Glomerular and serum immunoglobulin G subclasses in IgA nephropathy

CLINICAL

IMMUNOLOGY

Glomerular P. AUCOUTURIER,*

AND

IMMUNOPATHOLOGY

51,

338-347 (1989)

and Serum lmmunoglobulin IgA Nephropathy R. C. MONTEIRO,?

L. H. No&t

G Subclasses J. L.

in

PRRUD’HOMME,*

AND P. LESAVREt” VNSERiU U25 and Department of Nephrology, HGpital Necker, 161 rue de SPvres, 75015 Paris, France, and “Laboratory of Immunology and Immunopathology (CNRS UA 1172). Poitiers University Hospital, 86021 Poitiers, France The distribution of human IgG subclasses among mesangial glomerular deposits of 11 patients with IgA nephropathy (IgA-N) was examined by indirect immunofluorescence with subclass-specific mouse monoclonal antibodies (mAb). A subclass restriction was observed with mesangial deposits containing almost exclusively IgGi (81% of the studied biopsies) and IgG, (64%). IgG, was present in only 1 out of the 11 cases studied and IgG., was never found to be present, although seven different anti-&G, mAb were used. In addition, serum levels of total IgA and IgG, as well as serum IgG subclass levels, were measured in 21 patients with IgA nephropathy by an indirect competitive immunoenzymatic assay using mAb. It was noted in IgA-N patients, but not in normal individuals, that there was signiticant positive correlation between total IgA and IgG serum levels which was entirely due to a positive correlation between total serum IgA and IgG, levels. This study provides no explanation for the subclass restrictions observed but suggests that (i) the presence of IgA-IgG,-IgG, in mesangial deposits may be secondary to an antigenic stimulation, possibly viral, and (ii) the positive correlation between IgA and 0 1989 Academic IgC+ serum levels may result from an increased T helper function. Press, Inc.

INTRODUCTION

Previous reports on the distribution of the four IgG isotypes among glomerular deposits in human glomerulonephritis have been published with various results (l-6). We have recently shown that the glomerular deposition of the four IgG subclasses differs according to the type of glomerulonephritis (GN) studied: a marked subclass restriction was observed both in idiopathic membranous nephropathy and in anti-glomerular basement membrane nephritis (7). It is notable that IgG,, which is present only in smaIl amounts in serum, was highly present in these two types of GN. In the present study we determined serum IgG subclass levels and IgG subclass distribution among glomerular deposits in IgA nephropathy (IgA-N) using subclass-specific mouse monoclonal antibodies (mAb). Mesangial IgA deposits contain predominantly, if not exclusively, IgA, subclass (8, 9,lO). In addition to these IgA deposits, which constitute the hallmark of this GN, IgG are deposited in the same mesa&al localization in about one-third of the biopsies studied. Although not constant, the mesangial IgG deposits may represent an important part of the immune response in this disease, as has been shown in a murine model (11). i To whom reprint requests should be addressed. 338 0090-1229/W $1.50 Copyright 0 1989 by Academic Press, Inc. Au rights of reproduction in my fom reserved.

IgG

SUBCLASSES

IN

IgA

339

NEPHROPATHY

In contrast with Russel et al. (10) who reported that IgGr, IgG,, and IgG4 were present in the mesangium in biopsies from patients with &A-N, we report here a clear restriction of IgG subclass distribution, the mesangial deposits in IgA-N containing almost exclusively IgG, and IgG,. Furthermore, the determination of serum IgG subclass levels in IgA-N did not reveal major imbalances but a significant increase in the IgA serum level unexpectedly positively correlated to the IgG, serum level. PATIENTS

Three groups of patients were studied. Group 1: Primary IgA nephropathy (Berger’s disease), including 27 Caucasianpatients (22 men and 5 women aged 15 to 74 years, mean 37.6 years) with biopsy-proven IgA-N corresponding to the more recent and successively seen patients in our department. None had signs of systemic lupus erythematosus (SLE), liver disease, Henoch-Schbnlein purpura (HSP), or other systemic diseases. Kidney biopsies of 11additional patients with IgA-N were retrospectively selected among the biopsies cryopreserved in our laboratory, on the basis of the presence of mesangial IgG deposits in addition to IgA. Those biopsies were studied for IgG subclass distribution among the deposits. Group 2: 8 patients under 15 years with active HSP nephritis and with mesangial IgA deposits demonstrated by kidney biopsy. Group 3: 8 patients with alcoholic liver cirrhosis. Control sera: 35 age- and sex-matched controls randomly chosen from a group of 122Caucasianblood donors whose IgG subclasslevels had been previously studied (12). METHODS Antibodies. Fluorescein-conjugated goat or rabbit polyclonal antisera specific for human y, OL,and l.~chains were purchased from Hyland laboratories (Costa Mesa, CA) or Behringwerke (Marburg, West Germany). Murine mAb specific for the four human IgG subclasseswere used for the analysis of glomerular deposits and serum level determinations (Table 1). Rhodamine-tagged rabbit F(ab’)* (prepared in our laboratory) and fluorescein-conjugated affinity-purified goat antibodTABLE No. 1 2 3 4 5 6 7 8 9 10 I1 12

