Immunology 1991 73 491-497
ADONIS
001928059100187Q
The measurement of respiratory burst induced in polymorphonuclear neutrophils by IgA and IgG anti-gliadin antibodies isolated from coeliac serum W. W. STEWART & M. A. KERR Department of Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee Acceptedfor publication 23 April 1991
SUMMARY The properties of IgA and IgG anti-gliadin antibodies from the serum of patients with coeliac disease have been compared. The antibodies were quantified by ELISA using microtitre plates coated with crude gliadin fractions. Their specificity was confirmed by immunoblotting. Heat-treated sera containing IgA antibodies stimulated chemiluminescence when added together with neutrophils to microtitre plates coated with crude gliadin. Sera containing only IgG antibodies were less efficient. When IgA and IgG were purified from a serum containing both classes of anti-gliadin antibodies, each of the preparations was able to stimulate neutrophil chemiluminescence in plates coated with gliadin. Although the yield of anti-gliadin antibody determined by ELISA was high, the ability of the purified immunoglobulins to stimulate neutrophil chemiluminescence was much less than that of the unfractionated serum. This loss of activity was shown to be due to the ability of each class of antibody to potentiate the activity of the other in the whole serum. INTRODUCTION Coeliac disease is characterized by damage to the mucosa of the small intestine brought about by ingestion of certain cereals. The toxic components reponsible for this adverse response have been identified as wheat gliadins, components of the storage protein gluten, and related proteins termed prolamines from barley, rye and possibly oats.'2 Gliadins exist as some 40 different polypeptides which can be classified into four groups, a, IJ, y and co, depending on their mobility on electrophoresis at acid pH.3 Both IgA and IgG antibodies to gliadin components have been detected in coeliac sera, with the former being suggested to be more diagnostic of coeliac disease.4-8 Attempts to correlate disease activity with the presence of circulating antibodies to individual gliadins have been inconclusive,9 12 although there is a tendency for them to be specific for the toxic fractions of gliadin, a, /5 and y. "1'31'4 The mechanism by which mucosal damage is brought about by these proteins is uncertain, but the enhancement of immune response towards gliadins'5 and the resulting high titres of anti-gliadin antibodies in the sera of coeliac patients suggest that it may be immune complex mediated. It has also been shown that, while on a normal diet, there is a large gluten-dependant infiltration of lymphocytes, mast cells, basophils, eosinophils and plasma cells into coeliac intestinal mucosa.'6 This provides the opportunity
for further tissue damage brought about by cell-mediated cytotoxic reactions towards IgA- and IgG-gliadin complexes. We have recently purified an IgA receptor from human neutrophils and showed that this receptor will recognize serum and secretory IgA with high affinity.'7 Binding of artificially aggregated IgA to this receptor stimulated degranulation and a respiratory burst which could be measured by chemiluminescence. The ability of IgA to stimulate the neutrophil respiratory burst was found to be greater than that of an equivalent amount of IgG,'8 which demonstrates the potential of IgA-containing complexes to cause tissue damage in vivo. The same IgA receptor has also been shown to be present on monocytes and related cell lines. 19 Coeliac disease is an inflammatory disease characterized by the presence of circulating IgA and IgG antibodies of known specificity. We have therefore chosen coeliac disease as a model with which to compare the ability of pathologically important IgA and IgG antibodies to induce a respiratory burst in neutrophils. MATERIALS AND METHODS Patients sera Serum samples were obtained from 17 coeliac patients (five male, 12 female, age 16-54) who were not on a gluten-free diet. These samples were stored at -200 until required. Normal serum was collected from 10 healthy volunteers (three male, seven female, age 19-49), pooled and also stored at - 20°. Heat treatment of sera involved incubating at 560 for I hr.
Correspondence: Dr M. A. Kerr, Dept. of Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD9 ISY, U.K.
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Purification of IgA1- and IgG-containing anti-gliadin antibody activity IgA I and IgG were purified from normal human serum as described recently.20 By employing essentially the same method, but scaled down for FPLC separation (Pharmacia, Uppsala, Sweden), IgA I and IgG were purified from the serum of coeliac patients which had been shown to contain anti-gliadin antibodies. This was done by ammonium sulphate precipitation (to 50% saturation) of the serum (I ml) followed by desalting on a PD-10 gel filtration column (Pharmacia). The peak protein fraction was then passed down an anion exchange column (MonoQ; Pharmacia) equilibrated in 50 mM Tris/HCI, pH 8-0, the material passing unretarded through the column being pure IgG. After elution with a 0-0-5 M linear gradient of NaCl, the IgA-containing fractions were passed down a small column of Jacalin-Sepharose (Vector Labs, Burlingame, CA) equilibrated in PBS (phosphate-buffered saline, 8 5 mm sodium phosphate/ 0-15 M NaCl, pH 7-1). Pure IgAI was eluted from this column with 0-8 M D +galactose. Analysis of the purified IgG or IgA by gel filtration on Superose 6 failed to detect the presence of aggregated anti-gliadin antibodies.
Isolation ofpolymorphonuclear neutrophils Neutrophils were isolated from heparinized blood from healthy volunteers by density centrifugation by the method of English & Anderson2' as described by Albechtsen, Yeaman & Kerr.22 The cells were washed in PBS before being counted and analysed on a Coulter Counter. The preparation typically contained less than 1% and 10% contaminating lymphocytes and red blood cells, respectively. SDS-PAGE and immunoblotting SDS-PAGE was performed according to Laemmli23 using 515% linear gradient gels to achieve separation. Proteins were visualized with Coomassie Brilliant Blue R-250 (0 5% in 40% methanol/10% acetic acid). Transfer of separated proteins to nitrocellulose and subsequent immunoblotting was carried out according to Towbin, Staehelin & Gordon.24 Briefly, the nitrocellulose containing the blotted protein was cut into strips and blocked for 1 hr in 5% skimmed milk powder/PBS. The strips were then developed with sera or purified immunoglobulin, diluted in the same buffer, for a further hour before being washed in PBS and incubated with Sigma IgA- or IgG-specific alkaline phosphatase conjugate (1/500 dilution in 5% milk powder/PBS). The blots were developed using 5-bromo-4-
chloro-3-indolyl phosphate (5BCIP). ELISA for IgA and IgG anti-gliadin The ELISA used for the detection of antibodies to gliadin was modified from that described by Volta et al.7 Microtitre plates were coated with 150yl of a crude gliadin preparation (Sigma) at a concentration of 0-2 mg/ml in 0 005 M carbonate buffer, pH 10-2. After incubation overnight at room temperature the plates were washed three times in 0-1% Triton/PBS and blotted dry. The sera or purified antibodies were diluted in 5% normal sheep serum (NSS) in 0-1% Triton/PBS and 100 p1 added to the gliadin-coated wells for 1 hr at 37°. The wells were then washed a further three times in the same Triton buffer and alkaline phosphatase-conjugated goat antibody specific for either human IgA or IgG (Sigma, St Louis, MO), was added at a dilution of 1/1000 in 0-1% bovine serum albumin (BSA) in 0-1%
IgA
IgG
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l 2 3 4 5 6 7 8 91011 121314151617N
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Figure 1. Detection of anti-gliadin antibodies in the sera of patients with coeliac disease. Serum from 17 coeliac patients together with pooled normal serum (N) was assayed at a 1/200 dilution for IgA and IgG antigliadin antibodies by ELISA as described in the Materials and Methods. The OD"'" is indicated. The horizontal line on each graph shows 2-5 x the value given by pooled normal serum (solid bar).
