Binding sites for carrier-immobilized carbohydrates in the kidney: implication for the pathogenesis of HenochSchönlein purpura and/or IgA nephropathy
Anna
edivá1,
Karel Smetana, Jr.2,
Josef Stejskal3,
Ji
ina Bart
ková1,
Fu-Tong Liu4,
Nicolai V. Bovin5 and
Hans-Joachim Gabius6
1 Institute of Immunology, 2nd Faculty of Medicine, Charles University,
2 Institute of Anatomy, 1st Faculty of Medicine, Charles University,
3 Institute of Pathology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic,
4 La Jolla Institute for Allergy and Immunology, San Diego, CA, USA,
5 Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia and
6 Institute for Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Munich, Germany
Correspondence and offprint requests to:
Anna
edivá, Institute of Immunology, 2nd Faculty of Medicine, Charles University, V úvalu 84, CZ-150 18 Prague 5, Czech Republic.
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Abstract
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Background. HenochSchönlein purpura is a common vasculitis of childhood affecting the skin, joints, gastrointestinal tract, and kidney. The mesangial deposition of IgA1 is the most critical factor for the prognosis of patients with this disease. The aberrant glycosylation of the IgA1 subclass with the absence of terminally located galactose and presence of only
-N-acetylgalactosamine in O-linked oligosaccharides in the hinge region of IgA1 represents a prominent difference from the normal IgA1. These alterations prompt the supposition that the sugar part may guide IgA deposition by recognition of endogenous lectins on the mesangium.
Methods. Owing to the limited knowledge about the expression of carbohydrate-binding sites in the human kidney we initiated the study of this aspect with a class of tools which are suitable to map the lectinome of cells. Employing biotinylated neoglycoconjugates, glycosaminoglycans, and sulphated polysaccharides we monitored the presence of accessible carbohydrate-binding sites in control kidneys represented by tumour-free areas of kidneys with Grawitz tumour and in biopsies from patients with HenochSchönlein purpura-associated IgA nephropathy.
Results. Using frozen sections, no expression of any tested carbohydrate-binding site(s) was observed in the endothelial and the mesangial cells in glomeruli of the control kidneys as well as in the biopsies from HenochSchönlein purpura IgA nephropathic kidneys, in contrast to the tubules. The N-acetylgalactosamine-binding sites were expressed only in the inner layer of Bowman's capsule of 20% of glomeruli of the control kidney from one patient with Grawitz tumour and one biopsy from a patient with HenochSchönlein purpura-associated IgA nephropathy. However, the macrophages in the glomeruli of patients with IgA nephropathy and interstitial macrophages from both studied groups, i.e. without and with IgA nephropathy, harbour capacity to recognize carrier-immobilized
-N-acetylgalactosamine. Access to this binding site for the neoligand conjugate can be blocked by the monoclonal antibody MEM-18 recognizing CD14 antigen.
Conclusion. The possibility for a participation of macrophage deposition of IgA1 in mesangium via a lectin mechanism involving this binding capacity warrants further studies.
Keywords:
-N-acetylgalactosamine; CD14; Henoch Schönlein purpura; IgA nephropathy; lectin macrophage
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Introduction
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HenochSchönlein purpura (HSP) is the most common vasculitis of childhood, affecting the skin, joints, gastrointestinal tract, and kidneys. Kidney involvement is the most serious and limiting factor in the clinical outcome of children with HSP. Despite the fact that the disease has been known for more than 150 years [1] its aetiology still remains ill defined. Histopathological examinations in the kidney have revealed mesangial proliferation accompanied by the predominantly mesangial deposition of IgA. An almost identical picture of glomerular pathology is seen in IgA nephropathy [2,3]. The actual mechanism of IgA deposition in the kidneys is unknown. Deposited IgA is of the polyclonal IgA1 subclass characterized by a high proportion of
light chains, which has an important source in plasma cells located in the bone marrow [4]. Besides these observations, the tonsillar lymphocytes harvested from patients with IgA nephropathy produce IgA that directly interacts with mesangium in frozen sections [5].
