Evidence for an autologous immune complex pathogenic mechanism in acute poststreptococcal glomerulonephritis

Kidney International, Vol. 14 (1978). pp. 501—510

Evidence for an autologous immune complex pathogenic mechanism in acute poststreptococcal glomerulonephritis RAWLE M. McINT0sH,* RAFAEL GARCIA, LIRIM0 RuBlo, DANA RABIDEAU, JAMES E. ALLEN, RONALD I. CARR, AND BERNARDO RODRIGUEZ—ITURBE Divisions of Nephrology, Clinical Immunology and Infectious Diseases, University of Colorado Medical Center, Denver, Divisions ofAllergy and Clinical Immunology, National Jewish Hospital and Research Center, Denver, Colorado, and the Renal Service, Department of Medicine, Hospital Universitario and UniversidaddelZulia, Maracaibo, Venezuela

cours de l'APSGN—dès le 8ème jour de l'infection streptococcique—sont en faveur du role de l'anti-IgG dans Ia pathogénie

Evidence for an autologous immune complex pathogenic mechanism in acute poststreptococcal glomerulonephritis. Clinical and

experimental studies have suggested a role for antiimmuno-

immune de I'APSGN. Quoique notre étude soit en faveur de

globulins in the pathogenesis of glomerulonephritis associated with streptococcal infection. We attempted to localize anti-IgG in the kidneys of 22 patients who had renal biopsies performed 7 to 66 days after the onset of acute poststreptococcal glomerulonephritis (APSGN). In addition to routine localization of immunoglobulins and C3, specimens were stained with neuraminidasetreated, heat-aggregated, and unmodified IgG. Twelve biopsies showed staining with fluorescein-labeled human neuraminidasetreated IgG. A lesser number gave positive staining with the other IgG preparations. Following treatment of slides with 0.01 M citrate buffer, seven previously negative biopsies showed positive staining with neuraminidase-treated IgG. The demonstration

l'hypothése selon laquelle l'anti-IgG a partir d'une IgG modifiée par le produit streptococcique, la formation de cet auto-anticorps contre de l'IgG incorporé dans un complexe exogéne (anticorpsantigène streptococcique) ne peut pas être exclue.

of anti-IgG by these methods was possible in 19 out of 22 biopsies. It correlated best with the presence of C3 and IgG, and to a lesser extent with 1gM, in the renal glomerulus. These resuits and our previous finding of elevated levels of serum antiIgG early in the course of APSGN, and as early as 8 days follow-

ing the antecedent streptococcal infection, support a role for anti-IgG in the immune pathogenesis of APSGN. Although our series of studies support the hypothesis that anti-IgG is formed to an IgG modified enzymatically by streptococcal product, the formation of this autoantibody to IgG incorporated in an exogenous (streptococcal antigen-antibody) complex can not be ruled out.

Preuve d'un mécanisme pathogénique mettant en jeu les immuns complexes autologues dans Ia glomerulonéphrite aiguë poststreptococcique. Les etudes cliniques et expérimentales ont suggéré un role des antiimmunoglobulines dans Ia pathogenie de Ia

glomerulonephrite associée a une infection streptococcique. Nous avons essayé de localiser l'anti-IgG dans les biopsies rénales de 22 malades, biopsies réalisées 7 a 66jours après le début d'une glomérulonéphrite post-streptococcique (APSGN). En plus de Ia localisation habituelle des immunoglobulines et do C3, les échantillons ont été traités par de l'IgG traitée par Ia neuraminidase, ou agrégée par Ia chaleur, ou non modifiée. Dans 12 biopsies une fluorescence a été observée pour l'IgG traitée par Ia neuraminidase. Des images positives pour les autres préparations dIgG ont été observées dans un moms grand nombre de biopsies. Sept biopsies initialement negatives pour l'IgG traitée par Ia neuraminidase sont devenues positives aprCs traitement par un tampon citrate 0,01 M. La mise en evidence de l'anti-IgG par ces méthodes a été possible pour 19 biopsies sur 22. Cette presence est bien correlée avec celle de C3 et d'IgG et a moindre degre d'IgM. Ces résultats, ainsi que notre constation antérieure de concentrations sériques élevées d'anti-IgG précocément au

