Immune regulation of immunoglobulin production in IgA-nephropathy

CLINICAL

1MMUNOLOGY

Immune

AND

IMMUNOPATHOLOGY

23,430-436

(1982)

Regulation of lmmunoglobulin in IgA-Nephropathy**l

Production

FERNANDOG.COSIO, SYLVIALAM, AYOOLAO.FOLAMI,~ MARYELLENCONLEY,~ AND ALFRED F. MICHAEL Department

of Pediatrics.

University of Minnesota.

Minneapolis.

Minnesota 55455

Serum immunoglobulin concentrations, percentage surface IgA-positive peripheral blood lymphocytes, and PWM- and Con A-stimulated immunoglobulin production in vitro were evaluated in patients with IgA nephropathy and normal controls. Elevated levels of serum IgA and IgM were present in 46 and 62% of patients, respectively, but the absolute concentrations of IgA, and IgA, were not different from normal values. In comparison with controls, patients with IgA nephropathy demonstrated similar numbers of surface IgG- and IgA-positive peripheral blood lymphocytes and after stimulation with PWM and Con A their lymphocytes produced similar amounts of IgG, IgA, and IgM. Separate analysis of a subgroup of patients with severe disease demonstrated signiticantly higher IgG and IgM production after PWM stimulation. However, serum immunoglobulin levels were not different. The number of Ig-bearing lymphocytes and the in vitro production of IgA is normal in IgA nephropathy. Excessive production of IgG and IgM in vitro is limited to patients with severe disease.

INTRODUCTION

IgA nephropathy or Berger’s disease is one of the most common forms of primary glomerular disease in humans. The hallmark of the disease is the presence of IgA deposits in the glomerular mesangium, usually associated with deposition of other immunoglobulins and complement components (1). It has been postulated that IgA-containing immune complexes (2) and/or polymeric IgA (3) play a role in the pathogenesis of this disease. More recently, an increased number of surface IgA-positive peripheral blood lymphocytes (4) and defective suppressor mechanisms for IgA production (5) have been demonstrated, but the relationship of these findings to the pathogenesis of the disease is unclear. Immunohistochemical studies using heterologous antisera have suggested the presence of IgA, and IgA, in glomerular deposits (6). However, studies carried out with highly specific monoclonal reagents demonstrated only IgA, and not IgA, (7). This, in concert with the absence of secretory component and J chain (8), suggests that the IgA, is not polymeric or secretory IgA. In the present study, we have evaluated the number of surface IgA-positive peripheral blood lymphocytes and the regulation of IgA production in vitro in a group of patients with kidney biopsy proven IgA nephropathy. Our results demonstrate that the number of surface IgA-positive peripheral blood lymphocytes, the immune regulation of IgA production in vitro, * This article is dedicated to Robert A. Good on the occasion of his 60th birthday. 1This work was aided by grants from the National Institutes of Health (AI10704 and AM25518). the Minnesota Medical Foundation (KRF-2-81), the Arthritis Foundation, and the Minnesota Vikings Children’s Fund. * Department of Pediatrics, University College Hospital, Ibadan, Nigeria, West Africa. 3 Department of Immunology, Children’s Hospital, Philadelphia, Pennsylvania. 430 0090-1229/82/050430-07$01,00/O Copyright All rights

@ 1982 by Academic Press. Inc. of reproduction in any form reserved.

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and the absolute serum concentrations of IgA, and IgAB are normal. In addition, we have confirmed previous reports demonstrating high serum concentrations of IgA and IgM in some patients with this disease. MATERIALS

