appeared to have phagocytosed eosinophils as part of a clearance process. The KO mice also had a strongly polarized Th2 immune response as judged by elevated levels in the lungs of messenger RNA (mRNA) transcripts for IL-4, IL-5, IL-6, and IL-13, but decreased levels of mRNA for interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In addition, cells recovered by bronchoalveolar lavage from the airways of these mice secreted a Th2-biased profile of cytokines upon restimulation in vitro with parasite antigen. In contrast, wild-type C57BL/6 or KO mice treated with recombinant IL-12 had a polarized Th1 phenotype with elevated levels of IFN-gamma and TNF-alpha mRNA in the lungs, and an airway cell population that secreted abundant IFN-gamma. Very few MGCs were detected in these mice, and there was an absence of pulmonary eosinophilia. We conclude that the formation of MGCs in our model is promoted in the absence of IL-12 and is linked instead to the abundance of Th2 cytokines, notably IL-4 and IL-13.

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2-type characteristics.

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1-type lymphocytes. To investigate a possible role for IL-12 in the induction of protection, we have compared the immune response of IL-12 p40-deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN-gamma increased IL-4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL-4 and IL-5 but little IFN-gamma, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL-12 and Th1-mediated immune responses. This conclusion is reinforced by the administration of rIL-12 to KO mice immediately after vaccination which led to increased IFN-gamma and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL-12-independent pathway, possibly mediated by Th2 cells.

Interleukin-12 and protective immunity to schistosomes.

The attenuated vaccine against Schistosoma mansoni induces Th1-mediated protective immunity and we have sought to identify a role for IL-12 in this model. Elevated levels of IL-12 (p40 mRNA) were detected in the lymph nodes (LN) and the lungs of vaccinated mice, whilst treatment of vaccinated mice with anti-IL-12 antibodies decreased the ratio of IFN gamma:IL-4 secreted by in vitro-cultured LN cells. However, there was only marginal abrogation of the level of resistance in these mice. Soluble antigens from the lung-stage of the parasite (SLAP) appeared to be efficient stimulators of IFN gamma and IL-12 secretion. These antigens when used to immunise mice in conjunction with IL-12 as an adjuvant, elicited a polarised Th1 response with abundant IFN gamma secretion but no IL-4. This immunisation regime also induced significant protection against reinfection, whereas inoculation of mice with SLAP alone did not. The induction of a dominant Th1 response using SLAP + IL-12 probably operates via IFN gamma production by natural killer (NK) cells stimulated by IL-12, since in vivo ablation of NK cells using anti-NK1.1 antibody reduced CD4(+)-dependent IFN gamma production from cultured LN cells by over 97%. Nevertheless, in mice with a genetic disruption of the IFN gamma receptor, administration of SLAP + IL-12 induced levels of IFN gamma equal to those in wild-type mice, thus showing that in this model IL-12 can directly prime T cells independent of IFN gamma. Clearly, IL-12 has a critical role in protective immunity to schistosomes and it may aid the development of an effective vaccine against this disease.

Interleukin-12 and the host response to parasitic helminths; the paradoxical effect on protective immunity and immunopathology.

In general, helminth infections are associated with the development of dominant Th2-mediated immune responses which may be host protective but can also be the cause of immunopathology. Interleukin 12 (IL-12) is known to be a potent inhibitor of Th2 immune responses and as such it might be expected to have an important modulatory role in helminth-induced immune responses. In this review, we discuss the effect of IL-12 on susceptibility to infection, protective immunity and immunopathology, in the context of exposure to a range of helminths including intestinal nematodes, filariae and schistosomes. It is apparent that the effects of IL-12 are complex and can be beneficial as well as detrimental for the host. The precise role of IL-12 depends upon a number of factors including the type of helminth and the specific tissue involved in the inflammatory response.