Circulating immune complexes in idiopathic glomerular disease

Kidney International, Vol. 21(1982), pp. 507—512

Circulating immune complexes in idiopathic glomerular disease STEPHEN A. CAIRNS, R. ANSON LONDON, and NETAR P. MALLICK Department of Renal Medicine, Manchester Royal Infirmary, Manchester, England

Circulating immune complexes in idiopathic glomerular disease. Circulating immune complexes (CIC) could be found in the majority of 271 sera from 131 patients with idiopathic minimal change, membranous and mesangial proliferative glomerulonephritis when a combination of CIC assays detecting different properties of dC were used. In neither individual patients nor in any of the three groups as a whole did CIC

than a proportion of the CIC present in pathological sera [31. We have, therefore, used four assays detecting immunochemically distinct CIC in a survey of a large group of adults with

levels reflect the state of the renal lesion. No correlation was found between the class of immunoglobulin in the CIC and that deposited in

findings in minimal change, membranous and mesangial proliferative GN. The results of an assay for IgA CIC in patients with the Henoch-SchOnlein syndrome and other IgA nephropathies were compared with those from patients with other mesangial proliferative lesions and a small number of patients with membranous and minimal change disease.

the kidney. With the exception of minimal change disease in which non-

idiopathic GN. An attempt has been made to correlate the levels, pattern, and size of CIC found with the renal biopsy

Clq binding IgG CIC predominated, a range of dId was found in the patients examined. The pattern of did detected did not allow different forms of renal disease to be distinguished. IgA dId could be found in mesangial proliferative glomerulonephritis both with and without IgA deposition and in some patients with membranous and minimal change disease, as well as in a high proportion of sera from 12 patients with the Henoch-Schönlein syndrome. CIC size was estimated in six patients, but only in one did a specific size of complex predominate. The CIC which may be found in the majority of sera from patients with idiopathic glomerulonephritis provide little information of clinical value; no direct relationship can be demonstrated between the CIC found and the renal lesion.

Methods The patients were derived from those in a long-term study of proteinuria being conducted in our department. All were seen at intervals of four months or less. Patients with other conditions

Immuns complexes circulants au cours des glomerulopathies idiopathiques. Des immuns complexes circulants (ICC) ont été trouvés dans Ia majorit des 271 serums provenant de 131 malades atteints de glomérulonéphrites idiopathiques avec lesions minimes, extra-membraneuse, et proliferative endocapillaire en utilisant une sCrie de dosages des ICC dCtectant leurs diverses propriétés. Chez aucun malade individuellement, ni dans aucun des 3 groupes dans leur ensemble, les concentrations d'ICC ne reflétaient le degre de lesion rénale. II n'a pas Cté trouvé de relation entre Ia classe d'immunoglobuline constituant les ICC et celle déposCe dans le rein. Exception faite des lesions glomei-ulaires minimes ofl des ICC a IgG ne liant pas le Clq predominaient, tout un ensemble d'ICC a été trouvé chez les malades étudiés, et leur composition n'a pas permis de distinguer plusieurs types de néphropathies. Des ICC a IgA ont eté trouvés au cours de glomérulonCphrites prolifératives endocapillaires avec ou sans dCpôt d'IgA et de quelques glomérulonCphrites extramembraneuses ou a lesions glomerulaires minimes, aussi

known to cause GN were excluded. An assessment of renal function was made by measurements of serum creatinine concentration, creatinine clearance, 24-hour urine protein excretion, and counts of cells and casts in 2-hour urine collections. No patient had a serum creatinine concentration greater than 200 moles/liter at the start of the study. The diagnosis was based on a renal biopsy with, in the case of

minimal change disease, a response to steroid therapy. The biopsies were examined by light and electron microscopy and by indirect immunofluorescence for IgG, 1gM, IgA, and complement components. Three groups of idiopathic patients were studied: (1) Minimal change disease. Twenty-four patients with minimal mesangial proliferation, no significant immunofluorescent deposits and only a fusion of foot processes on electron