AND

METHODS

Patient selection. Peripheral blood was obtained from 15 patients with IgA nephropathy and 23 controls. The group of patients included nine males and six females ages from 11 to 40 years (mean 22). The control group included 11 males and 12 females ages from 18 to 37 years (mean 26). Informed consent was obtained from ail subjects. The diagnosis of IgA nephropathy was based upon a clinical history of recurrent macroscopic or persistent microscopic hematuria; the absence of systemic diseases, liver disease, or anaphylactoid purpura; and the presence of mesangial deposits of IgA usually associated with other immunoglobulins (Ig) and complement components. Cell separation. Whole blood was diluted with an equal volume of media RPM1 and centrifuged in a Ficoll- Hypaque gradient. Mononuclear cells were recovered from the gradient interphase, washed three times with RPM1 and 20% horse serum (HS), and stained for surface immunoglobulins and/or established in culture. Surface immunoglobulins. Fluorescein-labeled F(ab’)z goat anti-human IgG, IgA, IgM, and IgD (Kallestad Laboratories, MN) were used for staining lymphocyte surface Ig using methods previously described (9). Monocytes were identified by latex bead ingestion and excluded from cell counting. At least 200 cells were counted in each slide and results were expressed as percentage surface immunoglobulin-positive cells. Cell cultures. Two million mononuclear cells were placed in round bottom sterile plastic 12 x 75 tubes (Falcon) at a concentration of 1 x lo6 cells/ml in RPM1 with 20% HS. Tubes were loosely capped and incubated for 7 days in an incubator at 37°C with 5% CO,. By titration of mitogen we determined that pokeweed mitogen (PWM, Grand Island Biological Co.), at a concentration of 15 wl/ml, produced maximal stimulation of Ig production and concanavalin A (Con A, Pharmacia), at a concentration of 30 pg/ml, produced maximal suppression of PWM-stimulated Ig production. The same lot numbers of both mitogens were used throughout this study. Three sets of cultures were established for each patient and control: unstimulated, PWM stimulated, and PWM and Con A stimulated. At the end of 7 days supematants were separated by centrifugation and kept at -70°C for subsequent determinations of Ig concentration. Cell viability, as determined by Trypan blue exclusion, was 70 to 80% at the end of the culture period. Determination of immunoglobulin concentrations. The concentrations of IgG, IgM, and IgA in the culture supernatants were determined by a sensitive fluorometric assay previously described (Immunofluor, Bio-Rad) (IO). The percentage suppression of immunoglobulin production induced by Con A was calculated by the following formula: % Suppression

=

IgPWM

- Ig (PWM IgPWM

-t Con A)

432

COSIO ET AL.

where IgPWM represents Ig produced in PWM-stimulated cultures and Ig (PWM + Con A) represents Ig produced in cultures stimulated with PWM and Con A. Serum Ig concentrations were determined by the same fluorometric method. Levels of serum IgA, and IgAz were evaluated in three patients evaluated for in vitro Ig production and 10 additional patients by radioimmunoassay (lOa). Student’s f test was used for data analysis. RESULTS Lymphocyte Surface Immunoglobulins Surface immunoglobulins were determined in 12 patients and 18 controls. There were no significant differences in the percentage surface immunoglobulin G, A, M, or D-positive lymphocytes between both groups (Table 1). Serum Immunoglobulins Six of 13 IgA nephropathy patients (46%) had elevated concentrations of IgA (>250 mg/dl) and 8 of 13 (62%) demonstrated elevated concentrations of IgM (>200 mg/dl). Serum IgG concentrations were within normal limits in all 13 patients tested (Table 2). The serum level of IgA, was 90.8 + 48.5% (mean k SD) of standard pool (controls 92.9 k 40.0) and that of IgA, was 82.2 k 63.2% of standard pool (control 90.2 t 40). There were no statistically significant differences between patients and controls. In Vitro Immunoglobulin

Production

In unstimulated cultures, the production of IgG, IgA, and IgM was significantly higher in patients than in controls (Table 2). Analysis of the data revealed that in 10 of the 15 patients, unstimulated production of Ig was similar to normal controls: However, one patient demonstrated high unstimulated production of all three Ig, one patient high production of IgG and IgA, one patient high production of IgG, and two patients high production of IgM. After PWM stimulation, there were no statistical differences in the amounts of IgG, IgA, or IgM produced by patients or control’s cells (Table 2). In cultures of patients and controls, IgM was produced in highest amounts and IgG in lowest. None of the cultures demonstrated isolated increased production of IgA or IgG; however, cultures from four of the 15 patients and two of the 23 controls demonstrated isolated increases in IgM production in response to PWM stimulation. There was no difference in the percentage suppression of IgG, IgA, and IgM TABLE PERCENTAGE

1

SURFACE Ig-PosITwE LYMPHOCYTES IN CONTROLS PATIENTS WITH IgA NEPHROPATHY

Group

W

IgA

Control (18)” IgA nephropathy (12Y’

2.8 2 0.6 2.1 2 0.25b

1.50 -r- 0.21 1.75 k 0.12

n Number of controls or patients. b Values represent means + SEM.

