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Infection and Immunity, September 2007, p. 4456-4462, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00633-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antibodies Elicited by the Bovine Lungworm, Dictyocaulus viviparus, Cross-React with Platelet-Activating Factor

Frans N. J. Kooyman,^* Erik de Vries, Harm W. Ploeger, and Jos P. M. van Putten

Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

Received 4 May 2007/ Returned for modification 3 June 2007/ Accepted 18 June 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Parasite N-glycans may play an important role in helminth infections. As antibodies from Dictyocaulus viviparus-infected calves strongly react with N-glycans, we investigated the characteristics of the major immunodominant glycoprotein (GP300) of this parasite. Probing of worm extracts with various lectins demonstrated unique binding of GP300 to wheat germ agglutinin. Analysis of lectin-purified GP300 revealed that the glycan was substituted with phosphorylcholine and reacted with the phosphorylcholine-specific antibody TEPC-15. Competitive enzyme-linked immunosorbent assay with GP300-coated plates and GP300-specific immunoglobulin G (IgG) in conjunction with free phosphorylcholine or TEPC-15 demonstrated that antibodies from infected calves recognized phosphorylcholine on GP300. Additional assays showed that these antibodies cross-reacted with the phosphorylcholine moiety present on platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a proinflammatory mediator of the host. Heavily infected calves contained high levels of serum GP300-specific IgG1 but low levels of IgA and IgG2 and showed a reduced influx of eosinophils in the lungs, all consistent with a neutralization of PAF activity. In conclusion, we demonstrated that D. viviparus infection elicits GP300-specific antibodies that cross-react with PAF and may neutralize PAF function, thus limiting the development of a protective response as well as parasite-induced host pathology.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Helminths are well adapted to their host and have evolved sophisticated strategies to avoid or modulate the host immune defense. Although the exact mechanisms via which helminths subvert the host responses are not completely understood, they typically induce an anti-inflammatory environment (22). At higher worm burdens, however, this may not be sufficient to prevent disease, as exemplified by the nematode Dictyocaulus viviparus, the etiologic agent of parasitic bronchitis in cattle. This parasite is ingested as larvae that, after penetration of the intestinal wall, migrate via the lymph nodes and the blood circulation to the lungs, where they mature into adult worms. Eggs produced by theses adults are coughed up, swallowed, and excreted in the feces as first-stage larvae. In the lungs, pathology develops due to the influx and activation of eosinophils and mast cells that cause restriction of the airways and a collapse of the alveoli, resulting in edema and emphysema (16).

Since during natural D. viviparus infection the host ultimately generates a protective immune response (18), prevention against this nematode infection through vaccination may be feasible. Serum transfer experiments in calves indicate that protection is accomplished via immunoglobulins (Igs) (17). Antibody responses against nematodes are often directed against glycoconjugates (5, 28). Immunologically important typical features of nematode glycans include core (1,3) fucosylation, instead of core (1,6) fucosylation as in mammals, and the decoration of complex type N-glycans with phosphorylcholine (PC). The presence of both core (1,3) fucose and PC cause the glycans to be highly immunogenic (1, 6, 9, 31), while core (1,3) fucose may also give rise to strong allergic reactions.

In search for protection-inducing antigens of D. viviparus, we recently identified several glycoproteins that were highly immunogenic. The antibody response was almost exclusively directed against the N-glycan moieties (19). The most immunodominant antigen in adult excretory-secretory (ES) products was a high-molecular-weight glycoprotein that appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as a double band of 200 to 300 kDa (GP300). The nature of the immunodominant glycan, however, was not determined. In the present study, we purified GP300 by use of lectin chromatography and identified the nature of the major immunodominant epitope. In addition, we demonstrate that GP300 shares immunoreactivity with the mammalian inflammatory mediator platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). It is hypothesized that this shared immunoreactivity results in down-regulation of the inflammatory responses against D. viviparus.

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Parasites. Adult lungworms were collected from lung washings at day 35 of infection as described previously (7). ES products from these adults were obtained after overnight incubation (19). To obtain adult soluble and water-insoluble extracts, 1 volume of adult worms was homogenized in 2 volumes of phosphate-buffered saline (PBS) containing complete protease inhibitor (Roche, Germany) in a Dounce homogenizer. After centrifugation (3,000 x g, 20 min, 4°C) the pellet was resuspended in another 2 volumes of PBS with protease inhibitor and again collected by centrifugation. Both supernatants were combined and stored as soluble extract at –80°C. The pellet was resuspended in 1 volume of PBS, and whole extract was precipitated with trichloroacetic acid-acetone (19). Dry pellet was extracted with 1 volume of urea buffer (8 M urea in 10 mM Tris, pH 7.4; 20°C, 30 min) while shaking. After centrifugation (3,000 x g, 20 min, 4°C), the supernatant was collected as the water-insoluble fraction and stored at –80°C.

