materials and methodsKeywords: Echinococcus granulosus, immune evasion, recombinant antigen, chemotaxis, IgG4, Th1/Th2 cytokines
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Copyright © 2005 British Society for Immunology Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape GenBank Accession number AY874524. Correspondence: Dr Alessandra Siracusano, Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. E-mail: siracusano/at/iss.it Accepted August 12, 2005.  This article has been cited by other articles in PMC. | |||||||||||||||||||
Abstract The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-γ (P = 0·001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection. Keywords: Echinococcus granulosus, immune evasion, recombinant antigen, chemotaxis, IgG4, Th1/Th2 cytokines | |||||||||||||||||||
Introduction Cystic echinococcosis (CE) causes a long-lasting infection that affects humans and animals, thus posing a serious public health problem worldwide [1,2]. The immune response to Echinococcus granulosus varies widely within human and animal hosts [3]. In humans, E. granulosus induces humoral and cellular responses. Most patients with CE produce various classes of serum antibodies. Although none of these antibodies are associated with protection they are valuable diagnostic indicators. Studies designed to assess the IgG-subclass response in human CE show that IgG4 antibodies correlate well with active disease, whereas IgG1 antibodies correlate with inactive disease [4]. IgG subclasses recognize different hydatid cyst fluid antigens: the IgG1 subclass preferentially recognizes antigen 5 whereas IgG4 recognizes antigen B [5–7]. This differential antigen recognition is important in making the clinical diagnosis of CE and also in studying parasite survival mechanisms. In particular, because IgG4, a subclass normally associated with prolonged, chronic infections, is neither cytophilic nor complement fixing it may help the parasite to evade the host immune response [8–10]. Conversely, by competing directly with IgE, it could protect the host against anaphylactic responses [11,12]. Studies on the cellular immune response to E. granulosus in humans have shown that a critical feature in controlling development of the disease is an appropriate Th1/Th2 cytokine ratio. Although the Th2 response benefits the parasite, whereas the Th1 response benefits the host, their mechanisms of action remain unclear despite intensive investigation [13–15]. In parallel to these insights into the cellular interactions in infection, understanding of parasite immunomodulation is emerging, at the molecular level. Molecular research has already identified numerous E. granulosus-derived immunoregulatory molecules [16,17]. All of these molecules have known or hypothesized roles in immune interference (innate and acquired immunity) causing chronic disease to develop and progress. These findings suggest that already during the early stages of disease, the parasite begins to create the anti-inflammatory environment that best favours its survival [13,18]. During active disease, some native and recombinant E. granulosus antigens can elicit a predominant Th2 response, typically IL-4 production and IgE and IgG4 responses [19–22]. Seeking to identify and characterize other molecules that behave as immunomodulatory antigens, in this study we screened an E. granulosus cDNA library with IgG4 from patients with active CE. We isolated a protein that we named EgTeg because it was located in the tegument of the protoscolex and on the germinal layer of cyst wall. To characterize the immunological properties of this protein we assessed its ability to interfere with the early inflammatory response and its role in acquired immunity. For this purpose we first evaluated the effects of EgTeg on random motility, chemotaxis, and the oxidative metabolism of polymorphonuclear cells (PMN) from uninfected controls. Second, by cytometric analysis we evaluated intracellular EgTeg-driven-Th1 (IFN-γ) and Th2 (IL-4) cytokines in peripheral lymphocytes from patients with CE. Finally, we assessed by immunoblotting (IB) total IgG and IgG subclass responses to EgTeg in patients with CE, patients with other parasitoses and healthy controls; and analysed the IB results in patients grouped according to the clinical stage of disease and presence of allergic manifestations. | |||||||||||||||||||
