Increased intestinal intra-epithelial T lymphocytes in primary glomerulonephritis
A role of oral tolerance breakdown in the pathophysiology of human primary glomerulonephritides?
Guy Rostoker1,,
Jean-Charles Delchier2 and
Marie-Thérèse Chaumette3
1 Service de Néphrologie et de Dialyse, Hôpital Claude Galien, Quincy sous Sénart;
2 Service d'Hépatologie et de Gastro-entérologie, and
3 Departement de Pathologie, Centre Hospitalier Universitaire Henri Mondor, Créteil, France
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Abstract
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Background. There is increasing evidence that some organ-specific and generalized autoimmune diseases in humans might be related to a breakdown of oral tolerance. We explored this hypothesis in human primary glomerulonephritides. We prospectively counted intraepithelial T lymphocytes in the duodenal mucosa (as a marker of rupture of oral tolerance), together with IgA1 and IgA2 mucosal plasma cells, in patients with primary glomerulonephritides.
Methods. We investigated eight adults with immune-complex glomerulopathy (membranous nephropathy+membranoproliferative glomerulonephritis), 16 adults with an idiopathic nephrotic syndrome, and 25 adults with IgA nephropathy. Patients with glomerulonephritides were compared to two control groups: group 1 consisted of nine healthy adults; group 2 comprised five adults with coeliac disease before dietary gluten withdrawal or during a clinical relapse related to gluten ingestion. (The latter disease is associated with increased numbers of intraepithelial T lymphocytes, and a breakdown of oral tolerance to gliadins is involved in the pathogenesis of coeliac disease). Duodenal fibroscopy was performed under neuroleptanalgesia. Four to six endoscopic biopsy specimens were taken from the second duodenum. Intraepithelial T lymphocytes were blindly counted on paraffin sections stained with haemateineosinsaffron (HES), within the epithelium of a villus in a zone with at least 100 cells. Mucosal IgA1 and IgA2 plasma cells were blindly counted in a mucosal tissue unit by using specific mouse monoclonal antibodies directed against IgA1 and IgA2, with alkaline phosphatase anti-alkaline phosphatase (APAAP) revelation. As values were not normally distributed, we used non-parametric analysis of variance with the KruskalWallis test, and compared median values by using the non-parametric MannWhitney test.
Results. Intraepithelial T lymphocytes were significantly more abundant in patients with primary glomerulonephritides and coeliac disease than in healthy controls (P<0.0001 in the KruskalWallis test): healthy controls, median 11 (range 4.6516); immune complex glomerulopathy, 27.45*** (1593); idiopathic nephrotic syndrome, 16.5** (926.5); IgA nephropathy, 26.10*** (11.347.5); coeliac disease, 55*** (2080) (*P<0.05; **P<0.01; ***P<0.005, MannWhitney test). No difference was found in the number of duodenal IgA1 and IgA2 plasma cells between controls and patients with primary glomerulonephritides. IgA1 and IgA2 plasma cells were increased in patients with coeliac disease.
Conclusion. The significant increase in intestinal intraepithelial T lymphocytes in primary glomerulonephritides suggests a pathophysiological role of oral tolerance breakdown.
Keywords: idiopathic nephrotic syndrome; IgA nephropathy; intra-epithelial T lymphocytes; membranoproliferative glomerulonephritis; membranous nephropathy; oral tolerance
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Introduction
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Oral tolerance is a state of specific immunological unresponsiveness to food and microbial antigens that are encountered in the gut and which, in other circumstances, are capable of inducing an active immune response [1]. This unresponsiveness is related to age and genetic background, the nature and amount of the antigens, the nature of the intestinal flora, and antigen-presenting cells; it is mediated by specific T suppressor cells secreting TGF-ß and IL4, clonal anergy and, possibly, secretory IgA and anti-idiotype antibodies [1]. Breakdown of oral tolerance has been implicated in the pathophysiology of coeliac disease and food-sensitive enteropathies [1]. Histological studies in these settings and in experimental models show increased T lymphocyte infiltration of the gut epithelium [1]. Recently, patients with juvenile rheumatoid arthritis were successfully treated with oral administration of chicken type II collagen [2]. There is increasing evidence that some organ-specific and generalized autoimmune diseases in humans might be related to a breakdown of oral tolerance [13].
