The Immunochemical Characterization of

Mesangial IgA Deposits

J. D. LOMAX-SMITH, BSc, MBBS,L. A. ZABROWARNY, G. S. HOWARTH, BSc (Hons),

A. E. SEYMOUR, MBBS, MD, andA. J. WOODROFFE, MBBS, MD

Mesangial deposits of IgA are found in IgA nephrop-athy, Schonlein-Henoch purpura (SHP) and in somepatients with alcoholic cirrhosis and systemic lupuserythematosus (SLE). In this study the authors charac-terized the mesangial IgA deposits in patients with theabove diseases using antiserums or monoclonal anti-bodies to A1, A2, J-chain and secretory component(SC), and examined SC binding in vitro. SC was not

PRIMARY IgA nephropathy, characterized by mes-angial deposits of IgA and C3 occurs most often inyoung men with synpharyngitic hematuria.1 Similarimmunopathologic findings are found in associationwith alcoholic cirrhosis,2 portal-systemic shunts,3dermatitis herpetiformis,4 and celiac disease'- allof which may be termed secondary IgA nephrop-athy-and in Schonlein-Henoch purpura (SHP).Mesangial IgA is also found in lupus nephritis (SLE),but other criteria readily differentiate this from IgAnephropathy.IgA is present in the circulation as monomeric

units, mainly of the A1 subclass, but at mucosal sitesit is secreted actively as A1 or A2 dimers6 polymerizedby J-chain and transported by binding to secretorycomponent (SC). The bowel is the main site of IgAproduction in the body.6 In secondary forms of IgAnephropathy, the mesangial deposits and the docu-mented increases in both circulating IgA and IgAclass immune complexes (CIC) might be accountedfor either by a defective mucosal barrier-with in-creased absorption of floral or dietary antigens andsystemic hyperimmunization -or by defective hepaticclearance of antigens, IgA polymers, or complexes(reviewed by Woodroffe et al7). The presence ofJ-chain, subclass A2 or SC in the mesangium wouldsupport a mucosal origin of the IgA. SC has rarelybeen identified in any form of mesangial IgA nephri-tis,8'9 and the reported presence of J-chain10 must be

From the Renal Unit, Royal Adelaide Hospital, Institute ofMedical and Veterinary Science, Department of Pathology, Universityof Adelaide, and Department of Histopathology,Queen Elizabeth Hospital

present, J-chain was ubiquitous, and A2 was found(with the use of monoclonal antibodies) rarely but withequal frequency in all groups. The SC binding capacityof the deposits differed between the groups and wasfound in 13 of 16 patients with alcoholic liver disease,3 of4 with SLE, 1 of 10 with primary IgA nephropathy,and none of 6 with SHP. (Am J Pathol 1983, 113:359-364)

qualified because this is also a normal component ofIgM, which often accompanies the IgA deposits. TheIgA subclass studies so far reported have beencontradictory. 10-12

It has been suggested that primary IgA nephrop-athy is mediated by the deposition of IgA polymersfrom the circulation.1314 The capacity to bind free SCis an important method of distinguishing polymericimmunoglobulins containing J-chain from mono-mers.15 Several groups have shown mesangial IgAdeposits to bind free SC14'16'17 or be of polymericsize. 18 Egido et al14 showed SC binding capacity in 16of 20 biopsies with primary IgA nephropathy, com-pared with 6 of 7 with SHP and none of 3 with SLE.Subsequently Bene et al16 have shown SC bindingcapacity in all of a series of 15 patients with IgAnephropathy. Patients with alcoholic liver diseaserepresent a group of patients where a mucosal originfor the mesangial IgA deposits may be predicated.' Insupport of this, Sancho et al17 found SC bindingcapacity in all of 6 acid eluates from autopsy kidneys

Supported in part by a grant from the National Healthand Medical Research Council.

Accepted for publication June 28, 1983.Address reprint requests to Dr. A. J. Woodroffe, Renal

Unit, Royal Adelaide Hospital, North Terrace, Adelaide,S.A., Australia, 5000.