Calves. Nineteen worm-free Holstein-Friesian female calves of 4 months of age were used. Sixteen calves were infected at day 0 with 30 (n = 8) or 500 (n = 8) L3 larvae. The remaining calves served as the challenge control group (n = 3) and remained worm free until infected at day 35, when all animals received a challenge infection of 1,000 L3 larvae. After slaughter at day 56, worm counts from the lungs were performed as described previously (7), and protection against challenge infection was calculated based on worm counts. Blood samples were taken throughout the experiment, and the sera were stored at –20°C until use. Day 35 sera from the eight animals infected with 500 L3 larvae were pooled and used as the positive sera throughout the study. Pooled control (negative) sera were obtained from the same animals at day 0. All experimental procedures were approved by the ethical committee on animal experimentation of Utrecht University.

Collection of BALF. Bronchoalveolar lavage fluid (BALF) was collected as described previously (26). After centrifugation (2,000 x g, 10 min, 4°C) and measurement of the protein concentration, BALF supernatants were stored at –20°C until use. The BALF pellet was resuspended in PBS, and the number of cells was counted (microcell counter CC-108; Sysmex). Giemsa staining was performed on cytospins to determine the number of eosinophils, alveolar macrophages, neutrophils, and lymphocytes; 200 cells were scored. Cell numbers were expressed per mg of recovered BALF protein.

SDS-PAGE and Western blotting. Electrophoresis and Western blotting were performed as described previously (19). Blots were incubated with pooled positive or negative bovine sera (1:1,000 dilution) and subsequently with anti-IgG1 monoclonal antibody (mAb) (mca 627, 1:1,000 dilution; Serotec) and alkaline phosphatase (AP)-conjugated goat anti-mouse Ig (1:2,000 dilution; DAKO). The mAb TEPC-15 (1:1,000 dilution; Sigma) served as a positive control for the detection of PC-containing glycoproteins (21) and was used in combination with goat anti-mouse Ig-AP (1:2,000 dilution; DAKO). AP activity was determined using 5-bromo-4-chloro-3-indolylphosphate (BCIP)-NBT (Sigma) as a substrate. For detection of lectin binding glycoproteins, blots were incubated with biotinylated wheat germ agglutinin (WGA), Lens culinaris agglutinin, and concanavalin A (ConA) (all from Pierce) at a concentration of 1 µg/ml in Tris-buffered saline (TBS) containing 0.1% gelatin and 0.05% Tween 20. ConA binding was performed in the presence of 1 mM CaCl₂ and 1 mM MnCl₂. Biotinylated lectins were detected with streptavidin-horseradish peroxidase conjugate (1 µg/ml; Pierce). Reactivity was visualized with DAB (3,3'-diaminobenzidine tetrahydrochloride) as the substrate.

WGA chromatography. Ten milliliters of the water-insoluble protein fraction stored in 8 M urea, 10 mM Tris was diluted with 30 ml of TBS to 2 M urea. After the removal of precipitates (3,000 x g, 5 min, 20°C), the supernatant was added to 2 ml of drained WGA-agarose (Sigma) and preequilibrated with TBS containing 2 M urea in a 50-ml tube. After 2 h of end-over-end rotation (20°C) and the removal of the supernatant (unbound fraction), the WGA-agarose with bound glycoproteins was transferred to a disposable polystyrene column (Pierce) and washed with 40 ml of TBS containing 2 M urea. Elution was performed with 10 ml of TBS containing 6 M urea and 1 M N-acetylglucosamine (GlcNAc), and 1-ml fractions were collected. Eluted fractions that contained protein (Bradford protein assay) were analyzed on SDS-PAGE gels, pooled, concentrated on a YM10 Centriprep (Amicon) to 1 µg/µl of protein, and stored at –20°C until use.

Affinity purification of anti-GP300. WGA-purified GP300 (100 µg) was coupled to 0.33 g of CNBr-activated Sepharose 4B according to the protocol of the manufacturer (Amersham). Five-milliliter portions of negative or positive pooled sera were diluted with equal volumes of PBS, and 100 µl of 0.5 M EDTA was added to inhibit calcium-dependent binding of C-reactive protein. The sera were incubated (4°C for 16 h or 20°C for 3 h) with the immobilized GP300 in a 50-ml tube by use of an end-over-end rotator. The supernatant (unbound fraction) was removed, and the GP300-Sepharose with bound antibodies was transferred to a disposable polystyrene column (Pierce). After being extensively washed with PBS to remove all unbound material, the bound fraction was eluted with 8 ml of 0.2 M glycin, pH 2.8. Fractions (1 ml) were directly neutralized with 100 µl of 1 M Tris (not pH adjusted). Protein-containing fractions (fractions 1 to 3) were pooled and concentrated on a Centricon YM30 (Amicon) to a final volume of 0.5 ml. Enzyme-linked immunosorbent assay (ELISA) using GP300-coated plates indicated that >90% of the anti-GP300 reactivity of the positive serum was recovered in the bound fraction. The reactivity of the negative pooled sera towards GP300 was too low to determine the percentage of recovery. Part of the purified anti-GP300 (0.5 mg/ml in PBS) was biotinylated with EZ-Link sulfo-N-hydroxysuccinimide-biotin (Pierce) according to the instructions of the manufacturer.