Clone NL16 HP6014 GOM2 ZG4 C3.8.34 RJ4 HP6020 HP6022 HP6023 HP6025 HP6026 HP6006

Origin Seward Dr. C. Seward Seward Dr. M. Seward Dr. C. Dr. C. Dr. C. Dr. C. Dr. C. Dr. C.

B. Reimer

D. Cooper B. B. B. B. B. B.

Reimer Reimer Reimer Reimer Reimer Reimer

1 Specificity

Ref.

ylFc y2Fab y2Fc y3 hinge

(13, 1% (14) (13, 15) (13, 14) (15) (13, 14) (14) (14) (14) (14) (14) (14)

Y3 y4Fc v4Fd y4Fc y4Fc y4Fc y4Fd y4Fd

340

AUCOUTURIER

ET AL.

ies to mouse Ig (Behringwerke, Marburg, West Germany) absorbed with solubilized human IgG were used to reveal the mouse mAb. Zmmunojluorescence studies. Biopsy specimens were frozen in isopentane cooled by liquid nitrogen. Two micrometer sections were obtained in a cryostat and incubated for 30 min in a moist chamber with mAb diluted l/50 (mAb No. I, 2, 4, 6 and 7-12 when indicated) and then with the anti-mouse Ig conjugate. A further threefold dilution (l/150) of the mAb did not significantly change the fluorescence reaction, suggested excess conditions at the working dilution. Total serum ZgA and ZgG, and ZgG subclass levels. IgG subclass levels were determined in coded samples from 43 patients without knowledge of the clinical context, by competitive indirect immunoenzymatic assay with the mAb 14 presented in Table 1, as previously described (12). The subclass specificity of these six mAb, which has been previously reported (13-17), was further confirmed in our laboratory by ELISA (and Western blot) with a panel of purified human monoclonal IgG (12). Normal values were established in the study of 122 sera from normal adult blood donors (12). Total serum IgG and IgA levels were determined by laser nephelometry (Behringwerke). The analyses were performed in blind fashion, with clinical histories obtained after completion of serum Ig level determination. RESULTS

Distribution of ZgG subclasses among mesangial deposits in primary ZgA nephropathy. The distribution of IgG subclasses among mesangial deposits in IgA nephropathy clearly appears restricted to IgG, and IgG, (Fig. 1). The absence of IgG, was observed using mAb no6 (see Table 1). Since the absence of IgG, was contradictory with the results obtained by Russell et al. (IO), we used six additional anti-&G, monoclonal antibodies kindly provided by C. B. Reimer (mAb 0-12) (14) (see Table 1) to study five biopsies from IgA-N patients (patients No. 1,5,7,8,9). IgG, was not detected in these cases with any of the seven anti-&$,.

10025 -4 42 503 8

lg6 sllbcbsses

106 slibckm

FIG. 1. Distribution of IgG subclasses among glomerular deposits in IgA-N (11 biopsies). (Left) a, Positive; Cl, negative by immunofluorescence studies. (Right) Results are expressed as the percentage of positive biopsies for one given igG subclass.

IgG

SUBCLASSES

IN

IgA NEPHROPATHY

341

Conversely, all these seven mAb led to the detection of IgG., in two biopsies from membranous nephropathy patients who were used as positive controls, since we have previously shown the presence of IgG., in such cases (7). Representative immunofluorescence micrographs of biopsy No. 7 are presented in Fig. 2. No correlation was observed between the IgG subclasses identified and the presence of mesangial complement proteins (Clq absent and C3 present in the 11 biopsies studied), the degree of mesangial lesions, and the clinical status (creatininemia, proteinuria, hematuria). Serum ZgG subclass levels. Mean serum Ig concentrations in IgA-N patients (Table 2) were not significantly different from those of normal controls, except for IgA (Z’ < 0.001); the IgGz serum level was elevated individually in two patients who also had high IgA levels. No other IgG subclass imbalance was found in this group. Elevated serum IgG, and IgG, were also noted in patients with alcoholic cirrhosis, with no evident link between serum IgG, and IgA; however, statistical analysis in this group (as well as in HSP) could not be performed because of the small number of subjects. The existence of increased IgG, concentrations in some patients with IgA-N led us to look for correlations between different IgG isotypes in this disease. Linear regression analysis of the total IgA and IgG subclass levels showed some peculiarities in the group of patients with IgA-N. Total IgG showed a clear link with serum IgA, which was not found in normal controls (Fig. 3). This correlation was only due to IgG, levels since no significant correlation could be demonstrated between IgA and the other IgG subclass or their sum (Table 3). DISCUSSION