Concerning the biochemical features of the reactive IgA molecules, abnormalities in their charge have been detected in patients [4,6]. Moreover, changes in the glycosylation of IgA molecules have been reported. Similar to the other immunoglobulins, human IgA1 contains N-linked glycan chains, which appears to be of relevance for the modulation of binding of the target antigen and in the mediation of effector functions [7]. IgA1 also belongs to the class of serum glycoproteins, which bear O-linked carbohydrates. These carbohydrate chains are located in the hinge region of IgA [8]. The altered glycosylation of O-linked carbohydrates, namely reduced terminal galactosylation, was repeatedly reported in IgA nephropathy [912]. This disorder seems to be linked to a reduced activity of a ß-1,3-galactosyltransferase in B lymphocytes [13]. Interestingly, the stimulation of murine B cells by interleukins 4 and 5 also induced altered terminal glycosylation of IgA in vitro [14]. With respect to further processing of the O-linked carbohydrate chains it is known that the proportion of sialylated IgA1 is lower in patients with IgA nephropathy than in controls [15]. The IgA1 molecules with altered glycosylation form aggregates and bind to the surface of the cultured mesangial cells [11,12,16]. As an alternative to a profound influence of glycan chains on IgA1 conformation to explain this behaviour, it is principally reasonable to assume that these sugar chains may serve as ligands for endogenous lectins [17,18].
To evaluate this hypothesis, it is essential to have information available on the capacity of different compartments in the human kidney to bind carbohydrate ligands. Since the monitoring of the expression of carbohydrate-binding site(s) in the human kidney has so far not been performed, the presented study focused on the question whether suitable carbohydrate ligands can bind to the mesangium of human kidney. This question was addressed histochemically by employing biotinylated neoglycoconjugates, a natural glycosaminoglycan, and sulphated polysaccharides as tools for the visualization of carbohydrate-binding sites in the tumour-free area of human kidney and kidneys of patients with HSP-associated IgA (HSP/IgA) nephropathy. This technique enables us to visualize binding sites, which are neither blocked by endogenous ligands nor are harmed by the steps of tissue processing [1922].
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Materials and methods
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The binding sites for carbohydrates were examined on frozen biopsy specimens from 11 children with HSP/IgA nephropathy and tumour-free sections of kidneys from five patients with tumours. Frozen sections (710 µm thick) were used for glycohistochemical analysis after fixation with absolute methanol (4°C), absolute acetone (4°C), or 2% (w/v) paraformaldehyde in PBS (4°C, pH 7.2) for 5 min. The IgA deposits were visualized by FITC-labelled swine-anti human IgA immunoglobulin (Sevac, Prague, Czech Republic). Human galectin-3 (Gal-3) was detected by the anti-human monoclonal antibody (A1D6 antibody, [23]) at a concentration of 20 µg/ml. CD14 antigen was determined using monoclonal anti-human antibodies MEM-15 and MEM-18 at the same concentration.
After extensive washing with PBS, the immunohistochemical procedures were performed. The endogenous peroxidase activity was blocked by preincubation of specimens with 5% (v/v) methanol solution of hydrogen peroxide for 15 min. Commercial peroxidase-labelled swine-anti-mouse antibody (Sevac, Prague, Czech Republic) was employed in the second step. The specific binding was visualized using the diaminobenzidine tetrahydrochloridehydrogen peroxideurea developing system (Sigma FastTM, Sigma, Prague, Czech Republic). The results of immunocytochemical reactions after methanol or acetone fixation were visualized employing FITC-labelled swine anti-mouse antibody (Sevac, Prague, Czech Republic). Control reactions were performed employing anti-human involucrin antibody (Sigma FastTM, Sigma, Prague, Czech Republic) instead of the first-step antibody and by the incubation of samples with the second-step antibody only. The glycohistochemical reaction employing the biotinylated probes, namely a panel of neoglycoconjugates, the glycosaminoglycan heparin as well as sulphated polysaccharides sulphated fucan and and sulphated galactan at a concentration of 20 µg/ml (TBS with 5 mM of Ca2+) was used for the visualization of carbohydrate-binding sites (Table 1
) [2431]. The specificities of probes used are shown in Table 1
including abbreviations. The peroxidase-labelled avidin (ABComplex, Dako, Glostrup, Denmark) and TRITC-labelled extravidin (ExtrAvidin, Sigma, Prague, Czech Republic) were used as the second-step reagent. The results of glycohistochemical reactions using peroxidase-labelled avidin were visualized with Sigma FastTM (Sigma FastTM, Sigma, Prague, Czech Republic).