Deglycosylation of glycoproteins. Protein deglycosylation was carried out by diluting 200 µg of the water-insoluble protein fraction or 10 µg of GP300 in Tris-urea buffer in four volumes of 50 mM K₂HPO₄, 25 mM EDTA (pH 7.0). SDS and 2-mercaptoethanol were added to final concentrations of 0.2% and 0.5%, respectively. Samples were boiled for 5 min, and 10% Triton X-100 was applied to a final concentration of 2%. After cooling of the samples to room temperature, 1 µl (1 µl = 1 U) of recombinant peptide-N-glycosidase F (PNGase F; Roche) was added per 100 µg of protein. Deglycosylation was allowed overnight at 37°C and terminated by boiling for 3 min. Mock-treated samples (all components except PNGase F) were incubated simultaneously in all cases.

Detection of glycosylation. Mock- and PNGase F-treated extracts were run on SDS-PAGE gels. Staining of glycoproteins was done with a Pro-Q emerald 300 glycoprotein gel and blot stain kit (Molecular Probes) according to the manufacturer's instructions. After being stained for glycoproteins, the same gel was stained with silver to visualize total protein contents.

GP300 competitive ELISA. Affinity-purified and biotinylated anti-GP300 (0.05 µg Ig/ml) was mixed with different concentrations of potential inhibitory Igs (100 to 0.003 µg/ml), incubated for 1 h at 37°C, and transferred to ELISA plates coated with 0.05 µg/ml of GP300 as described previously (19). Binding of anti-GP300-biotin was detected with 1 µg/ml of streptavidin-horseradish peroxidase (Pierce) and 3,3',5,5'-tetramethylbenzidine (15 min). Color development was stopped with H₂SO₄, and optical density at 450 nm (OD₄₅₀) was measured in a Ceres UV 900C plate reader.

PAF ELISA. Antibody binding to PAF was carried out as described previously (27) but with modifications for the bovine system. Purified PAF (Sigma) was stored in ethanol (EtOH; 20 mg/ml) at –20°C. Maxisorp plates (Nunc) were coated (16 h, 4°C) with 25 µl/well of PAF in EtOH (200 µg/ml). After evaporation of the EtOH, the plates were washed once with TBS, blocked with dilution buffer (TBS containing 0.1% gelatin), and incubated with serial dilutions of anti-GP300. Ig isotype-specific detection was performed using anti-IgG1 (mca 627, 1:200 dilution; Serotec) or anti-IgA (mca 628, 1:200 dilution; Serotec) in dilution buffer and goat anti-mouse Ig-AP (D0486, 1:1,000 dilution; DAKO). The PC-specific antibody TEPC-15 (Sigma) (21) and the negative control antibody IE7 (20) served as controls. All incubation steps were performed for 1 h at 20°C. Antibody binding was determined with a pNPP kit (Pierce), and OD₄₀₅ was measured in a Ceres UV 900C plate reader.

GP300 and ES-specific ELISA. Antibodies in bovine sera (1:1,000 dilution) directed against GP300 were detected with the GP300-specific ELISA. ELISA plates were coated with WGA-purified GP300 that received PNGase F or mock treatment (0.2 µg/ml). Binding of Igs was determined using one of the following isotype-specific mAbs in combination with goat anti-mouse Ig-AP (D0486, 1:2,000 dilution; DAKO) and the pNPP kit: anti-IgG1 (mca 627; Serotec), anti-IgA (mca 628; Serotec), and anti-IgG2 (mca 626; Serotec) (all at 1:1,000 dilution). Reactivity was quantified using a Ceres UV 900C plate reader. The above-described procedure was also used to detect ES-specific antibodies, except that in these assays, plates were coated with 2 µg/ml ES and BALF was diluted to a concentration of 50 µg/ml of protein.

Statistical analysis. Correlations were calculated with Spearman's nonparametric correlation test from the SPSS software package (version 10.01.0).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Extraction of GP300 from adult worms. GP300 has previously been identified as an immunodominant antigen of the ES product of adult lungworm (19). In order to obtain an amount of material sufficient for purification and further analysis of GP300, adult worms were fractionated into water-soluble and -insoluble extracts and tested for the presence of GP300. SDS-PAGE and Western blotting followed by probing with IgG1 from sera from infected animals showed that the water-insoluble fraction contained a double, high-molecular-weight band, typical for GP300 (Fig. 1A).

