VI INICAL.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
lmmunopathology Glomerulonephritis MICHELINE Unite
10,
477-492 (1978)
of Membranoproliferative with Subendothelial Deposits (Type I MPGN)
LEVY, MARIE-CLAIRE GUBLER, MIREILLE AGNES BEZIAU, AND REN~E HABIB
de Recherches sur les Maladies Hhpital Necker-Enfants
du Mi!abolisme chez I’Enfant Malades. Paris. France
SIGH, (INSERM),
Received December 6, 1977 Immunofluorescence microscopy (IF) studies were carried out in 36 patients with Type I MPGN and showed the constant presence of deposits containing C3. Based on the presence or the absence of immunoglobulins (I&, two main patterns were observed. Complement profiles (30 patients) and C3NeF activity (20 patients) were studied in the two groups defined by IF. Our findings indicate an activation of the complement system through the classical pathway even in the group characterized by the absence of Ig. Such an activation suggests the role of immune complexes in the pathogenesis of the disease. Because of the presence of C3NeF activity in occasional patients, a superimposed activation through the alternative pathway is not unlikely. In two additional patients, one with predominant IgA within the deposits and one with CZ-deficiency, the complement system is probably activated through the alternative pathway.
INTRODUCTION In 1965, Gotoff et al. (1) and West et al. (2) emphasized the persistence of hypocomplementemia in patients presenting with membranoproliferative glomerulonephritis (MPGN). Hypocomplementemia was initially interpreted as related to activation of the complement system through the alternative pathway. In fact, low C3 was not invariably present (3). In addition, some discrepancies with respect to the pathway of complement activation were noted. They were likely due to the late recognition by most authors of the two variants of MPGN (4). In 1973, we stressed the fact that MPGN with dense intramembranous deposits, or dense deposit disease (Type II), should be differentiated from MPGN with subendothelial deposits (Type I) on the basis of morphological characteristics as well as of immunopathological features (5). In dense deposit disease (Type II MPGN) the glomerular basement membranes are thickened by an abnormal, refringent material which, by electron microscopy, appears very electron-dense and is located within the lamina densa. These “deposits” are also found in the basement membranes of Bowman’s capsule and tubules. In MPGN with subendothelial deposits (Type I MPGN), thickening of the capillary walls is due to an interposition of mesangial matrix and cells between the endothelium and the basement membrane producing a double contour appearance. By electron microscopy, mesangial- subendothelial deposits are clearly demonstrated in all cases. Subepithelial deposits are frequently found and the association of subendothelial and subepithelial deposits may lead to a disruption of the basement membrane. 477 0090-1229/78/0104-0477$01.00/O Copyrtght All right,
Q 1978 by Acildr,n,c Press. Inc. of reproducrmn in any form resewed
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The results of immunopathological studies have clearly shown that an alternative pathway of complement activation is involved in dense deposit disease. now widely accepted as a disease entity (6). In contrast, complement activation is still a matter of discussion in MPGN with subendothelial deposits. We wish, herein, to report the complement profiles and the measurements of C3NeF in patients affected with Type I MPGN, grouped according to their characteristics by immunofluorescence (IF), and to speculate about the pathogenetic mechanisms these results suggest. PATIENTS
AND METHODS
Thirty-eight children presenting with Type I MPGN were studied by immunofluorescence microscopy. Repeat biopsies were obtained in four patients. The interval between onset and the first renal biopsy is reported in Fig. 1. The diagnosis was based on light (38 cases) and electron microscopy (21 cases). Definition of the lesion included the following features: (a) proliferation of mesangial cells; (b) increase in basement membrane material in the axial stalks; (c) ingrowth or interposition of the mesangium between the lamina densa and the capillary wall with “tramendothelial layer producing the picture of a “split” track” or double contoured basement membrane: and (d) presence of deposits either subendothelial or paramesangial (beneath the basement membrane covering the mesangial stalks) or truly mesangial (within the mesangial stalks). In addition, in 11 patients, there were subepithelial deposits unevenly distributed along the capillaries, either assuming the shape of large “humps” or small and interspersed with “spikes”. In seven cases, the subepithelial deposits were contiguous or in continuity with subendothelial deposits, and the interposed basement membrane showed more or less extensive disruption and replication of the lamina densa. The presence of both subendothelial and subepithelial deposits as well as the basement membrane alterations were clearly seen in silver impregnation (7). In five instances, approximately half of the glomeruli showed the typical features of MPGN, whereas in the remaining ones only minimal glomerular abnormalities were noted (focal MPGN). Kidney tissue was snap-frozen in isopentane Immunojluorescence microscopic. precooled in liquid nitrogen. Cryostat sections were cut at 2-3 pm, fixed in
FIG.
1. Interval
between
onset
and first
renal
biopsy.
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acetone for 15 min. dried, and washed with phosphate-buffered saline (PBS), pH 7.2, The slides were incubated in a humid chamber at room temperature with fluorescein-isothiocyanate-labeled antisera for 30 min. The sections were carefully washed with PBS and mounted in buffered glycerol. Monospecific antisera to human IgA, IgG, IgM, C3 (Hyland Laboratories), albumin, and fibrin (Behringwerke Laboratories) were used in all cases. Antisera to Clq, C4, and C3PA (factor B) (Behringwerke Laboratories) were used respectively, in 25, 28. and 23 cases. Serum antiproperdin (kindly provided by Dr. McIntosh) was used in eight cases. In each case the composition, intensity, and localization of deposits were precisely determined. Very weak fixation (trace) was considered as negative. Endstage kidneys were excluded because interpretations derived from them may be misleading as far as pathogenesis is concerned. Not only could changes in the capillary permeability be responsible for the disappearance of some proteins, but also superimposed lesions such as hyalinosis favor the occurrence of deposits containing IgM, Clq, and C3, not related to the original deposits of MPGN. Plasma complement components. Clq, C4, C3, C3PA or Factor B, and C5 were measured by radial immunodiffusion (Hyland and Behringwerke Laboratories) in 32 cases. Serial measurements were obtained in 24 patients at different times of the disease. Normal values (mean ? 2 SD) were established in our laboratory with a group of normal children. Because of the wide variations observed with time, we did not purposely perform any statistical study and considered that longitudinal studies were more informative. Levels were called “fluctuant” when they crossed the line indicating mean -2 SD. C3 splitting activity and C3NeF activity. These activities were investigated in 20 patients, each by a different technique, and the results were compared in order to control their respective validity. Demonstration of C3 cleavage products by crossed immunoelectrophoresis (8). This technique is a qualitative assay detecting an eventual C3 splitting acti\,ity. The presence of a factor breaking down C3 in normal human serum was detected by incubation of the tested plasma with an equal volume of normal human serum for 1 hr at 37°C. The incubation mixture was then assayed for C3 degradation products by performing crossed immunoelectrophoresis: The first electrophoresis was in agarose ( 120 min, 5 V/cm); and the second was perpendicular to the first one, in agarose containing anti-human /3lC-@lA serum from Hyland Laboratories (180 min, 5 V/cm). In each assay the following controls were included: incubation of the same mixture at 4”C, incubation of normal human serum. incubation of the patient’s plasma at 37°C for 1 hr. Ethylenediaminetetraacetic acid (EDTA), 0.2 M, was added after 10 min of incubation in each specimen in order to reduce spontaneous breakdown of C3. Detection of the loss of the B antigen of native C3 (9). This technique is a quantitative assay allowing measurement of C3NeF acti\,itg. It was performed in presence of ethylene glycol bis @-aminoethyl ether)-N, N’-tetraacetic acid (EGTA) which has a high affinity for CaS+ therefore blocking the classical pathway.