microscopy; (2) Membranous GN. Thirty-six patients with thickening and spiking of the glomerular basement membrane on light microscopy, predominantly subepithelial dense deposits on electron microscopy and positive immunofluorescence confined to the basement membrane; (3) Diffuse mesangial proliferative GN. Sixty-two patients with diffuse mesangial hypercellularity and positive immunofluorescence confined to the mesangium with no antecedent history of an acute glomerulonephritis. Three sub-groups were identified: (a) Predominant 1gM deposition : 30 patients; (b) Berger's IgA nephropathy: 13

bien que dans un grand nombre de serums de malades atteints de purpura rhumatoIde. La taille des ICC a été estimée chez 6 malades, mais chez un seul prCdominaient des ICC d'une taille specifique. Les ICC retrouvés chez Ia plupart des malades atteints de glomérulopathie idiopathique offrent donc peu d'informations d'intérêt clinique. Ii n'a pas été dCmontré de rapport direct entre les ICC trouvCs et Ia lesion rCnale.

A large proportion of adult human glomerulonephritis (GN) is believed to be immune complex mediated [1]. Animal experimental evidence indicates that the deposition of complexes preformed in the circulation may lead to a range of histological types of glomerulonephritis (GN) [2]. Assays designed to detect such circulating immune complexes (CIC) in human sera all have limitations of specificity and sensitivity which prevent any one assay from detecting more

507

Received for publication March 6, 1981 and in revised form July 8, 1981 0085—2538/82/0021—0507 $01.20

© 1982 by the International Society of Nephrology

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patients, recurrent macroscopic hematuria and prominent mesangial IgA deposition; (c) significant mesangial proliferative on light microscopy but little or no mesangial IgA or 1gM deposition : 19 patients. Henoch-SchOnlein Syndrome (HSS). Twelve patients with the syndrome, all of whom had had extrarenal manifestations, purpura, arthropathy, and/or alimentary involvement at some stage, have been studied. Eleven had renal involvement, and

sera. The mean intra-assay variation of multiple assays of these standards was less than 4%. The interassay variation increased with an increasing degree of inhibition but did not exceed 10%. The average deviation from the mean of a series of aggregated IgG preparations in the L0 assay was 8%.

nine of these had a renal biopsy. All the biopsies showed

The fourth assay used was a solid phase Clq technique (Sc) [6]. Polystyrene tubes were coated with human Clq and EDTA-

mesangial proliferation and mesangial IgA deposition. Renal disease activity. Remission was defined as a resolution of edema and/or hematuria, proteinuria < 300 mg/24 hours and

The LG, Lc, and LA assays were sensitive to less than 1 p.g/ml of aggregated immunoglobulin in decomplemented serum.

treated serum added. After incubation and washing, the Clqbinding IgG containing CIC were estimated quantitatively by an absence of cells and casts from the urine. Relapse was counting the binding of radiolabelled antihuman IgG. Of the believed to have occurred when edema or hematuria recurred; panel of 60 normal sera 90% caused the binding of < 0.4 p.g of 24-hour urine protein excretion exceeded 1 g and/or a urine anti-IgG/ml of test serum. This binding was taken as the upper deposit became abnormal. Declining renal function was defined limit of normal. The intra- and interassay variations of the Sc as a continuing rise of the serum creatinine concentration in at assay were similar to the latex assays; it was sensitive to less least three consecutive serum samples. than 10 p.g of aggregated IgG in EDTA-treated serum. Blood samples were taken from all patients for CIC measureImmune complex size was estimated by Sepharose 6B colments. Sequential samples were taken particularly from pa- umn chromatography. Serum decomplemented as for the latex tients in relapse, those with evidence of continuing disease assays was run on a column calibrated with proteins of known activity and those with declining renal function. molecular weight. Each fraction collected between the void Blood was kept at 37° C after venesection until separated, volume ( > 4 x 106 daltons), and the albumin peak was within 2 hours, and the serum was then snap-frozen and stored concentrated back to the starting volume by ultrafiltration and in multiple aliquots at —70° C. The sera were assayed as soon assayed for LG complexes. Of six normal sera run on the after venesection as feasible, and in all cases within three column, no fraction between the void volume and the elution months. Aliquots of serum, having once been thawed for assay, were discarded. Assay techniques. IgG and IgA containing and Clq binding complexes were measured by the latex agglutination inhibition technique described by Levinsky and Soothill [41 with minor modifications. The assays were performed on sera decomple-

