sign in sign up
<<
Home , Glomerulonephritis, Glomerulonephrosis, Lupus nephritis

Journal of Clinical Investigation Vol. 45, No. 2, 1966

Acute Poststreptococcal : Immune Deposit Disease * ALFRED F. MICHAEL, JR.,t KEITH N. DRUMMOND,t ROBERT A. GOOD,§ AND ROBERT L. VERNIER || WITH THE TECHNICAL ASSISTANCE OF AGNES M. OPSTAD AND JOYCE E. LOUNBERG (From the Pediatric Research Laboratories of the Variety Club Heart Hospital and the Department of Pediatrics, University of Minnesota, Minneapolis, Minn.)

The possible role of immunologic mechanisms in the in acute glomerulonephritis have also acute poststreptococcal glomerulonephritis was revealed the presence of discrete electron dense suggested in 1908 by Schick (2), who compared masses adjacent to the epithelial surface of the the delay in appearance of after glomerular (11-18). injection of heterologous serum to the latent pe- The purpose of this paper is to describe immuno- riod between scarlet and onset of acute glo- fluorescent and electron microscopic observations merulonephritis. Evidence in support of this con- of the kidney in 16 children with acute poststrepto- cept is the depression of serum complement during coccal glomerulonephritis. This study demon- the early stages of the disease (3) and glomerular strates 1) the presence of discrete deposits of yG- localization of immunoglobulin. Immunofluores- and fl3c-globulins along the glomerular basement cent studies have revealed either no glomerular membrane and its epithelial surface that are similar deposition of a-globulin (4) or a diffuse involve- in size and location to the dense masses seen by ment of the wall (5-9). Seegal, Andres, both thin section microscopy and electron micros- Hsu, and Zabriskie (10) demonstrated the pres- copy; 2) the characteristic and unique specificity of ence of 7 S y-globulin, /31c-globulin, and strepto- this lesion in acute poststreptococcal glomerulo- coccal in the glomeruli of most patients , its difference from other glomerular dis- with this disease. By studies employing ferritin- eases, and the similarity to experimental antigen- labeled , these could be demon- complex nephritis; and 3) the resolution strated in the mesangium, between the endothelial of these deposits during recovery from the disease cells, and within and adjacent to the basement and their absence from the glomeruli 10 years af- membrane. Electron microscopic observations of ter epidemic acute poststreptococcal glomerulo- nephritis. * Submitted for publication May 3, 1965; accepted No- vember 4, 1965. Methods Aided by grants from the U. S. Public Health Service (AI-02168 and HE-05662), the American Heart Associ- Patients. The major portion of the study concerned ation, and the Cardiovascular Clinical Research Center sixteen children with acute poststreptococcal glomerulo- (HE-06314). nephritis having the characteristic clinical course and re- Presented in part at the American Society for Clinical nal as well as prior P-hemolytic streptococcal Investigation, Fifty-sixth Annual Meeting, May 4, 1964, and elevated or rising antistreptococcal anti- Atlantic City, N. J. (1). body titers. A total of 22 renal were performed t Established Investigator, American Heart Associa- on these patients by the method previously described tion. (19) and at the times indicated in Table I. As part of t Present address: Dept. of Pediatrics, McGill Uni- a separate follow-up study (20) 10 years after well- versity, Montreal Children's Hospital, Montreal, Canada. documented acute poststreptococcal glomerulonephritis § American Legion Memorial Heart Research Profes- (Red Lake, Minnesota, epidemic of 1953), additional re- sor of Pediatrics and Microbiology. nal biopsies were performed on 14 children and studied Address requests for reprints to Dr. Robert A. Good, by fluorescent microscopy. Dept. of Pediatrics, University of Minnesota Medical Electron and fluorescent microscopic observations of School, Minneapolis, Minn. 55455. the kidney were also made in one patient with 11 Established Investigator, American Heart Association. nephritis and one with Goodpasture's disease. Present address: Dept. of Pediatrics, Center for Health Histologic methods. Part of each was placed Sciences, University of California, Los Angeles, Calif. in 10% buffered formalin (pH 7.35) for standard paraffin 237 238 MICHAEL, DRUMMOND, GOOD, AND VERNIER embedding and sectioning and subsequently stained with man fibrinogen was prepared from human Fraction I 5 hematoxylin-eosin, azocarmine, and periodic-acid Schiff. according to the method of Laki (30, 31). 