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Equal volumes of 0.1 M MgC 1, and 0.1 M EGTA were mixed to obtain a stock solution of 0.1 M Mg2+-EGTA, pH 7.5. Mg*+- EGTA (100~1) was incorporated in normal human serum (1 ml). The plasma to be tested was then incubated at 37°C for 30 min with that mixture. The final concentration of Mg”+ -EGTA was 5 mM. At the end of the incubation, EDTA, 0.08 M, pH 7.5, was added. The cleavage of C3 was measured by the loss of the B antigen of native C3 by radial immunodiffusion against monospecific antiserum (Central Laboratorium van de Bloedtransfusiednst, Holland). Measurements of B antigen performed in pure and diluted (1:2, 1:4) normal human serum allowed the determination of a reference curve. B antigen was measured in the mixture before (concentration 1) and after incubation (concentration 2). The percentage of conversion was given by the formula: (concentration 1 - concentration concentration 1
2) X 100
These two techniques were performed on 275 samples in a wide variety of renai and nonrenal diseases. An evident C3 splitting activity was always demonstrated on crossed immunoelectrophoresis when the percentage of conversion was > 25%. We therefore considered 25% of the conversion as the limit of C3NeF activity. When the percentage of conversion was < 25%, splitting activity was most often absent. RESULTS I. Immunojluorescence
Study
The results of immunofluorescence microscopy were heterogeneous with respect to the distribution as well as the composition of the deposits. The exact location of deposits along the GBM was often difficult to evaluate. In the majority of cases, they seemed to be located on the internal side of the GBM. In eight cases, however, the epithelial side of the GBM had a hump-like appearance, presumably due to the presence of numerous subepithelial deposits recognized by light microscopy and electron microscopy. Deposits were present within the mesangial areas in 20 cases. Variations in the abundance and intensity of each protein were observed. Though deposits could vary from one capillary loop to another, the general appearance of all glomeruli looked similar except for the live cases of focal MPGN. Deposits of C3 were always found in constrast to the variable presence of immunoglobulins (Ig). Two main groups were therefore differentiated according to the presence or absence of Ig (Table 1 and Figs. 2 and 3). Group I. In 26 biopsy specimens, the characteristic feature was the association of deposits containing both C3 and Ig. On the basis of the presence or absence of mesangial deposits two patterns were observed: (i) In 15 biopsy specimens, deposits containing Ig and C3 were present along the GBM while no deposits could be found within the mesangial areas. They were more or less spread along the GBM and frequently outlined a lobulated tuft. In the
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GLOMERULONEPHRITIS
TABLE IMMUNOFLUORESCENCE
1 MICROSCOPY
Proteins IgM
Clq
C4
C3
P
B
717
919
lYl5
l/l
o/7
lO/ll 919 O/10 O/7 O/l O/l 111 l/l
7/9 O/8 O/l l/l
ll/ll lO/lO l/l l/l
l/4 o/9 313 O/7 o/o o/o o/o o/o
I%A
1s
Group 1 Without mesangial deposits
s/15
15115 13/15
With mesangial deposits Group 2 Predominant IgA C2 Deficiency
4/11 O/l0 l/l O/l
IV11 O/l0 l/l l/l
F 1111.5 7111 5110 O/l O/l
majority of cases, deposits containing Ig roughly paralleled those of C3. In two cases, however, C3 appeared very abundant, whereas deposits of Ig were segmental. Contrasting with the constant presence of IgG and the frequent presence of ,IgM, IgA was rarely found. Deposits reacting with Clq and C4 antisera were observed in the nine specimens studied, and their location was similar to that of Ig. Properdin was present along the capillary loops in the only case studied. Some segmental and focal deposits of fibrin were frequently seen. The hump-like appearance of the GBM was observed in two cases. In two cases of Focal MPGN, the “nonproliferative glomeruli” exclusively contained mesangial deposits. (ii) In 11 biopsy specimens, deposits containing Ig and C3 were present along the GBM, but in addition, deposits of C3 were observed within the mesangial areas. More or less diffuse deposits of Ig paralleled deposits of C3 along the GBM in five cases. In the remaining six, Ig was present on segments of GBM whereas deposits containing C3 were nearly diffuse. IgG was constantly found, and IgA was rarely present. Deposits of Clq and C4 were observed in the 10 cases studied in a location similar to that of Ig. Within the mesangium, deposits of C3 varied from scattered, intensely bright granules to tiny, diffusely spread deposits, giving a cloudy appearance to the mesangial areas. Deposits of properdin were observed in one of the four cases studied in a location similar to C3. Deposits containing fibrin were found either along the capillary loops (two cases) or both along the capillary loops and within the mesangium (four cases). In one case, a diffuse hump-like appearance of the GBM was observed. In the “nonproliferative“ glomeruli of two cases of focal MPGN, no deposits could be detected in one case, whereas mesangial deposits were observed in the second. In addition, small, segmental deposits of C3 were seen along some tubular basement membranes in one biopsy specimen. Group 2 (10 cases). Ten biopsy specimens were characterized by the presence of C3 while Ig and early reacting complement components were absent in all cases. The localization of deposits containing C3, however, varied, and different aspects were observed. In two cases segmental deposits outlined the periphery of the tuft but no deposits were seen within the mesangial areas. In four cases, the deposits appeared numerous and scattered within the tuft; they seemed to be within the mesangium and along the capillary loops. In the four remaining cases, the deposits
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Frc;. 2. Immunofluorescence microscopy. Anti-C3 serum. MPGN with subendothelial deposits. (a) 1. The deposits outline the periphery of the tuft, IgG, Clq. and ~4 were present in the Same location. NO mesangial deposits are present. (b) Group 2. Deposits are present both along the capillary walls and within the mesangium. No fixation of Ig and early acting complement components is observed. Group
were abundant both outlining the periphery of the tuft and located within the mesangial areas, giving an intense staining to the whole tuft. In addition, in these four cases, the GBM had a diffuse hump-like appearance. Deposits containing fibrin and properdin were present in a location similar to that of C3. In one case of
MEMBRANOPROLIFERATIVE
Fxc. 3. Immunofluorescence
microscopy.
GLOMERULONEPHRITIS
483
Anti-C3 serum. MPGN with subendothelial depos ,its. clearly located in the subendothelial zone. (b) the capillary walls and the granular depo sits
G lroup 1. High magnification. (a) The deposits appear N ote the hump-like appearance of the deposits along w rithin the mesangial areas.
fcxal MPGN the deposits were small and purely mesangial in the “nonprolife ratiive” glomeruli. In two instances, segmental deposits of C3 were also seen alew some tubular basement membranes.