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FIG. 1. Extraction of GP300. (A) Western blot demonstrating the reactivities of ES (lane 1), water-insoluble extract (lane 2), and water-soluble extract (lane 3) of adult worms (7.5 µg protein/lane) with IgG1 from D. viviparus-infected calves. Sera from noninfected animals did not react (not shown). (B) Total protein profile (DB71) and WGA reactivity of the water-insoluble extract.

Lectin binding properties of GP300. Purification of GP300 may be achieved using lectin affinity chromatography. To investigate this, the reactivity of the glycoprotein with different types of lectins was determined using Western blotting. Blots containing the insoluble proteins from adult worms and probed with biotinylated lectins Lens culinaris agglutinin and ConA yielded several positive bands, but neither lectin reacted exclusively with GP300 (data not shown). In contrast, biotinylated WGA specifically recognized the GP300 protein doublet (Fig. 1B), indicating that WGA affinity chromatography may enable one-step purification of the antigen.

Purification of GP300 using WGA affinity chromatography. To purify GP300, adult insoluble extract was applied to WGA-agarose. After extensive washing to remove unbound materials, bound glycoproteins were eluted with TBS containing 6 M of urea and 1 M of GlcNAc. Analysis of all fractions by SDS-PAGE and silver staining (Fig. 2A) demonstrated that, as expected, the eluted fractions contained only the double band characteristic of GP300. Western blotting with sera from infected animals (Fig. 2B) confirmed that the eluted fractions contained the immunodominant glycoprotein, while virtually no GP300 was detected in the unbound fraction. Based on the amount of recovered protein, it was estimated that 100 mg of water-insoluble protein applied to the WGA affinity column yielded 90 µg of purified GP300.

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FIG. 2. Purification of GP300. GP300 was purified from water-insoluble extract by use of WGA affinity chromatography and analyzed by SDS-PAGE and silver staining (A) and Western blotting with IgG1 from sera of infected animals (B). Materials loaded onto the gel: starting material (lanes 1), WGA-unbound fraction (lanes 2), and WGA-bound proteins (lanes 3).

To further confirm the identity of the purified material, the purified glycoprotein was submitted to treatment with PNGase F. This enzyme removes the N-linked glycan moieties of the proteins that lack core (1,3) fucosylation (29). Emerald 300 glycan staining showed that the enzyme treatment completely removed the carbohydrate moiety from the protein, demonstrating that the glycan was N linked without core (1,3) fucosylation (Fig. 3A). Silver staining showed that deglycosylation reduced the size of the purified protein from 390 and 310 kDa to 320 and 240 kDa, respectively (Fig. 3B), indicating that the protein is heavily glycosylated.

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FIG. 3. Posttranslational modifications on GP300. For glycan detection, purified GP300 was PNGase F (+) or mock (–) treated, run on SDS-PAGE gels (0.5 µg protein/lane), and stained with emerald glycan stain (A) and then with silver (B). For detection of PC, WGA-purified GP300 (0.5 µg protein/lane) (C) and water-insoluble (insol.) extract (7.5 µg protein/lane) (D) were separated by SDS-PAGE, blotted, and probed with the anti-PC mAb TEPC-15.

The N-glycans of GP300 contain PC. Carbohydrate analysis on GP300 by use of mass spectrometry did not yield a resolution sufficient to determine the structure of the glycan moiety (data not shown), possibly due to the presence of highly charged posttranslational modifications. One charged component previously identified on glycans of nematodes is PC. To investigate the possible presence of PC on GP300, blots with GP300 were probed with the PC-specific mAb TEPC-15. The antibody clearly recognized GP300 and deglycosylation resulted in the loss of reactivity, indicating that the PC was attached to the glycan moiety (Fig. 3C). Probing of blots containing the complete adult insoluble fraction with TEPC-15 showed exclusive reactivity with GP300 (Fig. 3D), suggesting that the substitution with PC is unique to GP300.

Immunogenicity of the PC attached to GP300. To investigate whether antibodies elicited by lungworm infection also recognized the PC moiety on GP300, a competitive ELISA was developed. For this purpose, GP300-specific antibodies were affinity purified from sera from infected calves and biotinylated. These antibodies specifically recognized GP300 (Fig. 4, insert). In the competitive ELISA with plates coated with purified GP300, the binding of biotinylated GP300-specific antibodies to the antigen was measured in the presence of various concentrations of the PC-specific antibody TEPC-15. As shown in Fig. 4, TEPC-15 caused >90% inhibition of binding of GP300-specific antibodies, while no inhibition of binding was observed for the control mAb IE7 (20). Inhibition was also obtained with the pooled sera from which the GP300-specific antibodies were derived but not with pooled sera obtained before infection from the same animals (Fig. 4). Furthermore, binding of both TEPC-15 and the biotinylated GP300-specific antibodies was inhibited in the presence of 1 mM of free PC but not with 1 to 10 mM of phosphorylethanolamine or phospho-L-serine (not shown). Together, the data indicate PC as the major immunodominant epitope on GP300 in infected calves.