The control sections were incubated with competitive inhibitors for ascertaining the sugar specificity of reaction and with second-step reagents only to exclude signal generation by the binding of the mannose-rich glycoproteins avidin and horseradish peroxidase as constituents of the kit to endogenous mannose receptors [32]. Some sections were counterstained by haematoxylin and all specimens were routinely dehydrated by alcohol and mounted in Solacryl (Medika, Prague, Czech Republic). Double-labelling experiments for the simultaneous detection of IgA and binding sites for
-GalNAc and CD14 were performed in acetone-fixed specimens using fluorescently labelled second-step reagents. The processed specimens were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA) and examined using Nikon-Optiphot-2 fluorescence microscope employing standard filter blocks.
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Results
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The results of the study are summarized in Tables 2 and 3
. Due to the indicated potential relevance for the clinical syndrome the localization of detectable binding sites for
(ß)-GalNAc moieties is presented as figures.
The results of glycohistochemical reactions do not appear to be substantially influenced by the fixation procedure used and independent on the labelling procedure, i.e. using the fluorescently and peroxidase-labelled second-step reagents. A high level of cytoplasmic expression of carbohydrate-binding sites in cells of renal tubules of all types was found in kidney with and without HSP-associated IgA nephropathy (Figures 1 and 2
). However, no binding of tested probes was detected on the surface or in the cytoplasm of endothelial and mesangial cells of studied glomeruli in samples from both the studied groups of patients under these conditions (Figure 1
). The only exception in this respect was the inner layer of the Bowman's capsule, where binding sites for
(ß)-GalNAc were seen in approximately 20% of glomeruli from one patient with Grawitz tumour and one patient with IgA nephropathy (Figure 1
). Nuclei of endothelial as well as mesangial cells displayed the expression of binding sites for the sulphated polysaccharides sGal and sFucan and not for Hep-S in both groups (not shown). All the studied biopsies from the patients with IgA nephropathy exhibited a variable level of deposition of IgA to the glomerular mesangium (Figure 1
).
The interstitium of kidney with and without IgA nephropathy was infiltrated by CD14+ macrophages which are reactive for
(ß)-GalNAc (Figure 2
). The visualization of such binding sites in macrophages was completely inhibited by incubation with the MEM-18 antibody and partially inhibited by the MEM-15 antibody recognizing the CD14 antigen (Figure 2
). The macrophages harbouring the
-GalNAc-binding sites were also observed in glomeruli affected by the IgA mesangial deposition. The double-labelling experiments revealed that these macrophages were strongly positive for IgA (Figure 1
). Interestingly, these macrophages expressed galectin-3, a known binding partner for IgE and IgM.
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Discussion
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The precise delineation of the aetiology of both HenochSchönlein purpura and IgA nephropathy continues to warrant in these efforts despite evident progress [2,3]. A major question concerns the definition of the causative agent for IgA nephropathy. Due to this manifestation of IgA1 deposits the biochemical characteristics of IgA (isotype, light chain, origin, charge, glycosylation, elevated levels) have been carefully studied. Since it is probable that one or several examined features will explain this distinct behaviour of the IgA1, it is remunerative to try and correlate defined biochemical features of the altered IgA1 to putative molecular mechanism(s) how IgA1 may accumulate in this site. An aberrant glycosylation of IgA1 molecules has repeatedly been observed in the given line of reasoning. This feature may entail the reaction crucial for pathogenesis of the disease. Besides acting as a spatial factor in guiding the folding of a nascent glycoprotein, the carbohydrate chains themselves may harbour ligand properties for endogenous lectins adding to the potential of the peptide part to be engaged in recognitive interactions [33]. Indeed, this concept of glycan functionality is being exploited to target drugs by tailor-made carbohydrate ligands as part of neoglycoconjugates [18,34,35].