IgA nephropathy (IgAGN) is characterized by the presence of IgA and C3 in the mesangium, and is the commonest form of primary glomerulonephritis world-wide; it is thought to be an IgA immune-complex nephritis [4]. The idiopathic nephrotic syndrome (INS) consists of minimal-change disease, IgM nephropathy, and segmental sclerosis, and is believed to be a T cell disorder with excessive secretion of various uncloned cytokines [4]. Membranous nephropathy (MN) and membranoproliferative glomerulonephritis (MPGN) are thought to be mediated by IgG immune complex formation either in situ in glomeruli or in the blood stream [4]. In these primary glomerulonephritides, the efficacy of oligo-antigenic diets and specific food-antigen avoidance, together with the observed increase in intestinal permeability and in serum IgA1 reactivity to food and air-borne antigens and self-antigens, and the increased levels of salivary and serum secretory IgA, have raised the possibility that the mucosal immune system plays a part in their pathophysiology [512]. Furthermore, experimental IgAGN was recently induced in mice by oral immunization with ovalbumin plus cyclophosphamide, an agent able to disrupt oral tolerance [13]. In this study, we tested the hypothesis that a breakdown of oral tolerance might be involved in the pathogenesis of human primary glomerulonephritides, by counting intraepithelial T lymphocytes in the duodenal mucosa of these patients, together with IgA1 and IgA2 mucosal plasma cells.
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Subjects and methods
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Patients and controls
We prospectively studied eight adults with immune complex glomerulopathy (IC-GN) (MN+MPGN), 16 adults with INS, and 25 adults with IgA nephropathy. Patients with IgA nephropathy were divided into three groups according to the clinical subtype: recurrent macroscopic haematuria, mild histological lesions and minor or no urinary anomalies between episodes of macroscopic haematuria and normal renal function (group I; n=6); permanent proteinuria (>0.5 g/24 h), microscopic haematuria, normal or near-normal renal function and moderate histological lesions (group II; n=8); and heavy proteinuria, severe histological lesions, and a low glomerular filtration rate (group III; n=11). The renal disorders were diagnosed according to usual histological criteria and the patients were studied in an active phase of the disease (during a nephrotic period or relapse in INS and IC-GN; in the month following macroscopic haematuria or during a proteinuric period associated with microscopic haematuria in IgA-GN), before treatment with corticosteroids and immunosuppressive drugs (INS, IC-GN), angiotensin-converting-enzyme inhibitors or polyclonal immunoglobulins, or suppression of infectious foci (IgAGN) [4]. As abnormal numbers of intestinal intraepithelial T lymphocytes and of IgA1 and IgA2 plasma cells have been reported in some diseases, we also studied two control groups: group 1 consisted of nine healthy adults matched for age and sex with patients, who had functional upper abdominal complaints but no organic disease or renal anomalies (normal renal function, no proteinuria, and normal urinary leukocyte and erythrocyte counts); group 2 was composed of five adult patients with coeliac disease before dietary gluten withdrawal or during a clinical relapse related to gluten ingestion (breakdown of oral gliadin tolerance has been demonstrated in coeliac sprue). None of the patients or controls was taking non-steroidal anti-inflammatory drugs or were alcohol abusers. All the patients and controls gave informed consent to the study, which received local ethics committee approval.
Duodenal fibroscopy was performed under neuroleptanalgesia. Four to six endoscopic biopsy specimens were taken in the second duodenum. Half the tissue specimens were fixed in Bouin's fluid and embedded in paraffin, while the others were snap-frozen in liquid nitrogen and stored at -70°C until cryostat preparation of 2-µm sections. Paraffin sections were stained with haemateineosinsaffron (HES).