Triton/PBS. After a further incubation of 1 hr at 370 the wells were washed as before and developed using p-nitrophenylphosphate (2 mg/ml) in 0 1 M sodium bicarbonate buffer, pH 10-2, containing I mM MgCl2. The absorbance at 405 nm was measured after 30 min. Assays were carried out in duplicate and mean values taken. Results were compared to those obtained with a pooled normal serum control. The intra-assay variation was less than 5%. Interassay variation using the same reagents but carried out on different days was less than 6%. Chemiluminescence studies Immunoglobulins bound to wells. These studies were performed on a Dynatech Microlite ML1000 luminometer using chemiluminescence microtitre plates (Dynatech, Billinghurst, Sussex, U.K.) by a method described previously.'8 IgA and IgG complexed to gliadin. These studies used a modification of the above method. Wells were coated with crude gliadin (Sigma) at a concentration of 0-2 mg/ml in 0-005 M carbonate buffer, pH 10-2, by incubating 150 yl of the solution overnight at room temperature. After washing three times in PBS, 100 pl of either heat-treated sera (1/200 dilution in 0-1% BSA/PBS) or IgA or IgG preparations, diluted appropriately in the same buffer, were added to the wells -in triplicate and incubated at 37° for 2 hr. After washing the wells several times in PBS, lucigenin (100 pl, 0-1 mg/ml; Sigma) in HBSS (Gibco, Grand Island, NY)/BSA (1 mg/ml; Sigma) was added followed by neutrophils (50 p1 of I x 106/ml) in HBSS/BSA. Chemiluminescence was measured at regular intervals for I hr. RESULTS Measurements of IgA and IgG antibodies against gliadin in the sera of coeliac patients
Sera from 17 coeliac patients were assayed for anti-gliadin activity. The level of these antibodies having IgA and IgG isotype was determined by an ELISA specific for each. The results of these assays, which include control sera, are presented
493
Anti-gliadin antibodies in coeliac disease N
1
2
3
4
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Figure 2. Neutrophil chemiluminescence induced by binding of antigliadin antibodies to surface-bound gliadin. Purified neutrophils were added to gliadin-coated mictotitre plates which had been incubated with heat-treated coeliac sera at a 1/200 dilution. Respiratory bursts were elicited by serum 1 (-), 2 (-), 3 (-) and 5 (*) as measured by lucigeninenhanced chemiluminescence. No such bursts were observed with serum 11 (A), 15 (0), 4 (0), 7 (>) or pooled normal serum (+) at the same dilution.
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Figure 4. Detection of anti-gliadin antibodies by immunoblotting. Immunoblotting was performed on crude gliadin which had been separated into its different components by SDS-PAGE on a 5-15% gradient gel. Pooled normal serum (N) or coeliac sera 1, 2, 3, 4, 5 or 6 were used at 1/25 dilutions and developed for IgA and IgG anti-gliadin antibodies as indicated (A and G). IgA and IgG purified from serum 1 (lanes a and b, respectively) or an equivalent amount of IgA and IgG isolated from pooled normal serum (lanes c and d, respectively) were also blotted and developed using the appropriate conjugate (A or G). Lanes e and f were blotted with the dilution buffer only before being developed with IgA (A) or IgG (G)-specific conjugate. The gel strip beside the immunoblot indicates the protein bands picked up by Coomassie staining of the gliadin preparation run on the same the SDS gel.
After incubating the plates as described in the Materials and Methods, chemiluminescence bursts were observed after addition of neutrophils (Fig. 2). The two sera with the highest levels of IgA anti-gliadin (sera 1 and 2) gave the greatest respiratory burst. The size of the burst correlated well with the IgA antigliadin titres and but not with the titres of IgG anti-gliadin (Fig. 3). Serum 15, which had the same IgG titre as 1 but lacked detectable IgA, gave a negligible burst at this dilution. Isolation of IgAl and IgG with specificity for gliadin from coeliac
Figure 3. The correlation between IgA or IgG anti-gliadin antibody levels and induction of neutrophil chemiluminescence. The maximum chemiluminescence (Clmas) induced in neutrophils by gliadin-coated microtitre plates incubated with a 1/200 dilution of heat-treated coeliac sera is plotted against the amount of IgA (-) or IgG (0) anti-gliadin antibody measured by ELISA (OD405).
serum
in Fig. 1. When IgA anti-gliadin was determined, eight out of the 17 sera were positive, having an OD405 which was greater than 2-5 times that for pooled normal serum at 1/200 dilution. For IgG anti-gliadin estimations, measured at the same dilution, the number of positive sera was 12. Sera that were positive for IgA anti-gliadin were always positive for the IgG antibody, although several IgA-negative sera had positive IgG titres. None of the sera was positive for the IgA antibody alone.
IgA I and IgG were isolated from I ml of the coeliac serum I as described in the Materials and Methods. The purity of the proteins was confirmed by SDS-PAGE and radial immunodiffusion (RID). Both the IgA and IgG showed specificity towards gliadin as determined by ELISA and immunoblotting. Gliadin antibodies in the IgAI pool (II ml, 0-08 mg/ml by RID) were detected at a titre of 1/100 (0-0008 mg/ml). Similarly, antibodies specific for gliadin in the IgG pool (6 ml, 1-3 mg/ml by A280) were detected at a 1/1000 dilution (0-0013 mg/ml). This suggested that in the serum the same percentage of the total IgA and IgG was specific for gliadin, i.e. (11 x 0-008) mg IgA and (6 x 0-0013) mg IgG, and implied that the total amount of IgG specific for gliadin was some 7-8/0 88 (8 6) times greater than the amount of IgA with the same specificity.
Neutrophil respiratory bursts induced by heat-treated coeliac sera Heat-treated sera from coeliacs with high or low anti-gliadin IgA and IgG titre or pooled normal serum, were added to gliadin-coated chemiluminescence plates at a 1/200 dilution.