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FIG. 4. Competition for GP300 binding between anti-GP300 and TEPC-15. Binding of biotinylated GP300-specific antibodies to GP300-coated ELISA plates was measured in the presence of the anti-PC mAb (TEPC-15) or sera from infected calves (pos). Sera from noninfected animals (neg) and a nonrelevant mAb (NR) served as controls. The inhibition of anti-GP300 binding to the antigen was expressed as the percentage of binding in the absence of inhibitors. Data are means of two independent assays with each measurement performed in duplicate. The insert shows the reactivity of biotinylated GP300-specific antibodies with PNGase F-treated (+) or mock-treated (–) GP300, as determined by Western blotting.

Cross-reactivity of GP300-specific antibodies with PAF. PC-substituted structures from many pathogens are known to exert immunomodulatory effects (10), but mammals themselves also produce PC-containing molecules such as the inflammatory mediator PAF. The finding that lungworm infection elicits PC-specific antibodies led us to investigate whether these antibodies cross-react with PAF and thus perhaps neutralize PAF function. Direct binding of GP300-specific antibodies to PAF was investigated by ELISA with PAF-coated plates. This showed that affinity-purified anti-GP300 derived from sera from infected calves cross-reacted with PAF in a dose-dependent fashion (Fig. 5). The sera contained anti-PAF antibodies of both the IgG1 and IgA isotypes. Importantly, sera from the same calves obtained prior to infection did not contain anti-PAF antibodies (Fig. 5). These results clearly demonstrate that GP300 and PAF share PC epitopes and that lungworm infection elicits cross-reactive antibodies.

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FIG. 5. Cross-reactivity of anti-GP300 with PAF. PAF-coated ELISA plates were incubated with serial dilutions of affinity-purified anti-GP300 derived from noninfected (day 0) and infected (day 35) calves. The PC-specific TEPC-15 antibody served as a positive control. Both IgA and IgG1 isotype reactivities were determined. Data represent the absorbances (mean OD405 values for duplicate wells) from one experiment representative of three.

Correlation of anti-GP300 with parasite-specific responses. Among many other biological effects, PAF is known to stimulate IgG2 and IgA production and to attract eosinophils. Thus, it is plausible that the generation of GP300-specific antibodies that cross-react with PAF may result in reduced IgG2 and IgA (but not IgG1) responses and a reduced influx of eosinophils in the infected lung. We analyzed the anti-GP300 levels in sera of calves that received either no dose, a low dose, or a high dose of L3 larvae (parasitological data are shown in Table 1) . For all three groups of animals, infection resulted in the generation of GP300-specific antibodies of all tested isotypes (IgA, IgG1, and IgG2) (Fig. 6). These antibodies were directed mainly against the glycan moiety, as the reactivity against the protein backbone of GP300 was generally low after primary infection. Even after subtraction of the reactivity against the protein backbone, which (as expected) increased upon challenge of the animals (19), IgG1 was the most reactive isotype against the glycan moiety, and its level increased during the entire duration of infection in all groups. Anti-GP300 IgG1 levels were highest for the group that initially received the highest dose, while this group showed the lowest GP300-specific IgA and IgG2 levels at the end of the experiment (Fig. 6). Similarly, ES-specific IgA, but not IgG1, levels in BALF were lower in the high-dose group at the end of the experiment (ES-specific IgG2 levels in BALF were below the detection limit) (Fig. 7). Comparison of the eosinophil influxes in BALF samples of both groups revealed in addition that the influx of eosinophils, but not of macrophages, in BALF was lower in the high-dose than in the low-dose group of animals (Fig. 7). When both groups were combined, we found a significant negative correlation of GP300-specific IgG1 levels at day of challenge (day 35) with ES-specific IgA levels (P < 0.01) and the number of eosinophils (P < 0.05) after challenge (day 54). Such correlations were found neither for alveolar macrophage numbers nor for ES-specific IgG1 levels. All these observations are consistent with the hypothesis that anti-GP300 IgG antibodies neutralize PAF activity, resulting in reduced IgG2 and IgA titers and a reduced influx of eosinophils into the lung. A mechanism that down-regulates IgG2 but not IgG1 is of particular interest, as the IgG2/IgG1 ratio is correlated with protection against infection (Fig. 8).

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TABLE 1. Parasitological data from infection experiment

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FIG. 6. Isotype-specific recognition of GP300 by sera from lungworm-infected calves. ELISA plates coated with PNGase F-treated (dotted part of the bar) or mock-treated (total bar) purified GP300 were incubated with sera from individual calves collected at days 14, 35, and 54 postinfection and probed for binding of IgG1, IgG2, and IgA. All measurements were performed in duplicate. Results represent means + standard errors of the means (SEM) of the OD405 values for each group.