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Two additional cases did not fit in any of the two groups mentioned above. In one case, as in group 1, deposits outlined the periphery of the tuft and were not seen within the mesangium, but contained predominantly IgA and small amounts of IgG and C3, whereas Clq and C4 were absent. In the second case, IF revealed diffuse and intense staining for IgG, Clq, C4, and C3 with a hump-like pattern along the peripheral capillary walls and on coarsely granular deposits within the mesangium. Repeat biopsies were obtained in four cases belonging to group 1 (two cases) and group 2 (two cases). There was no significant change in the composition and distribution of the deposits observed between the two specimens. II. Complement System The data obtained from the immunofluorescence microscopy study demonstrated the presence of Ig and of early acting complement components, suggestive of complement activation through the classical pathway in group 1, whereas the results of IF were not conclusive in group 2. These findings led us to analyze the results of measurements of plasma complement components (C lq, C4, C3, and C5) separately for group 1 and group 2. Measurements of C3PA were also’ performed in all patients. They appeared either in the low limits of normal range or below. Urinary loss of C3PA is known to be marked in patients with heavy proteinuria (10). The low levels of C3PA observed in patients presenting with
FIG. 4. Follow-up studies of complement components (Clq, dothelial deposits (Group 1.) C3 levels arc most often fluctuant, mean levels + 2 SD are indicated b.y the horizontal lines.
C4, C3, and CS). MPGN with subenand C4 levels are frequently low. The
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MPGN cannot, therefore, be taken as evidence for any given pathway of complement activation. Group I (21 cases) (figs. 4 and 6). Two patients had persistently low values of C3. Clq levels were persistently normal and C4 was fluctuant, as was C5. C3NeF activity was detected in both. Eleven patients had fluctuant levels of C3. Levels of Clq were low (three cases), fluctuant (five cases), or normal (three cases). Levels of C4 were low (two cases), fluctuant (six cases), or normal (three cases). Four patients had fluctuant C5 and two had persistently normal levels. C3NeF activity was detected on two samples in one of the six patients investigated. Four patients had persistently normal values of C3. Three showed persistently normal levels of C lq and one had fluctuant Clq. Fluctuations of C4 were observed in three and normal values in one. Two patients had normal values of C5, and levels were fluctuant in one. No C3NeF activity was detected in the serial samples obtained in three patients. Four patients had only one measurement of C3. Two patients had normal C3 and low C4. In another patient, low levels of C3, Clq, and C5 as well as C3NeF activity were noted, whereas C4 was normal. The remaining patient had normal C3, Clq, C4, and C5, but a high C3NeF activity. Group 2 (nine cases) (figs. 5 and 6). Four patients had persistently very low levels of C3. Three showed fluctuations of Clq, and one had persistently normal
FIG. 5. Follow-up studies of complement components (Clq, C4, C3, and CS). MPGN with subendothelial deposits (Group 2.) C3 levels are frequently low. Clq and C4 levels are most often below the normal range. The mean levels 4 2 SD are indicated by the horizontal lines.
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levels. They all had fluctuant levels of C4. Three had low values of C.5, and one had fluctuant C5. C3NeF activity which was not detected in serial samples obtained in two patients was fluctuant in one and reached persistently high levels in the remaining patient. Two patients had fluctuations of C3 and CS. Both had normal values of C lq and C4. C3NeF activity was detected in none of them. Three patients had only one measurement of C3. One had normal C3 and C4. Two had low values of C3 and normal Clq and C4. One of these had a low C5; C3NeF activity was not demonstrated. The patient with predominant IgA had normal Clq, C4, and C3. In the remaining patient, C2 hemolytic activity was not detectable. Serial determinations by radial immunodiffusion showed normal C4, C3, and C5, and fluctuations of Clq. DISCUSSION
It has become clear in the past few years that the condition referred to as membranoproliferative glomerulonephritis (MPGN) may be differentiated into two main variants depending on the location of deposits in the glomerular capillary walls: MPGN with subendothelial deposits (Type I MPGN) and dense deposit disease (Type II MPGN). Immunopathological studies have confirmed the distinctive features of these two types. In dense deposit disease, immunofluorescence microscopy reveals a specific and homogeneous pattern characterized by the presence of round nodules, intensely stained with anti-C3 serum, in the mesangial areas, whereas fixation of C3 along the glomerular basement membranes is weaker, appearing as a continuous or discontinuous line (6). C3 levels are nearly always persistently low, contrasting with normal levels of Clq and C4 (1 1 - 13) as well as C5 (Levy et nl., unpublished observations). Both immunofluorescence and complement studies have clearly shown that complement is exclusively activated through the alternative pathway. This activation is likely due to a circulating factor, the C3 nephritic factor (C3NeF), which seems detectable in nearly all patients (12). The pathogenesis of dense deposit disease, however, is still a matter of speculation. In contrast, in MPGN with subendothelial deposits immunofluorescence tindings (14-28) and complement profiles (11, 12. 13,24,28,29) are variable. In order to elucidate the pathway of complement activation in this type of MPGN, we have analyzed the results of measurements of complement components and C3NeF in groups of patients presenting similar characteristics by immunofluorescence. In all cases, C3 was present along the capillary walls, whereas Ig and early complement components were either present (group 1) or absent (group 2). In 11 cases of group 1 and eight cases of group 2 there were, in addition, mesangial deposits of C3. When present 1%), properdin was seen in a location similar to that of C3 in both groups. With the present state of knowledge, it is not possible to determine whether the patterns observed by IF are related to well-defined varieties of Type I or merely reflect different stages of the disease. No correlation could be established between the composition of the deposits and the date of the renal biopsy (Fig. 1). Probably the use of more repeat biopsies will lead to definite conclusions. In group 1, there was an evident relationship between the presence of Ig and that of early reacting complement conponents. Studies of the complement system
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FIG. 6. Follow-up studies of C3NeF activity in Group 1 and 2. C3NeF activity is expressed as the loss of the B antigen of native C3. Twenty-five percent of the conversion is considered as the inferior limit of C3NeF activity. C3 splitting activity on crossed immunoelectrophoresis is represented by A. Absence of C3 splitting activity is represented by 0. Note the constant presence of C3 splitting activity when the percentage of conversion is > 25% and the eventual presence of a C3 splitting activity when the percentage of conversion is < 25%.