Intraepithelial T lymphocytes (IEL) were blindly counted by one of us (M-TC) on HES-stained paraffin sections within the epithelium of a villus in a zone with at least 100 cells, according to Otto with slight modifications [14]; results were expressed as the number of IEL divided by the sum of the number of epithelial cells and IEL nuclei. A minimum of 300 cells were counted in each sample.
Mucosal IgA1 and IgA2 plasma cells were blindly counted by one of us (M-TC) in a mucosal tissue unit, as described by Westberg [15], with modifications; the unit contained the axis of a villus and the underlying intercryptic chorion. A few sections were not cut perpendicular to the mucosa, ruling out the use of this technique; in these cases IgA1 and IgA2 plasma cells were counted on a linear theoretical surface. Immunohistochemical studies were based on mouse monoclonal antibodies directed against IgA1 (clone 1-155-1) and IgA2 (clone 14-3-26) at a dilution of 1:100, obtained from Becton Dickinson (USA), and revealed with the Dakopatts alkaline phosphatase anti-alkaline phosphatase (APAAP) kit (Dakopatts, Denmark) [16]. The specificity of these monoclonal antibodies for IgA subclasses has been demonstrated elsewhere [17]. Alkaline phosphatase activity was determined after incubation in fast Red TR (1 mg/ml; Sigma, USA) and naphthol AS-TR phosphate solution (0.2 mg/ml, Sigma), containing levamisole (0.24 g/ml, Sigma) to block endogenous alkaline phosphatase activity.
As values were not normally distributed, we used non-parametric analysis of variance with the KruskalWallis test (GraphPad Instat, USA) [18]. In case of significant differences, parameters were compared to those of healthy controls, by using the two-tailed non-parametric MannWhitney test (GraphPad Instat, USA) [19]. Variables are expressed as medians and ranges. Differences with P values lower than 0.05 were considered significant [18].
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Results
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With the exception of those from patients with coeliac disease, none of the samples showed villous atrophy. Intraepithelial T lymphocyte numbers were significantly increased in patients with primary glomerulonephritides and in those with coeliac disease (P<0.0001, KruskalWallis test) (Table 1
) (Figure 1
). Interestingly, all the clinical subtypes of IgA nephropathy showed the same order of significant increase in intraepithelial T lymphocytes relative to the healthy controls (Table 1
) (Figure 1
).
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Table 1. Intraepithelial T lymphocytes and IgA1 and IgA2 plasma cells in duodenal mucosa of patients with primary glomerulonephritis
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Fig. 1. Intra-epithelial T lymphocytes (IEL) in primary glomerulonephritides. ICGN, immune complex glomerulonephropathy; INS, idiopathic nephrotic syndrome; IgAGN, IgA nephropathy. Group I, recurrent macroscopic haematuria, mild histological lesions and minor or no urinary anomalies, and normal renal function; group II, permanent proteinuria (>0.5 g/24 h), microscopic haematuria, normal or near normal renal function, and moderate histological lesions; group III, heavy proteinuria, severe histological lesions, and decline in the glomerular filtration rate. Values are mean+SEM. **P<0.01, ***P<0.005, MannWhitney test.
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No difference was found in the number of IgA1 and IgA2 plasma cells between patients with primary glomerulonephritis and controls (Table 1
). IgA1 and IgA2 plasma cell numbers were significantly increased in patients with coeliac disease (P<0.001, MannWhitney test) (Table 1
). As IgA1 and IgA2 plasma cells were increased to the same degree, the ratio of IgA1 plasma cells divided by the sum of IgA1 and IgA2 plasma cells was unmodified in coeliac disease, whereas it was normal in primary glomerulonephritides (Table 1
).