W 9.50 2 1.10 9.41 -c 1.51

AND

W 11 2 1.85 8.83 + 1.20

Total 21.6 r 2.85 18.8 * 2.20

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TABLE 2 SERUM Ig AND in Vitro Ig PRODUCTION IN PATIENTS WITH IgA NEPHROPATHYANDCONTROLS In vitro Ig production (ngi2 x lo6 cells) class

Serum Ig (mgidl)

Unstimulated

PWM

Con A (% suppression)

G A M

910 k 172” 184 + 24 182 k 36

150 2 24 108 2 20 116 2 25

700 2 112 816 t 169 1450 t 295

69 5 5 71 2 5 80 + 5

G A M

626 k 68 234 i- 28 224 k 40

304 2 9Ob 247 T 82* 239 2 82”

797 + 188 1045 2 309 2315 t 538

64+5 70 5 7 76 k 5

k Group Controls

Patients

’ Values represent means 2 SEM. * Compared with controls P < 0.05.

production induced by Con A between patients and controls (Table 2). None of the patients demonstrated isolated lack of suppression of IgA although in one patient Con A stimulation produced only minimal suppression of IgA (16%) and IgG (22%) production. Clinical

Correlations

On the basis of clinical and pathological parameters known to affect the prognosis of the disease (1 l), seven patients were thought to have severe IgA nephropathy and eight patients mild disease. All seven patients in the severe group demonstrated proteinuria which was >3 g/24 hr in four of seven. Five of the seven demonstrated decreased creatinine clearance although the highest serum creatinine in the group was 2.5 mg/dl. Six patients demonstrated severe disease on kidney biopsy with diffuse proliferation, focal necrosis, and crescents in one case. Three patients on the mild group and three in the severe group had elevated serum IgA levels. Elevated serum IgM levels were present in four patients with mild disease and in three patients with severe disease. Compared with patients with mild disease, patients with severe disease appeared to have higher production of Ig in PWM-stimulated cultures; however, these differences did not reach statistical significance. There were no differences in unstimulated cultures or in Con A suppression between patients with mild or severe disease (Table 3). Compared with controls, patients with severe disease produced significantly higher IgM (P < 0.01) and IgG (P < 0.025) in response to PWM; although production of IgA appeared higher the difference was of borderline statistical significance (0.1 < P > 0.05) (Fig. 1). No differences in unstimulated Ig production or in Con A suppression were observed between patients with severe disease and controls. In one patient in vitro Ig production was determined during remission and during an episode of gross hematuria. During remission, IgG and IgA production was higher than controls in unstimulated cultures and failed to suppress in re-

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COSIO ET AL. SERUM

Ig CONCENTRATIONS PATIENTS

AND

WITH

MILD

TABLE 3 In Vitro IMMUNOGLOBULIN PRODUCTION AND SEVERE IgA NEPHROPATHY

IN

In vitro production (ng/2 x 10” cells) Severity of disease

PWM

PWM + Con A (f suppression)

276 k 120 195 k 90 236 5 121

557 t 191 671 + 352 1910 k 520

592 10 69 2 9 77 rt 9

196 k 90 271 + 141 244 f 119

1676 t 608’ 1529 it 529 3011 + 1008d

72 k 4 7117 75 f 5

Ig class

Serum Ig (mddl)

Unstimulated

Mild (8)n

G A M

701 k 79b 227 f 35 247 k 45

Severe (7)

G A M

539 2 100 242 k 54 223 2 47

(1Number of patients. b Values represent means c SEM. c Compared with controls (Table 2) P < 0.025. d Compared with controls (Table 2) P < 0.01.

sponse to Con A. IgM production was similar to controls. During a subsequent episode of gross hematuria, patient’s cells demonstrated higher than controls unstimulated production of IgG, IgA, and IgM and failed to respond to both PWM and Con A. This patient was thought to have mild IgA nephropathy and had no clinical or serologic evidence of systemic lupus erythematosus. DISCUSSION The results of the present study indicate that the number of surface IgA-positive lymphocytes in peripheral blood from patients with IgA nephropathy is similar to

F

ontrol

Mild

Severe

IgmNeDhropathy

FIG. 1. PWM stimulated Ig production disease (0) and severe disease (0).