in this group (Fig. 4) showed that C3 levels could be persistently low but were most often fluctuant and, when low at onset, could rise to normal levels. In some instances, however, they were persistently normal. C5 levels usually paralleled C3
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levels. Clq and C4 levels were often low or in the lower limits or normal range. These findings clearly indicate activation of complement through the classical pathway. The absence of Ig and early complement components characterizing group 2 has already been reported (24,30,31). In order to explain these findings, the following hypotheses, based on the fact that Ig may disappear on sequential biopsies (31), have been proposed: loss of immunoglobulins; presence of small amounts not detectable with fluorescent-conjugated antibodies; determinants of Ig, Clq, and C4 undetectable because of extensive or early deposition of C3; and intermittent presence of material composing the deposits, with degradation of previously deposited material (10, 31, 32). The finding of exclusively C3-containing deposits in early biopsies (Fig. 1) and the recurrence in renal allografts of the same deposits in the same location (33), however, suggests that we are dealing with a distinct process. Complement profiles in this group were similar to those observed in group 1. Low or fluctuant levels of the early components were seen as well as low levels of C5 (Fig. 5). Our studies, in contradiction with some reports (24), indicate that although glomerular deposits do not contain Clq and C4, the complement system has been activated through the classical pathway. Group 2 therefore seems clearly differentiated from dense deposit disease with which it could be confused because of the predominant presence of C3. Electron microscopy (EM) study did not demonstrate any distinguishing feature between group 1 and group 2, but the presence of abundant, truly mesangial, electron-dense deposits was well correlated with the presence of mesangial deposits containing C3. It has been recently suggested by Strife et al. (34) and Anders et al. (35) that a third type of MPGN (Type III MPGN) can be identified on the basis of the following EM findings: presence of subendothelial and subepithelial deposits, together with basement membrane disruption. According to the data presented by these authors, the deposits in Type III MPGN seem to contain predominantly C3. Of the seven cases in our series presenting with a pattern corresponding to the so-called Type III, four belonged to group 1 and three to group 2. It seems, therefore, that Type III MPGN does not correspond to any definite immunopathological group as far as IF and complement profiles are concerned. Several techniques are available for the determination of C3 splitting activity, but they do not allow a distinction between the classical and alternative pathways. In the presence of EGTA, which blocks the classical pathway, C3NeF activity may be demonstrated. Some investigators have shown either C3 splitting activity or C3NeF activity in MPGN but the distinction between Type I and Type II was rarely taken into account (36-38). In Type I MPGN, C3 splitting activity was detected in three of nine patients (13) and in 10 of 22 patients (28) and was not present in the patients reported by Strife et al. (34). Williams et nl. (12) demonstrated the presence of C3NeF activity in only five of 29 patients with Type I MPGN. Our experience confirms the occasional finding of C3NeF activity in such patients and shows that on serial tests, there may be wide variations either in the presence or in the percentage of conversion (Fig. 6). Among the 20 patients investigated, C3NeF activity, expressed as the loss of the B antigen of native C3, was present at least once in five patients belonging to group 1 and in two patients
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GLOMERULONEPHRITIS
489
belonging to group 2, whereas it was almost consistently found in patients with dense deposit disease (10/13). In seven patients, we demonstrated a C3 splitting activity on crossed immunoelectrophoresis, whereas percentage of conversion was low. Our interpretation is that in those samples splitting activity might be due to classical pathway activation. Immunopathological studies in patients with Type I MPGN show, therefore, definite involvement of the classical pathway. Not only do they suggest the role of immune complexes in the pathogenesis of the disease, but they allow a better understanding of complement activation. Reduced C3 levels observed in many patients have been shown by metabolic studies to be the result of increased C3 fractional catabolic rates, reduced synthesis rates, and increased extravascular distribution (39). C5 levels seem to parallel C3 levels, and it may be suggested that low levels of C5 are related to the activity of the classical pathway C5 convertase. The presence of properdin (10, 34) in the glomerular deposits in some cases and the low levels of serum properdin (12, 13, 34) show that this protein is involved in this pathway. Properdin might stabilize the so-called amplification convertase (40) resulting from the interaction of B, D, and C3b, the major cleavage fragment of C3 due to the action of the classical pathway convertase. Because of the presence of C3NeF activity in occasional patients, we suggest a superimposed C activation through the alternative pathway (41). The factors triggering C3NeF in these patients remain to be determined. Immune complexes are likely responsible for the classical pathway activation and the induction of glomerular injury. Indeed, the morphological picture of MPGN with subendothelial deposits may be found (although other types of glomerulopathies are also observed) in various diseases where the presence of circulating immune complexes has been clearly established, and antigens have been identified: coagulase negative staphylococcus or corynebacterium bovis in shunt nephritis (42-44); hepatitis viral antigen in glomerulopathy-associated chronic viral hepatitis (45-48); DNA in lupus nephritis (49); renal tubular epithelial antigen in patients with sickle-cell disease (50); al anti-trypsin in glomerulopathy associated with cirrhosis and al anti-trypsin deficiency (51). MPGN with subendothelial deposits appears, however, most often as “idiopathic”. Circulating immune complexes have been detected in only 20 to 50% of the cases, depending on the techniques used (52-56). It is possible that complexes are either intermittently present in the circulation and escape detection unless the patient is followed serially or are present in relatively small amounts, requiring more sensitive techniques in order to be detected. Immune responsiveness might play a role; the detection of a specific B-lymphocyte allo-antigen in 77% of patients with MPGN demonstrates the desirability of further immunogenetic investigations (57). The development of chronic immune complex glomerulonephritis has been suggested to be related to defective clearance of antigen by antibody (58). A complement deficiency could predispose to infection resulting in immune complex glomerulonephritis. Reports of MPGN with subendothelial deposits occurring in patients with complement deficiency are exceptional (59-61). Moreover, the pattern by IF is unusual: In one of our patients and in the patient described by Kim et al. (61), both presenting with a C2-deficiency, a marked abundance of deposits containing Ig and early complement components
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ET AL.
was observed along the capillary walls as well as within the mesangium. In such cases, although deposits contain Clq and C4, the complement system is also activated through the alternative pathway. In occasional patients with Type I MPGN with predominant IgA in the subendothelial deposits, and with a pattern which may be compared to anaphylactoid purpura nephritis (62) and to glomerulonephritis associated with cirrhosis of the liver (63), the complement system does not seem to be activated through the classical pathway. ACKNOWLEDGMENTS The patient with C2 deficiency was under the care of Pr. Mathieu, Hopital Bretonneau, Paris. C? hemolytic activity was measured by Dr. Peltier, Hopital Lariboisiere. Paris. We wish to thank Pr. Berthoux from Lyon for his technical advice and Mireille Lacoste, Colette Monnier, and Catherine Grandhomme for technical assistance. This work was supported by a grant from INSERM (CRL 76.5.1825).
REFERENCES 1. Gotoff, S. P.. Fellers, F. X.. Vawter. G. P., Janeway, C. A., and Rosen, F. S., Nrn, El)gl. J. Med. 273, 524, 1965. 2. West, C. D., McAdams, A. J., McConville, J. M.. Davis, N. C., and Holland, N. H., J. Pedint. 67, 1089, 1965. 3. Cameron, J. S., Ogg, C. S.. White, R. H. R., and Glasgow. E. F., C/in. Nephrol. 1, 8, 1973. 4. Royer, P., Habib, R., Vermeil, G., Mathieu, H., and Alizon. M., Ann. Pediar. 38, 173, 1962. 5. Habib, R., Kleinknecht, C., Gubler, M. C., and Levy, M., C/m. Nephrol. 1, 194. 1973. 6. Habib, R., Gubler. M. C., Loirat, C., Ben Maiz, H.. and Levy, M., Kidney Int. 7, 204. 1975. 7. Movat, H. Z., Amer. J. Clin. Pnthol. 35, 528. 1961. 8. Peters, D. K., Martin, A., Weinstein, A., Cameron, J. S.. Barratt. T. M.. Ogg, C. S., and Lachmann. P. J., C/iv. Exp. Ir~7n7~7ol. 11, 3 11, 1972. 9. Freyria, A. M.. Berthoux, F. C., Tremblay, M.. and Traeger, J., 111 “Proceeding of the VIth International Congress of Nephrology”. Florence, abstr. 370, 1975. IO Michael, A. F., and McLean, P. H., 112“Advances in Nephrology” (J. Hamburger, J. Crosnier. M. H. Maxwell, Eds.), pp. 49-66, Year Book Medical Publishers, Chicago, 1974. Il. Habib, R., Loirat, C., Gubler, M. C., and Levy, M., It7 ‘Advances in Nephrology” (J. Hamburger, J. Crosnier, and M. H. Maxwell, Eds.), pp. 109- 136, Year Book Medical Publishers, Chicago, 1974. 12. Williams, C. D., Peters, D. K., Fallows, J.. Petrie, A., Kourilsky, 0.. Morel-Maroger, L., and Cameron, J. S., C/in. Exp. Immunol. 18, 391. 1974. 13. Ooi, Y. M., Vallota, E. H., and West, C. D., Kidney Inr. 9, 46, 1976. 14. Berger, J., Yaneva, H., and Antoine, B., It7 “Advances in Nephrology” (J. Hamburger. J. Crosnier, and M. H. Maxwell. Eds.), pp. 11-30, Year Book Medical Publishers, Chicago, 1971. 15. Bariety, J.. Druet, P., Loirat, P.. and Lagrue. G., Pnrhol. Biol. 19, 259. 1971. 16. Mandalenakis. N., Mendoza, N., Pirani. C. L., and Pollak, V. E., Medicine 50, 319, 1971. 17. Michael, A. F., Westberg, N. G., Fish, A. J., and Vernier, R. L. J. Exp. Med. 134, 208, 1971. 18. Morel-Maroger, L., Leathem, A.. and Richet, G.. Amer. J. Med. 53, 170. 1972. 19. Churg, J., and Grishman C.. Ann. Int. Med. 76, 479. 1972. 20. Burkholder, P. M.. Hyman, L. R., and Krueger, R. P. In “Glomerulonephritis” (Kincaid-Smith P., Mathew T. H. and Becker E. L., Eds.), pp. 557-589, Wiley, New York, 1973. 21. Germuth, F. G., and Rodriguez, E., “Immunopathology of the Renal Glomerulus,” Little, Brown, Boston, 1973. 22. Pollak, V. F.. Mendoza, N., and Pirani, C. L.. In “Glomerulonephritis” (Kincaid-Smith P., Mathew T. H., and Becker E. L.. Eds.), pp. 633-640, Wiley, New York, 1973. 23. Bohle, A., Gartner, H. V., Fischbach, H., Bock, K. D., Edel, H. H., Frotscher, U., Kluthe, R., Monninghoff. W., and Scheler F., Virchou,s Arch. A 363, 213, 1974.