Patients, materials and methods Blood samples Blood samples were obtained from 69 patients with CE (53 with cysts in the liver, 2 with cysts in the lung, 1 with cysts in brain, 1 with cysts in muscle, 1 with cysts in the kidney, 1 with cysts in the spleen and 10 with cysts in multiple sites), from 10 subjects with other parasitoses (6 with schistosomiasis, 2 with cysticercosis, 2 with trichinellosis), 6 patients with cystic lesions, and from 21 healthy donors. Hydatid cysts were classified according to the WHO sonographic classification [23] as type CE1 (unilocular, simple cysts), type CE2 (multivescicular, multiseptate cysts), type CE3 (unilocular cysts that may contain daughter cysts with detachment of laminated membrane), type CE4 (heterogeneous or hyperechoic degenerative contents) and type CE5 (calcified cysts). Cyst types CE1 and CE2 reflect active disease (developing cysts, usually fertile); cyst type CE3 reflect the transitional stage of disease (cysts starting to degenerate); cyst types CE4 and CE5 reflect inactive disease (degenerating or totally or partially calcified cysts). All procedures were approved by the local Ethical Committee and all participants gave their informed consent to the study.Production of recombinant EgTeg The cDNA library was prepared as previously described [24]. The cDNA clones were screened with a pool of five sera from patients with clinically confirmed CE in the active stage (cyst types CE1-CE2) at the time of serum sampling and with strong reactivity in an E. granulosus specific IgG4 ELISA (OD280 > 2). The nucleotide sequence of the cloned cDNA insertion was sequenced with an automated sequencer (ABI prism 310 Collection (PE). The selected cDNA clone was subcloned into the Bam HI/Kpn I site of the QIA express vector, pQE31. The 6X fusion protein was expressed in Escherichia coli SG130009 cells, purified by affinity of NI-NTA resin for the 6Xhistidine tag and eluted under denaturing conditions (urea) according to the supplier's (Qiagen, GmbH, Hilden, Germany) instructions. To purify the protein after electrophoresis on 12·5% SDS-PAGE the part of gel below 21·5 marker was removed and protein was eluted from polyacrylamide strips with a Model 422 Electro-Eluter Module (Bio-Rad, Richmond, CA, USA) at 10 mA/tube for 3 h at 4 °C as recommended by the manufacturer. SDS was removed through a Sephadex G-10 column (Pierce, Rockford, IL, USA). Before the protein was used to immunize mice, it was dialysed in PBS for two days at 4 °C. After dialysis the protein was divided into aliquots and kept at −80 °C for subsequent use in serological tests. To remove endotoxins, before the use in cellular cultures, the protein was purified by affinity chromatography through a Detoxi-gel affinity pack column (Pierce). Protein concentration was determined by the Bio-Rad Bradford protein assay (Bio-Rad). The quantitative chromogenic Limulus amebocyte lysate test (QLC-1000 BioWhittaker, Inc, Walkersville, MD, USA) conducted according to the manufacturer's instructions detected no measurable endotoxins in the EgTeg preparation.Computer analysis To analyse sequence assembly we used the software developed by the Genetics Computer Group by searches of the GenBankTM data base. To identify sequences similar to EgTeg we used the BLAST network server at the National Center for Biotechnology Information (NCBI). To compare multiple sequences we used CLUSTAL for T-COFFEE (1·41) program. To analyse the transmembrane protein topology prediction we used the hidden Markov model (TMHMM). For evaluating cell migration through micropore filters we used the method of computer-assisted image analysis, already described elsewhere [25]. Using the MIF Bioinformatics program we identified two predicted antigenic peptides containing 20 and 19 amino acid sequences (Peptide 1 YEGVWQVVILTGSYSAFSAY; peptide 2 PERLFHFKFGR FVVLVWQS). The sequenced peptides were purchased from Primm (Primm s.r.l., Milan, Italy).Immunohistochemistry For immunolocalization, material derived from sheep hydatid fluid was used to reveal binding sites of a mouse polyclonal antiserum specific for the recombinant protein EgTeg on protoscoleces. Fluid was either deposited on electrostatically charged glass slides (Superfrost plus, Manzel-Glaser, Germany) and dried at room temperature or centrifuged at 500× g. The pellet obtained was fixed in 10% neutral buffered formalin and embedded in paraffin. Five-µm thick sample sections were loaded on the above cited slides and dew axed in histolene; after rehydration, sections were heated by microwaving in 10 mm