501

The renal injury following infection with nephritogenic streptococcus is generally considered to have an immune-complex pathogenesis [1—4]. There

is, however, considerable controversy as to the nature of the antigen(s) involved and the characteristics of the antigen-antibody interaction [1]. The finding of cryoglobulins in the sera of these patients, and particularly, the demonstration of sialic acid depletion of the IgG component of these cryoproteins, led to the postulation that such alteration in the structure could account for an antigenic transformation of autologous IgG [5—7]. Demonstra-

tion in rabbits of the nephritogenic potential of autolgous IgG thus modified by streptococcus and, more specifically, by neuraminidase, suggested a possible determinant role for this material in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN) [8,9]: antiglobulin response to altered IgG could result in the formation of circulating or glomerular-fixed immune complexes. Recently, we have demonstrated anti-IgG in the serum of the majority of the patients with APSGN, *Dr. McIntosh died on September 20, 1978. Received for publication December 8, 1977; and in revised form May 10, 1978. 0085-2538/78/0014—0501 $02.00

© 1978 by the International Society of Nephrology.

502

McIntosh et a!

and found that these autoantibodies appear very

mined by agglutination using latex particles coated

early in the course of the disease [10—111. The pur-

with IgG and by immunoabsorbent using IgG

pose of this communication is to report the incidence and nature of antiglobulins in the glomerulus of patients with acute poststreptococcal glomerulonephritis. Methods

Twenty-two patients (12 males and 10 females) are the subject of this study. The age of the patients ranged between 2 and 18 yr; only one patient was

older than 11 yr of age. There was evidence of a preceding streptococcal infection in all patients as

shown by elevated antistreptolysin 0 and antihyaluronidase titers and streptozyme test. Skin infection anteceded nephritis in 16 patients, pharyngeal infection in three, and both skin and throat in-

coupled to CNBR activated sepharose [10, 11]. The serologic studies were performed on 119 pa-

tients admitted to the hospital with well-documented APSGN [10,11]. The 22 patients reported here were selected from the total group and repre-

sent those patients from whom renal tissue was available for study.

Renal biopsy studies. A portion of the renal biopsy specimen was fixed in 10% buffered formalin

and 4- paraffin sections stained with hematoxylin and eosin (H&E) and periodic acid Schiff reagent (PAS) for light microscopy. A part of the tissue was

fixed in gluteraldehyde for electron microscopy. The remainder of the specimen was snap-frozen in dry ice and acetone and stored at —70° C for immu-

nohistologic studies. Immunofluorescent studies

fections in three patients. The time of onset of

were performed on 4-es cryostat sections with fiuo-

streptococcal infection and the time of the first sign or symptom of nephritis were recorded. All patients

rescein isothyocyanate (FITC)-conjugated rabbit antisera to human IgG (RAH IgG), 1gM (RAH 1gM), and C3 (RAH C3). In addition, unmodified

were admitted to the Hospital Universitario de Maracaibo, Venezuela, with an acute nephritic syn-

drome (hematuria, urinary red cell casts, hypertension, and edema). After clinical and serologic studies were done, percutaneous renal biopsy was obtained following informed consent. In addition to clinical and laboratory evaluation of renal disease, titers of antistreptolysin 0 (Hyland Laboratories reagent kit), antihyaluronidase (Bacto-Difco Reagent

Kit), and streptozyme (Wampole rapid slide test) were measured. Serum levels of IgG, 1gM, IgA, and C3 were quantitated by immunodiffusion. Circulat-

ing immune complexes were measured by the Clq binding assay of Zubler, Nydegger, and Perrin [12] with Clq labeled iodine 125 (12I) and polyethylene glycol. The method of Clq determination has been

human IgG (HIgG-u) aggregated human IgG (HIgGagg) and neuraminidase-treated (sialic-acid-deplet-

ed) human IgG (HIgG-N) were conjugated with FITC and used to stain the frozen section by conventional direct immunofluorescent staining methodology. FITC-conjugated unmodified rabbit IgG (RIgG-u) and FITC rabbit aggregated (RIgG-agg) were also used for staining purposes. In all specimens in which fluorescent deposits of IgG preparations were not observed, the sections were treated with 0.01 M citrate buffer (pH, 3.2) for 1, 4, and 12 hr at 26° C. The immunofluorescent staining was then repeated. Renal biopsy specimens from 97 patients with a variety of renal diseases (see