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FIG. 7. Analysis of BALF of infected animals. (Left) The numbers of eosinophils (top) and alveolar macrophages (bottom) present in BALF at various times during D. viviparus infection were determined. Cell numbers are expressed per mg BALF protein (means + SEM per group). (Right) ES-specific IgA (top) and IgG1 (bottom) levels in BALF (50 µg/ml of protein) collected at the indicated days postinfection were determined by ELISA. Values are the means + SEM per group. neg, negative.

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FIG. 8. Correlation between protection against infection and the GP300-specific IgG2/IgG1 ratio at day 54. The ratio of the GP300-specific IgG2 and IgG1 levels was calculated from the data presented in Fig. 6 (mock-treated GP300). The correlation between the ratio and protection (r2 = 0.762) was statistically significant (P < 0.05) for the group receiving the high primary infection dose. There was no correlation between the IgG2/IgG1 ratio and protection for the group that received the low primary dose (not shown).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we purified and characterized the major immunodominant glycoprotein (GP300) of the nematode D. viviparus. GP300 binds to the lectin WGA, is substituted with PC, and lacks core (1,3) fucosylation. Interestingly, the PC group elicits anti-GP300 antibodies in infected calves that cross-react with the PC moiety of the proinflammatory mediator PAF. Neutralization of PAF by anti-GP300 antibodies is proposed as a possible mechanism for the observed down-regulation of IgG2 and IgA levels and the reduced influx of eosinophils during lungworm infection.

Western blotting indicated that GP300 was the only WGA-reactive molecule of D. viviparus. This allowed the successful purification of GP300 by lectin affinity chromatography. Purification required, besides 1 M of GlcNAc, the presence of 6 M of urea in the WGA elution buffer, possibly due to the high affinity of GP300 for WGA or the formation of larger protein complexes (not shown). The urea requirement during purification may explain why previous attempts to purify glycoproteins of D. viviparus with WGA affinity chromatography have not been successful (3). The binding of GP300 to the lectin indicates the involvement of GlcNAc and/or NeuNAc residues. As NeuNAc residues have thus far never been identified on N-linked glycans of nematodes, we expect that the WGA reactivity of GP300 is mediated via GlcNAc residues.

GP300 is also unique in that it was the only glycoprotein of D. viviparus that carried PC. This constituent was identified through its reactivity with the PC-specific mAb TEPC-15. Haslam et al. (12) characterized by mass spectrometry N-glycans released from detergent extracts of whole adult D. viviparus. Many complex type structures were found, but PC-containing structures were not reported. For other nematodes, PC-linked N-glycans have been demonstrated and found to be all of the complex type with or without core (1,6) fucosylation and carrying between two and six GlcNAc residues. None of these structures were core (1,3) fucosylated. For distantly related nematodes like the filaria Acanthocheilonema viteae (13), Trichinella spiralis (23), and the free-living nematode Caenorhabditis elegans (4), PC has been reported to be linked to an N-acetylhexosamine, most likely GlcNAc. Because of the reactivity of GP300 with WGA, it seems plausible that in D. viviparus PC is also attached to a GlcNAc residue. This suggests that WGA purification may be a good tool to purify PC-substituted N-glycans from other nematodes as well.

Our results clearly indicate that D. viviparus infection in calves elicits PC-specific antibodies, and, interestingly, that these antibodies cross-react with the proinflammatory mediator PAF. PAF is a pleiotropic mediator influencing a broad range of cells. In the lungs, it induces vascular permeability, bronchoconstriction, and airway hyperreactivity; patients with asthma, edema, and sepsis show increased levels of PAF (30). In human peripheral blood mononuclear cells, PAF stimulates IgG2 but not IgG1 production, and the effect is neutralized by the PC-specific antibody TEPC-15 (15). In the present study, TEPC-15 served as a positive control in all our experiments and exhibited binding characteristics towards GP300 and PAF similar to those of the bovine GP300-specific antiserum. This suggests that anti-GP300 may also be able to neutralize PAF activity. Indeed, in experimental infections the groups of animals with the highest levels of the putative neutralizing antibody (GP300-specific IgG1) at the day of challenge had the lowest levels of IgG2 (and IgA) after challenge. Furthermore, we found a negative correlation between GP300-specific IgG1 levels at the day of challenge and local parameters such as the influx of eosinophils and ES-specific IgA in BALF after challenge. This was not the case for the influx of alveolar macrophages or ES-specific IgG1 levels in BALF. These observations are consistent with the notion that anti-GP300 may neutralize PAF activity, as both IgA production (14, 25) and eosinophil influx (32) are PAF dependent.