4) Human Light microscopy was carried out on 21 of the 22 biopsies. fibrin was prepared from human fibrinogen; 25.0 mg of A total of 12 to 30 glomeruli were evaluated in 17 of the fibrinogen was diluted in isotonic saline to a final con- 21 biopsies; in each of the remaining specimens, five to centration of 5.0 mg per ml, and then 10.0 ml of 0.075 M six glomeruli were present. sodium chloride in phosphate buffer (pH 6.1) and 0.5 ml Another portion of the biopsy specimen was immedi- of bovine thrombin were added. After standing for 1 ately cut into cubes (0.5 mme) under a drop of buffered hour, the clots were washed with distilled , lyophi- (1%o) osmic acid solution, dehydrated by standard tech- lized, and suspended in particulate form in 0.15 M saline. niques, and embedded in Vestopal-W1 polyester resin. Antisera were also prepared against 5) human Frac- Sections 0.5 to 1.0 j in thickness cut from these blocks (Cohn Fraction V),6 6) rabbit y-globulin (Cohn were stained by either the Wright-Giemsa (21) or basic tion II), and 7) heat-killed and ground-cell suspension fuchsin method (22) and examined under oil immersion of nephritogenic, type 1, group A, fi-hemolytic strepto- light microscopy. Very thin sections for electron micros- coccus. 8) Extracellular products of the same strepto- copy were stained with either uranyl acetate (23) or lead coccus were prepared according to the method of Wan- citrate (24) and examined with either an RCA EMU-3D namaker (32) and concentrated by ammonium sulfate or a Phillips EMU-200 at original precipitation or by pervaporation. magnifications of 2,500 to 75,000 times and enlarged Preparation of antibody. Antibody to these human anti- photographically as desired. gens was prepared in rabbits. Immunizations were car- Immunofluorescent methods (25-27) were carried out ried out with alum-precipitated human 'yG-globulin and on tissue that had been frozen in isopentane albumin, fibrin and Pic-globulin incorporated in complete in liquid nitrogen, fixed to a small cellulose sponge,2 and Freund's adjuvant,8 and a saline solution of fibrinogen. sectioned in a Lipshaw cryostat. Fluorescent staining of Alum-precipitated rabbit y-globulin was injected into unfixed tissue was carried out according to the method goats. The specificity of the antisera was shown by im- of Ortega and Mellors (28). In some instances an indi- munoelectrophoresis of the immunizing antigen and whole rect technique was also employed. Inhibition of fluores- serum developed against the antisera. Antisera showing cence was carried out by pretreating the section once or more than one major precipitin band were not used. twice with unlabeled antiserum. A horse anti-'yM-globulin serum,9 employed in these The sections were viewed in a Zeiss microscope with studies, formed one major line on immunoelectrophoresis an HB-200 light source or a mercury lamp (GE-AHG) against normal serum; an additional faint line was also mounted in a Scopicon water-cooled unit. The ultra- seen in the a2-globulin .region. violet activating and heat absorption filters were UG-1 Antisera to the various streptococcal preparations were and KG-1, respectively. The barrier filters consisted of obtained by immunizing rabbits three times weekly for OG-4 and a 2A Wratten (Kodak). Photographs were a total of 2 months with 0.5 ml of antigen in solution or taken with an Exacta camera using Kodak Tri-X or suspension. Antigen preparations were injected intra- high speed Ektachrome film. The intensity and amount venously during the first month and subcutaneously dur- of fluorescence were arbitrarily graded as negative, trace, ing the second month of immunization. The development 1+, 2 +, and 3 +. Six to 18 glomeruli were available of antibodies to multiple in these crude vaccine for evaluation from each of the 22 biopsies. materials was demonstrated by the appearance of a num- Preparation of antisera. Antisera were prepared against ber of lines of precipitation in a double-diffusion agar the following antigens: 1) Human -yG-globulin was iso- system. lated by DEAE Sephadex A-50 chromatography; 3 im- The ammonium sulfate-precipitated globulins were di- munoelectrophoresis of this antigen against antihuman alyzed and then conjugated with fluorescein-isothiocya- serum revealed a single precipitin line. 2) Human Pic- nate 10 (33, 34) with 0.015 g per g of . The la- globulin, a part of the third component of complement, beled antiserum was then passed through a column of was prepared from 400 ml of fresh human serum by the Sephadex G-25 to remove the free fluorescein (35, 36). column chromatographic method of Muller-Eberhard, Antisera were stored as small portions at - 220 C until Nilsson, and Aronsson (29); the 8,c-globulin isolated used. Immediately before use, the antisera were ab- formed one precipitin line with both antihuman serum and with known rabbit anti-Pic-globulin serum.4 3) Hu- 5 Provided by the American Red Cross and Dr. Rich- of 1 ard von Korff, Department Biochemistry, University Martin Jaeger Co., Geneva, Switzerland. of Minnesota, Minneapolis, Minn. 2 no. 1, obtained from Histomed, Paterson, Onkosponge 6 Cutter's normal human serum albumin, Cutter Labora- N. J. tories, Berkeley, Calif. 8 Some of the -yG-globulin used was obtained from Im- munology, Inc., Lombard, Ill. 7 Rabbit y-globulin (Fraction II), Nutritional Bio- 4 During the initial part of this investigation the rab- chemical Corp., Cleveland, Ohio. bit anti-Pie serum was kindly supplied by Dr. H. Muller- 8 Difco Laboratories, Detroit, Mich. Eberhard, Scripps Clinic and Research Foundation, 9 Hyland Laboratories, Los Angeles, Calif. La Jolla, Calif. 10 Baltimore Biological Laboratories, Baltimore, Md. ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS: IMMUNE DEPOSIT DISEASE 239

