Pigeon fanciers' lung: identification of disease-associated carbohydrate epitopes on pigeon intestinal mucin

Abstract

Pigeon intestinal mucin, a complex high molecular weight glycoprotein, is a key antigen in the development of pigeon fanciers' lung (PFL). We have studied the specificity of antibodies to mucin in patients with PFL and asymptomatic antibody-positive individuals. Extensive papain digestion, which removes the non-glycosylated regions of the mucin leaving the heavily glycosylated ‘bottle brush’ regions, resulted in a 600-fold decrease in IgG3 antibody titres with little effect on IgG1 and IgG2 titres. This suggests that IgG1 and IgG2 are directed against the region rich in O-linked sugar chains whilst the majority of the IgG3 is directed against epitopes which are proteinase-sensitive. Lectin mapping of the carbohydrates present on pigeon intestinal mucin demonstrated high levels of exposed n-acetyl neuraminic acid, n-acetyl galactosamine and n-acetyl glucosamine, with lower levels of fucose and some galactose. Sera from pigeon fanciers inhibited binding of lectins specific for n-acetyl neuraminic acid, n-acetyl galactosamine, internal n-acetyl glucosamine and fucose. Sera from people with PFL, compared with sera from asymptomatic antibody-positive fanciers, had significantly higher titres of antibody that inhibited binding of four lectins specific for n-acetyl galactosamine and one fucose-specific lectin, suggesting that these sugars may play a dominant role in disease-associated epitopes. The results suggest that different IgG subclasses recognize different epitopes on mucin and that the epitopes recognized by the major subclasses are present on the O-linked oligosaccharides. Further, the carbohydrate-specific anti-mucin antibodies produced by PFL patients may differ in their specificity from those found in asymptomatic individuals.

INTRODUCTION

Pigeon intestinal mucin is an important antigen in pigeon fanciers' lung (PFL) [1,2], a form of extrinsic allergic alveolitis (EAA). In these diseases hypersensitivity reactions occur after inhalation of various organic dusts, resulting in both local and systemic symptoms occurring some 4–6 h after exposure. The pathogenesis of PFL is unclear and multiple mechanisms may be involved, but the time course for development of symptoms, the presence of extremely high antibody titres together with evidence for complement activation within the lung and an early neutrophilic alveolitis are all consistent with the symptoms of acute disease being mediated by immune complexes which form at the alveolar epithelial surface [3]. The ability of these immune complexes to induce disease is likely to be influenced by both the antibody isotype response to the causative antigen(s) and the biochemical nature of this antigen(s).

It is unclear why some pigeon fanciers who develop high titres of antibodies against pigeon antigens remain asymptomatic, whilst other develop PFL. In a recent study of the antibody responses of pigeon fanciers to pigeon intestinal mucin it was shown that, whilst high titres of IgG antibodies were present in both symptomatic and some asymptomatic individuals, subjects with PFL had significantly higher titres of IgG1 subclass antibodies compared with asymptomatic antibody-positive individuals [4]. This suggests that differences in the IgG subclass response to pigeon intestinal mucin are important in the development of disease.

Mammalian intestinal mucins are complex glycoproteins with a mol. wt of approximately 10 million, comprising 70–80% carbohydrate [5,6]. The mucin protein core contains two distinct regions based on resistance to proteolytic digestion: the highly glycosylated ‘bottle brush’ regions, which are rich in serine, threonine and proline, and regions accessible to proteolysis where glycosylation is sparse or absent. Little is known of the structure or biochemical nature of avian intestinal mucins, but in both pigeon and quail these have the same physical characteristics as mammalian mucins with respect to their density and fragmentation pattern after digestion by proteinases [1,7]. Quail mucin also has a similar amino acid and sugar composition to mammalian mucins, with a high proportion of n-acetylgalactosamine, indicative of O-glycosidic linkages [7].

In this study we have investigated the IgG1, IgG2 and IgG3 responses of symptomatic and asymptomatic antibody-positive pigeon fanciers against both intact mucin and the highly glycosylated carbohydrate-rich ‘bottle brush’ fragments following proteolysis. We demonstrate that different subclasses show specificity for different regions of the molecule. Further, we demonstrate differences in the ability of sera from symptomatic and asymptomatic groups to compete with a variety of lectins for carbohydrate binding sites on pigeon intestinal mucin.