volume of monomeric IgG caused more than 5% inhibition and inhibition below 5% in these fractions of pathological sera was therefore ignored. The LG assay was sensitive to a wide range

mented by treatment with EDTA to release Clq; Clq was

insensitive to detect CIC in column fractions. Statistical methods. Correlations between results of the four assays and between CIC levels and parameters of renal disease were sought using Pearson's correlation coefficient and Kendall's rank correlation.

absorbed on a Sepharose 4B gel coated with IgG. This step also removed rheumatoid factors which would have interfered in the assays and, because in the process sera were diluted 1:10, the concentration of monomeric immunoglobulins was reduced to a level at which they did not interfere in the assays. IgG CIC (IG) were detected by their inhibition of the agglutination by rabbit IgM-anti-IgG of 1.15 p. diameter, 0.8 p, latex coated with IgG and IgA CIC (LA) by the inhibition of latex-IgA agglutination by rabbit 1gM anti-IgA. Clq-binding CIC (Lc) were detected by

their inhibition of the agglutination of latex-IgG by purified

of sizes of LgG aggregates but the L assay was relatively insensitive to small (< 1 x 106 daltons) aggregates and thus proved unsuitable for CIC sizing. The Sc assay was too

Results Two hundred and seventy-four sera from 134 patients, including 37 sera from 12 HSS patients, were examined. Not all were

assayed by each CIC method. The LG assay was applied to almost all sera and the Lc and Sc assays to as many of the sera

human Clq [5]. In each case the inhibition was estimated as was possible within three months of collection. The LA assay quantitatively by counting the unaggregated latex particles in a was applied to the sera from patients with HSS and IgA Coulter counter and expressing the result as the percentage nephropathy and to a selection of sera from patients with other inhibition of the agglutination achieved in the absence of serum.

Chylomicra, which might interfere in this counting process because of their size, were excluded by setting the thresholds

mesangial proliferative, membranous and minimal change disorders. The results of the four assays are detailed in Table 1.

In sequential sera from individual patients, levels of CIC

on the counter so that particles of < 0.6 p.3 (chylomicra —0.4 p.3)

fluctuated from normal to abnormal; the number of individuals

were not counted. The limits of normality of the assays were set using a panel of 60 normal sera. The concentration of the aggregant, rabbit 1gM or human Clq, was adjusted thus altering the sensitivity of the assay until 90% of the panel caused less than 20% inhibition of agglutination; this level was taken as the upper limit of normal.

whose sera were persistently positive or negative was small. The actual levels of CIC in idiopathic GN were low. Most positive sera caused less than 40% inhibition in the latex assays (Fig. 1). Correlation coefficients were calculated between each

Reproducibility of the assays was monitored using known

group.

concentrations of column-purified, heat aggregated immunoglobulin, and multiple aliquots of known positive and negative

of the assays in each group of patients, but a significant correlation was not found between any of the assays in any Minimal change disease. Thirty-three sera from 24 patients were examined. Of these, 52% contained LG but only 25 and

509

Immune complexes and glomerulonephritis

CIC assay LG

L

S

LA

Any assay

45 (33) 54 (24)

19 (26) 25 (20)

12 (25) 16 (19)

42 (12) 71 (7)

64 62.5

44 (85)

40 (58) 53 (36)

18 (68) 26 (31)

42 (36)

70 (36)

67 (15)

67 94

52 (68) 74 (30)

57 (42) 80 (20)

13 (56) 19 (26)

47 (30) 47 (17)

75 87

61(18)

27 (11) 38 (8)

20 (15)

(13)