citrate buffer, pH 6·0, for a total of 15 min, essentially as described by Munakata and Hendricks [26]. Both kinds of slides (containing whole protoscolices or sections) after three washes in phosphate-buffered saline (PBS), were covered with bovine serum albumin (1% in PBS) and incubated at room temperature overnight with mouse polyclonal antiserum specific for the recombinant protein EgTeg obtained with standard procedures, or with preimmune mouse serum, both at the dilution of 1 : 50. After washings, slides were incubated with a FITC-labelled anti-mouse IgG (Santa Cruz Biotechnol., Inc., CA, USA), at 1–100 dilution, at room temperature for 45 min, and were then washed and mounted with antifade (Vectashield, Vector, Burlinghame, CA, USA) to be observed later with a fluorescence microscope (Axioplan, Leica, Milan, Italy).Polymorphonuclear cell separation Peripheral blood mononuclear cells (PBMC) were separated from plasma by Lymphoprep (Nyegaard & Co., Oslo, Norway) density gradient centrifugation by the method of Boyum [27] and polymorphonuclear cells (PMN) were obtained as previously described [28]. The cell viability, checked by trypan blue exclusion, was always greater than 99%. The cell suspension consisted of 97–98% neutrophils and only 2–3% eosinophils. To avoid up-regulation of the nonspecific surface receptors, all the isolation steps were performed at 4 °C [29].Chemotactic assay PMN from each of three uninfected controls were divided into six aliquots and diluted in PBS Dulbecco solution to a final cell concentration of 1 × 106/ml: Aliquots were cultured with the following preparations: recombinant EgTeg protein at a concentration of 0·5, 1 or 1·5 µg/ml; peptide 1 (YEGVWQVVILTGSYSAFSAY) at a concentration of 1 µg/ml; peptide 2 (PERLFHFKFGRFVVLVWQS) at a concentration of 1 µg/ml; a non parasite-related recombinant protein (Nedd5, Accession No. Q15019) [30], expressed and purified in our laboratory following the same protocol described above, at 1·5 µg/ml concentration; and without antigen, and all were tested in triplicate. Chemotaxis and random migration were assayed as previously described [31].Superoxide anion production by PMN Superoxide dismutase-inhibitable ferricytochrome C reduction was studied using the method previously described [32]. Cells were preincubated for 5 min at 37 °C with the antigens described above in the chemotaxis section and with BSA in PBS, pH 7·2, at the same concentrations as recombinant protein (controls). The stimulating agents was f-formyl methionyl leucyl phenylalanine (f-MLP) 10−6 M.Proliferation assay Proliferation was assayed by the established procedure [33]. In all experiments, besides cultures with EgTeg (10 µg/ml), cultures with phytohaemagglutinin (2 µg/ml) and cultures without antigen were set up as positive and negative controls. Net counts per minute (cpm) of triplicate cultures were determined and the proliferative response was expressed as stimulation indices (ratio between the mean cpm in stimulated and unstimulated cultures). The mean value of stimulation indices in healthy blood donors, plus 2 SD., was taken as the threshold level for a positive reaction (cut off value = 2).Detection of intracellular IFN-γ, and IL-4 in peripheral T lymphocytes PBMC from patients and controls were cultured in 96-well round bottom (3 × 106 cells/ml) plates in the presence of EgTeg (10 µg/ml) or medium alone. After 5 days recombinant human rIL-2 was added (20 U/ml) (Roche Diagnostics, Mannheim, Germany). After 10 days PBMC were transferred to 96-well round bottom plates in the presence of autologous monocytes previously pulsed with EgTeg (10 µg/ml) or medium alone. To inhibit protein transport in the cytoplasm, cells were incubated with brefeldine A (Sigma Chemical Co. St Louis, MO, USA) for 4 h. To determine IFN-γ and IL-4 in the cytoplasm of peripheral T lymphocytes, activated and inactivated samples were divided into aliquots (3 × 105 cells/tube) and stained with 10 µl of peridinin chlorophyll protein (PerCP)-conjugated CD3 specific mAb (BD Biosciences, San Jose, CA, USA) for 20 min at room temperature, and treated with 2 ml of fluorescence-activated cell sorted (FACS) lysing solution (BD Biosciences). After incubation for 10 min, samples were centrifuged and incubated with FACS permeabilizing solution for 10 min at room temperature in the dark. The cells in sample tubes were washed and incubated with 20 µl of fluorescein isothiocyanate (FITC)-conjugated IFN-γ or phycoerythrin (PE)-conjugated IL-4 specific MoAb (Becton Dickinson Co., CA, USA) for 30 min at room temperature in the dark. FITC-conjugated mouse IgG2a and PE-conjugated mouse IgG1 were used as controls. After washing, cells were diluted in 1% paraformaldehyde and analysed by flow cytometry using the FastImmune Cytokine System (BD Biosciences). The typical forward and side scatter gate for lymphocytes together with a CD3+ gate (logical gate) were set to exclude contaminating cells from the analysis. Data were obtained on a three-colour Becton Dickinson FACScan flow cytometer and the percentages of IFN-γ- and IL-4- producing cells were determined with CellQuest software (BD Biosciences).Immunoblotting Immunoblotting (IB), after 12·5% SDS-PAGE in reducing conditions, was performed as previously described [34]. In brief, EgTeg was used as antigen at the concentration of 3 µg/lane and was revealed by human sera diluted 1 : 50 for IgG and IgG subclass detection and 1 : 10 for IgE, detection. A goat anti-human IgG horseradish peroxidase conjugate serum (Bio-Rad, Richmond, CA, USA) was used as second serum to determine total IgG, and a goat anti-human IgE peroxidase labelled serum (Cappel, Cochranville, PA, USA) to determine IgE. Monoclonal mouse anti-human IgG1, -IgG2, -IgG3, or -IgG4 (BD Biosciences) were used to determine IgG subclasses and a goat anti-mouse IgG peroxidase-labelled serum (Bio-Rad) was used as third antibody. 3,3′-didiaminobenzidine dihydrochloride (Sigma Chemical Co) was used as substrate. For the immunolocalization, protoscoleces and hydatid fluid were used as antigen as described by Margutti et al. [24].Immunoblotting inhibition To inhibit specific IgG4, the human serum pool used in the screening was diluted 1 : 50 in PBS-Tween 20 and incubated overnight at RT with 2 mg/ml of the two peptides (Peptide 1 YEGVWQVVILTGSYSAFSAY; peptide 2 PERLFHFKFGR FVVLVWQS).according to the method reported by Huang et al. [35]. Unabsorbed sera and sera absorbed with EgTeg and with a non parasite-related recombinant protein (Nedd5, Accession No. Q15019) were included as controls. Strips were incubated overnight at RT with the mixtures and then developed as described in the IB section. The results were analysed by laser densitometry GS 700 (Bio-Rad).Statistical analysis Fisher's exact test and chi-square analysis were used to evaluate differences between percentages and Student′t-test to evaluate differences between arithmetic means. Differences with a confidence interval of 95% or higher were considered statistically significant (P ≤ 0·05).Nucleotide sequence accession number The sequence described in this work was deposited in the GenBank data base under accession number AY874524. | |||||||||||||||||||
Results Molecular characterization of the E. granulosus tegumental protein Immunological screening of an E. granulosus protoscolex cDNA library, using a pool serum obtained from patients with active CE disease and high IgG4 titres, identified a strongly reactive cDNA clone, designated EgTeg. DNA sequence analysis showed that EgTeg was not a full-length cDNA, because neither an initiator (ATG) codon nor a Kozak sequence was found at the beginning of the 5′ terminus. The cDNA sequence contained 476 bp, a termination codon at position 315 and a polyadenylation signal starting at position 451. The amino acid sequence predicted from the open reading frame is 105 residues long with a deduced molecular mass of 12 kD, and a predicted isoelectric point of 6·7. The molecular size and the purity of the predicted partial expressed protein was confirmed by 12% SDS-PAGE and immunoblotting (Fig. 1). We named the partial predicted portion of the protein EgTeg because BlastP analysis showed that its amino acid sequence scored highest against a family of Schistosoma proteins termed tegumental associated proteins. EgTeg had 34·5% identity with the 22·6 tegumental antigen of Schistosoma japonicum, 31% with the 22·6 tegumental antigen of S. mansoni, 28·6% with the 21·7 tegumental antigens of S. japonicum and S. mansoni and 26% with the 20·8 tegumental antigen of S. mansoni. A search for possible conserved putative domains in the database disclosed a dynein light chain type 1 domain. Transmembrane protein topology prediction analysis (using the hidden Markov model (TMHMM)) showed that the region within the 66–84 amino acids, between the hydrophilic intracellular N-terminal region and extracellular C-terminal region, exhibited high hydrophobicity (Fig. 2).
