Modulation of dendritic cell differentiation and cytokine secretion by the hydatid cyst fluid of Echinococcus granulosus

Abstract

Chronic infection by Echinococcus granulosus results in establishment of fluid-filled cysts (hydatid cysts) in liver or lungs of infected hosts, which can escape destruction by the host immune system for long periods. This study explores the modulation by hydatid cyst fluid of the in vitro human monocyte to dendritic cell (DC) transition induced by granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Addition of the fluid to adherent peripheral blood monocytes cultured in GM-CSF/IL-4 stimulates release of prostaglandin E₂ (PGE₂) and IL-6. Exposure of differentiating DC to the fluid during the 7-day culture in GM-CSF/IL-4 impairs their subsequent ability to secrete IL-12, IL-6 or PGE₂ in response to lipopolysaccharide (LPS) stimulation. This inhibition is not dependent on the initial release of PGE₂. The presence of hydatid cyst fluid also modulates the phenotype of the cells generated during culture, resulting in increased CD14 expression and decreased expression of CD1a. Finally, hydatid fluid can stimulate predifferentiated DC to mature, as evidenced by release of IL-12 and IL-6, and by up-regulation of class II major histocompatibility complex and CD86. The possible role of dendritic cell modulation in regulating the host immune response to hydatid cysts is discussed.

Introduction

Echinococcus granulosus is a small flatworm that infects domestic and wild dogs. The egg produced by the adult form releases an oncosphere that will, in most cases, establish itself in the liver or lungs of the intermediate host (which can be a wide variety of mammals, including man). There the oncosphere will differentiate, in a few weeks, into a fluid-filled cyst (hydatid cyst) often establishing a long-term chronic infection.¹ The cyst may contain protoscoleces that can develop either in the adult tapeworm in dogs or in a secondary cyst in a suitable intermediate host.

Much effort has been invested in recent years to determine the host immune response in early and chronic hydatid cyst infection (reviewed in ^2–5) and whether the parasite can modulate it. However, the results are not yet clear cut or conclusive. Increased levels of interleukin (IL)-4, IL-5 and immunoglobulin E (IgE) in patients with liver hydatidosis have been reported.^6–9 Similar results have been shown in mice experimentally infected with live protoscoleces^10,11 suggesting that the host is driven to a T helper 2 (Th2)-like response. On the other hand, Rigano et al.¹² have shown that antigen-stimulated peripheral blood mononuclear cells (PBMC) from hydatid patients expressed higher levels of interferon-γ (IFN-γ) than controls, although differences were not statistically significant. Furthermore, in a prolonged experimental secondary hydatidosis in mice Haralabidis et al.¹³ observed that in the initial 4 months of infection serum levels of tumour necrosis factor-α (TNF-α), IFN-γ, IL-6, IL-10 and specific IgG1 and IgG3 were higher than controls. After that period IL-6 and TNF-α decreased and IL-10 increased in concentration. However, studies with a closely related parasite, Echinococcus multilocularis, have shown that IL-12 and TNF-α and/or lymphotoxin-α were crucial to inhibit larval growth indicating that a Th1 response is beneficial to the host.^14,15 To corroborate this suggestion it was shown that patients that responded to chemotherapy had increased levels of IL-12 p35, IFN-γ and TNF-α messenger ribonucleic acids (mRNAs) in contrast to non-responders.¹²

Parasitic helminths use many mechanisms in their quest to evade the host immune system (for a review see 16). A wide variety of immunomodulatory activities have been attributed to hydatid cyst fluid (HF) or subfractions of it, including cytotoxicity,^17,18 polyclonal activation of B and T cells,^19–23 down-regulation of CD4 and CD8 expression in T cells in vivo and thymocytes in vitro, IL-1, IL-2 and IL-6 mimicry.²⁴ A carbohydrate-rich fraction from protoscoleces has also been shown to induce mouse splenocytes to secrete IL-10.²⁵

The ability of some helminths either to produce themselves or to induce host production of prostanoids may similarly play a role in immunomodulating the host response.²⁶ Among these the prostanoid prostaglandin E₂ (PGE₂) could be of vital importance as it can favour a T helper (Th) cell type 2 response²⁷ which might not be efficient against some tissue parasites. Schistosoma mansoni skin stage schistosomula, for example, have been shown to induce a PGE₂-dependent IL-10 production by keratinocytes driving the host to a more Th2 type response.²⁸

Dendritic cells (DC) are the most important and efficient professional antigen-presenting cells (APC). DC are present in a wide variety of tissues where they are able to take up antigen and later present them to and activate T cells in secondary lymphoid organs. They can induce both immunity and tolerance and are the only APC able to activate naïve resting T cells.^29,30 Most important, depending on their own state of polarization/differentiation DCs have also the ability to bias Th cells into type 1 or 2.³¹ The ability to regulate DC differentiation may therefore provide an important mechanism whereby Echinococcus granulosus can modulate the host immune response. The aim of this study was to determine whether hydatid fluid stimulates PGE₂ production by monocytes, and whether this in turn influences the differentiation of monocyte-derived dendritic cells (MDDC) which is driven by culture in GM-CSF/IL-4.