The specificity of IgA and IgG anti-gliadin antibodies determined by immunoblotting To further characterize the binding of IgA and IgG antibodies to gliadin, immunoblotting was performed on the crude gliadin
W. W. Stewart & M. A. Kerr
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0*4 02 60 50 30 40 20 10 (min) Figure 5. Neutrophil chemiluminescence induced by purified IgA or IgG anti-gliadin antibodies. IgA (a) or IgG (b) purified from serum 1 were incubated on microtitre plates with (i) and without (ii) a gliadin coating. The IgA was used undiluted (A) or at dilutions of 1/2, 1/4 (0), 1/8 (A), 1/16 (0) and 1/32 (0); IgG was used undiluted (A) or at dilutions of 1/5 (U), 1/10 (0), 1/20 (A), 1/40 (0) and 1/80 (0). IgA and IgG isolated from pooled normal serum (-0-) was also used at a concentration equivalent to that of the undiluted immunoglobulins isolated from serum 1. Time
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IgAl 1:40 1:20 1:10 IgG 1:40 1:20 1:10 only' only IgG IgAI dilution dilution Figure 6. Co-operation between IgA and IgG anti-gliadin antibodies in the stimulation of neutrophil chemiluminescence. (a) IgA 1 anti-gliadin, diluted 1/4 (-) and 1/8 (A), were co-incubated on gliadin-coated plates with IgG anti-gliadin at 1/10, 1/20 and 1/40 dilutions. The open circles (0) indicate the respiratory burst induced by the IgG dilutions on their own. (b) IgG anti-gliadin was incubated at 1/5 dilution (0) in the presence of IgAl anti-gliadin diluted 1/10, 1/20 and 1/40. The open squares (0) indicate the respiratory burst induced by the IgA dilutions on their own. I
preparation using the purified IgAI and IgG pools as well as six sera with the highest titres of anti-gliadin antibodies (including serum 1) and pooled normal sera (Fig. 4). All sera were diluted 1/25. No IgA and only a trace of IgG anti-gliadin was detected in pooled normal serum. IgA anti-gliadin was detected to varying degrees in the coeliac sera, as was IgG antigliadin. No obvious pattern of bands was observed for the coeliac sera, with a range of protein bands being recognized between 35,000 and 70,000 MW by both IgA and IgG anti-
coeliac
gliadin. The specificity of the IgA I and IgG isolated from serum I (lanes a and b, respectively) agreed with the blotted whole serum. The IgG from this serum did differ slightly from the other sera in that it appeared to recognize bands mostly in the 35,00040,000 MW region. In agreement with the results of the ELISA, the level of IgA antibodies in the sera other than 1 and 2 appeared to be much less than the IgG antibodies.
Microtitre plates coated with gliadin were incubated with IgA and IgG isolated from coeliac serum 1 at different dilutions and then neutrophils were added (Fig. 5ai and aii). Chemiluminescence bursts were only observed with IgA I when used undiluted (80 yg/ml) or at a 1/2 dilution (40 yig/ml) and with IgG when used undiluted (1-26 mg/ml) or at 1/5 dilution (0-252 mg/ml, only a slight burst). No chemiluminescence bursts were obtained when the same concentrations of IgA 1 and IgG purified from normal serum were incubated with blocked gliadin-coated plates. The binding of the anti-gliadin antibodies was therefore shown to be antigen-specific. The same concentrations of the IgA and IgG pools were also incubated for the same length of time in chemiluminescence wells which had not been coated with gliadin (Fig. Saii, bii). Under these conditions, the immunoglobulins bind non-specifically to the plates. For any concentration, the chemiluminescent burst elicited by bound IgA was always greater than for IgG, consistent with our previous findings. If the plates were blocked with I mg/ml BSA prior to addition of the immunoglobulin, no immunoglobulin bound and no chemiluminescence burst was elicited.'8
Anti-gliadin antibodies in coeliac disease The Cl/rn, for undiluted IgA on gliadin-coated plates (0 4) the same as the C/max induced by a 1/16 dilution (5 yg/ml) incubated for the same length of time on a uncoated well. Similarly, the Clmax for undiluted IgG on gliadin (0-2) was approximately equivalent to a 1/160 dilution (8 pg/ml) on uncoated wells. Since the response to antigen-coated plates was much less than that for plates coated non-specifically with immunoglobulin of the same isotype it can be concluded that it is the amount of bound antibody which is the limiting factor on the gliadin-coated plates. The response to either purified IgG or IgA anti-gliadin antibody was in fact less than expected. In the whole serum a dilution of 200-fold which would give an IgA concentration of 5 pg/ml and IgG of 50 Mg/ml still gave a major response on gliadin-coated plates. It is clear that activity is lost upon fractionation of the serum. However, ELISA showed no significant loss of either IgG or IgA anti-gliadin antibody.
was
Co-operation between Fc receptors in response to IgA and IgG anti-gliadin antibodies To investigate the possibility that this apparent loss of activity was due to a co-operative effect of the two classes of antibody in the whole serum, IgA1, purified from coeliac serum 1, was incubated at different concentrations with gliadin-coated plates in the presence and absence of different concentrations of IgG, purified from the same serum. In previous experiments, these concentrations of the IgG pool had been shown not to elicit a respiratory burst and this was again found to be the case [Fig. 6a (--- )]. A clear enhancement of the burst elicited by each concentration of the IgAI was observed when the IgG was coincubated in the gliadin coated plates (Fig. 6a). Similarly, when the IgG anti-gliadin pool was incubated on gliadin plates at a 1/5 dilution, co-incubation with IgA anti-gliadin, at dilutions which on their own did not induce a respiratory burst, resulted in enhancement of the IgG response (Fig. 6b). DISCUSSION anti-gliadin antibodies (AGA) are of circulating levels Elevated characteristic of coeliac disease, the amount of antibody correlating with disease activity."' Several studies have examined the usefulness of these antibodies as a screen for active coeliac disease and an indicator for small intestinal biopsy.8 25 It was concluded by several investigators that IgA (AGA) rather than IgG (AGA) has the greater diagnostic value."7 This conclusion was supported by the detection of 'false positive' antibodies to gliadin, measured by ELISA and immunofluorescence which were all of the IgG isotype.7 In our study 70% and 47% of the coeliac sera from untreated patients were positive for IgG and IgA AGA, respectively. The relative titres of IgA and IgG AGA have been reported to vary in each coeliac patient4"'I and the data presented in this study are in agreement with this, although it does not support the observation that the specificities of IgA and IgG AGA in each patient are identical." By comparing blots of six of our coeliac sera for IgA and IgG AGA specificity, differences in the pattern of bands recognized for each patient were observed for the two isotypes. However, if the blots for the six different patients were compared, a similar range of discrete bands was recognized by both IgA and IgG AGA. It has previously been reported that coeliac sera exhibited antibody binding to discrete groups of gliadin bands. Kieffer et
495