Localization A mouse serum raised against the recombinant protein localized EgTeg specifically in the tegument of protoscolex, either using whole structures (Fig. 3A) or sections and on the internal germinal membrane (Fig. 3C). No EgTeg-antibody staining was observed in sections incubated with preimmune mouse serum (Fig. 3B). Immunoblotting of proscolex extract and hydatid fluid, revealed with the mouse polyclonal antiserum specific to EgTeg, showed two strong bands at 27 and 12 kD in the protoscolex extract and one weak band at 12 kD in the hydatid fluid (Fig. 3D).
Effect of EgTeg on neutrophil random migration and chemotaxis EgTeg, peptide 1, peptide 2 and an irrelevant recombinant control protein left PMN random migration statistically unchanged, yielding a final plane similar to that in the control (data not shown). Conversely, EgTeg strongly inhibited PMN chemotaxis, so that final planes differed significantly in EgTeg-treated PMN at the various concentrations (0·5 µg/ml 118·3 ± 9·8; 1 µg/ml, 107·8 ± 14·8; 1·5 µg/ml 101·7 ± 9·8 and controls, 155 ± 16·4 µg/ml; P < 1 × 10−3) (Fig. 4). Like EgTeg, peptide 1 (YEGVWQVVILTGSYSAF SAY) significantly inhibited PMN chemotaxis (P < 1 × 10−3); whereas peptide 2 (PERLFHFKFGRFVVLVWQS) and the non parasite-related recombinant protein (Nedd5, Accession No. Q15019) did not.
Effect of EgTeg on PMN superoxide anion production None of the EgTeg concentrations (0·5, 1 and 1·5 µg/ml), no peptide 1, peptide 2 and a non parasite-related recombinant protein (Nedd5, Accession No. Q15019) tested affected PMN superoxide anion production in response to f-MLP (data not shown).Cellular immune response In 8 (42%) of 19 patients’ PBMC EgTeg induced a proliferative response. Stimulation indices ranged from 0·8 to 12·7 and the mean stimulation indices significantly differed (P = 0·04) in patients with inactive and active cysts (Fig. 5). Analysis of intracellular cytokines in the CD3+ T lymphocytes showed a significantly higher mean percentage of IL-4 producing cells than IFN-γ producing cells in all the 10 patients tested, independently from disease activity (IL-4 mean 51·7% ± 2·9; IFN-γ 0·9% ± 0·16; P = 0·001) (Fig. 6).
Humoral immune response to EgTeg IB analysis of the 69 sera from patients with CE showed that 50 (73%) sera contained total IgG specific to EgTeg and 46 (66%) contained IgG4 specific to EgTeg. Sera from 27 (39%) of 69 patients contained IgG1 specific to EgTeg and 35 (50%) contained IgGE specific to EgTeg. In 19 (27%) patients with CE, IB detected IgG2 and in 3 (4%) patients IgG3 specific to EgTeg (Table 1). All sera from patients with schistosomiasis and cysticercosis contained IgG and IgG4 specific to EgTeg, whereas sera from patients with trichinellosis did not. IB detected IgG and IgG1 specific to EgTeg in 3 (50%) of 6 patients with cystic lesions. Of the 21 sera from healthy subjects, 15 (71%) contained IgG specific to EgTeg and 6 (29%) of 15 contained IgG1. None of the sera from healthy subjects or patients with cystic lesions contained IgG4 or IgE specific to EgTeg.
The percentage of patients whose sera tested positive for total IgG, IgG1, IgG4 and IgE specific to EgTeg was similar in patients with active, transitional and inactive disease (Table 2). The percentage of patients whose sera tested IgG2-positive was higher, though not significantly higher, in the group with inactive disease than in those with active disease (63%versus 36%). Conversely, all the patients whose serum IgG3 reacted to EgTeg (27%) had active disease.
IB analysis disclosed a higher percentage of total IgG and IgG subclass and IgE EgTeg-immunoreactivity in patients with allergic manifestations than in patients without but the differences did not reach statistical significance (Table 2). IB inhibition analysis showed that peptide 2 (PERLFHFK FGRFVVLVWQS) strongly blocked IgG4 immunoreactivity to EgTeg (85% inhibition), whereas peptide 1 (YEGVWQV VILTGSYSAFSAY) induced only 27% inhibition (Fig. 7).