Table 5) were studied by the same immuno-

detailed in a previous communication (RoDRIGuEzITURBE et al: Submitted for publication). Clq was isolated from normal human serum radiolabeled with 125J and reacted with the test sera. Separation of free from complex bound '251-Clq was achieved by selective precipitation with polyethylene glycol. CIq binding activity (C1qBA) was calculated as the percent of 1251-Clq precipitated. All samples were test-

histologic methods using the identical antisera. The methodology used for immunohistologic observations has been described previously [13—16]. Preparation of human IgG reagents (HIgG). Immunoglobulin G (IgG) was isolated from the serum. of healthy United States males by precipitation with ammonium sulfate, ion exchange chromatography on diethylaminoethyl cellulose (DEAE), and Seph-

ed in duplicate. Serum samples from apparently normal healthy individuals were included on each test run. The procedure was also performed react-

adex G-200 gel filtration chromatography. The serum donors had no history of recent illness, no antibodies to streptococcal enzymes (streptolysin 0, hyaluronidase, streptozyme), and they had nor-

ing '251-Clq with varying amounts of aggregated IgG

(aggregated by heat and precipitation with sodium sulfate). CIqBA was measured in over 100 apparently healthy subjects. The mean binding obtained was 9.5 2.6. Therefore, the mean + 2 SD was 14.7, and in our laboratories Clq greater than 15% is considered abnormal. Rheumatoid factor was deter-

mal urine analyses and endogenous creatinine clear-

ances. Donors' serum levels of C3 and immunoglobulins (G, M, and A) were normal, and no Clq binding activity was detected in the serum. Rheumatoid factor was not detected in their sera. The IgG preparation was characterized by immu-

503

Poststreptococcal glomerulonephritis: Immunology

noelectrophoresis, Ouchterlony double diffusion in agar gel, and cellulose acetate electrophoresis. No other serum proteins were detected in the material.

Results

The serologic data on the 119 patients from whom the cases in this study were selected are the subject of separate communications [10,11,17] and are summarized in Table 1. All 22 patients in the study had markedly elevated levels of serum IgG and 1gM. Serum IgA levels were elevated in six patients. The C3 levels were depressed in all patients, and Clq binding activity was elevated in 10 cases. Rheumatoid factor titers were elevated in all patients (Table

Part of the IgG solution was conjugated with FITC, and a final concentration of 20 mg/ml was used for immunohistochemical studies. Part of the IgG was treated with neuraminidase as described

previously [9] and neuraminidase-treated IgG (HIgG-N) purified by ammonium sulfate precipitation and gel chromatography using Sephadex G 200 [9]. No neuraminidase was detected in the prep-

2).

aration. The HIgG-N was conjugated with FITC,

Renal morphology showed acute, diffuse, proliferative glomerulonephritis in all patients. No altera-

and a concentration of 20 mglml was used for immunofluorescent studies.

Immuoglobulin U was aggregated by heating at 63° C for 20 mm followed by precipitation with sodium sulfate. The aggregated IgG was separated by chromatography on Sephadex G-200 and Sepharose

Table 1. Summary of serologic findings in acute poststreptoccal glomerulonephritis

6B. Only the fraction in the exclusion volume of Sephadex G-200 was used. This fraction appeared as a single peak on Sepharose. The IgG was con-

No. of patients

Percent

Antistreptococcal ABa

81

IgG (> 1600 mgIlOO ml)

68

IgM(> lSOmg/lOOmI) IgA (> 300 mg/100 ml)

77 21 65 45

98.8 93.2 98.7 26.9 85.5 55.5

82 73 78 78

106

89.2

119

Serologic findings

jugated with FITC, and a concentration of 20 mg/mI of the material (HIgG-agg) was used for fluorescent studies. Preparation of rabbit IgG reagents (RJgG). Fluorescein-conjugated unmodified rabbit IgG (RIgG-u) and fluorescein-conjugated rabbit aggregated IgG

f

t

Total no. of patients studied

C3 (< 100 mgIlOO ml)

CIqBA" (> 15%)

Rheumatoidfactor(> 1:32)

positive

76

80

a Evidence of streptococcal infection was indicated by the following: streptozyme 1:100; antistreptolysin 0> 1:125 Todd units for patients under 5 yr of age, and > 1:250 Todd units for patients aged 5 yr and older; antihyaluronidase > 1:125; or combination. b CIqBA = Clq-binding activity.

