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

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 × 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 × 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 × 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 × 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).

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).

• Open in new tab
• Download powerpoint

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.

• Open in new tab
• Download powerpoint

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 α(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 α(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.

• Open in new tab
• Download powerpoint

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.

• Open in new tab
• Download powerpoint

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.

• Open in new tab
• Download powerpoint

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).

• Open in new tab
• Download powerpoint

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.

• Open in new tab
• Download powerpoint

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.

• Open in new tab
• Download powerpoint

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).