61(18) 62 (13)

100

Sera (32)

41(32)

38 (24)

20 (25)

14 (8)

Patients (19)

37 (19)

37 (19)

22

(14)

17 (6)

31(23) 38 (8)

64 (33)

73

83 (12)

92

Minimal change Sera (33) Patients (24) Membranous Sera (85) Patients (36) Mesangial proliferative (1) 1gM

Sera (68) Patients (30) (2) Berger's IgA Sera (19) Patients (13) (3)

85

Others

25

(12)

95

56 53

Henoch-SchOnlein syndrome Sera (37)

45 (27)

36

Patients (12)

78

50

(9)

(25) (8)

L,

The table represents the results of four CIC assays, LG, S, and LA in idiopathic ON, subdivided by histological appearances, and in the Henoch-Schönlein syndrome. Both the proportion of patients having at least one positive result and the proportion of positive sera in each group is shown. Results are expressed as the per cent positive; actual numbers of patients and sera involved are in parentheses.

16% contained Lc and Sc CIC, respectively. In at least one assay (including LA), 63% of the sera were positive, and CIC

in none was a relationship found between activity of disease and

could be found at some time in 62.5% of patients.

LA CIC. This type of CIC could be found in most patients with HSS. Levels could be related to the activity of disease in two patients in relapse but not in two others, in one of whom levels never exceeded 20%, the upper limit of normality. Levels

Five patients were seen in relapse but in only one was a relationship apparent between CIC levels and the duration of relapse or proteinuria. In the group as a whole CIC levels did

dc.

not correlate with either the time from the onset, the most

as high as those found in relapse were seen in others in

recent relapse, or with the degree of proteinuria. Indeed, of 22 sera from 16 patients taken 12 months or more after the last relapse when all were proteinuria-free and all treatment had been withdrawn, 50% of the sera contained LG CIC. Membranous GN. CIC could be found by at least one method (including LA) in 67% of 85 sera from 36 patients. Sera from all but four patients contained CIC at some time. The indolent nature of the disorder limited the observations of the relationship of CIC levels to changes in the state of disease in individual patients. Two patients were followed into remission and in two proteinuria was halved during the followup. A fifth patient was followed into a relapse. In none of these five patients was a relationship apparent between the results of any CIC assay and the state of the disease. In the group as a whole, the results of the CIC assays did not correlate with a degree of proteinuria or with the length of time elapsed from the onset or the relapse of the renal lesion. CIC levels were no higher in 23 sera from 6 patients with declining renal function than in patients with stable renal function. Mesangial proliferative GN. Of the 119 sera examined 73% contained CIC in at least one assay, and of the 62 patients in this group 49 had CIC at some stage. A similar high incidence of a variety of CIC was found in the I{SS patients, 9 of whom had mesangial proliferative lesions. No relationship emerged between dC assay results and renal disease activity assessed as the duration of disease, degree of

remission. LG CIC were frequently found in HSS. The coexistence of LA and LG CIC did not correlate with renal disease activity and LA and LG CIC were found together during relapse in one patient without renal involvement. The incidence of LA CIC was equally high in patients with IgA nephropathy but levels again proved to be a poor marker of activity. LA CIC were found in only one of the two patients followed through a relapse of hematuria. A relationship was not apparent between the assay results and the state of the disease. The frequency and levels of LA CIC were as high in patients in remission as in those patients in relapse.

LA CIC were also found, not only in patients with other mesangial proliferative lesions, but also in a small sample of membranous and minimal change sera. Levels of LA CIC alone or in association with other CIC did not allow a clear distinction to be drawn between any of the disease groups examined. Renal immunofluorescence. LG CIC results were compared

with renal IgG deposition in patients with membranous and mesangial proliferative GN. IgG deposition was subjectively graded, without knowledge of the CIC results, by scoring the immunofluorescence of renal biopsy material as negative to 4+. No correlation was found between LG CIC levels and this score in either group of patients.