al.9 found that IgG AGA exhibited high titres for a and 3 fractions of gliadin separated on ion-exchange chromatography. Friis et al.14 observed that the specificity of IgG AGA in patients with untreated coeliac disease was towards polypeptides in the y-fraction, having molecular weights of 35,000 and 45,000 with antibodies against the a and P fraction being secondary to mucosal damage. We have reported a range of IgG specificity between 35,000 and 75,000 MW for 5/6 of the blotted sera with the pattern of blotting for the sixth sera (1) being concentrated between 35,000 and 45,000 MW. The IgA AGA from the same serum did not show this pattern. Only the two sera with highest IgA AGA titres (1 and 2) showed intense IgA bands, ranging from 35,000 to 75,000 MW when blotted. Our results are therefore consistent with the range of specificities reported in other publications suggesting that no pattern of gliadin specificity is characteristic of coeliac disease. The ability of immune complexes to trigger degranulation and a respiratory burst in neutrophils is well documented, the responses being brought about via receptors for the Fc portion of immunoglobulins which are present on the cell surface.26 28 Interaction of IgA and IgG Fc domains with their receptors on the neutrophil surface results in the release of granular enzymes into the surrounding environment which in vivo would lead to tissue damage.'8 29 IgA and IgG complexes also have the ability to elicit a respiratory burst in neutrophils with the resulting release of highly reactive superoxide creating the potential for further damage to the local tissue.'8'30 We have previously shown that the ability of different immunoglobulins to induce this respiratory burst can be measured by coating the proteins to microtitre plates and following the increase in lucigenin-enhanced chemiluminescence after addition of purified neutrophils.'8 In applying this technique to the specific IgA 1 and IgG antibodies purified from the coeliac serum we were able to determine that both were capable of producing a respiratory burst in neutrophils at equivalent concentrations. The burst for IgA 1 was always greater than the burst for an equivalent amount of IgG, indicating greater potential for tissue damage. This is in spite of the fact that the predominant subclasses of IgG anti-gliadin antibodies have been reported to be IgGl and IgG3, the subclasses binding with highest affinity to the neutrophil Fcy
receptors.3' When whole coeliac serum was used to stimulate neutrophils, a 1/200 dilution of heat-treated serum I produced a significant respiratory burst (Clmax 0-17), while significant bursts were only observed for the IgAl (Clmax 0-4) and IgG (Clmax 0-2) pools when used undiluted. The IgA I and IgG concentrations of these pools were estimated to contain 1/1 1 and 1/6 the concentrations in serum 1, respectively. Hence they would have to be diluted a further 20 and 30 times to have the same IgA and IgG content as the 1/200 dilution of serum 1. No chemiluminescence burst was observed when neutrophils were incubated with these dilutions of immunoglobulin. One possible explanation for the lower activity of the purified immunoglobulins was that in the serum the two were able to act in synergy. Our results show clearly that there is a synergistic effect between the two classes of antibody in stimulating the neutrophil chemiluminescent burst. The marked dependence of the size of the chemiluminescence burst on the IgA AGA titre must reflect this synergistic effect. The level of IgA AGA in sera I and 2 is apparently high enough to enable
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them to elicit a respiratory burst at a 1/200 dilution but is too low in the other sera, even though their IgG AGA titres are as high or indeed higher than sera 1 or 2. Co-operation between FcRot and FcRy has previously been demonstrated by Fanger, Goldstine & Shen.32 Secretory IgA has also been shown to be able to potentiate the effect of IgG in promoting antibodydependent cell cytotoxicity (ADCC) by human neutrophils, monocytes and lymphocytes.33 We have demonstrated the usefulness of chemiluminescence assays carried out in microplate format for the detection of specific antibodies with pathologic potential. These assays are as sensitive and simple to perform as many conventional immunoassays. Our results suggest that it might be useful to use the chemiluminescence assay described to screen coeliac patients for potentially harmful IgA anti-gliadin antibodies, althoug a more extensive clinical investigation using a large sample of coeliac sera is required to fully evaluate the assay. The pathological significance of the efficient stimulation of neutrophils by IgA anti-gliadin antibodies upon binding to surface-bound antigen is not clear. Coeliac disease is characterized by chronic inflammatory infiltrates rich in lymphocytes, containing cells of the monocyte/macrophage lineage but poor in neutrophils. Monteiro et al.'9 have clearly demonstrated the presence of an identical IgA receptor on neutrophils and monocytes; the role of this monocyte receptor therefore warrants investigation. The role of the neutrophil might, however, be more important in the condition associated with coeliac disease termed dermatitis herpetiformis (DH). A high percentage (85%) of DH patients have associated gluten-sensitive enteropathy and skin lesions associated with the disease have been shown to contain granular deposits of IgA13435 at the junction of the dermis and the epidermis. A similar high percentage of patients with coeliac disease show some skin involvement36. IgA antigliadin antibodies are present in these patients and immunoblotting analysis has shown them to be of similar specificity to those found in coeliac sera. The involvement of these antibodies in the granular deposits found in DH patients has been implied from the improvement in skin lesions brought about by a gluten-free diet. DH lesions are characterized by an intense neutrophil infiltrate in the papillae. IgA deposits are frequently heavier in adjacent, uninvolved skin suggesting loss in the lesions due to phagocytosis.
ACKNOWLEDGMENTS This work was supported by a project grant from the Arthritis and Rheumatism council and by funds from the Cunningham Trust for purchase of the luminometer. We are grateful to Mr R. Fawkes and S. Macpherson for assistance in the preparation of the figures and to Mrs H. Cowper for preparing the manuscript.
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J.H. (1989) Anti-gliadin specificity for gluten-derived peptides toxic to coeliac patients. Clin. exp. Immunol. 76, 384. STERN M., FISCHER K. & GRUTTNER R. (1979) Immunofluorescent serum gliadin antibodies in children with coeliac disease and various malabsorptive disorders. Eur. J. Pediatr. 130, 165. FRIIS S.U., NOREN O., SJOSTROM H. & GUDMAND-HOYER E. (1986) Patients with coeliac disease have a characteristic gliadin antibody pattern. Clinica. Chimica. Acta, 155, 133. STROBER W. (1986) Gluten-sensitive enteropathy: a nonallergic immune hypersensitivity of the gastrointestinal tract. J. Allergy clin. Immunol. 78, 202. FREEDMAN A.R., MACARTNEY J.C., NELUFER J.M. & CICLITIRA P.J. (1987) Timing of infiltration of T lymphocytes induced by gluten into the small intestine in coeliac disease. J. clin. Pathol. 40, 741. MAZENGERA R.L. & KERR M.A. (1990) The specificity of the IgA receptor purified from human neutrophils. Biochem. J. 272, 159. STEWART W.W. & KERR M.A. (1990) The specificity of the human IgA receptor (FcRa) determined by measurement of chemiluminescence induced by serum or secretory IgAl or IgA2. Immunology, 71, 328. MONTEIRO R.C., KUBAGAWA H. & COOPER M.D. (1990) Cellular distribution, regulation, and biochemical nature of an Fca receptor in humans. J. exp. Med. 171, 597. LOOMES L.M., STEWART W.W., MAZENGERA R.L., SENIOR B.W. & KERR M.A.' (1990) Purification and characterisation of human immunoglobulin IgA 1 and IgA2 isotypes from serum. J. immunol. Meth. (in press). ENGLISH D. & ANDERSEN B.R. (1974) Single-step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J. immunol. Meth. 5, 249.
22. ALBRECHTSEN M., YEAMAN G.R. & KERR M.A. (1988) Characteri-
zation of the IgA receptor from human polymorphonuclear leucocytes. Immunology, 64, 201. 23. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680. 24. TOWBIN H., STAEHELIN T. & GORDON J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
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29.
30.
sheets: procedure and some application. Proc. nail. Acad. Sci. U.S.A. 76,4350. KHOSHOO V., BHAN M.K., PURI S., JAIN R., JAYASHREE S., BHATNAGAR S., KUMAR R. & STINTZING G. (1989) Serum antigliadin antibody profile in childhood protracted diarrhoea due to coeliac disease and other causes in a developing country. Scand. J. Gastroenterol. 24, 1212. FANGER M.W., SHEN L., GRAZIANO R.F. & GuYRE P.M. (1989) Cytotoxicity mediated by human receptors for IgG. Immunol. Today, 10, 92. SHEN L., GRAZIANO R.F. & FANGER M.W. (1989) The functional properties of FcyRI, II and III on myeloid cells: a comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors. Mol. Immunol. 26, 959. KERR M.A. (1990) The structure and function of human IgA. Biochem. J. 271, 285. HENSON P.M., JOHNSON H.B. & SPIEGELBERG H.L. (1972) The release of granule enzyme from human neutrophils stimulated by aggregated immunoglobulins of different classes and subclasses. J. Immunol. 109, 1182. GORTER A., HIEMSTRA P.S., LEIJH P.C.J., VAN DER SLUYS M.E., VAN DEN BARSELAAR M.T., VAN Es L.A. & DAHA M.R. (1987) IgA- and secretory IgA-opsonized S. aureus induce a respiratory burst and
31.