(RIgG-agg) were prepared in a manner similar to that described for the human IgG counterparts. No antibodies to human serum proteins or streptococcal enzymes were detected in the rabbit serum.

Table 2. Serological data on the 22 patients in the study'

Patient No.

RF

CIqBA

1:2048 1:128 1:64

7.65

170

1024

14.85 6.8 27.81 8.0 20.01 34.1 6.8

256 2048 1024 2048 1024

<10 <10 50

1:4096 1:1024 1:1024 1:256 1:4096 1:16

340 680 280 340 340 280

54 70 30 30

1:128 1:1024 1:512 1:128

40

171

56 60 50

1:512 1:64 1:1024 1:512 1:64 1:1024 1:64

174 175

75 35

1:64

C3

122 128

50

131

75

134 135 139 141

90 40 50

150

155 157 158

160 162

164 165 166 167 169 170

a C3,

85

50 55 54

1:256

35.59

ASO

340

29.3

680 340 340

28.5

680

8.4 21.6 16.6 21.6 12.5 6.06 6.9 19.4

960

5.1

480 480

11.1

11.98

680 960 340 340 480 480 240

AH

>

256 2048 2048 1024 1024 1024

2048 2048 2048 2048 1024 512 2048 512 1024 1024

SZ

Site of infection

1:400 1:400 1:400

36

6

1:200

5

30

23

1:600

T,S T,S

20

8

30

10

21 14 21

7

1:200 1:600 1:200 1:500 1:200 1:400

1:600 1:400 1:800 1:800 1:400

Days from infection

Days from nephritis

5

25

12

T 5

28

U

S 5

T 5 5 5

1

19

U

8

30

19

28

T

14

15 10 14

5

21

T

11

5

U

1:800

T,S

27

9

1:400 1:200 1:200

5

26 24

15

1:800 1:400

S

5 5 5

5

U

30

8 8 18

13

7

19

rheumatoid factor (RF), Clq binding (CIqBA), antistreptolysin 0 (ASO), antihyaluronidase (AH), and streptozyme (SZ), were performed at the time of admission to the hospital. Site of infection is: S. skin; T, throat. Days from onset of infection by history and onset of clinical signs of nephritis represent the interval between these times and obtaining serological data. U is unknown.

Mcintosh et a!

504

tions suggestive of chronicity were observed. The results of immunohistologic studies are shown in Table 3. The interval of time that elapsed from the biopsy to the first sign or symptom of infection and nephritis is also noted in Table 3. There was no relationship between the immunohistologic results and the site of infection, age, sex, apparent duration of nephritis, or time elapsed from streptococcal infection. All biopsies had positive stainings with antihuman C3. Twenty biopsies had positive staining for IgG, and 15 were positive for 1gM. Twelve biopsies had positive staining with HIgG-N. All the patients showing positive staining with HIgG-N or HIgG-agg, also showed similar fluorescent deposits of IgG and C3. Immunoglobulin M deposits were demonstrated in 9 of 12 patients with positive HIgG-N staining. On the other hand, all biopsies but one, showing glomerular 1gM deposition, showed HIgG-N-positive staining. Unmodified human IgG fixed in the glomeruli of nine biopsies; all of them had HIgG-Npositive staining. Only one patient showed positive HIgG-N staining and negative results with HIgG-u

ing with HIgG-N, seven were also positive with RIgG-u.

In general, staining with HIgG-N was the most prominent, staining with HIgG-u was somewhat less, and the staining with HIgG-agg was more com-

monly focal, segmental, and weaker. Figs. 1—6 shows immunohistologic staining of representative glomeruli from patient #171. Following treatment of the negative biopsy specimens with citrate buffer, 1gM and IgG were not localized in those patients in whom these immuno-

globulins were not detected by direct immunofluorescence prior to elution. However, After a 12hr elution period, it was possible to show HIgG-N fixed in the glomeruli of seven out of nine patients that were previously negative. The same was true for six out of nine patients with respect to HIgG-u.

