IgA-Gliadin antibodies, IgA-containing circulating immune complexes ...

Nephrol Dial Transplant (1999) 14: 1875–1880

Nephrology Dialysis Transplantation

Original Article

IgA-Gliadin antibodies, IgA-containing circulating immune complexes, and IgA glomerular deposits in wasting marmoset syndrome Carsten Schroeder, Awad A. Osman1, Dirk Roggenbuck2, and Thomas Mothes1 German Primate Centre, Go¨ttingen, 1Institute of Clinical Chemistry and Pathobiochemistry of the University of Leipzig, Leipzig and 2Dr Fooke Laboratories, Neuss, Germany

Abstract Background. Marmosets in captivity are highly susceptible to wasting marmoset syndrome ( WMS ), the aetiology of which is still not fully determined. Methods. The level of IgA-gliadin antibodies (IgAAGA), of IgA-containing circulating immune complexes (IgA-CIC ), and the degree of glomerular IgA deposits were compared between marmosets suffering from WMS and animals not affected by the disorder. Results. Both IgA-AGA and IgA-CIC were demonstrable in all groups of monkeys investigated. IgAAGA and IgA-CIC were significantly higher in monkeys with WMS than in non-affected animals. There was a significant correlation between the glomerular IgA-deposition and titre of IgA-AGA. The group of marmosets strongly positive for glomerular IgA deposits comprised significantly more animals suffering from WMS than the group without deposits. In the diet of the animals a considerable amount of gliadinlike cereal proteins was assayed. Conclusions. There are several parallels between the human disorders (coeliac disease and IgA-nephropathy/Berger∞s disease) and the changes observed in WMS. It should be further investigated if WMS in marmosets is a suitable animal model for both human diseases. Key words: Wasting marmoset syndrome, gliadin antibodies, immune complexes, nephropathy

Introduction Marmosets (Callitrichidae) are small New World monkeys well suited for laboratory research in biomedicine and behavioural science due to their close relationship to man and their low space and food requirements. In captivity, however, the animals are very susceptible to Correspondence and offprint requests to: Prof. Dr Thomas Mothes, Institute of Clinical Chemistry and Pathobiochemistry of the University of Leipzig, Paul-List-Str. 13–15, D-04103 Leipzig, Germany.

wasting marmoset syndrome ( WMS), characterized by failure to thrive, generalized weakness, often progressing to death [1–3]. Chronic enteritis [4,5], diarrhoea [2], colon carcinoma [6 ], and glomerulopathy [7] with striking similarities to IgA-nephropathy in humans [8] have been described in various members of the callitrichidae. The diet of marmosets in captivity is considered as one possible cause for enteritis and WMS [4,9]. Recently, a beneficial effect of a gliadin-free diet on animals with WMS was reported [10]. In susceptible humans, gliadins from wheat and related proteins from other cereals represent the aetiological factor for coeliac disease [11,12] with damage of the small intestinal mucosa, diarrhoea, increased anti-gliadin (AGA) and connective tissue antibodies [13], and frequently enhanced circulating immune complexes (CIC ) [14]. The antibodies involved include the IgA class. The incidence of glomerular IgA deposits in humans with coeliac disease is increased [15]. Furthermore, in man a relation between gliadins and primary IgA nephropathy is considered [16,17]. In this disorder, IgA is deposited in the glomerular mesangium, which was found positive for food antigens in a significant fraction of patients [18], and frequently IgA-CIC and antibodies against dietary antigens including AGA are present in the serum [19–21]. Intestinal permeability is often enhanced [22]. In a group of patients with elevated IgA-CIC and food antibodies, gluten-free diet decreased in most cases CIC and antibody levels and reduced proteinuria and microscopic haematuria [17,23,24]. As yet there is no evidence for a causal involvement of gliadins in WMS and if IgA-AGA and IgA-CIC occur in marmosets with WMS. In this study, IgAAGA and IgA-CIC class in marmoset with and without WMS were measured and IgA-AGA correlated to the degree of renal IgA deposition in marmosets.

Materials and methods Source of sera Sixty marmosets (Callithrix jacchus) from the German Primate Centre (GPC, Go¨ttingen, Germany), eight from

© 1999 European Renal Association–European Dialysis and Transplant Association

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Bayer AG ( Wuppertal, Germany), and four from the Institute of Pharmacology and Toxicology of the Free University (Berlin) were investigated. The animals of both sexes ranged in age from 6 months to 8 years. Animals from the GPC were fed on Ssniff marmoset pellets (Ssniff, Soest, Germany) ad libitum, supplemented by rusks, crisp breads, pasta, oat flakes, fruits, vegetables, milk products, and animal protein (eggs, insects, poultry). Animals from Bayer AG were fed on Mazuri pellets (Code 808–616, SDS, Essex, UK ) ad libitum, supplemented by oat flakes, milk products, almonds, raisins, and bananas. The animals from the Free University Berlin were fed on Altromin marmoset pellets (Code 0633, Altromin, Lage, Germany) ad libitum, supplemented with fruits, oat flakes, eggs, and milk products. The diagnosis of WMS was made according to history (recurrent diarrhoea), clinical findings (weight loss, dehydration, poor hair), and necropsy reports (chronic enteritis). Marmosets having developed typical signs of WMS were classified as ‘wasters’. For comparison, three rhesus monkeys (Macaca mulatta), three cynomolgus monkeys (Macaca fascicularis), two baboons (Papio hamadryas), one mandrill (Mandrillus sphinx), and one langur (Presbytis entella) were included, all from GPC. These Old World monkeys ranged in age from 10 to 25 years. They were fed on Ssniff-Pellets, supplemented by fruits, vegetables, and milk products. Venous blood was collected immediately before sacrificing the animals and sera were stored at −20°C until use.