Similarly, LA CIC levels were compared with renal IgA deposition in all those patients with mesangial proliferative lesions, including nine HSS patients in whom LA CIC had been

proteinuria, or the decline in renal function in this group of measured, but no relationship was found between LA CIC and patients. Seven patients were seen in relapse and remission but

renal IgA deposition.

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CIC size. Sera from two patients from each of the minimal change, membranous, and 1gM mesangial proliferative groups, all of whom had clinically and biochemically active disease and high levels of LG dC, were column-fractionated and LG CICestimated. In neither minimal change, (Fig. 2), nor membranous GN, (Fig. 3), did a specific CIC size predominate. In one patient

with mesangial proliferative GN, who had particularly high (80% inhibition) serum LG CIC levels and whose disease followed an unusually rapid course, complexes slightly smaller than 1gM (9 x i05 daltons) predominated. The second patient with mesangial proliferative GN had no such peak of a specific complex size (Fig. 4). Discussion

CIC levels correlate with disease activity in such systemic immune complex diseases as systemic lupus erythematosus [7— 9] and in acute poststreptococcal GN [10, 11]. Certain sizes of

CIC appear to have particular pathogenic significance in both SLE and acute poststreptococcal GN [7, 8, 11]. It has been

suggested that immunochemical characteristics of CIC are important in determining the manifestations of disease in, for example, HSS [12] and Wegener's granulomatosis [13]. Our study was undertaken to determine whether or not similar considerations apply to an adult population with idiopathic ON.

Results of the application of CIC assays in idiopathic ON have been variable. A major source of this variation appears to be the assay used. Certain assays, for example, latex agglutination inhibition, [141 platelet aggregation [15—17] and lymphocyte

EAC-rosette inhibition [18—20] have been more frequently positive than others such as the Raji cell radioimmunoassay [17, 21—241 and 1251 Clq-binding assays [17, 21, 24—27]. This may be

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2. CIC size in two patients with minimal change disease in relapse. The elution profile of serum proteins from a Sepharose 6B column is shown as a continuous line with the void volume (> 4 x 106 daltons) to the left and albumin peak to the right. The positions at which monomeric 1gM and IgG leave the column are indicated. Each fraction collected was assayed in the LG assay, and inhibition in excess of 5% was plotted as a vertical bar. Fig.

the result of the varying capacities of these assays to detect the type of complexes present because all current methods detect only a limited range of complexes. The spectrum of CIC present in idiopathic GN sera is indicated by the high proportion of sera which were positive in at least one of the battery of assays we used, but only a minority of the sera tested were positive by any single technique. The lack of correlations between our assay results indicates that each is detecting a substantially different portion of the CIC pool. The low levels of CIC found may have contributed to this lack of correlation and accounts for the low

incidence of CIC detected by the Sc assay which proved relatively insensitive. Only in minimal change disease did a specific pattern of CIC emerge. Our finding of non-Clq binding, IgG complexes extends to an adult population, an observation first made in children by Levinsky et al [14]. Unlike these authors and others from the

same group [18], however, we have not been able to demonstrate a link between the levels of CIC found and the activity of the renal lesion. Experiments with the autologous immune complex model of membranous GN have demonstrated the potential importance

511

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the

nephrotic syndrome; the

of in situ immune complex formation in its pathogenesis. This evidence has been taken, together with reports of a lack of CIC in human membranous GN [2 1—23, 26, 28—30], to suggest a similar mechanism of mediation of the human disease [31]. However, CIC may be found in patients with membranous GN [17, 19, 24, 32, 33], and we have found them in the majority of our patients by using multiple assays. Neither pattern nor levels of CIC proved to be a useful guide to disease activity either in individual patients or in the group as a whole. The finding of CIC in membranous nephropathy, in the absence of evidence of a direct role in the renal lesion, leaves open the question of its pathogenesis. HSS may be mediated by IgA complexes [12]. CIC have also been found in other IgA nephropathies [34]. We found such complexes in a high proportion of sera from patients with both HSS and IgA nephropathy. They were also present, however, in a proportion of sera from each of the other histological types of ON examined. LA CIC levels proved to be less than perfect markers of disease activity in HSS, and no relation could be found between LA CIC and activity in Berger's disease. It has been suggested that the coexistence of IgG and IgA CIC results in renal injury in HSS [12]. We have not found this pattern to be exclusive to HSS nephritis, and we have seen it in a patient with