MATERIALS AND METHODS

Sera and clinical status

Sera were collected from pigeon fanciers and classified according to the presence or absence of the clinical symptoms of PFL and of circulating precipitating antibodies to pigeon serum and pigeon droppings as previously described [4]. Sera from 98 antibody-positive individuals were selected for further study. Of these, 48 were classified as Group A (symptomatic, precipitating antibody-positive) and 50 were classified as Group B (asymptomatic, precipitating antibody-positive) [4]. Patients in Group A were considered to have classic PFL. All of the samples were from males and there were no significant differences between Groups A and B with respect to age or degree of exposure to pigeons.

Antigens

Pigeon intestinal mucin was collected from 50 freshly killed pigeons and purified by caesium chloride density gradient centrifugation as previously described [4]. The purified mucin was stored as aliquots at −80°C.

Papain-digested mucin (PDM) ‘bottle brush’ segments were produced as previously described [1].

Assays for specific antibodies

IgG and IgG subclass antibodies to intact pigeon intestinal mucin and PDM were quantified by ELISA as previously described [4]. Plates were coated with optimal concentrations of each antigen (0.3 μg/ml and 3 μg/ml carbohydrate, respectively).

Inhibition ELISA

Competition ELISA were performed, where free antigen was added to inhibit the binding of IgG1, IgG2 and IgG3 to antigen-coated plates, as described previously [4]. Pigeon intestinal mucin and PDM were used to inhibit the binding of 16 sera, selected for high titres of IgG1, IgG2 and IgG3, to pigeon mucin.

Lectin mapping of pigeon intestinal mucin

The glycosylation of pigeon intestinal mucin was investigated by a modification of the enzyme-linked lectin assay (ELLA) [8]. Assays were carried out on Nunc-immuno Maxisorp 96-well plates coated with pigeon intestinal mucin (0.3 μg/ml carbohydrate) in 0.1 m sodium phosphate buffer pH 7.0. After washing three times in PBS–0.05% Tween, 100 μl of biotinylated lectin, at a range of concentrations in PBS–0.05% Tween–3% bovine serum albumin (BSA), were added to the wells (HAA and SJA; Sigma Chemical Co., Poole, UK; all other lectins Vector Labs, Peterborough, UK). The 19 lectins tested, their common abbreviations and their major sugar activity are shown in Table 1. Plates were incubated at room temperature for 2 h and then washed as above. ExtrAvidin–peroxidase conjugate (Sigma) was added to each well (100 μl/well of a 1:2000 dilution in PBS–Tween–BSA) and incubated for 1 h at room temperature. The plates were washed as previously and developed with OPD. The reaction was stopped after 30 min by the addition of 12.5% sulphuric acid, and plates were read in a MR 5000 Dynatech ELISA reader at 490 nm using Biolinx assay management software (Dynex Technologies, Inc., Chantilly, VA). A range of lectin concentrations was used from 2 to 2000 ng/ml. Results shown are the optical densities (OD) at 500 ng/ml of each lectin.

Table 1.

Lectins used in the study [14–16,20–32]

Competition ELLA

The ability of sera from symptomatic and asymptomatic antibody-positive fanciers to inhibit the binding of various lectins to antigen-coated plates was determined. Biotinylated lectins were diluted in PBS–0.05% Tween–3% BSA to concentrations previously determined to give an OD of approx. 1.0 in the mucin ELLA. Lectins were added to wells of mucin-coated ELISA plates containing a range of dilutions of sera from pigeon fanciers. After mixing the contents of each well plates were incubated for 2 h at room temperature. Plates were washed, incubated with ExtrAvidin–horseradish peroxidase (HRP) and then developed with OPD as above.

Absorbances were plotted against serum dilution and from this the titre calculated for each serum as the reciprocal of the dilution giving a 50% inhibition of the absorbance of lectin with no added sera (the higher the titre the less serum was needed to inhibit the lectin binding).

Statistical analysis

Statistical analysis was performed using the Mann–Whitney U-test for non-parametric data. All statistical calculations were carried out using a computer-based, commercially available statistics package (Minitab data analysis software). P < 0.05 was considered significant.

