Clin Exp Immunol 1995; 101:442-448

Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy

determined by in situ hybridization for J chain mRNA

S. J. HARPER, A. C. ALLEN, M.-C. BENE*, J. H. PRINGLEt, G. FAURE*, I. LAUDERt

& J. FEEHALLY Department of Nephrology, Leicester General Hospital, Leicester, UK, *Laboratoire d'Immunologie,

Faculte de Medicine de Nancy, Nancy, France, and tDepartment of Pathology, University of Leicester, Clinical Sciences Building,

Leicester Royal Infirmary, Leicester, UK

(Acceptedfor publication 2 May 1995)

SUMMARY

The origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significantproportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chainexpression within lymphoid tissue in IgAN have utilized antibodies which other investigators havefound to be non-specific. To address this problem, we have developed an in situ hybridization(ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patientswith IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii)combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were

identified in germinal centres, and within the subepithelial and interfollicular zones. A greater

number of J chain mRNA-positive cells were found in the germinal centres of patients (mean57.7+4-6 cells/105 /m2) compared with controls (mean 36-9 ± 35 cells/105 /,m2) (P < 0.001).Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positiveinterfollicular IgA cells in patient tonsils (32 ± 34%) compared with controls (21 23%;P < 0 02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN.In addition, the finding of excess numbers of J chain-positive IgA-negative cells within germinalcentres suggests that an abnormality may be present at the B cell differentiation stage before IgAswitching. These results further highlight immune abnormalities within the tonsil as a centralfeature of abnormal polymeric IgA biology in this common form of glomerulonephritis.

Keywords J chain tonsils IgA nephropathy

INTRODUCTION

IgA nephropathy (IgAN) is a very common glomerular disease[1]. It is characterized and defined by the deposition of IgAwithin the glomerular mesangium [2]. The origin of mesangialIgA remains to be defined. The predominant molecular form ofthe IgA is still debated [3,4], but a significant proportion isbelieved to be dimeric. This view is supported by immunofluor-escent detection of J chain within mesangial deposits [5,6],secretory-component binding experiments [5] and elution stu-dies [6,7]. There is also considerable evidence of widespreadabnormalities in polymeric IgA (pIgA) biology in this disease(reviewed in [8]).

Dimeric IgA is composed of two monomeric IgA unitslinked by the 15-kD J or 'joining' chain [9]. Since dimeric IgAhas limited origins within the human immune system, a shifttowards this molecular form in a candidate tissue has been

Correspondence: Dr S. J. Harper, Department of Nephrology,Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW,UK.

interpreted as evidence for the origin of the IgA which may bedeposited in glomeruli. Lymphoid tissues (tonsils and bonemarrow aspirates) have therefore been investigated for theexpression of J chain [4, 10,11].

Increased J chain expression, and by implication dimericIgA production, has been reported in tonsils [10,11]. However,the methodology used in these studies has been open tocriticism, because the detection of J chain polypeptide iscomplicated by the site of J chain within the 3-dimensionalstructure of pIg. To expose J chain epitopes acid-urea dena-turation is required, and this also denatures other parts of theimmunoglobulin structure. Antibodies used in previous studiesof tonsil and bone marrow [4,11] have not been found to beentirely specific by other investigators, demonstrating a tend-ency to bind to denatured light chains ([10], and Kerr et al.,University of Dundee, UK, unpublished observations).Furthermore, comparison between immunohistochemical andin situ hybridization (ISH) techniques, for the demonstration oflight chain protein and mRNA, respectively, in tonsils andlymph nodes, has revealed that with the former detection of

44 1995 Blackwell Science442

J chain mRNA in tonsillar IgA cells

extracellular imunoglobulin, 'network' immunoglobulin andbackground staining to collagen within the mantle zone mayall obscure true patterns of cellular staining [12]. ISH detectionof mRNA avoids the problems associated with extracellulartarget, target uptake by dead or damaged cells or by cellsbearing Fc receptors, and possible non-specificity of antibodies.

We have therefore developed a non-isotopic ISH techniquefor the detection of J chain mRNA specifically within IgA cells[13]. We have applied this technique to duodenal laminapropria, and have reported a shift away from dimeric IgAproduction at this site in IgAN [14].

Here we describe a similar study of tonsils. In order toinvestigate dimeric IgA production within the tonsils in IgANwe used an ISH method adapted from that previously described[13]. This method specifically identifies cells expressing J chainmRNA, avoiding the potential problems of antibody-basedtechniques.