Materials and methods

Hydatid fluid

Hydatid fluid (HF) was obtained, in aseptic conditions, from bovine cysts (fertile and not fertile). HF from several cysts was pooled, aliquoted in 30 ml samples and frozen at −20° for further use. For all experiments HF was centrifuged at 15 000 g, filtered through a sterile 0·22 µm membrane and tested for the presence of endotoxin (Endotoxin kit – Timed gel formation, Sigma, Saint Louis, MO). Tests indicated that endotoxin levels in the fluid were, if any, below 100 pg/ml. Protein concentration of the pooled sample was 150 µg/ml measured using the Bradford assay (Bio-Rad, Hemel Hempstead, UK). The protein content was analysed using 12·5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Fig. 1) and showed a characteristic complex protein banding pattern³² including a major band at 65 000 MW corresponding to bovine albumin, and bands at around 38 and 28 000 MW likely corresponding to subunits of the major Echinococcus antigen 5.³³

Figure 1.

Protein composition of HF. HF (15 µg total protein) was fractionated on a 12·5% polyacrylamide denaturing gel and stained using Coomassie Blue.

Generation of MDDC

MDDC were obtained by culture of peripheral blood mononuclear cells (PBMC) isolated by density gradient (Lymphoprep, Nycomed Pharma, Oslo, Norway) from 60 ml of heparinized blood from healthy volunteers. Cells were washed three times with Hanks' balanced salt solution (HBSS, Gibco, Paisley, UK), resuspended in RPMI-1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS), 50 µm 2-mercaptoethanol, 2 mm l-glutamine, and 100 IU/ml penicillin/streptomycin (complete medium) and seeded in a six-well tissue culture plate at a concentration of approximately 1·5–2·0 × 10⁷ cells/well (3 ml/well). Plates were incubated for 2–3 hr at 37° in an atmosphere of 5% CO₂ after which non-adherent cells were removed. Adherent cells were then cultured in 3 ml/well of fresh complete medium supplemented with 50 ng/ml human recombinant IL-4 (Prepotech, London, UK and from Schering-Plough, Madison, NJ) and 100 ng/ml human recombinant GM-CSF (a gift from Dr S. Devereux, Department of Haematology, UCL Medical School, London, UK and from Schering-Plough). MDDC were obtained after incubation of these cells for 7 days at 37° in an atmosphere of 5% CO₂. The effect of HF on the differentiation of monocytes into DC was tested adding 100 µl of fluid per ml of complete medium supplemented with IL-4 and GM-CSF. This medium was added after removal of PBMC non-adherent cells at day 0 and cultures incubated for 7 days in the same conditions as described above.

Fluorescence-activated cell sorting (FACScan) analysis of MDDC

Monoclonal antibodies (mAbs) for the following cell-surface molecules were used: CD3 (mouse supernatant mAb UCH T1, IgG1, gift of Professor P. C. L. Beverley), CD19 (mouse ascites mAb BU12, IgG1, gift of D. Hardie), human leucocyte antigen-DQ (HLA-DQ) (mouse ascites mAb Ia3, IgG2a, gift of Professor R. Winchester), HLA-DR (mouse supernatant mAb L243, gift of Professor P. C. L. Beverley), CD14 (mouse supernatant mAb HB246, IgG2b, gift of Professor P. C. L. Beverley), CD86 (mouse supernatant mAb BU63, IgG1, gift of D. Hardie) and CD1a (NA1/34, IgG2a, a gift from Professor A. McMichael, John Radcliffe Hospital, Oxford, UK). Loosely adherent cells from the 7-day culture, as described above, were harvested, thoroughly washed with HBSS and further cultured for 48 hr in fresh complete medium containing either lipopolysaccharide (LPS; 100 ng/ml) or HF (100 µl/ml), but lacking IL-4 or GM-CSF. Cells were then harvested, washed and, for each mAb used, 2 × 10⁵ cells resuspended in complete medium supplemented with 10% rabbit serum, 0·1% sodium azide. After 15 min incubation at 4° 50 µl of primary antibody were added and the reaction extended for 30 more minutes. Cells were washed twice with HBSS supplemented with 10% rabbit serum, 0·1% sodium azide, resuspended in 50 µl of fluoroscein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) and incubated for 30 min at 4°. Cells were then washed twice in HBSS with 0·1% sodium azide and resuspended in HBSS containing 0·1% sodium azide and 2·5% formaldehyde. Data was acquired in a Becton-Dickinson flow cytometer (Becton-Dickinson, Mountain View, CA) and analysed using WinMDI version 2.8 software (Joseph Trotter, Scripps Research Institute, La Jolla, CA).