The results of the acid elution experiments are shown in Table 4. A variety of immunohistologic

patterns were observed: fine granular capillary walls, fine or coarse, granular, mesangial, or combi-

nations. In general, deposits with all fluorescent reagents showed similar patterns in the same patient. The results of immunohistologic studies on

and HIgG-agg.

Of those biopsies that presented positive stain-

controls are shown in Table 5.

Table 3. Immunohistologic data

Patient no. 122 128 131

134 135 139 141

150 155 157 158 160 162

Time from nephritis days

Time from infeetio&'

days

RAHIgG

36 23 24 23 66 7 32

NK

+ +

53

+ + + +

9

39

—

—

+ +

—

31 15 19

15

53 44

30 36 21

60 22 30 18

164

13 14

165

11

14 21 17

166 167 169 170

NK

NK

171

9 15 8 8

174

18

24 19 30

175

7

13

27

26

lmmunohistology with FITCconjugatedc

+

-4-

+ + —

+ + + + + + + +

RAHIgM —

+ —

+ —

+ + +

+ -I-

+ + + —

+

+ + + +

RAHC3

HIgG(N)

HIgG(agg)

+ + + +

+

— —

+ +

+ + + + + + + + +

+ +

+ + +

+ +

HIgG(u)

RIgG(agg)

RIgG(u)

—

—

—

—

—

ND

—

—

—

— —

—

—

+ + +

ND

+ + +

+

+

+ +

+

—

-I—

—

— —

+

+

+

—

—

+

+

+

—

+

+

—

—

—

— — —

— —

—

ND

ND

ND ND

-

—

ND

ND ND

ND ND

ND

-4-

— —

+ + + + + +

+ + +

--

+

+ — —

+

+ + +

ND +

—

— — —

+

+

ND

ND

ND

+

+

+

Interval of time is between onset of nephritis and renal biopsy. Interval of time is between onset of streptococcal infection and biopsy. Symbols are +,positive; —, negative. Abbreviations are: NK, not known, ND, not done; RAHTgG, rabbit antihuman lgG; RAHIgM, rabbit antihuman 1gM; RAHC3, rabbit antihuman C3; HIgG(N), neuraminidase-treated human IgU; HIgG(agg), aggregated human IgG; HIgG(u), unmodified human Igo; RIgG(agg), aggregated rabbit IgG; RIgG(u), unmodified rabbit IgU.

Poststreptococcal glomerulonephritis: Immunology

505

Fig. 1. Fluorescein isoihiocyanate (FITC)-conjugated rabbit antisera to human IgG, showing granular deposits along glomerular capillary walls and in the mesangium <32O).

Discussion

The latent period between streptococcal infection and the development of nephritis has long suggested a sequence of events similar to that of serum sick-

ness in the experimental animal [1—4]. Immunohistologic demonstration of granular deposits of immunoglobulins and complement components in the

glomerulus, and the demonstration of electrondense subepithelial deposits representing immunoglobulins and complement, as well as the depression of serum complement early in the disease, further support an immune-complex pathogenesis [1, 2, 4]. In addition, there is a more direct evidence in favor of the circulating immune complex hypothesis

since cryoprecipitable and Clq-binding immune

complexes have been shown to be present in the serum of patients with APSGN [7] (RoDRIGuEzITURI3E et a!, submitted for publication).

The nature of the association between streptococcal infection and nephritis led to early speculation that an exogenous antigen, related to streptococcus, was involved in the formation of pathogenic immune complexes. Several investigators, however, have attempted to localize soluble streptococ-

cal antigens in the glomeruli of patients with APSGN, but the results have been negative, difficult to repeat, or inconclusive [1, 18—20]. Treser et al have detected streptococcal antigenic material, distinct from M protein, in the glomeruli of early biopsy tissue from patients with APSGN [19, 20]. These investigators stress the importance of the