MATERIALS AND METHODS

SamplesTonsils were obtained from 12 patients with biopsy-provenIgAN (eight males, four females, mean age 20 years, range 8-54years). These samples had all been collected by the Laboratoired'Immunologie, CHU de Nancy, where they were sent byclinicians from the North-East and West of France [15].Twelve control samples were obtained from children andyoung adults requiring tonsillectomy (five males, seven females,mean age 5 2 years) who displayed no renal anomaly. Alltonsils from patients had been shown to display the alteredpartition of IgG- and IgA-containing plasma cells previouslydescribed [15], while the tissues from all controls containedpredominantly IgG plasma cells.

Sample collection and storageSamples were frozen in liquid nitrogen and maintained at-800C until studied. Samples were forwarded to the UK indry ice without a break in storage temperature.

Probe andprobe labellingDeoxyoligonucleotides (unlabelled sequences were kindlydonated by Pathway Laboratory Services Ltd, Leicester, UK)complementary to J chain mRNA [16] were 3'-labelled with thenucleotide analogue digoxigenin-lI-dUTP using a labelling kit(Boehringer Mannheim, Mannheim, Germany; 220-582). Thelabelling reaction was performed at 37°C for 2 h in 100 tlvolumes. Reaction mixture was as follows: probe cocktail,200 ng; 10 mm MnCl2, 10 pl; 1 mm digoxigenin-1l-dUTP, 2 3 Al;5 x buffer, 20 psl; terminal deoxynucleotidyl transferase, 1 ptl.Labelled probe was then purified through Sephadex G50 spuncolumns and probe labelling confirmed by test filters.

Non-isotopic ISHThe protocol for non-isotopic ISH on frozen tissue was adaptedfrom techniques we have previous reported for use with wax-embedded specimens [13,14,17,18]. RNase-free reagents andglassware (diethylpyrocarbonate (DEPC)-treated; Sigma,Poole, UK; D5758) were used throughout.

Fresh cryostat sections were cut onto three aminopropyl-triethoxysilane-coated slides and fixed for 10 min in freshlyprepared 4% paraformaldehyde in PBS/DEPC at 4°C. Slides

were then washed in PBS/DEPC (2 x 10 min) at roomtemperature. Labelled probe cocktail (50 ,l) was then pipettedonto a coverslip, the slide drained of excess fluid and allowed topick up the coverslip. A 2 h hybridization was used at 37°C.Probe cocktail was 20 ng/ml probe, 600 mm NaCl, 50 mm TrispH 7 5, 0-2% bovine serum albumin (BSA), 1% SDS, 1%polyvinylpyrrolidone (40 kD), 1% Ficoll (400 kD), 0 1%sodium pyrophosphate, 5 mm EDTA, 10% dextran sulphate,30% formamide. Post-hybridization washes were then as fol-lows: 2 x standard saline citrate solution (SSC)/30% forma-mide (2 x 10 min); 2- x SSC at room temperature (2 x 10 min);15 min in blocking solution. SSC = 150 mm NaCl, 15 mMtrisodium citrate pH 7 0; blocking solution = TBS, 3% BSA,0 1% Triton X-100.

Slides were incubated with alkaline phosphatase-labelledsheep polyclonal anti-digoxigenin antibody (Boehringer;1093274) 1:600 in blocking solution for 30 min. Specimenswere then washed in blocking solution (2 x 5 min), immersed inbuffer 3 (0-1 M Tris-HCl pH 9.5, 0-1 M NaCl, 0 05 M MgCl2)(1 x 10 min), and in substrate solution overnight (44 IlI nitroblue tetrazoleum (NBT; Sigma; 75 mg/ml in 70% dimethyl-formamide), 33 ,ul 5-bromo-4-chloro-3-indolyl phosphate(BCIP; Sigma; 50 mg/ml in dimethylformamide) in 10 mlbuffer 3). Finally, sections were washed in running tap waterfor 10 min, counterstained with Mayer's haematoxylin andmounted in aqueous mounting medium.

Combined immunofluorescence andfluorescent ISHThe protocol for combined immunofluorescence and fluores-cent ISH was identical to the non-isotopic ISH technique up tothe detection step. An FITC-conjugated anti-digoxigenin anti-body (Boehringer; 1207741) diluted 1:10 with sheep serum wasemployed at this stage in place of the alkaline phosphataseequivalent. Samples were washed and then flooded with a tetra-rhodamine isothiocyanate-labelled polyclonal rabbit anti-human IgA antibody 1:300 (Dako, High Wycombe, UK;R153) in blocking solution for 30 min. Samples were finallywashed and mounted with aqueous mount. IgA immunocyteswere then visualized under ultra-violet light (540 nm, red), andco-expression of J chain mRNA could then be determined byalteration of ultra-violet wavelength to 490 nm under which Jchain mRNA-positive cells could be observed (green) (see Fig. 1).