32.
33. 34.
35. 36.
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phagocytosis by polymorphonuclear leucocytes. Immunology, 61, 303. GALLAGHER R.B., CERVI P., KELLY J., DOLAN C., WEIR D.G. & FEIGHERY C. (1989) The subclass profile and complement activating potential of anti-gliadin antibodies in coeliac disease. J. clin. Lab. Immunol. 28, 115. FANGER M.W., GOLDSTINE S.N. & SHEN L. (1983) Cytofluorographic analysis of receptors for IgA on human polymorphonuclear cells and monocytes and the correlation of receptor expression with phagocytosis. Mol. Immunol. 20, 1019. SHEN L. & FANGER M.W. (1981) Secretory IgA antibodies synergize with IgG in promoting ADCC by human polymorphonuclear cells, monocytes and lymphocytes. Cell. Immunol. 59, 75. FRY L., SEAH P.P., HARPER P.G., HOFFBRAND A.V. & MCMINN R.M.H. (1974). The small intestine in dermatitis herpetiformis. J. clin. Pathol. 27, 817. HALL R.P. & LAWLEY T.J. (1985) Characterisation of circulating and cutaneous IgA immune complexes in patients with dermatitis herpetiformis. J. Immunol. 135, 1760. Scorr B.B., YOUNG S., RAJAH S.M., MARKS J. & LOWOWSKY M.S. (1976) Coeliac disease and dermatitis herpetiformis. Further studies on their relationship. Gut, 17, 759.
ADONIS
001928059100187Q
The measurement of respiratory burst induced in polymorphonuclear neutrophils by IgA and IgG anti-gliadin antibodies isolated from coeliac serum W. W. STEWART & M. A. KERR Department of Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee Acceptedfor publication 23 April 1991
SUMMARY The properties of IgA and IgG anti-gliadin antibodies from the serum of patients with coeliac disease have been compared. The antibodies were quantified by ELISA using microtitre plates coated with crude gliadin fractions. Their specificity was confirmed by immunoblotting. Heat-treated sera containing IgA antibodies stimulated chemiluminescence when added together with neutrophils to microtitre plates coated with crude gliadin. Sera containing only IgG antibodies were less efficient. When IgA and IgG were purified from a serum containing both classes of anti-gliadin antibodies, each of the preparations was able to stimulate neutrophil chemiluminescence in plates coated with gliadin. Although the yield of anti-gliadin antibody determined by ELISA was high, the ability of the purified immunoglobulins to stimulate neutrophil chemiluminescence was much less than that of the unfractionated serum. This loss of activity was shown to be due to the ability of each class of antibody to potentiate the activity of the other in the whole serum. INTRODUCTION Coeliac disease is characterized by damage to the mucosa of the small intestine brought about by ingestion of certain cereals. The toxic components reponsible for this adverse response have been identified as wheat gliadins, components of the storage protein gluten, and related proteins termed prolamines from barley, rye and possibly oats.'2 Gliadins exist as some 40 different polypeptides which can be classified into four groups, a, IJ, y and co, depending on their mobility on electrophoresis at acid pH.3 Both IgA and IgG antibodies to gliadin components have been detected in coeliac sera, with the former being suggested to be more diagnostic of coeliac disease.4-8 Attempts to correlate disease activity with the presence of circulating antibodies to individual gliadins have been inconclusive,9 12 although there is a tendency for them to be specific for the toxic fractions of gliadin, a, /5 and y. "1'31'4 The mechanism by which mucosal damage is brought about by these proteins is uncertain, but the enhancement of immune response towards gliadins'5 and the resulting high titres of anti-gliadin antibodies in the sera of coeliac patients suggest that it may be immune complex mediated. It has also been shown that, while on a normal diet, there is a large gluten-dependant infiltration of lymphocytes, mast cells, basophils, eosinophils and plasma cells into coeliac intestinal mucosa.'6 This provides the opportunity
for further tissue damage brought about by cell-mediated cytotoxic reactions towards IgA- and IgG-gliadin complexes. We have recently purified an IgA receptor from human neutrophils and showed that this receptor will recognize serum and secretory IgA with high affinity.'7 Binding of artificially aggregated IgA to this receptor stimulated degranulation and a respiratory burst which could be measured by chemiluminescence. The ability of IgA to stimulate the neutrophil respiratory burst was found to be greater than that of an equivalent amount of IgG,'8 which demonstrates the potential of IgA-containing complexes to cause tissue damage in vivo. The same IgA receptor has also been shown to be present on monocytes and related cell lines. 19 Coeliac disease is an inflammatory disease characterized by the presence of circulating IgA and IgG antibodies of known specificity. We have therefore chosen coeliac disease as a model with which to compare the ability of pathologically important IgA and IgG antibodies to induce a respiratory burst in neutrophils. MATERIALS AND METHODS Patients sera Serum samples were obtained from 17 coeliac patients (five male, 12 female, age 16-54) who were not on a gluten-free diet. These samples were stored at -200 until required. Normal serum was collected from 10 healthy volunteers (three male, seven female, age 19-49), pooled and also stored at - 20°. Heat treatment of sera involved incubating at 560 for I hr.
Correspondence: Dr M. A. Kerr, Dept. of Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD9 ISY, U.K.
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Purification of IgA1- and IgG-containing anti-gliadin antibody activity IgA I and IgG were purified from normal human serum as described recently.20 By employing essentially the same method, but scaled down for FPLC separation (Pharmacia, Uppsala, Sweden), IgA I and IgG were purified from the serum of coeliac patients which had been shown to contain anti-gliadin antibodies. This was done by ammonium sulphate precipitation (to 50% saturation) of the serum (I ml) followed by desalting on a PD-10 gel filtration column (Pharmacia). The peak protein fraction was then passed down an anion exchange column (MonoQ; Pharmacia) equilibrated in 50 mM Tris/HCI, pH 8-0, the material passing unretarded through the column being pure IgG. After elution with a 0-0-5 M linear gradient of NaCl, the IgA-containing fractions were passed down a small column of Jacalin-Sepharose (Vector Labs, Burlingame, CA) equilibrated in PBS (phosphate-buffered saline, 8 5 mm sodium phosphate/ 0-15 M NaCl, pH 7-1). Pure IgAI was eluted from this column with 0-8 M D +galactose. Analysis of the purified IgG or IgA by gel filtration on Superose 6 failed to detect the presence of aggregated anti-gliadin antibodies.