359

360 LOMAX-SMITH ET AL

and in all of 3 frozen sections from patients withmesangial IgA deposition and alcoholic liver disease.The present study reexamines the immunochemical

characteristics of the IgA mesangial deposits in pa-tients with primary IgA nephropathy, mesangial IgAnephritis secondary to alcoholic liver disease, SLE,and SHP. A1 and A2 subclasses were sought by con-

ventional indirect immunofluorescence (IIF) with theuse of sheep antiserums and by an avidin-biotin sys-

tem with the use of monoclonal antibody reagents. J-chain and SC were sought by IIF, and the ability ofthe IgA deposits to bind purified SC was alsoexamined.

Materials and Methods

The renal tissue selected for study consisted ofpostmortem specimens from 16 patients with alco-holic cirrhosis shown to have mesangial deposits ofIgA and C3 and renal biopsy specimens from patientswith primary IgA nephropathy (11), SHP (7) and SLE(5). Immunofluorescence-negative renal tissue fromautopsies (2) and biopsies (7) were examined ascontrols.

Tissue for IF obtained at autopsy within 24 hoursof death or immediately after removal of a needlebiopsy was snap-frozen in isopentane and liquidnitrogen. Unfixed frozen sections cut at 2 ji were

stained and examined with a Leitz Ortholux micro-scope fitted with Ploem epi-illumination, an HB 200mercury lamp as a light source, dichroic mirrors on

position 3, and BG 38 and K 510 filters. The reactionswere graded as 0 to 4 + by two of the authors (GSHand JDLS).

Direct IF was performed with fluorescein isothio-cyanate (FITC)-conjugated monospecific antiserumsto IgA (Wellcome, Beckenham, UK) and IgM (Hy-land-Travenol Laboratories, Costa Mesa, Calif).

IIF was performed with the use of 1:4 rabbit anti-human J-chain (Nordic Immunologic Laboratories,Tilberg, Netherlands) or 1:3 rabbit anti-human SC(Hoechst, Marburg, West Germany) that had beenabsorbed with 1:9 normal human serum (NHS). Thesections were then stained with FITC-conjugated goatanti-rabbit IgG (Dakopatts, Copenhagen, Denmark)that previously had been absorbed with 1:9 NHS. Therabbit antiserums were tested with sections fromhuman small bowel biopsies prepared according tothe method of Savilahti.19 Incubation of sections with1:3 normal rabbit serum was used as a control. Anti-serum to human J-chain stained the cytoplasm oflamina propria plasma cells, but not enterocytes. An-tiserum to human SC, however, stained both laminapropria plasma cell cytoplasm and enterocytes, the

latter in a "picket fence" distribution along lateralwalls and basal surfaces. After absorption with NHS,the SC antiserum stained only enterocytes.IgA subclasses were examined using 1:3 dilutions

of antiserums raised in sheep to A1 and A2 (Nordic)and stained with FITC-conjugated rabbit anti-sheepIgG (Wellcome) that had been absorbed with 1:9NHS. Staining was controlled by incubation with 1:3normal sheep serum. Sheep antiserums to A, and A2subclasses were tested on normal human jejunum andshowed similar bright 3 + staining of lamina propriaplasma cell cytoplasm. Neither antiserum stainedenterocytes. However, staining of an IgA, myelomabone marrow smear gave a 3 + reaction for A1 and a1 + reaction for A2.Monoclonal antibodies were obtained from a

mouse myeloma cell line, P3-X63-Ag8 653 hybridizedwith spleen cells from immunized BALB/C mice(IgAJ) or AJ mice (IgA2) (Becton Dickinson, Sunny-vale, Calif). The monoclonal antibodies were testedinitially by IIF with the use of FITC-conjugated rab-bit anti-mouse (Cappel, Cochranville, Pa) and later inan avidin-biotin amplification system. Subsequently,only the avidin-biotin system was used because thisclearly was more sensitive. Sections were incubatedwith the monoclonal antibody (20 jig/ml), biotiny-lated goat anti-mouse IgG (Tago, Burlingame, Calif)(500 Mg/ml) and then stained with FITC-conjugatedavidin (Becton Dickinson) (50 ,ug/ml protein).20 Stain-ing was controlled by incubation with 1:9 normalmouse serum. These reagents were tested on humansmall bowel biopsies and on human IgA1 myelomabone marrow smears.Murine monoclonal antibodies tested on normal

small intestine showed a 2 + reaction for large num-bers of lamina propria plasma cells stained for sub-class A1, and a 4 + reaction for lesser numbers ofplasma cells stained for A2. There was no reactivitywith enterocytes. The IgA, myeloma bone marrowsmear stained with monoclonal antibody to A1showed a bright cytoplasmic 4 + reaction, while forA2 there was faint nuclear but no cytoplasmicstaining.