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Discussion
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Intestinal intraepithelial lymphocytes belong to a unique T cell population interspersed between epithelial cells of both the small and large intestine. They are readily identifiable in paraffin-sections stained with HES [19,20]. They are located on the basolateral side of the epithelial layer, meaning that they would be exposed to a wide range of food and bacterial antigens [19,20]. A number of immunological roles have been proposed for intestinal intraepithelial T cells, including surveillance of the intestinal epithelial layer to detect and eliminate microbial pathogens (viruses, protozoa, and bacteria), removal of damaged and transformed epithelial cells, maintenance of epithelial integrity via growth factor secretion, and regulation of both local cellular and humoral immune responses [20]. Intestinal intraepithelial T lymphocytes are phenotypically heterogeneous. They can be classified into a number of subsets commonly distinguished on the basis of their expression
ß or 
T-cell antigen receptors and CD8 or CD4 co-receptors, and co-expression of accessory, co-stimulatory, adhesion, natural-killer-associated, and homing molecules [20,21]. Furthermore, strong phenotypical differences are observed between intraepithelial T cells isolated from the small and large intestines [20,21]. Intraepithelial T cells proliferate poorly in comparison to peripheral spleen or lymph node T cells, but have cytotoxic activity mediated both by perforin and granzyme release and the delivery of an apoptotic death signal via the Fas ligand; they also produce a wide array of cytokines (IL2, IL3, IL4, IL5, IL6, IFN
, lymphotactin) and have an oligoclonal TCR repertoire [20,21].
To our knowledge this is the first published study of intestinal intraepithelial T lymphocytes in human primary glomerulonephritides. The intraepithelial T lymphocyte counts in this study of adults with primary glomerulonephritides are validated by blinded evaluation, together with the comparison with healthy individuals and adults with coeliac disease. In this latter group, our results are in keeping with published data showing an increase in the number of intraepithelial T lymphocyte [19]. Immunochemical analysis of IgA subclass plasma cells in primary glomerulonephritis in this study is validated by blinded evaluation and by the fact that the results are similar to those in the literature on coeliac disease and IgA nephropathy (although studies of the latter disease involved few patients) [15,2224].
As intestinal intraepithelial T lymphocytes are phenotypically heterogeneous, it is tempting to postulate that the notion breakdown of oral tolerance may cover a wide range of clinical manifestations and pathogenetic mechanisms involving a peculiar phenotype of intestinal intraepithelial T cells.
Phenotypic studies of intraepithelial T lymphocytes in primary glomerulonephritides, aimed at characterizing T cell receptor expression, the oligoclonality of certain Vß genes, T cell differentiation antigens, and specific integrin expression [20,21], are required to identify the nature of the link between intraepithelial T lymphocyte infiltration and the type of glomerular disease and the increase in intestinal permeability shown in primary glomerulonephritides [9]. Such studies might shed light on the mechanisms leading to the J chain under-expression by duodenal IgA plasma cells of patients with IgA nephropathy [24]. Further studies are also required to distinguish intraepithelial T lymphocyte invasion due to hypersensitivity from that due to altered mucosal physiology resulting from glomerular disease or renal failure.
In conclusion, our results suggest a role of oral tolerance breakdown in the pathophysiology of primary glomerulonephritis. The triggering factor may be an infectious agent (a virus, bacterium, or parasite) or an environmental toxin. This may open new research avenues, and supports the use of aetiological treatments, especially new drugs able to restore oral tolerance, and dietary manipulations.
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Acknowledgments
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This work was supported by grants from Assistance Publique-Hôpitaux de Paris (Contrat d'Incitation à la Recherche Clinique), Association Quincy Recherche Clinique et Thérapeutique, Centre Hôspitalier Privé Claude Galien and Compagnie Génèrale de Santé. The authors are grateful to Mrs Sylvie De Paola, Sylvie De Marans and Mauricette Yovogan for their technical assistance.
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Notes
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Correspondence and offprint requests to: Dr Guy Rostoker, Service de Néphrologie et de Dialyse, Centre Hospitalier Privé Claude Galien, 20 route de Boussy Saint-Antoine, 91480, Quincy Sous Sénart, France. 
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Received for publication: 13. 4.99
Revision received 3.10.00.