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Discussion EgTeg, the new molecule we isolated by screening an E. granulosus cDNA library, located in the protoscolex tegument and on the germinal layer of the cyst wall is a tegumental protein probably related to parasite survival. Our study, one of the few studies to examine the antigens associated with the tegument of E. granulosus [36–38], shows that EgTeg is an immunomodulatory molecule: it inhibits chemotaxis, induces preferentially IL-4-positive T lymphocytes and noncomplement fixing antibodies. It therefore elicits a preferential Th2 immune response associated with chronic infection. During human CE, at the metacestode level, the tegument is not directly exposed to the immunological attack of the host because it is present in the internal germinal layer that gives origin to the protoscoleces. Fu et al. [38] reported that the tegumental protein EgA31 is a potent antigen during dog and human infection. Similar to EgA31, EgTeg induces a strong humoral response in patients with CE. The presence of EgTeg in the hydatid fluid, due to an unknown mechanism of secretion or to the release of the protein during the degeneration of the protoscoleces, could explain how the host immune response arises. The molecular analysis using BlastP showed that the EgTeg antigen has sequence homology with Schistosoma tegumental proteins and contains a dynein chain light type 1 domain. A study investigating how schistosomes maintain their protective tegument in the infected host has reported the molecular characterization of a 20·8 kD tegumental antigen with sequence similarity to cytoplasmic dynein light chains and tegumental associated antigens [39]. The sequence conservation in this restricted region could therefore indicate that the two proteins have a common function, EgTeg probably serving to maintain the protective tegument of E. granulosus within the infected host. The precise components involved in Echinococcus tegument maintenance call for further biochemical and immunological studies of EgTeg and dynein light chains. Our cellular studies showing that the new E. granulosus tegumental protein inhibited the immunological response of normal human neutrophils provide evidence that EgTeg influences the host inflammatory response and thus intervenes in parasite evasion mechanisms. The migration of neutrophils from the blood to inflammatory sites in tissues is a hallmark of the innate immune response. It is also a key event in the physiological defense against invading microorganisms and tissue damage. A search for the evading survival mechanisms used by E. granulosus identified as a chemotaxis inhibitory protein that blocks neutrophils, antigen B [40,41]. When an antigen invades the host it triggers neutrophil activation thus directly inducing cell chemotaxis, activating integrins, generating superoxide anions and releasing granule content. Our superoxide anion test results, showing that EgTeg, like antigen B, left cellular metabolism intact, suggest that EgTeg-induced inhibition of chemotaxis does not arise simply from the antigen's toxic effect. Neither does it arise from a cytoskeletal impairment because EgTeg-stimulated PMN fully retained their ability to migrate spontaneously. In migrating cells, the signal that regulates movement is initiated by cell-surface receptors that bind specific chemoattractants. The chemokine receptors vary widely in size and chemical properties, ranging from small molecules and peptides to protein ligands [42,43]. The precise mechanism by which E. granulosus molecules and in particular the peptide YEGVWQVVILTGSYSAFSAY inhibit PMN chemotaxis merits further study. Another finding strongly suggesting that the new molecule EgTeg indirectly controls the host inflammatory pathways is that it evokes a cellular response dominated by the production of the Th2 cytokine IL-4. The anti-inflammatory responses induced by helminths enable parasite survival within the host by limiting inflammatory responses that might otherwise be destructive. This controlled immune response, central to chronic helminth infection, may arise from signals received from the pathogen, because the chronic presence of metabolically active helminths challenges the immune system with an array of antigenic molecules [44,45]. EgTeg is not the only parasite molecule involved in the immunomodulation of the host immune response. Antigen B, the most abundant antigen present in hydatid fluid, and an important immunoregulatory molecule, acts by directly blocking neutrophil chemotaxis and by stimulating an immunopathology-associated Th2 cytokine polarization [40,41]. EgTeg, like antigen B, inhibits PMN recruitment and in T lymphocytes, induces elevated intracellular expression of IL-4 as well as scarce intracellular expression of IFN-γ. The increased percentage of EgTeg-driven CD3+ IL-4 + T lymphocytes that the sensitive assays of intracellular expression of this cytokine disclosed in our patients’ PBMC shows that these patients have an increased activation of the Th2 pathway (active disease). The