Controls and experimental designTo assess mRNA preservation and determine any anatomicaldifference in J chain mRNA expression within patient andcontrol biopsies, all specimens were initially studied usingnon-isotopic ISH. This method was then adapted to fluorescentISH, allowing simultaneous identification of IgA cells with acomplementary fluorochrome. Specificity of J chain mRNAsignal was confirmed by the use of appropriate negative con-trols on each section as previously listed. These were: omissionof probe, omission of anti-digoxigenin antibody, RNase Alpretreatment controls, random oligonucleotide cocktail sim-ilarly labelled (unlabelled sequences kindly donated by Path-way Laboratory Services Ltd), in addition to a non-homologous deoxyoligonucleotide cocktail with the same G-C content as the J chain probe [13,14,18,19].

Microscopy, cell counting and statistical analysisCell enumeration was performed on a Nikon Optiphot micro-

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444 S. J. Harper et al.

Fig. 1. Combined fluorescent in situ hybridization and immuno- cfluorescence on frozen tonsillar tissue. (a) IgA plasma cells visualizedunder UV (540 nm). (b) J chain mRNA-positive cells visualized underUV (490 nm). Cell 1, IgA-positive (a), J chain mRNA-negative (b); cell2, IgA-positive (a), J chain mRNA-positive (b); cell 3, IgA-negative (a),J chain mRNA-positive (b).

scope on coded specimens by a single observer who thereforewas unaware of the nature (control or patient) of all samples.After non-isotopic ISH examination of samples, germinalcentres with clearly defined mantle zones were identified andtotal cell counts performed with the aid of a grid-graticule in asystematic manner. Germinal centres that were incomplete,disrupted, crushed or within artefactual tissue folds were notincluded. The area of the germinal centre was determineddirectly from the slide at the time of counting by the use of aKontron Videoplanimeter system. Data are expressed asnumber of J chain-positive cells per 105 ,um2.

Cell enumeration after combined immunofluorescence andfluorescent ISH was conducted on at least 200 IgA cellsidentified in random fields. Only cells with discernible nuclei

Fig. 2. (a) Normal tonsillar germinal centre. D, dark zone; L, light zone;M, mantle zone. (Haematoxylin and eosin stain, wax-embedded sec-tion.) (b-d) Non-isotopic in situ hybridization for J chain mRNA onfrozen section. Nitro blue tetrazolium (NBT)/ 5-bromo-4-chloro-3-indolyl phosphate (BCIP) visualization, haematoxylin counterstain.(b) Germinal centres, J chain mRNA-positive cells concentrated inbasal light zone. (c) J chain mRNA-positive cells in subepithelial areas.(d) J chain mRNA-positive cells in interfollicular zone.

© 1995 Blackwell Science Ltd, Clinical and Experimental Immunology, 101:442-448

i! X _ .!S i fil_

J chain mRNA in tonsillar IgA cells

were counted. The co-expression of J chain mRNA in thesecells was then determined.

Statistical analysis was by unpaired t- and Mann-WhitneyU-tests.

RESULTS

Non-isotopic ISHNBT/BCIP sites of hybridization were seen staining cells in allcontrol and patient tonsil specimens. Sharp, clearly definedsignal contrasted with negligible background staining.Morphology was well preserved on all occasions. Negativecontrols yielded no signal on any occasion.

Germinal centres. Within the normal tonsil, germinal centresdevelop and subside in response to antigen stimulation. Maturegerminal centres are subdivided histologically into a dark zoneconsisting of proliferating B cell centroblasts, and a light zonecontaining predominately non-dividing centrocytes. Thesezones were surrounded by a perimeter of small compact pre-dominantly memory B lymphocytes known as the mantle zone(Fig. 2a).

J chain mRNA-positive cells were seen in all specimens inthe germinal centres, making up 0-24% (count of tworandomly chosen germinal centres from each specimen) of thenucleolated cells, depending on the plane of orientation of thesection through the follicle and the stage of activation. J chainmRNA-expressing cells were seen within both light and darkzones of the germinal centres. In sections cut through themiddle of follicles a zonal distribution of cells was seen inwhich J chain mRNA-positive cells tended to localize in a bandin the basal light zone-the junction of the light and dark zones(Fig. 2b). A greater number of J chain mRNA-positive cellswere present per unit area of germinal centre in patients (mean57*7 ± 4-6 cells/105 tm2) compared with controls (mean 36-9 +3-5 cells/105 [tiM2; p < 0-001 (unpaired t-test); Fig. 3). A similarnumber of germinal centres were examined in both patient (93)and control (92) groups. One control specimen had no germinalcentres suitable for cell counts to be performed.