Analysis of PGE₂, IL-6 and IL-12 production

Production of PGE₂, IL-6 and IL-12 was measured in culture supernatants taken at different intervals of time as follows. For the 7-day culture of PBMC, supernatants from days 1, 3 and 7 were analysed for the presence of PGE₂ and days 1 and 7 for IL-6. Concentration of the three molecules was also determined in the supernatants of further purified MDDC as follows. Loosely adherent cells generated at the end of the 7-day culture were harvested, washed three times with HBSS and resuspended in 2 ml of complete medium. Cells were then incubated with mAbs UCH T1 (anti-CD3) and BU 12 (anti-CD19) for 30 min at 4°, washed three times with HBSS and incubated with anti-mouse immunoglobulin-coated magnetic beads (Dynabeads, Dynal, Wirral, UK) for 45 min at 4°. Cells bound to Dynabeads were then attracted to and immobilized on the walls of the tube with the use of a magnet. Cells not bound to the beads were removed and transferred to another tube. With this procedure the MDDC population was further enriched (>80%). These cells were then resuspended in complete medium alone (no cytokines) or supplemented with either 100 ng/ml of LPS, or 100 µl/ml of HF and seeded in duplicates in a 96-well tissue culture plate at a concentration of 2 × 10⁵ cells/well. Plates were incubated at 37° in an atmosphere of 5% CO₂ and supernatants collected after 48 hr.

PGE₂ and IL-12 (p40) were detected by enzyme-linked immunosorbent assay (ELISA) using a kit from R & D Systems (Abingdon, UK) whereas for IL-6 an ELISA kit from BD Pharmingen (San Diego, CA) was used. ELISAs were performed according to manufacturers' instructions.

Statistical analysis

For comparisons with unequal variances (heteroscedasticity) logarithm of the cytokine concentrations was used. Statistical significance was determined by Student's t-test. Values of P less than or equal to 0·05 were considered significant.

Results

Cytokine and PGE₂ production by peripheral blood (PB) adherent cells cultured for 24 hr in GM-CSF/IL-4 and HF

Addition of HF (150 ng protein/µl) to PB adherent cells (>80% monocytes) cultured in GM-CSF/IL-4 (standard MDDC differentiation cultures) induced a rapid dose dependent release of PGE₂(Fig. 2a, left panel). The PGE₂ persisted throughout a subsequent 7-day culture period although the levels decreased slowly. No PGE₂ was detected in the HF itself (not shown). The PGE₂ inducing activity of the HF could not be attributed to contaminating endotoxin, because LPS in the HF was undetectable by the haemocyte agglutination test (<100 pg/ml); furthermore, the PGE₂-inducing activity of HF, in contrast to LPS, was heat labile and was destroyed by boiling for 5 min (not shown). PGE₂ production was completely inhibited by indometacin, confirming that HF acts by stimulating cyclo-oxygenase activity in the PB cells (Fig. 2a, right panel).

Figure 2.

HF induces the release of PGE₂ and IL-6 in PB adherent cells. (a) PB adherent cells were cultured in the presence of IL-4/G-CSF, and in the presence or absence of HF as shown. Supernatants were collected after 1, 3 or 7 days (left panel), or 1 day (right panel), and analysed for PGE₂ by ELISA. The PGE₂ concentration is significantly higher (P < 0·01) in the presence than absence of HF, at all concentrations and at all time points. In right panel, indometacin (indo, 10 µg/ml) with or without HF (100 µl/ml) was added to cultures as shown. The results show mean ± SEM, n = 3. Asterisk indicates P < 0·01 (compared to control). (b) PB adherent cells were cultured in the presence of IL-4/G-CSF, and in the presence or absence of HF (100 µl/ml) as shown. Supernatants were collected after 1 or 7 days (left panel), or 1 day (right panel), and analysed for IL-6 by ELISA. In right panel, indometacin (indo, 10 µg/ml) with or without HF (100 µl) was added to cultures as shown. The results show mean ± SEM, n = 3. Asterisk indicates P < 0·05 compared to controls.