MEMBRANOPROLIFERATIVE
74. Barbiano di Belgiojoso, G., Tarantino. C/in.
Nephrol.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
lmmunopathology Glomerulonephritis MICHELINE Unite
10,
477-492 (1978)
of Membranoproliferative with Subendothelial Deposits (Type I MPGN)
LEVY, MARIE-CLAIRE GUBLER, MIREILLE AGNES BEZIAU, AND REN~E HABIB
de Recherches sur les Maladies Hhpital Necker-Enfants
du Mi!abolisme chez I’Enfant Malades. Paris. France
SIGH, (INSERM),
Received December 6, 1977 Immunofluorescence microscopy (IF) studies were carried out in 36 patients with Type I MPGN and showed the constant presence of deposits containing C3. Based on the presence or the absence of immunoglobulins (I&, two main patterns were observed. Complement profiles (30 patients) and C3NeF activity (20 patients) were studied in the two groups defined by IF. Our findings indicate an activation of the complement system through the classical pathway even in the group characterized by the absence of Ig. Such an activation suggests the role of immune complexes in the pathogenesis of the disease. Because of the presence of C3NeF activity in occasional patients, a superimposed activation through the alternative pathway is not unlikely. In two additional patients, one with predominant IgA within the deposits and one with CZ-deficiency, the complement system is probably activated through the alternative pathway.
INTRODUCTION In 1965, Gotoff et al. (1) and West et al. (2) emphasized the persistence of hypocomplementemia in patients presenting with membranoproliferative glomerulonephritis (MPGN). Hypocomplementemia was initially interpreted as related to activation of the complement system through the alternative pathway. In fact, low C3 was not invariably present (3). In addition, some discrepancies with respect to the pathway of complement activation were noted. They were likely due to the late recognition by most authors of the two variants of MPGN (4). In 1973, we stressed the fact that MPGN with dense intramembranous deposits, or dense deposit disease (Type II), should be differentiated from MPGN with subendothelial deposits (Type I) on the basis of morphological characteristics as well as of immunopathological features (5). In dense deposit disease (Type II MPGN) the glomerular basement membranes are thickened by an abnormal, refringent material which, by electron microscopy, appears very electron-dense and is located within the lamina densa. These “deposits” are also found in the basement membranes of Bowman’s capsule and tubules. In MPGN with subendothelial deposits (Type I MPGN), thickening of the capillary walls is due to an interposition of mesangial matrix and cells between the endothelium and the basement membrane producing a double contour appearance. By electron microscopy, mesangial- subendothelial deposits are clearly demonstrated in all cases. Subepithelial deposits are frequently found and the association of subendothelial and subepithelial deposits may lead to a disruption of the basement membrane. 477 0090-1229/78/0104-0477$01.00/O Copyrtght All right,
Q 1978 by Acildr,n,c Press. Inc. of reproducrmn in any form resewed
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The results of immunopathological studies have clearly shown that an alternative pathway of complement activation is involved in dense deposit disease. now widely accepted as a disease entity (6). In contrast, complement activation is still a matter of discussion in MPGN with subendothelial deposits. We wish, herein, to report the complement profiles and the measurements of C3NeF in patients affected with Type I MPGN, grouped according to their characteristics by immunofluorescence (IF), and to speculate about the pathogenetic mechanisms these results suggest. PATIENTS
AND METHODS
Thirty-eight children presenting with Type I MPGN were studied by immunofluorescence microscopy. Repeat biopsies were obtained in four patients. The interval between onset and the first renal biopsy is reported in Fig. 1. The diagnosis was based on light (38 cases) and electron microscopy (21 cases). Definition of the lesion included the following features: (a) proliferation of mesangial cells; (b) increase in basement membrane material in the axial stalks; (c) ingrowth or interposition of the mesangium between the lamina densa and the capillary wall with “tramendothelial layer producing the picture of a “split” track” or double contoured basement membrane: and (d) presence of deposits either subendothelial or paramesangial (beneath the basement membrane covering the mesangial stalks) or truly mesangial (within the mesangial stalks). In addition, in 11 patients, there were subepithelial deposits unevenly distributed along the capillaries, either assuming the shape of large “humps” or small and interspersed with “spikes”. In seven cases, the subepithelial deposits were contiguous or in continuity with subendothelial deposits, and the interposed basement membrane showed more or less extensive disruption and replication of the lamina densa. The presence of both subendothelial and subepithelial deposits as well as the basement membrane alterations were clearly seen in silver impregnation (7). In five instances, approximately half of the glomeruli showed the typical features of MPGN, whereas in the remaining ones only minimal glomerular abnormalities were noted (focal MPGN). Kidney tissue was snap-frozen in isopentane Immunojluorescence microscopic. precooled in liquid nitrogen. Cryostat sections were cut at 2-3 pm, fixed in
FIG.
1. Interval
between
onset
and first
renal
biopsy.