Subepithelial and parafollicular zones. J chain mRNA-positive cells were also seen concentrated as a rim in thesubepithelial zone (Fig. 2c), although only a minority of thesections examined (two controls, four patients) included anyepithelium. In addition, cells were seen more diffuselythroughout the interfollicular areas (Fig. 2d); in these tworegions, morphologically plasmacytoid cells predominated.

Simultaneous immunofluorescence andfluorescent ISHThe distribution of J chain mRNA cells within the tonsilsections using a fluorescent detection system for the probelabel was identical to that described above. However, therewas significantly more background staining to the collagenwithin the tissue. This was eliminated by incubation of theFITC-labelled polyclonal sheep anti-digoxigenin antibody withsheep serum.

Combined immunofluorescence and fluorescent ISHallowed identification of the plasma cell type in which J chainmRNA was identified. Monomeric IgA cells (IgA-positive, Jchain-negative, Fig. la), dimeric IgA cells (IgA-positive, J chainmRNA-positive, Fig. la,b) and IgM, IgG, IgD or immature Bcells (IgA-negative, J chain mRNA-positive, Fig. lb) couldclearly be separated. This technique highlighted a number offeatures.

Germinal centres. In all specimens many cells within thegerminal centres were J chain-positive, but very few of thesestained positive for IgA ( < 1% in both controls and patients,P = NS, data not shown).

Subepithelial and parafollicular zones. Within the six speci-mens (two controls, four patients) which included regions ofepithelium, there were significantly more J chain mRNA-positive IgA cells in the subepithelial rim (55 8 ± 5%) than inthe interfollicular zone (37-8 + 4 8%) (P < 005, Mann-Whitney U-test) (Fig. 4). There were insufficient specimenscontaining epithelium to allow direct comparison of patientsand controls. However, there were significantly more J chainmRNA IgA cells within the tonsillar interfollicular zones inpatients (32 ± 3-4%) than in controls (21 + 2 3%) (P < 002,unpaired t-test) (Fig. 5).

DISCUSSION

We have previously reported a technique for the simultaneousdetection of native mRNA by non-isotopic ISH and identifica-tion of plasma cell type in formal-saline-fixed and wax-embedded tissue [13]. We have adapted the method for useon frozen tissue. Our experience suggests that this method maybe performed on tissue which has been stored for up to at least 5years.

The studies demonstrate for the first time the ISH identifi-cation of J chain mRNA in tonsillar cells in controls andpatients with IgAN. Positive cells are present in three distinctareas-germinal centres, subepithelial areas and in the inter-follicular zones.

Within the germinal centres non-isotopic ISH demonstrateda zonal distribution of J chain mRNA-positive cells concen-trated at the junction of light and dark zones similar to thatdescribed for light chain mRNA expression [12]. It has beenproposed that this is the micro-anatomical site of isotype classswitching and/or affinity maturation [12].

We present here new data suggesting an excess of J chain-positive germinal centre immunoblasts in IgAN. Since J chainexpression has been highlighted as a marker of clonal immatur-ity [20,21], our data suggest an anomaly of B cell maturationand development within germinal centres. This may be manifestby defects of isotype switching and affinity maturation. Indeed,we have previously reported reduction in IgA affinity to specificantigen in IgAN [22]. Furthermore, abnormal IgAl germinalcentre staining has been recently reported [23]. Further study ofthis micro-anatomical site is therefore warranted.

Combined immunofluorescence and fluorescent ISHrevealed an increase in J chain mRNA-positive IgA cellswithin the subepithelial area compared with the interfollicularzone. This highlights the need to perform detailed cell enumera-tion in distinct areas of the tonsil to be able to derive usefulinformation.

Although J chain is a feature of dimeric IgA and pentamericIgM, J chain expression has also been reported in cells which donot produce polymeric immunoglobulin (IgG, IgD) [10,24-26].It is in such cells that the presence of J chain has been linkedwith clonal immaturity [20,21]. J chain has also been reported inimmunoglobulin-negative cells in certain plasma cell dyscrasiasor lymphomas [27,28]. Furthermore, J chain-independentmultimeric IgM production has been demonstrated in rodent

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S. J. Harper et al.