effect of EgTeg on Th-cell activation receives support from antigen-driven PBMC proliferation. EgTeg induced in PBMC a very weak Th1-dependent proliferation, about 25-fold lower than that induced by sheep hydatid fluid (stimulation indices: mean 4·2 versus 107, data not shown). The significantly higher mean stimulation index found in PBMC from patients with inactive disease than in PBMC from patients with active disease needs further investigation to ascertain the role of EgTeg in the development of CE. A fundamental step in understanding parasitic evasion strategies is to identify antigens that elicit strong, but not detrimental, antibody responses. EgTeg induced a strong, but not detrimental, IgG4 response in a high percentage (66%) of sera from patients with CE. Immunization of mice with EgTeg also generated a prevalent Th2 immune response supported by high IgG1 and low IgG2a production (data not shown). These findings agree with evidence that because each immunoglobulin isotype has a distinct biological function, clearance of parasitic infection often depends on specific antibody classes and subclasses [10]. The role of IgG4 is to favour parasite invasion thus encouraging chronic disease. In this study, we based our serodiagnostic method on IB analysis because this relatively simple technique yields high diagnostic sensitivity in detecting E. granulosus antibodies in patients with CE [4,46]. Despite the high sensitivity (73%), the low specificity (44%) obtained by IgG-IB analysis precludes the use of EgTeg in IB. By contrast, IB yielded high specificity (89%) and acceptable sensitivity (65%) in detecting IgG4, suggesting that this IgG subclass might be used to confirm ultrasound documented cystic lesions suggestive of E. granulosus. Our finding that the E. granulosus protein EgTeg reacted strongly with sera from patients with schistosomiasis accords with the sequence homology between EgTeg and tegumental proteins from Schistosoma. The peptide PERLFHFKFGRFV VLVWQS, which inhibited IgG4 binding to EgTeg, lacks the 11 amino acid sequence (VWQV+ILTGSY) that has 100% similarity and 91% identity with the tegument membrane-associated antigens of S. mansoni and S. japonicum (Fig. 2). Use of this specific peptide instead of the whole EgTeg sequence in IB analysis could therefore improve immunodiagnosis. IgG subclass analysis, using either absolute values or subclass antigen recognition profiles, may be of value in post-treatment surveillance of CE [4,47]. In a previous study, we showed that E. granulosus antigen B preferentially bound IgG4 [5]. IgG4 antibody levels also decreased rapidly in successfully cured patients and correlated with chronic infection. In this study, in a long-term follow-up we tested a small number of patients with EgTeg and invariably found high titres (measured as optical density) of IgG4 specific to EgTeg showing that this antigen persisted during the infection (data not shown). We found no association between disease activity or allergic manifestations and the presence of total IgG, IgG subclasses or IgE. A recent study by others, who evaluated a post-treatment serological follow-up by IgG subclass antibody detection, reported that the specific IgG2 antibody response to crude hydatid cyst fluid correlated with disease activity [48]. The large number of patients with inactive disease (63%) who were IgG2-seropositive to EgTeg in this study invites future research into the possible role of the IgG2 antibody response specific to EgTeg as an immunological marker in the development of CE. Our finding that sera from a large number of patients with CE who had allergic manifestations (62%) contained IgE specific to EgTeg implies a possible role of EgTeg as an allergen. It therefore prompts further research into the specific antigen-induced mechanisms underlying allergic reactions in the human host. In conclusion, the sum effect of the changes induced by EgTeg on the host reactivity suggests that it might contribute to create the anti-inflammatory environment most favourable to E. granulosus survival. Recent studies, exploiting the ability of helminths to subvert the immune response, have provided important information on how parasites generate anti-inflammatory responses and envisage using pathogen products for immunotherapy [49]. Considering that EgTeg, the new E. granulosus protein we identified, is associated with the host anti-inflammatory response to a hydatid cyst, its biological importance during CE deserves investigation. Gene deletion, antisense or siRNA studies should allow us to confirm the role of this new molecule in the host-parasite relationship. | |||||||||||||||||||
Acknowledgments This work was supported by grant Istituto Superiore di Sanità n. C3MR to A. Siracusano and by grant Università di Pisa, Fondi di Ateneo to F. Bruschi. We are in debt to Dr A.Tomaselli (USL 5 Messina) for the kind gift of the hydatid fluid. The technical expertise of S. Mazzoni for immunohistochemical preparations was greatly appreciated. | |||||||||||||||||||
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