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acetone for 15 min. dried, and washed with phosphate-buffered saline (PBS), pH 7.2, The slides were incubated in a humid chamber at room temperature with fluorescein-isothiocyanate-labeled antisera for 30 min. The sections were carefully washed with PBS and mounted in buffered glycerol. Monospecific antisera to human IgA, IgG, IgM, C3 (Hyland Laboratories), albumin, and fibrin (Behringwerke Laboratories) were used in all cases. Antisera to Clq, C4, and C3PA (factor B) (Behringwerke Laboratories) were used respectively, in 25, 28. and 23 cases. Serum antiproperdin (kindly provided by Dr. McIntosh) was used in eight cases. In each case the composition, intensity, and localization of deposits were precisely determined. Very weak fixation (trace) was considered as negative. Endstage kidneys were excluded because interpretations derived from them may be misleading as far as pathogenesis is concerned. Not only could changes in the capillary permeability be responsible for the disappearance of some proteins, but also superimposed lesions such as hyalinosis favor the occurrence of deposits containing IgM, Clq, and C3, not related to the original deposits of MPGN. Plasma complement components. Clq, C4, C3, C3PA or Factor B, and C5 were measured by radial immunodiffusion (Hyland and Behringwerke Laboratories) in 32 cases. Serial measurements were obtained in 24 patients at different times of the disease. Normal values (mean ? 2 SD) were established in our laboratory with a group of normal children. Because of the wide variations observed with time, we did not purposely perform any statistical study and considered that longitudinal studies were more informative. Levels were called “fluctuant” when they crossed the line indicating mean -2 SD. C3 splitting activity and C3NeF activity. These activities were investigated in 20 patients, each by a different technique, and the results were compared in order to control their respective validity. Demonstration of C3 cleavage products by crossed immunoelectrophoresis (8). This technique is a qualitative assay detecting an eventual C3 splitting acti\,ity. The presence of a factor breaking down C3 in normal human serum was detected by incubation of the tested plasma with an equal volume of normal human serum for 1 hr at 37°C. The incubation mixture was then assayed for C3 degradation products by performing crossed immunoelectrophoresis: The first electrophoresis was in agarose ( 120 min, 5 V/cm); and the second was perpendicular to the first one, in agarose containing anti-human /3lC-@lA serum from Hyland Laboratories (180 min, 5 V/cm). In each assay the following controls were included: incubation of the same mixture at 4”C, incubation of normal human serum. incubation of the patient’s plasma at 37°C for 1 hr. Ethylenediaminetetraacetic acid (EDTA), 0.2 M, was added after 10 min of incubation in each specimen in order to reduce spontaneous breakdown of C3. Detection of the loss of the B antigen of native C3 (9). This technique is a quantitative assay allowing measurement of C3NeF acti\,itg. It was performed in presence of ethylene glycol bis @-aminoethyl ether)-N, N’-tetraacetic acid (EGTA) which has a high affinity for CaS+ therefore blocking the classical pathway.
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Equal volumes of 0.1 M MgC 1, and 0.1 M EGTA were mixed to obtain a stock solution of 0.1 M Mg2+-EGTA, pH 7.5. Mg*+- EGTA (100~1) was incorporated in normal human serum (1 ml). The plasma to be tested was then incubated at 37°C for 30 min with that mixture. The final concentration of Mg”+ -EGTA was 5 mM. At the end of the incubation, EDTA, 0.08 M, pH 7.5, was added. The cleavage of C3 was measured by the loss of the B antigen of native C3 by radial immunodiffusion against monospecific antiserum (Central Laboratorium van de Bloedtransfusiednst, Holland). Measurements of B antigen performed in pure and diluted (1:2, 1:4) normal human serum allowed the determination of a reference curve. B antigen was measured in the mixture before (concentration 1) and after incubation (concentration 2). The percentage of conversion was given by the formula: (concentration 1 - concentration concentration 1
2) X 100
These two techniques were performed on 275 samples in a wide variety of renai and nonrenal diseases. An evident C3 splitting activity was always demonstrated on crossed immunoelectrophoresis when the percentage of conversion was > 25%. We therefore considered 25% of the conversion as the limit of C3NeF activity. When the percentage of conversion was < 25%, splitting activity was most often absent. RESULTS I. Immunojluorescence
Study
The results of immunofluorescence microscopy were heterogeneous with respect to the distribution as well as the composition of the deposits. The exact location of deposits along the GBM was often difficult to evaluate. In the majority of cases, they seemed to be located on the internal side of the GBM. In eight cases, however, the epithelial side of the GBM had a hump-like appearance, presumably due to the presence of numerous subepithelial deposits recognized by light microscopy and electron microscopy. Deposits were present within the mesangial areas in 20 cases. Variations in the abundance and intensity of each protein were observed. Though deposits could vary from one capillary loop to another, the general appearance of all glomeruli looked similar except for the live cases of focal MPGN. Deposits of C3 were always found in constrast to the variable presence of immunoglobulins (Ig). Two main groups were therefore differentiated according to the presence or absence of Ig (Table 1 and Figs. 2 and 3). Group I. In 26 biopsy specimens, the characteristic feature was the association of deposits containing both C3 and Ig. On the basis of the presence or absence of mesangial deposits two patterns were observed: (i) In 15 biopsy specimens, deposits containing Ig and C3 were present along the GBM while no deposits could be found within the mesangial areas. They were more or less spread along the GBM and frequently outlined a lobulated tuft. In the
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481
GLOMERULONEPHRITIS
TABLE IMMUNOFLUORESCENCE
1 MICROSCOPY
Proteins IgM
Clq
C4
C3
P
B
717
919
lYl5
l/l
o/7
lO/ll 919 O/10 O/7 O/l O/l 111 l/l
7/9 O/8 O/l l/l
ll/ll lO/lO l/l l/l
l/4 o/9 313 O/7 o/o o/o o/o o/o
I%A
1s
Group 1 Without mesangial deposits
s/15
15115 13/15
With mesangial deposits Group 2 Predominant IgA C2 Deficiency
4/11 O/l0 l/l O/l
IV11 O/l0 l/l l/l
F 1111.5 7111 5110 O/l O/l
majority of cases, deposits containing Ig roughly paralleled those of C3. In two cases, however, C3 appeared very abundant, whereas deposits of Ig were segmental. Contrasting with the constant presence of IgG and the frequent presence of ,IgM, IgA was rarely found. Deposits reacting with Clq and C4 antisera were observed in the nine specimens studied, and their location was similar to that of Ig. Properdin was present along the capillary loops in the only case studied. Some segmental and focal deposits of fibrin were frequently seen. The hump-like appearance of the GBM was observed in two cases. In two cases of Focal MPGN, the “nonproliferative glomeruli” exclusively contained mesangial deposits. (ii) In 11 biopsy specimens, deposits containing Ig and C3 were present along the GBM, but in addition, deposits of C3 were observed within the mesangial areas. More or less diffuse deposits of Ig paralleled deposits of C3 along the GBM in five cases. In the remaining six, Ig was present on segments of GBM whereas deposits containing C3 were nearly diffuse. IgG was constantly found, and IgA was rarely present. Deposits of Clq and C4 were observed in the 10 cases studied in a location similar to that of Ig. Within the mesangium, deposits of C3 varied from scattered, intensely bright granules to tiny, diffusely spread deposits, giving a cloudy appearance to the mesangial areas. Deposits of properdin were observed in one of the four cases studied in a location similar to C3. Deposits containing fibrin were found either along the capillary loops (two cases) or both along the capillary loops and within the mesangium (four cases). In one case, a diffuse hump-like appearance of the GBM was observed. In the “nonproliferative“ glomeruli of two cases of focal MPGN, no deposits could be detected in one case, whereas mesangial deposits were observed in the second. In addition, small, segmental deposits of C3 were seen along some tubular basement membranes in one biopsy specimen. Group 2 (10 cases). Ten biopsy specimens were characterized by the presence of C3 while Ig and early reacting complement components were absent in all cases. The localization of deposits containing C3, however, varied, and different aspects were observed. In two cases segmental deposits outlined the periphery of the tuft but no deposits were seen within the mesangial areas. In four cases, the deposits appeared numerous and scattered within the tuft; they seemed to be within the mesangium and along the capillary loops. In the four remaining cases, the deposits
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ET AL
Frc;. 2. Immunofluorescence microscopy. Anti-C3 serum. MPGN with subendothelial deposits. (a) 1. The deposits outline the periphery of the tuft, IgG, Clq. and ~4 were present in the Same location. NO mesangial deposits are present. (b) Group 2. Deposits are present both along the capillary walls and within the mesangium. No fixation of Ig and early acting complement components is observed. Group
were abundant both outlining the periphery of the tuft and located within the mesangial areas, giving an intense staining to the whole tuft. In addition, in these four cases, the GBM had a diffuse hump-like appearance. Deposits containing fibrin and properdin were present in a location similar to that of C3. In one case of
MEMBRANOPROLIFERATIVE
Fxc. 3. Immunofluorescence
microscopy.