RESULTS

IgG subclass binding to intact mucin and PDM segments

The median IgG subclass titres for symptomatic (Group A) and asymptomatic (Group B) individuals against mucin and PDM are shown in Table 2. IgG1 titres against mucin and PDM were significantly higher in sera from symptomatic (A) compared with asymptomatic (B) pigeon fanciers (P = 0.0044 and 0.04, respectively). There were no significant differences in the IgG2 or IgG3 titres between the two groups against either antigen. IgG4 antibody titres to mucin have been shown to be extremely low [4], and were not investigated here.

Table 2.

IgG subclass responses to mucin and papain-digested mucin fragments

*Values are median titres. Median anti-mucin titres for unexposed individuals: IgG1 30, IgG2 8, IgG3 5, IgG4 5.†Numbers in parentheses represent values for first and third quartiles.‡Ratio of median titres intact mucin/papain-digested mucin.

The median IgG1 and IgG2 titres were between 2.3 and 3.4 times higher against mucin compared with PDM in both Groups A and B. Median IgG3 titres against mucin were 620 times greater in symptomatics and 530 times greater in asymptomatics than those seen against PDM. Only 3/48 symptomatic and 13/50 asymptomatic individuals had detectable (> 1/5) IgG3 titres against PDM segments (compared with 44/48 and 46/50 with a detectable IgG3 titre against intact mucin).

Inhibition ELISA

The ability of free mucin or PDM to inhibit binding of IgG subclass antibodies to mucin-coated ELISA plates was investigated. The results are shown in Fig. 1.

Fig. 1.

Inhibition of binding of IgG subclass antibodies to mucin-coated ELISA plates by (a) free mucin and (b) papain-digested mucin (PDM). ○, IgG1; □, IgG2; •, IgG3. Points represent the medians of eight sera.

There were no significant differences in the mean concentration of free mucin required for 50% inhibition of anti-mucin IgG1, IgG2 and IgG3 (170 ng/ml, 150 ng/ml and 175 ng/ml, respectively) and no significant differences between these concentrations and the concentration of PDM fragments needed for 50% inhibition of anti-mucin IgG1 and IgG2 (145 ng/ml and 235 ng/ml). However, significantly higher concentrations of PDM fragments were required to inhibit anti-mucin IgG3 (2600 ng/ml) compared with that required for inhibition of IgG1 and IgG2, or for inhibition of all three subclasses by undigested mucin (P < 0.03). These results are consistent with the reduced binding of IgG3 antibodies to PDM compared with undigested mucin shown above.

Lectin mapping of pigeon intestinal mucin by ELLA

The binding to pigeon intestinal mucin of the 19 lectins tested (Table 1) is shown in Fig. 2.

Fig. 2.

Binding of lectins to pigeon intestinal mucin as measured by enzyme-linked lectin assay (ELLA). Optical density (OD) values shown are for lectin concentrations of 500 ng/ml. Abbreviations and specificities of lectins are shown in Table 1.

Very little binding of the Gal-specific lectins EEL, GSL-I(B4) and PNA was seen, and there was no binding of the mannose-specific lectin NPL.

Of the lectins showing specificity for sialic acid, there was a strong reaction with MAL-I, specific for (α-2,3)NeuNAc, whilst there was no activity with EBL, specific for (α-2,6) NeuNAc.

The majority (five out of eight) of the GalNAc-specific lectins reacted very strongly with pigeon intestinal mucin, although both HAA and VVA, which specifically recognize GalNAc linked to serine or threonine, did not react.

A range of binding activities was seen with the fucose-specific lectins. Of these UEA, which recognizes type 1 H-blood group (Fuc α(1–2) Gal β(1–3) GlcNAc) was very weak, whilst LTL, which recognizes type 2 H-blood group (Fuc α(1–2) Gal β(1–4) GlcNAc), showed an intermediate activity. The strongest binding activity was by AAL, which recognizes α(1–6) as well as α(1–2) and α(1–3)Fuc groups (the latter may also be recognized by LTL).

There was strong binding by LEL, which binds internal GlcNAc and less binding of GSL-II, which recognizes terminal GlcNAc.