The distribution of the four IgG subclasses among glomerular deposits in human GN has been studied by several authors (l-7). Here we investigated the isotypes

FIG. 2. (a) Direct immunofluorescence study with IgA anti-serum. x310. IgA is detected in the mesangial area. (b) Direct immunofluorescence study with IgG antiserum. x310. IgG is detected in the same localization as IgA. (c) Indirect immunofluorescence with mouse monoclonal antibody directed against IgG,. IgG, is detected in the same localization as IgA and IgG.

342

AUCOUTURIER

ET AL.

TABLE TOTAL

SERUM

2

IgA AND IgG AND IgG SUBCLASS LEVELS IN PATIENTS WITH IgA HENOCH-SCH~NLEIN PURPURA, AND CIRRHOSIS

IgA

1gG bwhnl)

kG,

kG,

WA

mg/ml (% of total Ifi)

No.

(mg/ml)

IgA nephropathy (n = 27) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

9.3 6.3 6.2 3.8 3.7 4.6 2.0 3.9 2.3 3.0 2.6 3.3 3.4 2.4 5.7 4.8 4.5 3.8 4.5 4.3 3.4 2.5 4.7 4.3 3.1 4.2 5.0

20.0 12.3 10.4 9.5 14.9 5.8 10.1 11.0 6.2 15.4 7.8 10.4 13.3 5.2 13.5 8.5 9 6 8 10.5 10 9.5 11 12 10.5 10 11

7.6 5.0 5.5 5.05 8.5 4.25 5.75 7.7 4.25 8.7 5.85 9.6 7.5 2.75 7.6 7.0 5.8 3.7 5.1 7.7 5.5 7.4 6.7 7.3 7.25 6.5 6.5

Mean

4.13

10.43

6.37 (64)

+ SD

1.53

3.2

1.62 (11)

Henoch-Schiinlein purpura (n = 8) 28 29 30 31 32 33 34 35

3.8 1.9 8 1.9 5.5 1.7 2.5 2.7

6 2.5 7.5 2.5 5.5 6.5 6 6.5

Mean

3.5

5.37

5.56 (75)

+ SD

2.2

1.86

3.06 (8)

-

k’%

NEPHROPATHV.

3.55 2.0 5.0 3.5 4.15 7.7 11.5 7.1

(41) (32) (69) (55) (73) (62) (57) (64) (60) (58) (65) (67) (60) (55) (69) (70) (82) (71) (71) (73) (69) (85) (62) (72) (69) (58) (59)

(77) (76) (59) (70) (78) (84) (82) (74)

9.95 10.2 2.0 3.50 2.0 2.0 3.70 3.5 2.0 5.45 2.35 3.5 3.10 1.30 1.85 1.90 1.40 1.20 1.40 1.90 1.90 0.95 3.50 1.80 2.65 4.30 3.85

(54) (65) (25) (38) (17) (29) (37) (29) (28) (36) (26) (24) (25) (26) (17) (19) (18) (23) (19) (18) (24) (11) (32) (18) (25) (38) (35)

0.20 (1) 0.46 (3) 0.32 (4) 0.41 (4) 1.15 (10) 0.27 (4) 0.44 (4) 0.45 (4) 0.70 (10) 0.30 (2) 0.37 (4) 0.48 (3) 0.85 (7) 0.26 (5) 0.43 (4) 0.53 (5) 0.50 (6) 0.31 (6) 0.48 (7) 0.42 (4) 0.27 (3) 0.37 (4) 0.31 (3) 0.31 (3) 0.30 (3) 0.34 (3) 0.45 (4)

0.67 0.125 0.16 0.26 0.047 0.28 0.15 0.32 0.17 0.62 0.43 0.66 0.98 0.71 1.05 0.54 0.12 0.005 0.20 0.54 0.21 0.010 0.25 0.75 0.31 0.065 0.21

3.08 (28)

0.43

(4)

0.37

(4)

2.25 (12)

0.20

(2)

0.29

(3)