GLOMERULONEPHRITIS
483
Anti-C3 serum. MPGN with subendothelial depos ,its. clearly located in the subendothelial zone. (b) the capillary walls and the granular depo sits
G lroup 1. High magnification. (a) The deposits appear N ote the hump-like appearance of the deposits along w rithin the mesangial areas.
fcxal MPGN the deposits were small and purely mesangial in the “nonprolife ratiive” glomeruli. In two instances, segmental deposits of C3 were also seen alew some tubular basement membranes.
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ETAL.
Two additional cases did not fit in any of the two groups mentioned above. In one case, as in group 1, deposits outlined the periphery of the tuft and were not seen within the mesangium, but contained predominantly IgA and small amounts of IgG and C3, whereas Clq and C4 were absent. In the second case, IF revealed diffuse and intense staining for IgG, Clq, C4, and C3 with a hump-like pattern along the peripheral capillary walls and on coarsely granular deposits within the mesangium. Repeat biopsies were obtained in four cases belonging to group 1 (two cases) and group 2 (two cases). There was no significant change in the composition and distribution of the deposits observed between the two specimens. II. Complement System The data obtained from the immunofluorescence microscopy study demonstrated the presence of Ig and of early acting complement components, suggestive of complement activation through the classical pathway in group 1, whereas the results of IF were not conclusive in group 2. These findings led us to analyze the results of measurements of plasma complement components (C lq, C4, C3, and C5) separately for group 1 and group 2. Measurements of C3PA were also’ performed in all patients. They appeared either in the low limits of normal range or below. Urinary loss of C3PA is known to be marked in patients with heavy proteinuria (10). The low levels of C3PA observed in patients presenting with
FIG. 4. Follow-up studies of complement components (Clq, dothelial deposits (Group 1.) C3 levels arc most often fluctuant, mean levels + 2 SD are indicated b.y the horizontal lines.
C4, C3, and CS). MPGN with subenand C4 levels are frequently low. The
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GLOMERULONEPHRITIS
485
MPGN cannot, therefore, be taken as evidence for any given pathway of complement activation. Group I (21 cases) (figs. 4 and 6). Two patients had persistently low values of C3. Clq levels were persistently normal and C4 was fluctuant, as was C5. C3NeF activity was detected in both. Eleven patients had fluctuant levels of C3. Levels of Clq were low (three cases), fluctuant (five cases), or normal (three cases). Levels of C4 were low (two cases), fluctuant (six cases), or normal (three cases). Four patients had fluctuant C5 and two had persistently normal levels. C3NeF activity was detected on two samples in one of the six patients investigated. Four patients had persistently normal values of C3. Three showed persistently normal levels of C lq and one had fluctuant Clq. Fluctuations of C4 were observed in three and normal values in one. Two patients had normal values of C5, and levels were fluctuant in one. No C3NeF activity was detected in the serial samples obtained in three patients. Four patients had only one measurement of C3. Two patients had normal C3 and low C4. In another patient, low levels of C3, Clq, and C5 as well as C3NeF activity were noted, whereas C4 was normal. The remaining patient had normal C3, Clq, C4, and C5, but a high C3NeF activity. Group 2 (nine cases) (figs. 5 and 6). Four patients had persistently very low levels of C3. Three showed fluctuations of Clq, and one had persistently normal
FIG. 5. Follow-up studies of complement components (Clq, C4, C3, and CS). MPGN with subendothelial deposits (Group 2.) C3 levels are frequently low. Clq and C4 levels are most often below the normal range. The mean levels 4 2 SD are indicated by the horizontal lines.
486
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ET AL.
levels. They all had fluctuant levels of C4. Three had low values of C.5, and one had fluctuant C5. C3NeF activity which was not detected in serial samples obtained in two patients was fluctuant in one and reached persistently high levels in the remaining patient. Two patients had fluctuations of C3 and CS. Both had normal values of C lq and C4. C3NeF activity was detected in none of them. Three patients had only one measurement of C3. One had normal C3 and C4. Two had low values of C3 and normal Clq and C4. One of these had a low C5; C3NeF activity was not demonstrated. The patient with predominant IgA had normal Clq, C4, and C3. In the remaining patient, C2 hemolytic activity was not detectable. Serial determinations by radial immunodiffusion showed normal C4, C3, and C5, and fluctuations of Clq. DISCUSSION
It has become clear in the past few years that the condition referred to as membranoproliferative glomerulonephritis (MPGN) may be differentiated into two main variants depending on the location of deposits in the glomerular capillary walls: MPGN with subendothelial deposits (Type I MPGN) and dense deposit disease (Type II MPGN). Immunopathological studies have confirmed the distinctive features of these two types. In dense deposit disease, immunofluorescence microscopy reveals a specific and homogeneous pattern characterized by the presence of round nodules, intensely stained with anti-C3 serum, in the mesangial areas, whereas fixation of C3 along the glomerular basement membranes is weaker, appearing as a continuous or discontinuous line (6). C3 levels are nearly always persistently low, contrasting with normal levels of Clq and C4 (1 1 - 13) as well as C5 (Levy et nl., unpublished observations). Both immunofluorescence and complement studies have clearly shown that complement is exclusively activated through the alternative pathway. This activation is likely due to a circulating factor, the C3 nephritic factor (C3NeF), which seems detectable in nearly all patients (12). The pathogenesis of dense deposit disease, however, is still a matter of speculation. In contrast, in MPGN with subendothelial deposits immunofluorescence tindings (14-28) and complement profiles (11, 12. 13,24,28,29) are variable. In order to elucidate the pathway of complement activation in this type of MPGN, we have analyzed the results of measurements of complement components and C3NeF in groups of patients presenting similar characteristics by immunofluorescence. In all cases, C3 was present along the capillary walls, whereas Ig and early complement components were either present (group 1) or absent (group 2). In 11 cases of group 1 and eight cases of group 2 there were, in addition, mesangial deposits of C3. When present 1%), properdin was seen in a location similar to that of C3 in both groups. With the present state of knowledge, it is not possible to determine whether the patterns observed by IF are related to well-defined varieties of Type I or merely reflect different stages of the disease. No correlation could be established between the composition of the deposits and the date of the renal biopsy (Fig. 1). Probably the use of more repeat biopsies will lead to definite conclusions. In group 1, there was an evident relationship between the presence of Ig and that of early reacting complement conponents. Studies of the complement system
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FIG. 6. Follow-up studies of C3NeF activity in Group 1 and 2. C3NeF activity is expressed as the loss of the B antigen of native C3. Twenty-five percent of the conversion is considered as the inferior limit of C3NeF activity. C3 splitting activity on crossed immunoelectrophoresis is represented by A. Absence of C3 splitting activity is represented by 0. Note the constant presence of C3 splitting activity when the percentage of conversion is > 25% and the eventual presence of a C3 splitting activity when the percentage of conversion is < 25%.