To test this hypothesis in our context experimentally, carrier-immobilized carbohydrate structures or natural glycoconjugates offer a suitable class of tools. They allow monitoring of the presence of accessible sites in tissue sections. Since the reported abnormalities in the glycosylation of IgA molecule include its decreased sialylation and galactosylation, particularly lacking terminal galactose and thus exposing
-GalNAc, we have employed a panel of probes including
(ß)-GalNAc. We found binding sites for
(ß)-GalNAc in approximately 20% of glomeruli of one patient with Grawitz tumour as well as in a biopsy from one patient with IgA nephropathy. These detectable binding sites were not located in mesangium, which is the common site of IgA deposition, but in the inner layer of Bowman's capsule.
Concerning the nature of the probe, it should not be overlooked that it mimics the terminal (sugar) part of a glycopeptide. It is not a copy of the O-linked glycan in the Ig molecule. If recognition encompasses extended structural features including peptide motifs, this synthetic probe will probably reach only a degree of affinity. Under these circumstances it is also possible that such a probe may not compete successfully with high-affinity ligands in situ. Moreover, if a lectin-like molecule requires an extended contact to the carbohydrate ligand, then the complexity of the sugar part of a probe is to be increased, e.g. chemoenzymatic synthesis [19,22]. The glomeruli with mesangial IgA deposits were infiltrated by macrophages, which is a common feature of HSP-associated nephropathy [36]. They are highly positive for IgA and express the binding site(s) for
-GalNAc. Similarly, the subcapsular and interstitial macrophages from both studied groups of patients, i.e. with and without IgA nephropathy, revealed presence of binding sites with specificity for
-GalNAc. The expression of
-GalNAc-binding sites in macrophages was sensitive to the treatment with the anti-CD14 antibody blocking the binding of neoglycoprotein with
-GalNAc moieties to macrophages partially (MEM-15) or completely (MEM-18). This observation hints at the possibility of a putative participation of CD14 antigen or spatially closely associated molecule in the recognition of
-GalNAc. The lectin mechanism of the interaction between the CD14 and bacterial peptidoglycan including the blocking effect of MEM-18 has been reported [3740]. Other candidate lectins with reactivity to the Tn antigen (-O-
GalNAc) are e.g. galectin-3 and a member of the C-lectin family [17,41].
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Conclusion
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On the basis of the presented results a participation of macrophages in the aetiopathogenesis of the disease is intimated. The ability of macrophages to bind the
-GalNAc exposing neoglycoligand suggests a possible role of these cells in the transport of aberrantly glycosylated IgA1 to glomeruli in the course of IgA nephropathy using a mechanism different from Fc receptorFc fragment interaction. The IgA1 imported to the mesangium may serve as initiator for further invasion of monocytes and macrophages with IgA1 into the extravascular compartment of glomerulus, as it is known that the binding of IgA to human mesangial cells increases the production of chemokines stimulating the migration of macrophages and monocytes into the glomerulus [42]. The cytokine and chemokine milieu is crucial for IgA deposition as we were not able to prove the binding of aberrantly glycosylated IgA from HenochSchönlein patients to monocytes from healthy donors nor to monocytomacrophage human cell lines (data not shown).
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Acknowledgments
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This study was supported by the grant No 77/97/c/2.LF of the Grant Agency of the Charles University, grant of Internal Grant Agency of the Ministry of Health of Czech Republic IGA MZ 45533 and Complex Immunology Program 111300001 of the Ministry of Education of Czech Republic. The authors are grateful to Mrs Eva Vancová for excellent technical assistance.
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Received for publication: 17. 4.98
Accepted in revised form: 14. 7.99