The concentration of HF which released maximal PGE₂ as observed in panel (a) but minimal cytotoxicity (not shown) was chosen for further detailed study. In parallel to the production of PGE₂, 100 µl/ml HF also stimulated rapid release of IL-6 (Fig. 2b), although levels of IL-6 released varied widely between donors (Fig. 2b left and right panels). IL-6 levels were maximal at 24 hr after stimulation and decreased gradually throughout the culture period. The release of IL-6 was not inhibited by the presence of indometacin (Fig. 2b, right panel), suggesting IL-6 production was independent of PGE₂.

Cytokine and PGE₂ production by cells cultured for 7 days in GM-CSF/IL-4 and HF

Both PGE₂ and IL-6 have been suggested to drive DC differentiation towards a Th2-inducing phenotype. The ability of the adherent cells differentiated in the presence of IL-4/G-CSF and HF to produce IL-12, a hallmark of Th1-inducing activity, was measured (Fig. 3a). Monocytes cultured for 7 days in the presence of IL-4/G-CSF and HF had a greatly reduced ability to release IL-12 in response to LPS stimulation (Fig. 3a, left panel). This inhibitory activity of HF was not abrogated by indometacin (Fig. 3a, right panel), although this completely blocked PGE₂ production (Fig. 2a), suggesting that the IL-12 inhibition was independent of PGE₂.

Figure 3.

HF impairs the ability of DC to release IL-12, IL-6 and PGE₂. PB adherent cells were cultured in the presence or absence of HF (100 µl/ml) for 7 days as described in Methods. DCs were then harvested, and cultured for a further 48 hr in the presence or absence of LPS (100 ng/ml). Supernatants were collected and analysed for (a) IL-12 (b) IL-6 and (c) PGE₂ by ELISA. In the right hand panels indometacin (10 µg/ml) was added to the DC throughout the 7-day culture period. The results show mean ± SEM, n = 3. Asterisk (compared to no HF) and cross (not precultured compared to precultured) indicates P < 0·05.

Cells differentiated in the presence of HF also showed reduced ability to release IL-6 and PGE₂ in response to LPS stimulation (Figs 3b,c). The inhibition of IL-6 production was not affected by the presence of indometacin during the culture period (Fig. 3b, right panel), although, as expected, indometacin abolished almost all PGE₂ production (Fig. 3c, right panel). In contrast to the data presented in Fig. 2, the presence of indometacin during culture decreased IL-6 production in response to LPS (compare Fig. 3b, right and left panels), suggesting that under these conditions PGE₂ may itself drive some IL-6 production as proposed previously.³⁴

Phenotype of cells cultured in GM-CSF/IL-4 and HF

The total number of cells recovered from 7 day cultures containing HF was consistently less than in control cultures (by approximately 1/3, P < 0·01), suggesting that HF inhibited the survival and/or differentiation of DC in these cultures (although HF showed no acute toxicity on either DC or monocytes at these concentrations). The characteristics of the cells generated were therefore analysed in more detail by flow cytometry. Expression of HLA-DQ and DR was not affected by HF present for the seven day period (Fig. 4). HF pretreatment also did not affect the up-regulation of both HLA-DR and HLA-DQ induced by LPS. In contrast, HF present during culture increased expression of CD14 and decreased expression of CD1a. Cells cultured for 7 days in the presence of HF also failed to up-regulate CD86 expression in response to LPS stimulation.

Figure 4.

HF inhibits the differentiation of DC. PB adherent cells were cultured in IL-4/G-CSF and in the presence or absence of HF (100 µl/ml) for 7 days as described in Methods. The cells were harvested, purified by negative selection and cultured for a further 48 h in the presence or absence of LPS (100 ng/ml). The cells were then analysed for expression of a panel of cell surface molecules by flow cytometry as described. The left-hand panel shows the FL1 histograms from a representative experiment, with the cells cultured in the absence of HF shown with a solid line, and the cells cultured in the presence of HF with a dotted line. The right-hand panel shows the pooled results from three experiments, showing mean percentage positive cells for each marker, ±SEM, n = 3. Minus (–) and plus (+) signs refer to PBMC differentiated in the absence or presence of HF, respectively. Asterisk indicates P < 0,05 (not precultured compared to precultured).