HSS without nephritis and in other forms of ON, with and without IgA deposition. Patterns of CIC have not proved useful in distinguishing the subgroups of mesangial proliferative GN

and, as in membranous GN, although a variety of CIC could be found in the majority of the patients, levels did not prove to be useful indicators of activity. Our failure to demonstrate a relationship between the type of CIC found and the immunoglobulin deposited in the kidney is in agreement with other reports [19, 35]. What is present in the kidney at the time of the biopsy may differ from the material originally deposited, however, and, in our study, not all CIC assays were performed on sera taken at the time of the biopsy.

Animal experimental evidence suggests that only certain sizes of CIC are nephropathic [2]. Further evidence that the majority of the CIC we have found in GN patients are not linked

directly to the renal lesion is the failure to demonstrate a predominant size of CIC in five of the six sera we examined. Levinsky et al have found a similarly wide spectrum of CIC size in steroid responsive nephrotic syndrome [14] and HSS nephritis [12].

Low levels of a variety of CIC may be found in idiopathic GN. The pattern of distribution of different types of CIC does not allow different forms of UN to be distinguished, with the possible exception of minimal change disease, nor do overall levels of CIC provide useful information on the clinical state of the disease. The pool of dC present in these disorders appears to have little to do with the processes of renal injury. If these lesions are indeed CIC-mediated, then pathogenetic CIC may be present in amounts too small or be too rapidly removed from

512

Cairns et a!

the circulation to alter the size or characteristics of a pool of dC innocent of a direct role in renal injury. That the detected dC in idiopathic GN have resulted from generalized defects in

17. KASAI N, PARBTANI A, CAMERON JS, YEWDALL W, SHEPHERD P,

antigen handling is suggested by the observation of a prolonged

18. SMITH MD, BA1urr TM, HAYWARD AR, S0OTHILL JF: The inhibition of complement dependent lymphocyte rosette formation by the sera of children with steroid sensitive nephrotic syndrome and other renal diseases. C/in Exp Immunol 21:236—243, 1975 19. SMITH MD, VERROUST PL, ADAM C, GALCERAN M, MOREL-

and enhanced rise in CIC levels following food intake, compared to normal, in these patients [36].

VERROUST F: Platelet aggregating immune complexes and intra-

platelet serotonin in idiopathic glomerulonephritis and systemic lupus. C/in Exp Immunol, 43:64—72, 1981

MAROGER L: A study of the material inhibiting EAC-rosette formation in the sera of patients with nephropathies. C/in Exp

Acknowledgments Mrs. R. A. London was supported by a grant from the Manchester and North West Region Kidney Research Association. The histopathological examination of renal biopsy material was performed by Drs. G.

Immunol 30:364—369, 1977 20. GLUCKMAN JC, JACOB N, BEAUFILS H, BAUMELOU A, SALAH H,

GERMAN A, LEGRAIN M: Clinical significance of circulating immune complexes. Detection in chronic glumerulonephritis. Nephron 22:138—145, 1978

Williams and W. Lawler, Department of Pathology, University of Manchester.

Reprint requests to Dr. N. P. Ma/lick, Department of Renal Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, England

21. WOODROFFE AJ, BORDER WA, THEOFILOPOULOS AN, GOTZE 0, GLASSOCK RJ, DIXON FJ, WILSON CB: Detection of circulating

immune complexes in patients with glomerulonephritis. Kidney mt 12:268—278, 1977 22. TUNG KSK, WOODROFFE AJ, AHLIN TD, WILLIAMS RC, WILSON

CB: Application of the solid phase Clq and Raji cell radioimmune assays for the detection of circulating immune complexes in gbmerulonephritis. J C/in Invest 62:61—72, 1978

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