What might be the biological function of the parasite-induced anti-PC antibodies? Our findings indicate that the IgG2/IgG1 ratio (which may reflect the Th1/Th2 ratio) correlates with protection against D. viviparus infection (Fig. 8). Furthermore, lungworm disease manifests with clinical signs typically associated with PAF, such as edema, bronchoconstriction, and influx of eosinophils. Thus, it can be imagined that parasite-induced neutralization of PAF limits the development of protection (by limiting the IgG2 response) as well as the development of harmful inflammatory responses (2). The overall effect might be favorable to both the host and the parasite. Such a mutually beneficial effect of inhibition of the PAF response resembles observations with PAF receptor-deficient mice showing enhanced worm survival and decreased inflammation upon infection with the nematode Strongyloides venezuelensis (24). Finally, it should be noted that in addition to the development of PAF-reactive antibodies, PC-containing structures such as GP300 may also exert direct immunomodulating effects, as observed for ES-62, a filarial glycoprotein containing PC substituted to N-glycans (11). ES-62 interacts with B and T cells, dendritic cells, and macrophages, skewing the response to a Th2/anti-inflammatory phenotype. Recently, it was demonstrated that most but not all of these effects could be attributed to the PC moiety (8). Neutralization of PAF activity might be another previously unrecognized strategy of parasites to modulate the host response.

 
* Corresponding author. Mailing address: Department of Infectious Diseases & Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31 30 2531114. Fax: 31 30 2532333. E-mail: f.kooyman{at}vet.uu.nl

Published ahead of print on 2 July 2007.