in this group (Fig. 4) showed that C3 levels could be persistently low but were most often fluctuant and, when low at onset, could rise to normal levels. In some instances, however, they were persistently normal. C5 levels usually paralleled C3
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levels. Clq and C4 levels were often low or in the lower limits or normal range. These findings clearly indicate activation of complement through the classical pathway. The absence of Ig and early complement components characterizing group 2 has already been reported (24,30,31). In order to explain these findings, the following hypotheses, based on the fact that Ig may disappear on sequential biopsies (31), have been proposed: loss of immunoglobulins; presence of small amounts not detectable with fluorescent-conjugated antibodies; determinants of Ig, Clq, and C4 undetectable because of extensive or early deposition of C3; and intermittent presence of material composing the deposits, with degradation of previously deposited material (10, 31, 32). The finding of exclusively C3-containing deposits in early biopsies (Fig. 1) and the recurrence in renal allografts of the same deposits in the same location (33), however, suggests that we are dealing with a distinct process. Complement profiles in this group were similar to those observed in group 1. Low or fluctuant levels of the early components were seen as well as low levels of C5 (Fig. 5). Our studies, in contradiction with some reports (24), indicate that although glomerular deposits do not contain Clq and C4, the complement system has been activated through the classical pathway. Group 2 therefore seems clearly differentiated from dense deposit disease with which it could be confused because of the predominant presence of C3. Electron microscopy (EM) study did not demonstrate any distinguishing feature between group 1 and group 2, but the presence of abundant, truly mesangial, electron-dense deposits was well correlated with the presence of mesangial deposits containing C3. It has been recently suggested by Strife et al. (34) and Anders et al. (35) that a third type of MPGN (Type III MPGN) can be identified on the basis of the following EM findings: presence of subendothelial and subepithelial deposits, together with basement membrane disruption. According to the data presented by these authors, the deposits in Type III MPGN seem to contain predominantly C3. Of the seven cases in our series presenting with a pattern corresponding to the so-called Type III, four belonged to group 1 and three to group 2. It seems, therefore, that Type III MPGN does not correspond to any definite immunopathological group as far as IF and complement profiles are concerned. Several techniques are available for the determination of C3 splitting activity, but they do not allow a distinction between the classical and alternative pathways. In the presence of EGTA, which blocks the classical pathway, C3NeF activity may be demonstrated. Some investigators have shown either C3 splitting activity or C3NeF activity in MPGN but the distinction between Type I and Type II was rarely taken into account (36-38). In Type I MPGN, C3 splitting activity was detected in three of nine patients (13) and in 10 of 22 patients (28) and was not present in the patients reported by Strife et al. (34). Williams et nl. (12) demonstrated the presence of C3NeF activity in only five of 29 patients with Type I MPGN. Our experience confirms the occasional finding of C3NeF activity in such patients and shows that on serial tests, there may be wide variations either in the presence or in the percentage of conversion (Fig. 6). Among the 20 patients investigated, C3NeF activity, expressed as the loss of the B antigen of native C3, was present at least once in five patients belonging to group 1 and in two patients
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489
belonging to group 2, whereas it was almost consistently found in patients with dense deposit disease (10/13). In seven patients, we demonstrated a C3 splitting activity on crossed immunoelectrophoresis, whereas percentage of conversion was low. Our interpretation is that in those samples splitting activity might be due to classical pathway activation. Immunopathological studies in patients with Type I MPGN show, therefore, definite involvement of the classical pathway. Not only do they suggest the role of immune complexes in the pathogenesis of the disease, but they allow a better understanding of complement activation. Reduced C3 levels observed in many patients have been shown by metabolic studies to be the result of increased C3 fractional catabolic rates, reduced synthesis rates, and increased extravascular distribution (39). C5 levels seem to parallel C3 levels, and it may be suggested that low levels of C5 are related to the activity of the classical pathway C5 convertase. The presence of properdin (10, 34) in the glomerular deposits in some cases and the low levels of serum properdin (12, 13, 34) show that this protein is involved in this pathway. Properdin might stabilize the so-called amplification convertase (40) resulting from the interaction of B, D, and C3b, the major cleavage fragment of C3 due to the action of the classical pathway convertase. Because of the presence of C3NeF activity in occasional patients, we suggest a superimposed C activation through the alternative pathway (41). The factors triggering C3NeF in these patients remain to be determined. Immune complexes are likely responsible for the classical pathway activation and the induction of glomerular injury. Indeed, the morphological picture of MPGN with subendothelial deposits may be found (although other types of glomerulopathies are also observed) in various diseases where the presence of circulating immune complexes has been clearly established, and antigens have been identified: coagulase negative staphylococcus or corynebacterium bovis in shunt nephritis (42-44); hepatitis viral antigen in glomerulopathy-associated chronic viral hepatitis (45-48); DNA in lupus nephritis (49); renal tubular epithelial antigen in patients with sickle-cell disease (50); al anti-trypsin in glomerulopathy associated with cirrhosis and al anti-trypsin deficiency (51). MPGN with subendothelial deposits appears, however, most often as “idiopathic”. Circulating immune complexes have been detected in only 20 to 50% of the cases, depending on the techniques used (52-56). It is possible that complexes are either intermittently present in the circulation and escape detection unless the patient is followed serially or are present in relatively small amounts, requiring more sensitive techniques in order to be detected. Immune responsiveness might play a role; the detection of a specific B-lymphocyte allo-antigen in 77% of patients with MPGN demonstrates the desirability of further immunogenetic investigations (57). The development of chronic immune complex glomerulonephritis has been suggested to be related to defective clearance of antigen by antibody (58). A complement deficiency could predispose to infection resulting in immune complex glomerulonephritis. Reports of MPGN with subendothelial deposits occurring in patients with complement deficiency are exceptional (59-61). Moreover, the pattern by IF is unusual: In one of our patients and in the patient described by Kim et al. (61), both presenting with a C2-deficiency, a marked abundance of deposits containing Ig and early complement components
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ET AL.
was observed along the capillary walls as well as within the mesangium. In such cases, although deposits contain Clq and C4, the complement system is also activated through the alternative pathway. In occasional patients with Type I MPGN with predominant IgA in the subendothelial deposits, and with a pattern which may be compared to anaphylactoid purpura nephritis (62) and to glomerulonephritis associated with cirrhosis of the liver (63), the complement system does not seem to be activated through the classical pathway. ACKNOWLEDGMENTS The patient with C2 deficiency was under the care of Pr. Mathieu, Hopital Bretonneau, Paris. C? hemolytic activity was measured by Dr. Peltier, Hopital Lariboisiere. Paris. We wish to thank Pr. Berthoux from Lyon for his technical advice and Mireille Lacoste, Colette Monnier, and Catherine Grandhomme for technical assistance. This work was supported by a grant from INSERM (CRL 76.5.1825).
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MEMBRANOPROLIFERATIVE
74. Barbiano di Belgiojoso, G., Tarantino. C/in.
Nephrol.