0.65 0.40 2.50 1.0 1.10 1.80 1.60 1.70

0.28 (6.1) 0.13 (3) 0.60 (7) 0.39 (8) 0.25 (4) 0.73 (7) 0.47 (3) 0.38 (4)

0.10 0.095 0.33 0.11 0.20 0.22 0.48 0.41

(2)

(3) (4)

1.34 (19)

0.40

(5)

0.24

(3)

0.69 (6)

0.19

(2)

0.14

(1)

(23) (15.2) (30) (20) (19) (17) (11) (18)

(3) (1)

(2) (3) (0.5) (4) (1) (3)

(2) (4) (5) (5)

(8) (14) (10) (5)

(2) (0.1) (3) (5) (3) (0.1)

(2) (7) (3) (0.5)

c3

(3) (4)

(2) (3)

(2)

IgG

SUBCLASSES Table

Id

w

(mg/ml)

No.

Cirrhosis (n = 8) 36 37 38 39 40 41 42 43 Mean t SD Normal controls (II = 122) Mean 2 SD

IN IgA

343

NEPHROPATHY

2-Continued

NJ*

w*

(mg/ml) -

I&

mg/ml

IS,

(% of total IgG)

4.3 4.3 8.5 5.2 7.2 4.8 4.8 8

13 11.5 15 13.5 10.5 12 12 16.5

13.7 6.6 14.1 9.25 10.0 9.7 7.2 8.5

(90) (69) (78) (67) (83) (69) (58) (41)

0.95 2.25 3.0 3.50 1.05 3.10 4.60 11.1

(6) (23) (17) (25) (9) (22) (37) (54)

0.53 0.57 0.66 0.69 0.64 0.58 0.48 0.50

(3) (6) (4) (5) (5) (4) (4) (2)

0.062 0.12 0.21 0.38 0.29 0.59 0.09 0.45

5.89 1.72

13 1.96

9.88 2.74

(69) (15)

3.69 3.23

(24) (IS)

0.58 0.07

(4) (I)

0.27 0.18

(2)

2.98

9.81

6.84

(65)

2.75

(26)

0.37

(3)

0.54

(5)

1.17

1.96

1.75

(17)

1.26

(12)

0.14

(1)

0.48

(5)

(0.5) (1) (1) (3)

(2) (4) (0.5)

(2) (1)

of mesangial IgG which are codeposited with IgA in IgA-N in about 30% of the biopsies. We observed a subclass restriction with IgG mesangial isotypes being predominantly IgG, (82% of the biopsies studied) and IgG, (64%). It is possible that in this work the sensitivity of the indirect IF study might have differed for each of the IgG subclasses due to the individual properties of the anti-IgG subclass mAb. This possibility is unlikely, however, because of the striking differences we observed in IgG subclass distribution between IgA-N in this work and idiopathic membranous nephropathy (MN), de ndvo MN, anti-glomerular basement membrane nephritis, and SLE nephritis in our previous report (7). At variance with the work of Russell and colleagues (1 l), we did not detect mesangial IgG, deposits in

IgA-GN

l

.

l

l

.

8 0

a #

0

5

10 0 lgG2 (ma/ml)

5

FIG. 3. Correlation between IgA and lgGz serum levels in 27 IgA-N patients 0.54, P < 0.01) and in 35 controls (0) on the right (r = 0.02. not significant) rank-order correlation test.

10 (0)

on the left (r = using a Spearman

344

AUCOUTURIER

CORRELATION

BETWEEN

I&

0.70

SERUM

I&

TABLE 3 IgG SUBCLASSES AND IgA NEPHROPATHY 1+314

ND

P < 0.001

0.82 P < 0.001

0.08 NS 0.32

0.15 NS ND ND

NS

0.40 P < 0.05

ET AL.

IgA

LEVELS

IN

27PATIENTS

WITH

kA

k@.,

kG,

k&-h

0 NS

0.36

0.26

0.18

NS

NS

NS

0.54

0.06

0.15

P < 0.01

NS

NS

0.09 NS

0.05 NS

0.09 NS

0.004 NS

Note. Linear regression analysis; NS, not significant; ND, not determined.