Competition ELLA

Preliminary tests showed sera from pigeon fanciers could inhibit binding of 11 of the 15 lectins tested (data not shown). These lectins were further tested with the sera from 16 symptomatic and 16 asymptomatic antibody-positive individuals. The median IgG anti-mucin titre for these sera was not significantly different between the two groups, although median IgG1 titre was significantly higher in sera from symptomatic compared with asymptomatic fanciers (300 000 and 10 400, respectively).

The median serum titres able to inhibit 50% of the activity of 11 lectins are shown in Table 3. Sera from antibody-negative controls did not inhibit lectin binding to pigeon mucin. Some differences were seen between symptomatic and asymptomatic pigeon fanciers. Inhibition of binding of GSL-I, SJA, MPL, SBA (all GalNAc-specific) and LTL (fucose-specific) was achieved at significantly higher titres of symptomatic compared with asymptomatic sera, suggesting that people with PFL have higher titres of antibodies that compete for binding with these lectins. There were no statistical differences in the inhibitory activity of symptomatic compared with asymptomatic sera for the other six lectins. Individual data for two of the lectins, SJA and GSL-II, are shown in Fig. 3.

Table 3.

Inhibition of lectin binding to pigeon mucin by sera from pigeon fanciers

†Numbers are median titres with values for first and third quartiles in parentheses.Significant difference between symptomatic and asymptomatics: *P < 0.05; **P < 0.01.

Fig. 3.

Inhibition of binding of the lectins (a) SJA and (b) GSL-II to pigeon mucin by sera from symptomatic and asymptomatic pigeon fanciers. Points represents the serum titres giving 50% inhibition of lectin binding. Each point represents one individual and the bars indicate the median for the group.

For eight of the 11 lectins (AAL, GSL-II, MAL-I, LTL, MPL, SBA, GSL-I and SJA) there was a significant positive correlation between the serum anti-mucin IgG titre and the titre giving 50% inhibition of lectin binding. Three of the lectins (SBA, GSL-I and SJA) also showed a similar correlation with IgG1 anti-mucin titre.

DISCUSSION

Pigeon intestinal mucin, a high molecular weight and heavily glycosylated molecule, has been implicated as a key antigen in PFL [1,2,4]. In this study we report on the preliminary characterization of disease-associated carbohydrate epitopes on pigeon intestinal mucin.

Studies of antibody binding to proteolytically digested mucin indicate that the majority of the IgG1 and IgG2 is directed against the heavily glycosylated regions, whilst IgG3 antibodies react with papain-sensitive parts of the molecule. Proteolysis by papain has been shown to remove the exposed regions of the mucin protein core to leave highly glycosylated fragments with the remaining protein core protected by a substantial sheath of carbohydrate side chains (bottle brush segments) [5]. Although IgG1 and IgG2 antibody titres were somewhat lower against the digested mucin, this could be due to a reduced ability of the antigen to bind to the plastic ELISA plates. This idea is supported by the inhibition data, which show that, weight for weight, the papain-digested mucin was just as effective as the intact molecule in competing for IgG1 and IgG2 antibody binding to mucin-coated plates. These results suggest therefore that the majority of IgG1 and IgG2 antibody is specific for carbohydrate epitopes. In contrast, IgG3 reactivity against mucin was almost completely lost following proteolytic digestion, suggesting that these antibodies are predominantly directed against protein epitopes or possibly sugar chains in the sparsely glycosylated regions. Although subclass restriction in antibody responses is well documented [9,10], restriction of subclasses to epitopes within antigens has only occasionally been observed [11]. The reason why subclasses should differ in the epitopes recognized is unclear. With respect to the response to pigeon intestinal mucin, one possibility is the IgG1 and IgG2 antibodies to the heavily glycosylated regions arise through a T-independent (TI) response. Such responses are generally associated with polysaccharides, which contain multiple repeating epitopes and are resistant to degradation [12]: criteria likely to be fulfilled by the glycosylated regions of mucin. Moreover, TI responses in man are generally dominated by IgG2, with variable production of IgG1 [9]. The IgG3 antibody response to mucin might be T cell-dependent, an idea supported by the fact that this subclass is found in response to some protein antigens [9,13]. T-dependent antibody production requires that the antigen-specific B cells process and present the antigen to helper T cells: if intact mucin were unable to be processed effectively by B cells the TD response might be restricted to mucin fragments produced by the action of proteases present either in the lung or in the pigeon loft.