Anti-IgA antibodies Since no anti-marmoset and anti-Old World monkey IgA antibodies were available, rabbit anti-human IgA, a-chain specific (Dako, Hamburg, Germany) was tested for reactivity with monkey IgA using agarose immunodiffusion techniques [25]. Specificity of rabbit antibodies for IgA indicated by the supplier was checked by comparison with reactivity of rabbit anti-human IgM and rabbit anti-human IgG (Dako) in immune electrophoresis [26 ]. Marmoset immunoglobulins precipitated by rabbit anti-human IgA were comparable in position with the respective human immunoglobulins and clearly different from those recognized by rabbit anti-human IgM and rabbit anti-human IgG. There was no cross reaction of rabbit antibodies against IgA with IgM and IgG.

Determination of IgA-AGA A modification of a method previously described was applied [27]. In brief, microplates (Maxisorb, Nunc, WiesbadenBiebrich, Germany) were coated over night with a solution containing 50 mg gliadin/ml 70% ethanol, incubated 1 h at 37°C with 100 ml monkey serum (15100) and 2 h at 37°C with 100 ml rabbit anti-human IgA conjugated with peroxidase (1:2000) (Dako, Hamburg, Germany). Colour was developed by use of Seramun Blue/TMB substrate solution (Seramun, Dolgenbrodt, Germany) and optical density (OD) was read at 450 nm. A calibration curve was constructed using the positive control provided by Labmaster Ltd ( Turku, Finland ). Gliadin was obtained from wheat variety ‘Kanzler’ [28].

Determination of IgA-CIC Maxisorb microplates (Nunc) were coated over night with a solution containing 10 mg bovine conglutinin (Dr Fooke, Neuss, Germany)/ml veronal-buffered saline ( VBS, pH 7.2).

C. Schroeder et al.

After washing (0.1% Tween 20 in VBS) the plates were incubated 1 h at 37°C with monkey serum (1510), washed again, and incubated 1 h at 37°C with anti-human IgA conjugated with peroxidase (152000) (Dako) and colour was developed as described above. Suitability of conglutinin based assays for detection of IgA-CIC was recently shown [17].

Estimation of glomerular immunoglobulin-deposits For IgA-deposits, a modification of the immunofluorescence technique described previously [7] was used. Fresh renal tissues of marmosets from GPC obtained during necropsy were snap frozen in liquid nitrogen. Cryostat sections of these tissues were stained with FITC labelled polyclonal rabbit anti-human IgA (Dako) (1520 in phosphate-buffered saline). IgM-deposits were estimated according to [7]. For negative control, reactivity of rabbit anti-human IgG, cchain-specific (Dako) was checked.

Determination of endomysium antibodies These antibodies were assayed by indirect immunofluorescence methods using sections of monkey oesophagus (Mast Diagnostika, Reinfeld, Germany) [29]. For detection of bound monkey IgA, anti-human IgA conjugated with FITC (Dako) (1520 in phosphate-buffered saline) was used.

Determination of gliadin Pellets were milled and 100 mg dry powder were extracted with 5 ml 60% ethanol. Then 50 ml of the extract (diluted 151000) was incubated in the wells of microtitre plates (Flow, No. 76-381-04) over night. Gliadins and other ethanol soluble proteins coated to the wells were fixed with 100 ml formaldehyde (10%). Wells were then washed with buffer A (50 mM Tris, 150 mM NaCl, 5 mM NaN /l, 0.05% (w/v) 3 Tween 20, pH 10.2), blocked with buffer B (buffer A containing 0.5% Tween 20 and 10 g bovine serum albumin/l ), rinsed again with buffer A, and incubated for 1 h with 100 ml antigliadin serum (15400) obtained from rabbits after immunization with gliadin isolated from flour of wheat var. ‘Kanzler’. After incubation, wells were washed with buffer A and incubated 2 h with 100 ml pig anti-rabbit immunoglobulins conjugated with peroxidase (Dako, 152000). Wells were rinsed again with buffer A, and colour was developed by adding a solution containing o-phenylene diamine. The reaction was stopped by adding sulphuric acid and OD was read at 492 nm. For calibration of the assay, gliadin extracted from wheat cultivar Kanzler was solubilized in 60% ethanol and diluted to concentrations between 10 and 800 ng/ml.

Expression of results and statistical evaluation Results are presented in box plots extending from 25th to 75th percentiles and showing medians (solid lines). Tenth and 90th percentiles are indicated by capped bars. All data outside of these regions are presented. Statistical differences between the different monkey groups (P