Purified human SC was a gift from Dr. La Brooy,Department of Medicine, Royal Adelaide Hospital.The binding capacity of the SC preparation wastested by incubation at 200 jig/ml with a jejunal bi-opsy in a moist chamber followed by IIF staining forhuman SC. A control slide preincubated with phos-phate-buffered saline (PBS) was used to demonstratethe distribution of native SC in the section. Thejejunal section incubated with PBS and stained by IIFwith FITC anti-human SC showed localization of SCto the enterocyte basal and lateral walls. Sections

AJP * December 1983

NATURE OF MESANGIAL IgA DEPOSITS 361

incubated with SC and similarly stained showed en-terocyte SC but, in addition, bright staining ofmucosal plasma cell cytoplasm. The SC bindingcapacity of the mesangial IgA deposits in renal sec-tions was tested by incubation of adjacent sectionswith either SC or PBS followed by IIF for human SCas described above.The significance of differences in J-chain content or

SC binding between the disease groups and in relationto IgM was calculated with the use of a chi-square testwith a Yates' correction.A Mann-Whitney test for nonparametric samples

was applied to the incidence of SC binding capacityin primary IgA nephropathy deposits, compared withmesangial IgA deposits secondary to alcoholic liverdisease.

Results

The results of the renal IF studies are shown inTable 1. With the use of sheep antiserums, 10 of the11 patients with primary IgA nephropathy showedmesangial A,, and 9 showed A2. Monoclonal anti-bodies showed A, in the same 10 patients but A2 inonly 2. Of the 16 patients with alcoholic cirrhosis, thesheep antiserums showed 9 to have both A, and A2.With monoclonal antibodies, 15 had A, and 2 A2. Ofthe 7 patients with SHP, 4 showed staining for A, and2 for A2 when tested with the sheep antiserums. Withmonoclonal antibody, 5 had A,, but only 1 (Patient29) had A2 (Figure 1). Of the 5 patients with SLE,sheep antiserums showed A, in 4 and A2 in 4. Withmonoclonal antibodies, A, was shown in 5 and A2 in3. Overall, monoclonal antibody to A, was positive in35 of 39 patients, compared with 29 of 39 with thesheep antiserum. Monoclonal antibody for A2, how-ever, was positive only in 8 compared, with 25, of 39patients with the sheep antiserum.

J-chain reactions were present in 32 patients. Therewas no correlation between staining for IgM andJ-chain (P > 0.5). Twenty-six of 39 kidneys had mes-angial IgM staining: 7 of 11 patients with primaryIgA nephropathy, 13 of 16 patients with alcoholic cir-rhosis, all of those with SLE, and 1 of the 7 withSHP. Of these 26, 4 had no staining for J-chain. Ofthe 13 patients without IgM, 10 showed staining forJ-chain. J-chain staining was not found more fre-quently in patients with A2 subclass (P > 0.5).

Staining of the mesangium for SC was found in 29of 39 patients, but this was lost after preincubation ofthe antiserum with NHS in all but 1 case (Patient 12)where a trace remained.The SC binding capacity of the mesangial IgA

deposits is shown in Table 1. Thirteen of the 16 kid-

Figure 1 -Kidney from Patient 29, with Sch6nlein-Henoch purpura,stained with murine monoclonal antibody to A, (a) and A2 (b) anddemonstrated by IIF with biotinylated goat anti-mouse IgA and FITC-conjugated avidin. (x600)

neys from patients with alcoholic cirrhosis and mes-angial IgA deposits showed SC binding (Figure 2). Allof the 9 IF-negative control kidneys and 6 SHP kid-neys failed to bind SC, but biopsies from 3 of 4 pa-tients with SLE and 1 of 10 patients with primary IgAnephropathy bound free SC. There was an overallcorrelation between SC binding and IgM deposits (P< 0.05) but no difference in the frequency of IgM inthe patients with alcoholic cirrhosis, compared withprimary IgA nephropathy (P > 0.5), despite a signifi-cant difference in SC binding between these twogroups (P < 0.01). With the use of a Mann-Whitneytest for nonparametric samples, the difference in SCbinding capacity in patients with primary IgA ne-