Isolation ofpolymorphonuclear neutrophils Neutrophils were isolated from heparinized blood from healthy volunteers by density centrifugation by the method of English & Anderson2' as described by Albechtsen, Yeaman & Kerr.22 The cells were washed in PBS before being counted and analysed on a Coulter Counter. The preparation typically contained less than 1% and 10% contaminating lymphocytes and red blood cells, respectively. SDS-PAGE and immunoblotting SDS-PAGE was performed according to Laemmli23 using 515% linear gradient gels to achieve separation. Proteins were visualized with Coomassie Brilliant Blue R-250 (0 5% in 40% methanol/10% acetic acid). Transfer of separated proteins to nitrocellulose and subsequent immunoblotting was carried out according to Towbin, Staehelin & Gordon.24 Briefly, the nitrocellulose containing the blotted protein was cut into strips and blocked for 1 hr in 5% skimmed milk powder/PBS. The strips were then developed with sera or purified immunoglobulin, diluted in the same buffer, for a further hour before being washed in PBS and incubated with Sigma IgA- or IgG-specific alkaline phosphatase conjugate (1/500 dilution in 5% milk powder/PBS). The blots were developed using 5-bromo-4-
chloro-3-indolyl phosphate (5BCIP). ELISA for IgA and IgG anti-gliadin The ELISA used for the detection of antibodies to gliadin was modified from that described by Volta et al.7 Microtitre plates were coated with 150yl of a crude gliadin preparation (Sigma) at a concentration of 0-2 mg/ml in 0 005 M carbonate buffer, pH 10-2. After incubation overnight at room temperature the plates were washed three times in 0-1% Triton/PBS and blotted dry. The sera or purified antibodies were diluted in 5% normal sheep serum (NSS) in 0-1% Triton/PBS and 100 p1 added to the gliadin-coated wells for 1 hr at 37°. The wells were then washed a further three times in the same Triton buffer and alkaline phosphatase-conjugated goat antibody specific for either human IgA or IgG (Sigma, St Louis, MO), was added at a dilution of 1/1000 in 0-1% bovine serum albumin (BSA) in 0-1%
IgA
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Figure 1. Detection of anti-gliadin antibodies in the sera of patients with coeliac disease. Serum from 17 coeliac patients together with pooled normal serum (N) was assayed at a 1/200 dilution for IgA and IgG antigliadin antibodies by ELISA as described in the Materials and Methods. The OD"'" is indicated. The horizontal line on each graph shows 2-5 x the value given by pooled normal serum (solid bar).
Triton/PBS. After a further incubation of 1 hr at 370 the wells were washed as before and developed using p-nitrophenylphosphate (2 mg/ml) in 0 1 M sodium bicarbonate buffer, pH 10-2, containing I mM MgCl2. The absorbance at 405 nm was measured after 30 min. Assays were carried out in duplicate and mean values taken. Results were compared to those obtained with a pooled normal serum control. The intra-assay variation was less than 5%. Interassay variation using the same reagents but carried out on different days was less than 6%. Chemiluminescence studies Immunoglobulins bound to wells. These studies were performed on a Dynatech Microlite ML1000 luminometer using chemiluminescence microtitre plates (Dynatech, Billinghurst, Sussex, U.K.) by a method described previously.'8 IgA and IgG complexed to gliadin. These studies used a modification of the above method. Wells were coated with crude gliadin (Sigma) at a concentration of 0-2 mg/ml in 0-005 M carbonate buffer, pH 10-2, by incubating 150 yl of the solution overnight at room temperature. After washing three times in PBS, 100 pl of either heat-treated sera (1/200 dilution in 0-1% BSA/PBS) or IgA or IgG preparations, diluted appropriately in the same buffer, were added to the wells -in triplicate and incubated at 37° for 2 hr. After washing the wells several times in PBS, lucigenin (100 pl, 0-1 mg/ml; Sigma) in HBSS (Gibco, Grand Island, NY)/BSA (1 mg/ml; Sigma) was added followed by neutrophils (50 p1 of I x 106/ml) in HBSS/BSA. Chemiluminescence was measured at regular intervals for I hr. RESULTS Measurements of IgA and IgG antibodies against gliadin in the sera of coeliac patients
Sera from 17 coeliac patients were assayed for anti-gliadin activity. The level of these antibodies having IgA and IgG isotype was determined by an ELISA specific for each. The results of these assays, which include control sera, are presented
493
Anti-gliadin antibodies in coeliac disease N
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Figure 2. Neutrophil chemiluminescence induced by binding of antigliadin antibodies to surface-bound gliadin. Purified neutrophils were added to gliadin-coated mictotitre plates which had been incubated with heat-treated coeliac sera at a 1/200 dilution. Respiratory bursts were elicited by serum 1 (-), 2 (-), 3 (-) and 5 (*) as measured by lucigeninenhanced chemiluminescence. No such bursts were observed with serum 11 (A), 15 (0), 4 (0), 7 (>) or pooled normal serum (+) at the same dilution.
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Figure 4. Detection of anti-gliadin antibodies by immunoblotting. Immunoblotting was performed on crude gliadin which had been separated into its different components by SDS-PAGE on a 5-15% gradient gel. Pooled normal serum (N) or coeliac sera 1, 2, 3, 4, 5 or 6 were used at 1/25 dilutions and developed for IgA and IgG anti-gliadin antibodies as indicated (A and G). IgA and IgG purified from serum 1 (lanes a and b, respectively) or an equivalent amount of IgA and IgG isolated from pooled normal serum (lanes c and d, respectively) were also blotted and developed using the appropriate conjugate (A or G). Lanes e and f were blotted with the dilution buffer only before being developed with IgA (A) or IgG (G)-specific conjugate. The gel strip beside the immunoblot indicates the protein bands picked up by Coomassie staining of the gliadin preparation run on the same the SDS gel.
After incubating the plates as described in the Materials and Methods, chemiluminescence bursts were observed after addition of neutrophils (Fig. 2). The two sera with the highest levels of IgA anti-gliadin (sera 1 and 2) gave the greatest respiratory burst. The size of the burst correlated well with the IgA antigliadin titres and but not with the titres of IgG anti-gliadin (Fig. 3). Serum 15, which had the same IgG titre as 1 but lacked detectable IgA, gave a negligible burst at this dilution. Isolation of IgAl and IgG with specificity for gliadin from coeliac
Figure 3. The correlation between IgA or IgG anti-gliadin antibody levels and induction of neutrophil chemiluminescence. The maximum chemiluminescence (Clmas) induced in neutrophils by gliadin-coated microtitre plates incubated with a 1/200 dilution of heat-treated coeliac sera is plotted against the amount of IgA (-) or IgG (0) anti-gliadin antibody measured by ELISA (OD405).
serum
in Fig. 1. When IgA anti-gliadin was determined, eight out of the 17 sera were positive, having an OD405 which was greater than 2-5 times that for pooled normal serum at 1/200 dilution. For IgG anti-gliadin estimations, measured at the same dilution, the number of positive sera was 12. Sera that were positive for IgA anti-gliadin were always positive for the IgG antibody, although several IgA-negative sera had positive IgG titres. None of the sera was positive for the IgA antibody alone.
IgA I and IgG were isolated from I ml of the coeliac serum I as described in the Materials and Methods. The purity of the proteins was confirmed by SDS-PAGE and radial immunodiffusion (RID). Both the IgA and IgG showed specificity towards gliadin as determined by ELISA and immunoblotting. Gliadin antibodies in the IgAI pool (II ml, 0-08 mg/ml by RID) were detected at a titre of 1/100 (0-0008 mg/ml). Similarly, antibodies specific for gliadin in the IgG pool (6 ml, 1-3 mg/ml by A280) were detected at a 1/1000 dilution (0-0013 mg/ml). This suggested that in the serum the same percentage of the total IgA and IgG was specific for gliadin, i.e. (11 x 0-008) mg IgA and (6 x 0-0013) mg IgG, and implied that the total amount of IgG specific for gliadin was some 7-8/0 88 (8 6) times greater than the amount of IgA with the same specificity.