IgwNephropathy

in controls (0) and IgA nephropathy patients with mild

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normal controls. In addition, in vitro IgA production by mononuclear cells stimulated with PWM and/or Con A did not differ significantly between patients and controls. Several patients demonstrated elevated concentrations of serum IgA and IgM, but these values did not correlate with the severity of the disease. Serum concentrations of IgA, and IgAz were normal in these patients. Our results disagree with previous studies demonstrating a decreased number of IgG-bearing lymphocytes and an increased number of IgA-bearing peripheral blood lymphocytes in patients and family members of patients with IgA nephropathy (4, 12). The reason for these discrepant results is not clear but may be related to the use of non-Fab reagents for detection of cell surface Ig in prior studies. The present study has not confirmed the presence of defective suppressor mechanisms for IgA production (5). We have demonstrated that patients with severe disease demonstrate increased Ig production in response to PWM but the defect affected the production of IgG and IgM but not IgA. In addition, the increased Ig production was not caused by defective Con A suppressor mechanisms nor by an increased Ig production in unstimulated cells. The relation of this in vitro defect to the pathogenesis of the disease is unclear but could reflect the degree of proteinuria present in these patients. In one patient we demonstrated, on two occasions, defective regulation of Ig production; however, the defect affected IgA, IgG, and IgM. High unstimulated production of immunoglobulins and defective suppression of Ig production in response to Con A is reminiscent of the immunoregulatory defect previously demonstrated in patients with systemic lupus erythematosus (13), but this patient had no clinical or laboratory parameter suggestive of this disease. Thus, we have been unable to demonstrate any uniform defect in immunoregulation of Ig production in patients with IgA nephropathy. We have confirmed previous reports of elevated serum IgA and IgM concentrations in some patients with Berger’s disease (2). The fact that not all patients demonstrated these abnormalities may indicate individual variations of the disease or variations in Ig concentrations in different phases of the disease. It is of interest, however, that the absolute serum levels of IgA, and IgAz in IgA nephropathy were not different from normal even though immunohistochemical studies have demonstrated glomerular deposition of IgA, but not IgAz (7). The major source of serum IgA is the gut-associated lymphoid tissues (GALT) (14). Defective regulation of IgA production by the GALT could explain high serum IgA concentrations and the frequently observed association of episodes of gross hematuria with symptoms of mucosal inflammation. In animals, the production of serum IgA antibodies by the GALT is modulated by genetic and environmental factors: the mouse strain C,I-UHeJ demonstrates excessive production of serum IgA antibodies following oral immunization with sheep erythrocytes (15); the presence of Con A in the feed greatly increases the serum IgA antibody response to oral antigens in normal mice (16). It is possible that similar factors affect the GALT production of IgA in humans. A high prevalence of certain HLA antigens has been demonstrated in patients with IgA nephropathy (17) and recent studies in our laboratory have demonstrated an association with certain B-cell alloantigens (18). If hypersecretion of IgA by the GALT is present in patients with IgA nephropathy, the defect is not demonstrable in peripheral blood lymphocytes under the conditions of the present study.

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High production of IgA or high serum IgA levels do not explain the subsequent deposition of this protein in the glomerular mesangium. IgA immune complexes have not been demonstrated consistently in this disease (2), but this failure may be related to lack of highly specific and sensitive assays. Lopez Trascasa et al. (3) have demonstrated an increased amount of polymeric IgA on patients with IgA nephropathy; however, studies by Woodroffe et al. (2) have failed to demonstrate an increased serum concentration of polymeric IgA results which are in agreement with our own unpublished observations. ACKNOWLEDGMENTS We thank Mrs. Janice Aplin for secretarial assistance and Mr. Marshall Hoff for artistic assistance.

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