Vol. il3 * No. 3

362 LOMAX-SMITH ET AL

Table 1 - Mesangial Immunofluorescence Graded from 0 to 4+

Patientcategory

Primary IgAnephropathy

Mesangial IgA nephritisassociated with alcoholicliver disease

Sch6nlein-Henochpurpura

Systemic lupus erythematosus

NT, not tested. Nine control biopsies were negative in all tests used.

phropathy, compared with those with IgA nephritissecondary to alcoholic liver disease, was highly sig-nificant (P = 0.001).

Discussion

In this study we have examined by IF renal tissuefrom 39 patients with mesangial IgA deposits toelucidate the immunochemical nature of the IgA.With monoclonal antibodies, we showed that A, was

present in all but 4 of the 39 patients, with no sig-nificant differences between the 4 disease categoriesexamined. IgA2 was less common, occurring in only10-20O7o of patients with primary IgA nephropathy,mesangial IgA nephritis secondary to alcoholic cir-rhosis and SHP, and in 3 of the 5 patients with SLE.

A, and A2 subclasses have also been sought byConley et al,'I Andre et al," and Tomino et al,12 withconflicting results. Conley et al, using monoclonalreagents, examined 10 patients with primary IgA ne-

phropathy, 11 with SHP, and 9 with SLE. All were

positive for Al, but none showed A2 staining; and,while present, J-chain staining correlated only withthe intensity of IgM staining and not with that ofIgA. Andre et al found that rabbit antiserum (Nordic)to A, stained the mesangium of 5 of 10 patients withprimary IgA nephropathy, 8 of 9 with alcoholic cir-rhosis, and all of those with SHP and SLE. In con-

trast to Conley et al. Andre et al, using rabbit anti-serum to A2 (Nordic), demonstrated staining in all 10patients with primary IgA nephropathy, all 9 patientswith alcoholic cirrhosis, both of those with SHP, and

Patientnumber

1

234567891011

12131415161718192021222324252627

28293031323334

3536373839

Sheepantiserum

Al A2

3 23 33 23 12 10 01 02 13 41 13 1

4 4o o3 31 11 13 32 23 01 0o 01 10 10 00 01 11 1

0 04 42 10 01 00 01 0

4 34 32 11 00 1

IgA

33343334434

4422334321333333

2233322

44332

Murinemonoclonal

A1 A2

3 03 03 03 03 30 01 03 14 04 03 0

4 01 04 03 02 03 03 03 01 00 03 12 12 02 02 03 0

2 04 43 03 00 00 01 0

3 23 22 02 11 0

IgM

20

220

20

0

113

30

04313333134340

0

0

0

10

24334

J

3223

113

33

3

33

33

2

1

32

1

0

2

21

3

230

0

0

2

2

233

SCbindingcapacity

002000000NT0

3014430410334143

0NT00000

24NT10

AJP * December 1983

Vol. 113 * No. 3 NATURE OF MESANGIAL IgA DEPOSIrS 363

Figure 2-SC binding test performed on renal tissue from Patient 24,with mesangial IgA nephritis secondary to alcoholic cirrhosis, incu-bated with PBS (a) and human SC (b) at 200 mg/ml and demonstratedby IIF with rabbit anti-human SC and FITC-conjugated goat anti-rabbitIgG. (x800)

all but 1 with SLE. Tomino et al12 examined 7 patientswith primary IgA nephropathy using both Nordicantiserums and monoclonal reagents and found A,throughout, using both systems but A2 in only 2 in-stances using monoclonal antibody. Tomino et a121further examined 5 patients with SHP, finding A2 innone.We believe, like Tomino et al,12 that some of these

differences relate to the specificity of the reagents. Thesheep antiserum to A2 (Nordic) evaluated in this studycross-reacts with IgA, myeloma cells. The relativelyhigh incidence of mesangial A2 staining with thisantiserum was probably caused by this cross-reactiv-ity. We have more confidence in the monoclonal

reagents, particularly when used with the avidin-biotin method.