2000

1800

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Fig. 3. Scatter plot of numbers ofJ chain mRNA-positive cells/unit areaof 93 and 92 germinal centres in the tonsils of 12 patients with biopsy-proven IgA nephropathy (IgAN) and 11 controls, respectively. Mean isrepresented by the horizontal lines.

non-lymphoid transfected cell lines [29], and two IgA myelomashave been described in which J chain-free multimeric IgA was

found, although in these cases J chain was apparently replacedby either albumin or a,-antitrypsin (al,-AT) [30]. However, we

are aware ofno evidence to suggest that plasma cell synthesis ofJ chain-free dimeric IgA takes place in humans in vivo, either inhealth or any non-malignant clinical state. If such IgA was

produced it would probably be non-functional in the secretoryimmune sense. Indeed, experimental studies in a Chinesehamster ovary cell expression system (Atkin et al., manuscriptsubmitted) suggest that J chain may be essential to the produc-tion of dimeric IgA, in contrast to multimeric IgM [31].Transfection experiments in a mouse myeloma cell line, J558with a-heavy chain, both intact and without the J chain bindingterminal cysteine residues, support this proposal (Atkin et al.,manuscript submitted). This and other evidence support theview that J chain protein is required for the assembly andsecretion of dimeric IgA (reviewed in [32]) and is thereforecentral to the immune exclusion afforded by the secretoryimmune system [20]. J chain synthesis is controlled at thelevel of transcription [33,34] rather than translation, the datawe present therefore suggest a shift towards dimeric IgAproduction in the tonsils in IgAN.

In the mucosal immune system antigenic activation of Bcells takes place in the mucosa-associated lymphoid tissue.These cells then migrate via the lymphatic and systemic systems

0

P< 0*05 0

.

.

Interfollicularzone

Subepithelium

Fig. 4. Scatter plot of the proportion of J chain mRNA-positive IgAplasma cells in the subepithelial and interfollicular zones of the tonsilsof two controls and four patients with biopsy-proven IgA nephropathy(IgAN). Mean is represented by the horizontal lines.

to distant mucosal sites where terminal differentiation andimmunoglobulin synthesis take place [20,35], thereby armingthe whole mucosal system against an antigen which initiallymay be encountered only locally. Study of isotype and subclassvariations between plasma cells from different sites in themucosal immune system [36,37] has led to the proposal oftwo related but distinct mucosal immune systems in man; one

based on the Peyer's patch-lamina propria axis, the gastro-intestinal mucosal immune system; the other based on thepalatine and nasopharyngeal tonsil-airway mucosa axis, theairway-related mucosal immune system [38]. Cell migrationexperiments in animals would suggest that although they are

distinct, there is probably some overlap between the twosystems [39-41]; as indeed there may be between each ofthese mucosal systems and the systemic (marrow) IgA system[42].

The clinical association between acute mucosal infectionsand macroscopic haematuria in IgAN led us to study J chainmRNA expression within the duodenal lamina propria-themain synthetic site of the mucosal immune system [14]. Wefound a reduction in the proportion of IgA plasma cells and an

increase in the absolute numbers of IgG cells in duodenalbiopsies from patients with IgAN compared with controls.This contrasts sharply with the well described increase inIgA/IgG cell ratio in tonsils [15]. In addition, we found a

1995 Blackwell Science Ltd, Clinical and Experimental Immunology, 101:442-448

446

---- 99-- nv

J chain mRNA in tonsillar IgA cells 447

60-

50 0S

40 -

+

Z

E 30 -*Xl~~~~~~~ P< 0.02020CL

X X

0~~~~

*

10_

Control IgAN

Fig. 5. Scatter plot of the proportion of J chain mRNA-positive IgAcells in the interfollicular zone of tonsils from 12 patients with biopsy-proven IgA nephropathy (IgAN) and 12 controls. Mean is representedby the horizontal lines.

down-regulation of J chain mRNA specifically within the IgAcells in the duodenal lamina propria which contrasts with thetonsillar findings in this study. In the control groups from bothstudies the mean per cent of IgA plasma cells expressing J chainmRNA in duodenal lamina propria and the interfollicular zoneof the tonsils was 89-2% and 37-8%, respectively. These datasupport the concept oftwo distinct mucosal immune systems inman which may react independently due to intrinsic or envir-onmental differences. Our findings also support the notion ofaberrant mucosal immunity located primarily in the airway-related mucosal system in IgAN. This abnormality could beinnate to the B and T cells colonizing the tonsil, or secondaryexogenous immune influences.

In conclusion, this study is the first description of J chainmRNA detection in situ in tonsils. It provides new evidence ofabnormalities within tonsillar germinal centres which may underliemany features of IgAN. In addition, this work provides furtherevidence for compartmentalization ofthe mucosal immune system.

ACKNOWLEDGMENTS

S.J.H. is a Wellcome Trust Training Fellow, Grant 034937/91.

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