Neutrophil respiratory bursts induced by heat-treated coeliac sera Heat-treated sera from coeliacs with high or low anti-gliadin IgA and IgG titre or pooled normal serum, were added to gliadin-coated chemiluminescence plates at a 1/200 dilution.
The specificity of IgA and IgG anti-gliadin antibodies determined by immunoblotting To further characterize the binding of IgA and IgG antibodies to gliadin, immunoblotting was performed on the crude gliadin
W. W. Stewart & M. A. Kerr
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0*4 02 60 50 30 40 20 10 (min) Figure 5. Neutrophil chemiluminescence induced by purified IgA or IgG anti-gliadin antibodies. IgA (a) or IgG (b) purified from serum 1 were incubated on microtitre plates with (i) and without (ii) a gliadin coating. The IgA was used undiluted (A) or at dilutions of 1/2, 1/4 (0), 1/8 (A), 1/16 (0) and 1/32 (0); IgG was used undiluted (A) or at dilutions of 1/5 (U), 1/10 (0), 1/20 (A), 1/40 (0) and 1/80 (0). IgA and IgG isolated from pooled normal serum (-0-) was also used at a concentration equivalent to that of the undiluted immunoglobulins isolated from serum 1. Time
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IgAl 1:40 1:20 1:10 IgG 1:40 1:20 1:10 only' only IgG IgAI dilution dilution Figure 6. Co-operation between IgA and IgG anti-gliadin antibodies in the stimulation of neutrophil chemiluminescence. (a) IgA 1 anti-gliadin, diluted 1/4 (-) and 1/8 (A), were co-incubated on gliadin-coated plates with IgG anti-gliadin at 1/10, 1/20 and 1/40 dilutions. The open circles (0) indicate the respiratory burst induced by the IgG dilutions on their own. (b) IgG anti-gliadin was incubated at 1/5 dilution (0) in the presence of IgAl anti-gliadin diluted 1/10, 1/20 and 1/40. The open squares (0) indicate the respiratory burst induced by the IgA dilutions on their own. I
preparation using the purified IgAI and IgG pools as well as six sera with the highest titres of anti-gliadin antibodies (including serum 1) and pooled normal sera (Fig. 4). All sera were diluted 1/25. No IgA and only a trace of IgG anti-gliadin was detected in pooled normal serum. IgA anti-gliadin was detected to varying degrees in the coeliac sera, as was IgG antigliadin. No obvious pattern of bands was observed for the coeliac sera, with a range of protein bands being recognized between 35,000 and 70,000 MW by both IgA and IgG anti-
coeliac
gliadin. The specificity of the IgA I and IgG isolated from serum I (lanes a and b, respectively) agreed with the blotted whole serum. The IgG from this serum did differ slightly from the other sera in that it appeared to recognize bands mostly in the 35,00040,000 MW region. In agreement with the results of the ELISA, the level of IgA antibodies in the sera other than 1 and 2 appeared to be much less than the IgG antibodies.
Microtitre plates coated with gliadin were incubated with IgA and IgG isolated from coeliac serum 1 at different dilutions and then neutrophils were added (Fig. 5ai and aii). Chemiluminescence bursts were only observed with IgA I when used undiluted (80 yg/ml) or at a 1/2 dilution (40 yig/ml) and with IgG when used undiluted (1-26 mg/ml) or at 1/5 dilution (0-252 mg/ml, only a slight burst). No chemiluminescence bursts were obtained when the same concentrations of IgA 1 and IgG purified from normal serum were incubated with blocked gliadin-coated plates. The binding of the anti-gliadin antibodies was therefore shown to be antigen-specific. The same concentrations of the IgA and IgG pools were also incubated for the same length of time in chemiluminescence wells which had not been coated with gliadin (Fig. Saii, bii). Under these conditions, the immunoglobulins bind non-specifically to the plates. For any concentration, the chemiluminescent burst elicited by bound IgA was always greater than for IgG, consistent with our previous findings. If the plates were blocked with I mg/ml BSA prior to addition of the immunoglobulin, no immunoglobulin bound and no chemiluminescence burst was elicited.'8
Anti-gliadin antibodies in coeliac disease The Cl/rn, for undiluted IgA on gliadin-coated plates (0 4) the same as the C/max induced by a 1/16 dilution (5 yg/ml) incubated for the same length of time on a uncoated well. Similarly, the Clmax for undiluted IgG on gliadin (0-2) was approximately equivalent to a 1/160 dilution (8 pg/ml) on uncoated wells. Since the response to antigen-coated plates was much less than that for plates coated non-specifically with immunoglobulin of the same isotype it can be concluded that it is the amount of bound antibody which is the limiting factor on the gliadin-coated plates. The response to either purified IgG or IgA anti-gliadin antibody was in fact less than expected. In the whole serum a dilution of 200-fold which would give an IgA concentration of 5 pg/ml and IgG of 50 Mg/ml still gave a major response on gliadin-coated plates. It is clear that activity is lost upon fractionation of the serum. However, ELISA showed no significant loss of either IgG or IgA anti-gliadin antibody.