Editor: W. A. Petri, Jr.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Bardor, M., C. Faveeuw, A. C. Fitchette, D. Gilbert, L. Galas, F. Trottein, L. Faye, and P. Lerouge. 2003. Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 13:427-434.[Abstract/Free Full Text]
2
2. Bartemes, K. R., S. McKinney, G. J. Gleich, and H. Kita. 1999. Endogenous platelet-activating factor is critically involved in effector functions of eosinophils stimulated with IL-5 or IgG. J. Immunol. 162:2982-2989.[Abstract/Free Full Text]
3
3. Britton, C., G. J. Canto, G. M. Urquhart, and M. W. Kennedy. 1993. Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination. Parasite Immunol. 15:163-174.[Medline]
4
4. Cipollo, J. F., A. M. Awad, C. E. Costello, and C. B. Hirschberg. 2005. N-glycans of Caenorhabditis elegans are specific to developmental stages. J. Biol. Chem. 280:26063-26072.[Abstract/Free Full Text]
5
5. Dell, A., S. M. Haslam, H. R. Morris, and K. H. Khoo. 1999. Immunogenic glycoconjugates implicated in parasitic nematode diseases. Biochim. Biophys. Acta 1455:353-362.[Medline]
6
6. de Vos, T., and T. A. Dick. 1993. The mucosal and systemic response to phosphorylcholine in mice infected with Trichinella spiralis. Exp. Parasitol. 76:401-411.[CrossRef][Medline]
7
7. Eysker, M., J. H. Boersema, and W. M. Hendrikx. 1990. Recovery of different stages of Dictyocaulus viviparus from cattle lungs by a combination of a perfusion and a Baermann technique. Res. Vet. Sci. 49:373-374.[Medline]
8
8. Goodridge, H. S., S. McGuiness, K. M. Houston, C. A. Egan, L. Al-Riyami, M. J. Alcocer, M. M. Harnett, and W. Harnett. 2007. Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells. Parasite Immunol. 29:127-137.[CrossRef][Medline]
9
9. Harnett, W., M. R. Deehan, K. M. Houston, and M. M. Harnett. 1999. Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein. Parasite Immunol. 21:601-608.[CrossRef][Medline]
10
10. Harnett, W., and M. M. Harnett. 1999. Phosphorylcholine: friend or foe of the immune system? Immunol. Today 20:125-129.[CrossRef][Medline]
11
11. Harnett, W., I. B. McInnes, and M. M. Harnett. 2004. ES-62, a filarial nematode-derived immunomodulator with anti-inflammatory potential. Immunol. Lett. 94:27-33.[CrossRef][Medline]
12
12. Haslam, S. M., G. C. Coles, H. R. Morris, and A. Dell. 2000. Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode. Glycobiology 10:223-229.[Abstract/Free Full Text]
13
13. Haslam, S. M., K. H. Khoo, K. M. Houston, W. Harnett, H. R. Morris, and A. Dell. 1997. Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the excretory-secretory 62 kDa glycoprotein of Acanthocheilonema viteae. Mol. Biochem. Parasitol. 85:53-66.[CrossRef][Medline]
14
14. Huang, Y. H., L. Schafer-Elinder, H. Owman, J. C. Lorentzen, J. Ronnelid, and J. Frostegard. 1996. Induction of IL-4 by platelet-activating factor. Clin. Exp. Immunol. 106:143-148.[CrossRef][Medline]
15
15. Ishihara, Y., J. B. Zhang, S. M. Quinn, H. A. Schenkein, A. M. Best, S. E. Barbour, and J. G. Tew. 2000. Regulation of immunoglobulin G2 production by prostaglandin E₂ and platelet-activating factor. Infect. Immun. 68:1563-1568.[Abstract/Free Full Text]
16
16. Jarrett, W. F., F. W. Jennings, W. I. McIntyre, and W. Mulligan. 1957. The natural history of parasitic bronchitis with notes on prophylaxis and treatment. Vet. Rec. 69:1329-1339.
17
17. Jarrett, W. F., F. W. Jennings, W. I. McIntyre, W. Mulligan, and G. M. Urquhart. 1955. Immunological studies on Dictyocaulus viviparus infection. Passive immunisation. Vet. Rec. 67:291-296.
18
18. Jarrett, W. F., F. W. Jennings, W. I. McIntyre, W. Mulligan, and G. M. Urquhart. 1959. Immunological studies on Dictyocaulus viviparus infection. The immunity resulting from experimental infection. Immunology 2:252-261.[Medline]
19
19. Kooyman, F. N., H. W. Ploeger, J. Hoglund, and J. P. van Putten. 2007. Differential N-glycan- and protein-directed immune responses in Dictyocaulus viviparus-infected and vaccinated calves. Parasitology 134:269-279.[Medline]
20
20. Kooyman, F. N., A. P. Yatsuda, H. W. Ploeger, and M. Eysker. 2002. Serum immunoglobulin E response in calves infected with the lungworm Dictyocaulus viviparus and its correlation with protection. Parasite Immunol. 24:47-56.[CrossRef][Medline]
21
21. Leon, M. A., and N. M. Young. 1971. Specificity for phosphorylcholine of six murine myelomaproteins reactive with Pneumococcus C polysaccharide and ß-lipoprotein. Biochemistry 10:1424-1429.[CrossRef][Medline]
22
22. Maizels, R. M., A. Balic, N. Gomez-Escobar, M. Nair, M. D. Taylor, and J. E. Allen. 2004. Helminth parasites—masters of regulation. Immunol. Rev. 201:89-116.[CrossRef][Medline]
23
23. Morelle, W., S. M. Haslam, V. Olivier, J. A. Appleton, H. R. Morris, and A. Dell. 2000. Phosphorylcholine-containing N-glycans of Trichinella spiralis: identification of multiantennary lacdiNAc structures. Glycobiology 10:941-950.[Abstract/Free Full Text]
24
24. Negrao-Correa, D., D. G. Souza, V. Pinho, M. M. Barsante, A. L. Souza, and M. M. Teixeira. 2004. Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces fecundity in Strongyloides venezuelensis-infected mice. Infect. Immun. 72:1135-1142.[Abstract/Free Full Text]
25
25. Patke, C. L., C. G. Green, and W. T. Shearer. 1994. Differential effects of interleukin-2 and interleukin-4 on immunomodulatory role of platelet-activating factor in human B cells. Clin. Diagn. Lab. Immunol. 1:424-432.[Medline]
26
26. Soethout, E. C., V. P. Rutten, D. J. Houwers, H. S. de Groot, A. F. Antonis, T. A. Niewold, and K. E. Muller. 2003. Alpha4-integrin (CD49d) expression on bovine peripheral blood neutrophils is related to inflammation of the respiratory system. Vet. Immunol. Immunopathol. 93:21-29.[CrossRef][Medline]
27
27. Tektonidou, M. G., C. A. Petrovas, J. P. Ioannidis, P. G. Vlachoyiannopoulos, and H. M. Moutsopoulos. 2000. Clinical importance of antibodies against platelet activating factor in antiphospholipid syndrome manifestations. Eur. J. Clin. Investig. 30:646-652.[CrossRef][Medline]
28
28. Thomas, P. G., and D. A. Harn, Jr. 2004. Immune biasing by helminth glycans. Cell. Microbiol. 6:13-22.[CrossRef][Medline]
29
29. Tretter, V., F. Altman, and L. Marz. 1991. Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F cannot release glycans with fucose attached alpha 1-3 to the asparagine-linked N-acetylglucosamine residue. Eur. J. Biochem. 199:647-652.[Medline]
30
30. Uhlig, S., R. Goggel, and S. Engel. 2005. Mechanisms of platelet-activating factor (PAF)-mediated responses in the lung. Pharmacol. Rep. 57:206-221.[Medline]
31
31. van Die, I., V. Gomord, F. N. Kooyman, T. K. van den Berg, R. D. Cummings, and L. Vervelde. 1999. Core alpha1->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep. FEBS Lett. 463:189-193.[CrossRef][Medline]
32
32. Yamaguchi, S., M. Kagoshima, S. Kohge, and M. Terasawa. 1997. Suppressive effects of Y-24180, a long-acting antagonist for platelet-activating factor, on allergic pulmonary eosinophilia in mice. Jpn. J. Pharmacol. 75:129-134.[Medline]

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Infection and Immunity, September 2007, p. 4456-4462, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00633-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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