HF induces activation of MDDC

The data presented in Figs 1–4 focuses on the effects of chronic exposure to HF during the monocyte to DC transition. HF was also found to have effects on predifferentiated DC. HF induced up-regulation of the proportion of cells expressing CD86 (Table 1), consistent with DC maturation and analogous to that seen in the presence of LPS (Fig. 4). Although all the immature DC already expressed HLA-DQ and HLA-DR (>90%, Fig. 4 and Table 1), results indicate that HF induced an increased level of their expression as judged by MFI (Table 1). HF also induced release of PGE₂, IL-6 and IL-12 in differentiated DC (Fig. 5).

Table 1.

HF induces phenotypic changes in DC indicative of maturation

Surface antigen	Medium	HF
DQ	90 ± 6 (407 ± 160)	93 ± 6 (552 ± 306)
DR	95 ± 2 (714 ± 430)	96 ± 4 (1063 ± 657)
CD14	24 ± 4 (43 ± 22)	32 ± 19 (68 ± 29)
CD86	9 ± 4 (26 ± 8)	29 ± 21 (52 ± 28)
CD1a	71 ± 7 (133 ± 23)	74 ± 7 (172 ± 46)

DC were cultured in GM-CSF and IL-4 for 7 days, and then cultured for a further 24 hr in the presence of medium alone or HF. The results show mean percentage positive cells ± SEM, n = 3, and mean fluorescence intensity in parentheses.

Figure 5.

HF stimulates DC to release IL-12, IL-6 and PGE_2. DC were cultured for 7 days in GM-CSF/IL-4 in the presence or absence of HF (100 µl/ml), harvested, purified and cultured for a further 48 hr in the presence or absence of HF (100 µl/ml). Supernatants were harvested and assayed for IL-12, IL-6 and PGE₂ as shown. The results show mean ± SEM, n = 3. The pattern of changes was the same in all three experiments, although Student's t-test values did not reach significance, because of the high variation in quantity of cytokine produced between individuals.

Discussion

In this study, we document that HF from Echinococcus granulosus modulates both the cytokine production and the phenotype of PB adherent cells cultured in GM-CSF/IL-4, a widely studied model of DC differentiation.

HF was initially observed to stimulate a strong production of PGE₂ by PB monocytes cultured in IL-4/GM-CSF. PB adherent cells spontaneously release PGE₂ when cultured on plastic, but this production is almost entirely inhibited when the cells are cultured in IL-4/GM-CSF (Fig. 2), conditions which favour differentiation of a DC phenotype. Niiro et al.³⁵ have also shown that IL-4 blocks LPS-induced PGE₂ production by monocytes through the inhibition of cyclooxygenase-2 expression. We also observed the same effect in our laboratory (data not shown). HF overcomes this inhibition, however, stimulating cyclo-oxygenase activity even in the presence of these cytokines.

Induction of PGE₂ production by host cells is a common feature during infection with a variety of parasites, presumably contributing to the anti-pathogen inflammatory response. HF also induces the release of another inflammatory mediator, IL-6, a cytokine that has been shown to be released in response to PGE₂ in murine macrophages.³⁴ Some contribution of autocrine PGE₂ release to LPS-induced IL-6 was indeed observed (Fig. 3b, compare left and right panels), but HF-induced IL-6 release was independent of PGE₂ production.

In addition to inducing the rapid release of these inflammatory mediators, HF had long-term effects on DC differentiation under these culture conditions. Specifically, the cells cultured in the presence of HF had a greatly impaired ability to secrete IL-12, a key cytokine required for the stimulation of Th1 responses, in response to LPS stimulation. Inhibition of IL-12 production could be the result of PGE₂ desensitization, as described by Rieser et al.³⁶ and Kalinski et al.³⁷ Both groups demonstrated that DC obtained after culturing PBMC in the presence of PGE₂ produced reduced amounts of IL-12 in response to LPS stimulation. However, addition of indometacin to the cultures, while abrogating PGE₂ production, did not prevent inhibition of IL-12 production, suggesting that inhibition of cytokine release occurred by a PGE₂ independent pathway. Indeed, the inhibitory effects of HF were not restricted to the production of this Th1 cytokine, since release of both IL-6 and PGE₂ itself by DC, in response to LPS, was impaired after culture in HF. This global reduction in cytokine production may nevertheless favour the emergence of a Th2-like phenotype, because this pathway seems to act as the default in the absence of IL-12. Kane et al.³⁸ have recently shown that soluble molecules from Schistosoma mansoni eggs can inhibit murine DCs to secrete LPS-induced IL-12. Furthermore, these soluble molecules increased LPS-induced IL-10 release by DCs which was partially responsible for the observed reduction in IL-12 production. Hence, the regulation of IL-10, a well-described inhibitory cytokine produced by DC, will be a key question for future studies with E. granulosus hydatid fluid.