It has been suggested that staining for J-chain inpatients with IgA nephropathy merely representsactivity against J-chain in the associated IgM de-posits."0 The finding of mesangial J-chain staining in10 of the 13 IgM-negative cases and its absence in 4of the 26 IgM-positive cases does not support thissuggestion. This would imply the presence of J-chaincontaining IgA polymers in the deposits. Like otherworkers,89 we failed to demonstrate SC in the mes-angial deposits.The study of jejunal SC binding showed that SC

bound specifically to plasma cells, most of which inthis location might be expected to be producingdimeric IgA. With the use of this technique, SC bind-ing was observed in 13 of 16 kidneys from patientswith alcoholic cirrhosis, 1 of 10 patients with primaryIgA nephropathy, none of 6 patients with SHP, and3 of 4 with SLE. The ability of the IgA deposits inalcoholic cirrhosis to bind SC suggests that these arepolymeric."1 However, mesangial IgA is often accom-panied by IgM; and it is necessary to interpret SCbinding with some caution. Five primary IgA ne-phropathy patients and 2 with alcoholic cirrhosis hadsubstantial amounts of IgM, yet failed to bind SC. Incontrast, 2 patients with alcoholic liver disease but noIgM bound SC. There was a marked difference in theSC binding capacity of mesangial IgA deposits inprimary IgA nephropathy and alcoholic cirrhosis, al-though there was no significant difference in mes-angial IgM content.Our data show that SC binding is almost exclusive-

ly restricted to kidneys from patients with alcoholiccirrhosis and SLE. These findings differ from those ofother studies.14,1617 The reason for this is not known,nor can we explain the discrepancy between SC bind-ing and the presence of J-chain. Irrespective ofreagent specificity, we believe that our data demon-strate a functional difference between the IgA depos-its in primary IgA nephropathy and mesangial IgAdeposits secondary to alcoholic liver disease.

References

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2. Nochy D, Callard P, Bellon B, Bariety J, Druet P: As-sociation of overt glomerulonephritis and liver disease:A study of 34 patients. Clin Nephrol 1976, 6:422-427

3. Callard P, Feldman G, Prandi D, Belair MF, Mandet C,Weiss Y, Druet P, Benhamou JP, Bariety J: Immunecomplex type glomerulonephritis in cirrhosis of theliver. Am J Pathol 1975, 80:329-341

364 LOMAX-SMITH ET AL AJP * December 1983

4. Pape JF, Mellbye OJ, Oystese B, Brodwall EK: Glomer-ulonephritis in dermatitis herpetiformis. Acta MedScand 1978, 203:445-450

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9. Dobrin RS, Knudson FE, Michael AF: The secretoryimmune system and renal disease. Clin Exp Immunol1975, 21:318-328

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11. Andre C, Berthoux FC, Andre F, Gillon J, Genin C,Sabatier J-C: Prevalence of IgA2 deposits in IgA ne-phropathies. N Engl J Med 1980, 303:1343-1346

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15. Brandtzaeg P: Characteristics of SC-Ig complexesformed in vitro. Adv Exp Med Biol 1974, 45:87-97

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21. Tomino Y, Endoh M, Suga T, Miura M, Kaneshige H,Nomoto Y, Sakai H: Prevalence of IgAi deposits inHenoch-Schoenlein purpura (HSP) nephritis. Tokai JExp Clin Med 1982, 7:527-532

AcknowledgmentsThe authors wish to thank Dale Caville and Anna

Langen-Zueff for photograhic and graphic assistance; Mrs.Paula Cassidy for typing the final draft; Dr. A. R. Clark-son, Director of the Renal Unit, Royal Adelaide Hospital,for permission to use biopsy material from his patients andassistance in reviewing case histories; Dr. La Brooy for thegift of human secretory component; Ted Huber for his in-valuable help with statistical methods.