was
Co-operation between Fc receptors in response to IgA and IgG anti-gliadin antibodies To investigate the possibility that this apparent loss of activity was due to a co-operative effect of the two classes of antibody in the whole serum, IgA1, purified from coeliac serum 1, was incubated at different concentrations with gliadin-coated plates in the presence and absence of different concentrations of IgG, purified from the same serum. In previous experiments, these concentrations of the IgG pool had been shown not to elicit a respiratory burst and this was again found to be the case [Fig. 6a (--- )]. A clear enhancement of the burst elicited by each concentration of the IgAI was observed when the IgG was coincubated in the gliadin coated plates (Fig. 6a). Similarly, when the IgG anti-gliadin pool was incubated on gliadin plates at a 1/5 dilution, co-incubation with IgA anti-gliadin, at dilutions which on their own did not induce a respiratory burst, resulted in enhancement of the IgG response (Fig. 6b). DISCUSSION anti-gliadin antibodies (AGA) are of circulating levels Elevated characteristic of coeliac disease, the amount of antibody correlating with disease activity."' Several studies have examined the usefulness of these antibodies as a screen for active coeliac disease and an indicator for small intestinal biopsy.8 25 It was concluded by several investigators that IgA (AGA) rather than IgG (AGA) has the greater diagnostic value."7 This conclusion was supported by the detection of 'false positive' antibodies to gliadin, measured by ELISA and immunofluorescence which were all of the IgG isotype.7 In our study 70% and 47% of the coeliac sera from untreated patients were positive for IgG and IgA AGA, respectively. The relative titres of IgA and IgG AGA have been reported to vary in each coeliac patient4"'I and the data presented in this study are in agreement with this, although it does not support the observation that the specificities of IgA and IgG AGA in each patient are identical." By comparing blots of six of our coeliac sera for IgA and IgG AGA specificity, differences in the pattern of bands recognized for each patient were observed for the two isotypes. However, if the blots for the six different patients were compared, a similar range of discrete bands was recognized by both IgA and IgG AGA. It has previously been reported that coeliac sera exhibited antibody binding to discrete groups of gliadin bands. Kieffer et
495
al.9 found that IgG AGA exhibited high titres for a and 3 fractions of gliadin separated on ion-exchange chromatography. Friis et al.14 observed that the specificity of IgG AGA in patients with untreated coeliac disease was towards polypeptides in the y-fraction, having molecular weights of 35,000 and 45,000 with antibodies against the a and P fraction being secondary to mucosal damage. We have reported a range of IgG specificity between 35,000 and 75,000 MW for 5/6 of the blotted sera with the pattern of blotting for the sixth sera (1) being concentrated between 35,000 and 45,000 MW. The IgA AGA from the same serum did not show this pattern. Only the two sera with highest IgA AGA titres (1 and 2) showed intense IgA bands, ranging from 35,000 to 75,000 MW when blotted. Our results are therefore consistent with the range of specificities reported in other publications suggesting that no pattern of gliadin specificity is characteristic of coeliac disease. The ability of immune complexes to trigger degranulation and a respiratory burst in neutrophils is well documented, the responses being brought about via receptors for the Fc portion of immunoglobulins which are present on the cell surface.26 28 Interaction of IgA and IgG Fc domains with their receptors on the neutrophil surface results in the release of granular enzymes into the surrounding environment which in vivo would lead to tissue damage.'8 29 IgA and IgG complexes also have the ability to elicit a respiratory burst in neutrophils with the resulting release of highly reactive superoxide creating the potential for further damage to the local tissue.'8'30 We have previously shown that the ability of different immunoglobulins to induce this respiratory burst can be measured by coating the proteins to microtitre plates and following the increase in lucigenin-enhanced chemiluminescence after addition of purified neutrophils.'8 In applying this technique to the specific IgA 1 and IgG antibodies purified from the coeliac serum we were able to determine that both were capable of producing a respiratory burst in neutrophils at equivalent concentrations. The burst for IgA 1 was always greater than the burst for an equivalent amount of IgG, indicating greater potential for tissue damage. This is in spite of the fact that the predominant subclasses of IgG anti-gliadin antibodies have been reported to be IgGl and IgG3, the subclasses binding with highest affinity to the neutrophil Fcy
receptors.3' When whole coeliac serum was used to stimulate neutrophils, a 1/200 dilution of heat-treated serum I produced a significant respiratory burst (Clmax 0-17), while significant bursts were only observed for the IgAl (Clmax 0-4) and IgG (Clmax 0-2) pools when used undiluted. The IgA I and IgG concentrations of these pools were estimated to contain 1/1 1 and 1/6 the concentrations in serum 1, respectively. Hence they would have to be diluted a further 20 and 30 times to have the same IgA and IgG content as the 1/200 dilution of serum 1. No chemiluminescence burst was observed when neutrophils were incubated with these dilutions of immunoglobulin. One possible explanation for the lower activity of the purified immunoglobulins was that in the serum the two were able to act in synergy. Our results show clearly that there is a synergistic effect between the two classes of antibody in stimulating the neutrophil chemiluminescent burst. The marked dependence of the size of the chemiluminescence burst on the IgA AGA titre must reflect this synergistic effect. The level of IgA AGA in sera I and 2 is apparently high enough to enable
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them to elicit a respiratory burst at a 1/200 dilution but is too low in the other sera, even though their IgG AGA titres are as high or indeed higher than sera 1 or 2. Co-operation between FcRot and FcRy has previously been demonstrated by Fanger, Goldstine & Shen.32 Secretory IgA has also been shown to be able to potentiate the effect of IgG in promoting antibodydependent cell cytotoxicity (ADCC) by human neutrophils, monocytes and lymphocytes.33 We have demonstrated the usefulness of chemiluminescence assays carried out in microplate format for the detection of specific antibodies with pathologic potential. These assays are as sensitive and simple to perform as many conventional immunoassays. Our results suggest that it might be useful to use the chemiluminescence assay described to screen coeliac patients for potentially harmful IgA anti-gliadin antibodies, althoug a more extensive clinical investigation using a large sample of coeliac sera is required to fully evaluate the assay. The pathological significance of the efficient stimulation of neutrophils by IgA anti-gliadin antibodies upon binding to surface-bound antigen is not clear. Coeliac disease is characterized by chronic inflammatory infiltrates rich in lymphocytes, containing cells of the monocyte/macrophage lineage but poor in neutrophils. Monteiro et al.'9 have clearly demonstrated the presence of an identical IgA receptor on neutrophils and monocytes; the role of this monocyte receptor therefore warrants investigation. The role of the neutrophil might, however, be more important in the condition associated with coeliac disease termed dermatitis herpetiformis (DH). A high percentage (85%) of DH patients have associated gluten-sensitive enteropathy and skin lesions associated with the disease have been shown to contain granular deposits of IgA13435 at the junction of the dermis and the epidermis. A similar high percentage of patients with coeliac disease show some skin involvement36. IgA antigliadin antibodies are present in these patients and immunoblotting analysis has shown them to be of similar specificity to those found in coeliac sera. The involvement of these antibodies in the granular deposits found in DH patients has been implied from the improvement in skin lesions brought about by a gluten-free diet. DH lesions are characterized by an intense neutrophil infiltrate in the papillae. IgA deposits are frequently heavier in adjacent, uninvolved skin suggesting loss in the lesions due to phagocytosis.
ACKNOWLEDGMENTS This work was supported by a project grant from the Arthritis and Rheumatism council and by funds from the Cunningham Trust for purchase of the luminometer. We are grateful to Mr R. Fawkes and S. Macpherson for assistance in the preparation of the figures and to Mrs H. Cowper for preparing the manuscript.
REFERENCES 1. BAKER P.G. & READ A.E. (1976) Oats and barley toxicity in coeliac patients. Postgrad. Med. J. 52, 264. 2. ANAND B.S., PIRIS J. & TRUELOVE S.C. (1978) The role of various cereals in coeliac disease. Quart. J. Med. 185, 101. 3. FRIIS S.U. & SCHAFER-NIELSEN C. (1985) Separation of gliadin at pH 3.1 in a polyacrylamide gel suitable for blotting procedures. J. Biochem. Biophys. Meth. 10, 301.
4. UNSWORTH D.J., KIEFFER M., HOLBORow E.J., COOMBS R.R.A. & WALKER-SMITH J.A. (1981) IgA anti-gliadin antibodies in coeliac disease. Clin. exp. Immunol. 46, 286. 5. SAVILAHTI E., PERKKIO M., KALIMO K., VIANDER M., VAINIO E. &
REUNALA T. (1983) IgA antigliadin antibodies: a marker of mucosal
damage in childhood coeliac disease. Lancet, i, 320. 6. SCOTT H., FAUSA O., EK J. & BRANDTZAEG P. (1984) Immune response patterns in coeliac disease. Serum antibodies to dietary antigens measured by an enzyme linked immunosorbent assay (ELISA). Clin. exp. Immunol. 57, 25. 7. VOLTA U., LENZI M., LAZZARI R., CASSANI F., COLLINA A., BIANCHI F.B. & Pisi E. (1985) Antibodies to gliadin detected by immuno-
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