A broader action of HF on DC differentiation was supported by flow cytometry analysis of the resulting population. Although the cells generated in the presence of HF had high levels of class II HLA molecules similar to those of the control cultures, the population contained a higher proportion of cells expressing the macrophage/monocyte marker CD14, and a lower proportion expressing the DC marker CD1a. The results suggest that HF may deviate the differentiation process, which occurs in the presence of IL-4/G-CSF away from DC, towards a more monocytic/macrophage-like cell. This is consistent with the visual observation of star-shaped macrophage-like cells in the cultures containing HF (data not shown), which were never observed in control cultures. Jenne et al.³⁹ also studied the effect of a mixture of proteins of Echinococcus multilocularis (Em-Ag), the causative agent of alveolar echinococcosis, on the differentiation and maturation of monocyte-derived dendritic cells, but observed no alteration in the expression profile of CD83, CD86, CD80, CD40, or major histocompatibility complex class I and class II. However they did not analyse the expression of CD1a or CD14.

In addition to its action in impairing their differentiation and function, HF was found to directly activate DC when added directly to predifferentiated DC. This activity resulted in release of some IL-12, as well as high levels of IL-6 and PGE₂, and up-regulation of CD86 on the DC surface.

The identity of the DC-modulating activity in HF is the next key question which needs to be addressed. Indeed, it is unclear whether the various effects documented in this study are all the result of a single molecular entity, or several different molecules. Chomarat et al.⁴⁰ and Mitani et al.⁴¹ showed that even in the presence of IL-4 and GM-CSF, IL-6 can switch monocyte differentiation towards a macrophage rather than DC lineage. The effect of HF on DC differentiation may therefore be indirect, and mediated via IL-6, though this possibility has not yet been directly addressed.

The results of this study suggest that HF contains factors that can affect DC function, but that the effects may vary for acute and chronic exposure. Soluble factors from the HF may escape into the lymphatic system, and activate DC within draining lymph nodes to produce IL-12, IL-6 and PGE₂, and stimulate a mixed Th1/Th2 response to the parasite antigens. Once the hydatid cyst is fixed in a suitable host tissue, however, components of its fluid are likely to be released chronically into the pericystic microenvironment and stimulate a host inflammatory response, producing at least PGE₂ and IL-6. This microenvironment will tend to inhibit any infiltrating monocytes from differentiating into DC. Those few monocytes that manage to differentiate into DCs, will in turn be unable to express IL-12, and thus favour differentiation of naive T cells to become Th2-like. In support of such a model, antigen B (AgB), a major hydatid fluid component, stimulated the production of IL-4 and IL-13, but not IL-12 by PBMC from patients with hydatid cysts.⁴² T-cell lines specific to either antigen B or sheep hydatid fluid from patients with inactive cysts had a Th1 profile whereas in patients with transient or active cysts they observed a mixed Th1/Th2 or Th0 population.^7,43 Furthermore, treatment with recombinant IL-12 protected C57BL/6 J mice against secondary alveolar echinococcosis¹⁴ while after parasite chemotherapy IL-12 mRNA was detected almost exclusively in the patients treated successfully. Modulation of antigen-presenting cell function may therefore be one strategy whereby Echinococcus granulosus modulates the host immune response to ensure its continued survival.

Abbreviations

APC

antigen-presenting cell

DC

dendritic cell

ELISA

enzyme-linked immunosorbent assay

FITC

fluoroscein isothiocyanate

FCS

fetal calf serum

GM-CSF

granulocyte–macrophage colony-stimulating factor

HBSS

Hanks' balanced salt solution

HLA

human leucocyte antigen

HF

hydatid cyst fluid

IFN

interferon

IL

interleukin

LPS

lipopolysaccharide

mRNA

messenger ribonucleic acid

mAb

monoclonal antibody

MDDC

monocyte-derived dendritic cells

p40

p 40 subunit of IL-12

PB

peripheral blood

PBMC

peripheral blood mononuclear cells

PG

prostaglandin

TNF

tumour necrosis factor