IgA-N. Although we have no explanation for the difference observed, it is likely that in our study the absence of IgG, was real since the seven anti-IgG, mAb used here led to negative IF in IgA-N and to positive IF in two biopsies of patients with membranous glomerulonephritis where we earlier showed IgG, to be present (7). This study provides no explanation for the restrictions observed but it may favor the hypothesis that the IgA,-IgG,-IgG, deposited in IgA-N could represent antibodies to viral proteins known to be of IgG, and IgG, isotypes (llS-20). In this regard, it has recently been suggested that immune response to CMV may play a role in IgA-N (21). In contrast, antibodies to dietary proteins have been reported to be partially of the IgG, subclass (22), an isotype absent from mesangial deposits in IgA-N. The determination of IgG subclass serum levels in patients with IgA-N revealed that in addition to an increased IgA level already reported, a correlation between the IgA and the IgG serum levels exists. This correlation is of interest since it is not observed in normal controls (matched for age, sex, and racial origin) nor is it observed in other pathological conditions such as IgA deficiency (23,24), primary immune deficiencies (24, 25), and HIV infections (26). This correlation observed in IgA nephropathy was exclusively due to the correlation between IgA and IgG, serum levels. This is reminiscent of the data obtained by Russell ef al. (10) showing a highly significant positive correlation between IgA and IgG antibodies to casei’n in IgA-N patients but not in normal individuals, the IgG isotypes not being determined in this study. More generally, several observations have provided evidence in favor of a closely related regulation of IgA and IgGz expression in the serum. Indeed, while adult levels of IgM, IgG,, and IgG, are attained at the age of 1 or 2 years, IgA and IgG, slowly increase during childhood (27, 28); we recently demonstrated a similar evolution of Ig isotypes after allogenic bone marrow grafts (29). In “selective” IgA deficiency, the absence or low levels of serum IgG, occur in 16 to 25% of the patients (23,24,30-32); however, we found no linear correlation between IgA and IgG, levels in a group of 21 adults with IgA deficiency. In addition, IgG, deli-

IgG

SUBCLASSES

IN

IgA

NEPHROPATHY

345

ciency is often encountered in immune deficiencies where helper T cell function is decreased or defective, such as HLA class II deficiency, symptomatic HIV infections, ataxia-telangiectasia, and Di George syndrome (24-26, 33) (P. Aucouturier et al., manuscript in preparation); these situations in humans (34) are in accordance with the observation in the mouse that the expression of Ig isotypes whose constant region genes are located more “downstream” (3’ region of the CH cluster) are more dependent on T cell help than those with “upstream” (5’ region)-located genes (35). Thus, the significant correlation between IgGz and IgA present in IgA-N patients but not in normal individuals supports the hypothesis of a common dysregulation of Ig isotypes expression by bone marrow plasma cells (36) and may be related to the finding by some authors of increased function of peripheral blood T helper lymphocytes (37-39). Finally, the recent description of the presence of IgG class autoantibodies to glomerular antigens in IgA-N (40) may be relevant to the present study. It may thus be interesting to determine the isotypes of such autoantibodies. ACKNOWLEDGMENTS We are indebted to Dr. L. Halbwachs-Mecarelli for her assistance and critical discussions during this work. We thank Dr. C. B. Reimer (CDC, Atlanta, GA) and M. D. Cooper (UAB, Birmingham, AL) for the gift of mAb; Pr. J. P. Grtlnfeld and the medical staff of the Department of Nephrology (Necker Hospital, Paris, France); F. Lallemand and P. Nusbaum for expert technical assistance; and Mrs. D. Broneer for excellent editorial work. This work was supported in part by the Direction dela Recherche (Ministere de 1’Education Nationale) and Fondation pour la Recherche Medicale.

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6. McPhaul, J. J., and Dixon, F. J., Characterization of immunoglobulin G anti-glomerular basement membrane antibodies eluted from kidneys of patients with glomerulonephritis. J. Zmmunol. 107, 678-684, 7.

8. 9. 10. Il.

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Noel, L. H., Aucouturier, P., Monteiro, R. C., Preud’homme, J. L., and Lesavre P., Glomerular and serum immunoglobulin G subclasses in membranous and anti-glomerular basement membrane nephritis. Clin. Immunol. Immunopathol. 46, 186-194, 1988. Conley, M. E., Cooper, M. D., and Michael, A. F., Selective deposition of immunoglobulin A, in immunogloblin A nephropathy, anaphylactoid purpura nephritis, and system lupus erythematosus. J. Clin. Invest. 66, 1432-1436, 1980. Emancipator, S. N., Gallo, G. R., and Lamm, M. E., IgA nephropathy: Perspectives on pathogenesis and classification. Clin. Nephrol. 24, 161-179, 1985. Russell, M. W., Mestecky, J., Julian, B. A., and Galla, J. H., IgA-associated renal diseases: Antibodies to environmental antigens in sera and deposition of immunoglobulins and antigens in glomeruli. J. Clin. Immunol. 6, 74-86, 1986. Emancipator, S. N., and Lamm, M. E., The role of IgG, IgM and C3 in experimental murine IgA nephropathy. Semin. Nephrol. I, 286-288, 1987.

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