506

McIntosh et at

neously in cryoprecipitates isolated from patients with this disease [5—7]. The finding of anti-IgG activity in some of the cryoprecipitates led to the suggestion of a role for an autologous immune-complex mechanism in nephritis related to streptococcal in-

fection [6]. In this postulated mechanism, antibodies produced to IgG rendered autoimmunogenic by interaction with or modification by a streptococcal product or by incorporation into immune complexes, led to the formation of (anti-IgG) (IgG) path-

ogenic immune complexes. These autoantibodies could react with the neoantigen in the circulation in situ in the renal glomerulus. In addition, the autoantibody may also partially react with native host IgG. In a previous communication, we have reported

the isolation of an 1gM anti-IgG cryoprotein in which the IgG fraction lacked sialic acid [6]. Zinneman, Levi, and Seal isolated a similar cryoglobulin from a patient with essential cryoglobulinemia and showed that the 1gM fraction reacted with neuraminidase-treated (sialic-acid-depleted) IgG, whereas only a very weak reaction was observed with un-

Fig. 2. Gloinerulus stained wit/i FITC—conjugated rabbit antisera to human 1gM showing granular deposits along the glomerular basement membrane and in the mesangium (same patient #171);

(x282).

presence of this material in biopsies obtained early

in the course of APSGN, suggesting that later in the disease the antigenic sites are saturated with excess antibody. Antibodies against this water-soluble antigen fraction (endostreptosin) have been detected in the sera of patients with acute poststreptococcal glomerulonephritis [211. Elevated titers, however, are also present after uncomplicated streptococcal infections. Villarreal and Zabriskie have recently observed an extracellular protein unique to nephritogenic streptococci [221. It is noteworthy that none of the streptococcal antigens implicated have been identified in the subepithelial electron-dense deposits or "humps" that are so characteristic of the disease [17, 231.

We have reported the detection of serum cryoglobulins with biologic properties of immune complexes in all patients studied during the acute phase of APSGN [7]. Streptococcal immunoreactive material could only occasionally be found as a constituent of these cryoproteins by a variety of immunological techniques [5—7]. Moreover, streptococcal antigen and antibody were never detected simulta-

modified IgG [24]. We have shown that the sialic acid content of IgG was markedly decreased by incubation with a culture of nephritogenic streptococcus [25]. Autologous IgG modified either by incubation with this microorganism or by treatment

with neuraminidase was capable of inducing cryoglobulinemia, anti-IgG antibodies, and glomerulonephritis characterized by granular deposition of IgG and C3 [8, 9]. These events occurred in the absence of streptococcal infection, elevation of anti-

streptolysin 0 titer, or deposits of streptococcal material in the glomerulus. Other investigators have demonstrated the production of monoclonal cryoglobulins with anti-IgG activity in rabbits following immunization with streptococcal vaccines [261. Pre-

liminary studies by Davis, Baig, and Ayoub have shown that certain strains of streptococcus produce neuraminidase [27]. We have observed elevated titers of anti-IgG ac-

tivity as early as 10 days following the onset of streptococcal infection and on the day of onset of symptoms of nephritis in patients with APSGN [10,

11]. These studies suggest that the production of antiglobulins in APSGN may be related to the streptococcal infection and may be a primary event

in APSGN. It is possible, however, that the antiIgG is an antibody to IgG incorporated in an exogenous immune complex of streptococcal-related antigen and specific antibody. The fixation of neuraminidase-treated IgG to the glomerulus of patients in this study supports an au-

Poststreptococcal glomerulonephritis: Immunology

507

Fig. 3. Glomerulus stained with FITC-conjugated rabbit antisera to human C3 showing fine

granular deposits in the rnesangium and along the capillary basement membranes (same patient #171); (x320).

tologous immune mechanism in this disease. Reactivity was also observed with unmodified IgG and aggregated IgG; the fluorescence with these latter

reagents, however, was much less prominent. In previous studies, we have shown partial immunologic cross-reactivity between unmodified IgG and neuraminidase-treated IgG [9].

Elution studies permitted the demonstration of anti-IgG activity in previously negative biopsies. These studies suggest that, in some patients, complete saturation of antigen with antibody is taking place. The hypothesis that antiglobulins are produced in response to a neoautoimmunogen resulting from the

action of a streptococcal neuraminidase-like en-

zyme, is not unattractive. Anti-IgG antibodies have been found in the glomeruli of some patients with nephritis, and it has been suggested that their presence can exacerbate immune-complex renal disease [281. In addition, immunoglobulin complexes (IgG1gM, IgG-IgG) have been implicated in the patho-

genesis of the renal lesion in systemic lupus erythematosus [29], progressive systemic sclerosis [301, and essential cryoglobulinemia [32]. It is of interest that ultrastructural studies of glomeruli of

some patients with IgM-anti-IgG cryoglobulinemia have revealed subepithelial "humps" indistinguishable from those observed in APSGN [311. In our laboratories, we have looked routinely for antiglobulins in renal biopsy specimens. Excluding

508

McIntosh ci a!