The pigeon mucin bound four groups of lectins recognizing NeuNAc, Fuc, GalNAc and GlcNAc. The very strong activity of MAL-I, which recognizes chains terminating in α2–3NeuNAc, suggests that a large proportion of the oligosaccharide chains are sialylated. Interestingly, the fucose-binding lectins LTL and AAL reacted strongly, while UEA did not. LTL and AAL react with fucose on type 2 blood group H structures (Galβ1–4[Fucα1–3]GlcNAcβ1–3) [14], although the latter has a higher affinity for Fucα1–6GlcNAc [15]. Conversely, UEA is typically described to react with fucose on oligosaccharides terminating with a type 1 blood group H structure (Galβ1–3[Fucα1–3]GlcNAcβ1–3) [16]. It is unlikely therefore that the sugar chains of pigeon intestinal mucin contain oligosaccharides terminating in a type 1 blood group H structure: this is supported by the lack of activity shown by the Gal-specific lectins PNA, GSL-I(B4) and EEL. It has been recently demonstrated that LTL can also bind to the fucosylated lacdiNAc trisaccharide GalNAcβ1–4[Fucα1–3]GlcNAc [17], and it is possible that a similar structure with terminal GalNAc rather than Gal is present on pigeon intestinal mucin.

The mucin reacted with the majority of lectins recognizing GalNAc. The lectin VVA, which recognizes the GalNAc (α-1, Ser/Thr) structure characteristic of the attachment of the sugar chains to the protein core in mucins, does not bind. This would be in keeping with terminal GalNAc on the oligosaccharide chains being available for lectin binding whilst the GalNAc at the reducing end is masked. The absence of lectin-accessible structures involving terminal Gal is indicated by the lack of reactivity with EEL, GSL-I(B4) and PNA. This may suggest that the sugar chains are relatively short, as the backbone regions of mucin are normally made up of alternating Gal and GlcNAc residues [6]. This is similar to quail intestinal mucin, which has been reported to have relatively short side chains containing very little Gal [7].

Antibody-positive sera from pigeon fanciers inhibited the binding of lectins specific for (α2–3)NeuNAc, GalNAc, internal GlcNAc and Fuc. The finding that sera from antibody-negative controls did not inhibit the binding of lectins to mucin strongly suggests that this effect was due to mucin-specific antibodies in the sera of pigeon fanciers. This is further supported by the observation that, for the majority of lectins, there was a significant correlation between IgG anti-mucin titre and titre for inhibition of lectin binding.

Some interesting differences were apparent between symptomatic and asymptomatic antibody-positive individuals with respect to inhibition of lectin binding. This result could not be ascribed to differences in the IgG antibody titres between these two groups, suggesting that antibodies to particular epitopes may be associated with development of disease. Four such lectins were GalNAc-specific, whilst the fifth was fucose-specific, suggesting that such disease-associated epitopes may contain these sugars. From the known specificity of LTL and the lack of accessible Gal (as shown by failure to bind GSL-I(B4)) this fucose is probably linked to GlcNAc or GalNAc. Interestingly, there was a significant correlation between the IgG1 anti-mucin titre in sera and the inhibitory titre for three of these five lectins, suggesting that this isotype, which has been implicated in the development of PFL, may be responsible for the inhibition of these lectins.

It is clear from these studies that the antibody response of pigeon fanciers to pigeon intestinal mucin is predominantly carbohydrate-specific. Antibody responses to various glycoproteins are also considered to be important in the development of farmers' lung, a form of EAA resulting from reactions to Saccharopolyspora rectivirgula(formally Micropolyspora faeni), and analysis of antigens from this organism demonstrated that IgG2 antibodies reacted mainly with carbohydrate residues [18]. Furthermore, mannan and mannoproteins have been shown to be disease-associated antigens of Aspergillus umbrosus, the organism associated with farmers' lung in Finland [19]. Thus, carbohydrate-containing molecules may have a general role in the development of EAA. The apparent differences in fine specificity of anti-mucin antibodies between symptomatic and asymptomatic fanciers suggested by the lectin inhibition studies further confirm the importance of this antigen in the disease. We are currently engaged in detailed structural studies of these epitopes.