Fig. 4. Glomerulus stained with FITC-conjugated neuraminidase-treated human IgG showing predominant deposits along the capillary walls and in the mesangium similar to antihuman Igs and C3 (same patient #171); (x282).

Fig. 6. Glomerulus stained with FITC-conjugated human aggre-

gated IgG showing large but more sparse deposits pre-

dominantly in the mesangium and to a lesser extent along the glomerular basement membrane (same patient #171); (x282).

a few cases of the nephritis of systemic lupus erythematosus, we have failed to detect these autoantibodies.

It is quite possible that the immune injury in APSGN involves a variety of exogenous and endog-

Table 4. Immunohistologic findings after citrate buffer elution of biopsies that untreated failed to stain with neuraminidase-treated human IgG. .

Patient

no.

FITC-HIgG(N)

FITC-HlgU(u)

128

—

—

131

+ + + + + +

—

—

—

+

+

134 150 155

58 162 164 165

Fig. 5. Glomerulus stained with FITC-conjugated untreated rabbit igG showing large granular deposits in the mesangium and along the capillary loops (same patient #17/); (x282).

---

12 hr of acid elutiona Immunohistology after -----

-

+ + + +

a Symbols arc: +, positive; —, negative. (All specimens where negative were untreated or treated with citrate buffer for I to 4 hr). FITC-1-IIgG(N) is fluorescein isothyocyanate-corijugated

neuraminidase-treated human TgG. FITC-FTIgG(u) is fluorescein isothyocyanate-conjugated unmodified human lgG.

509

Poststreptococcal glomerulonephritis: Immunology Table 5. Immunohistologic studies on controls

No. of Patients

Classification

Diffuse

Anti-HIgM no. positive

Anti-HC3 no. positive

HIgG (n) no. positive

HIgG (u) no. positive

HIgG(a) no. positive

7

7

7

7

0

0

0

7 9

2 5

4

24

2 5 19

18

15

8 8

3

2 3

3

1

1

1

2

1

3

3

2 2

2

2

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 3 0 0 0 0 0

6 2

5 2

5 2

5 2

0 0

0 0

2

Idiopathic membranous nephropathy Proliferative nephritis Mesangial Focal

Lupusnephritis

Anti-HIgG no. positive

Minimal change nephrosis Focal schlerosis Amyloid Diabetic nephropathy Interstitial nephritis Membranous nephropathy associated with rheumatoid arthritis and gold therapy Cryoglobulinemia

1

enous immune-complex systems. Such a situation is

observed in the nephritis of systemic lupus erythematosus, where circulating as well as glomerular-fixed antigen-antibody interactions have been implicated. Acknowledgments

This work was supported by CONICIT Grant No. S4-141 (Venezuela), Grant from the Asociación

de Amigos del Riñón (Maracaibo), Grants in Aid from the American Heart Association, Colorado Heart Association, United States Public Health

Service Grants 5S01 RR 05357, GCR RR 69, RROO51, AMO 7135 and T3280 107028, Grant in Aid

from the Rocky Mountain Foundation. Part of this work was performed during Dr. McIntosh's tenure of Established Investigatorship from the American Heart Association. Reprint requests to Dr. D. Rabideau, Box c282, University qf Colorado Medical Center, 4200 E. 9th Avenue, Denver, Colorado 80262, U.S.A.

7 23 16 3

13 12

5

1

6. MCINTOSH RM, KAUFMAN DB, KULVINSKAS C: Cryoglobu-

lins: II. The biologic and chemical properties of cryoglobulins in acute poststreptococcal glomerulonephritis. mt Arch Allergy AppI Immunol 41:700—715, 1971 7. MCINTOSH RM, GRISWOLD WR, CHERNACK WR, WILLIAMS G, STRAUSS J, KAUFMAN DB, KOSS MN, MCINTOSH JR, Co-

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