o0 s be (U .5!(U bO (. 9

0 3 3c o0 cU U) ~t~t ol co Q 0 (U(I. 3: t C

4i W0 SC G 0 0 YCU -1 4i V

U) m 8. (Ud bo Ca e rxs>3 twf2== *_ 4.&CO 0 0 30

O

U) ) U 0 .0 . M .2 Cd Cd C (40 44 be tO be

.':g0'.0 .2 Vf

v v) veU) -( kZ Z

Cd co . Cd _ + b+ w + + *C.rU(U'.°(d N' - U)(U (U

.0 .44 . . .Z OU. *.

U) U) 0440 U) U) U) U) cu U - U,) O(U ~~. ._0 Cd Cd ci 00 4bO 40a be 4be U) U) ) z z z m

IV Id wo 0 . V *'r.0.X

O + + + 40 + ++ + ++ 75 .0 0 Ck a.o(U.0 .4 44. mm . Q0 C') eC'iC' Z C 0 44 - C.o ..acd v - U) be + + + + + w + + + "m VI + + + ++ ++ 0 E. m z C' - C4 - (' &doY 2

(U .- ..U 300a .- 0 be .0 -- v- V .- ^ vD - - Q _ .= Sc > = > (U c 4 > = = 0 = 0 * - C U- -, 1. Cd + be +bO fU U X + ++ @ +++ I + + + OUbO+ 0U + (4 m Z C ) - H "i (U-' 44.)C.(U o >,%) 0 l 40 --00n co0~a -U))'(400 ,/4 0Cd At 0 0 ) 0l I'd, O (4 vSL ~ .-_ 0 (4N N " --U) t- m -4-__-_4 0w >cd o vo- C

-.U)0 .0 (U rU vU( U 0C (U-E,OU U) (U( (Ur.o * R44U)-4 cU'

._ 0 _4 _ In oco 0o _4 C_. (U( c 240 MICHAEL, DRUMMOND, GOOD, AND VERNIER With the basic fuchsin technique the nodules were pale red or pink, whereas the basement membrane proper was intensely red.

II) Fluorescent microscopy A) Acute glomerulonephritis. The glomerular deposition of yG- and fl-globulins is summarized

FIG. 1. THIN SECTION (.5 ,) OF RENAL TISSUE OB- TAINED FROM PATIENT 6, 19 DAYS AFTER ONSET OF ACUTE GLOMERULONEPHRITIS, SHOWING A SEGMENT OF THE GLO- MERULUS. Discrete deposits are visible along the epi- thelial surface of the basement membrane (arrows) or appear to lie in the (dotted arrow). (Wright- Giemsa x 900.) sorbed twice with mouse powder.'0 Labeled anti- sera having low fluorescein to protein ratios were used as recommended by Goldstein, Slizys, and Chase (37) and Frommhagen and Martins (38). Results I) Light microscopy Light microscopy of standard 4-,u sections in all patients showed findings characteristic of acute poststreptococcal glomerulonephritis: glomerular endothelial proliferation, decreased erythrocytes in the glomeruli, polymorphonuclear leukocyte infiltration, segmentation, and in some cases crescents. Thin sections (Patients 1 to 4, 6, 10, 12). Al- though nodular masses seen along the basement membrane of glomerular of fluorescent and electron microscopy (see below) were not visible in routine 4-,u sections because of thickness of the tissue, these masses could be resolved by light microscopy with thinner sections. Examina- tion of sections of 0.5 to 1.0 pt, cut from plastic FIG. 2. RENAL BIOPSY OBTAINED FROM PATIENT 11, 23 DAYS AFTER ONSET. A) Discrete linear deposits of yG- blocks embedded for electron microscopy, disclosed globulin are present throughout the . A simi- nodular masses along the basement membranes lar morphologic picture is seen when the tissue is (Figure 1). Variable numbers of these masses stained with fluorescein-conjugated rabbit anti-Pic-globu- were seen in seven of seven cases (1 to 4, 6, 10, lin. (Fluorescent microscopy X 350.) B) Small seg- 12) studied by this technique. The masses stained ment of a glomerular capillary loop showing immune masses (m) of 'yG-globulin along the epithelial surface somewhat differently from the membrane itself. of the basement membrane (BM). The basement mem- With Wright-Giemsa stain, the masses were a brane stains weakly. The deposits in this photograph are deeper blue than the basement membrane proper. 2 to 4 g in diameter. (Fluorescent microscopy X 2,200.) ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS: IMMUNE DEPOSIT DISEASE 241 in Table I. In 12 of 16 patients, yG-globulin was demonstrated in the glomeruli of -the initial biopsy; f31c-globulin was detected in glomeruli of all pa- tients. Typically, the fluorescence was discrete and nodular, scattered diffusely throughout the glo- meruli (Figure 2A); linear areas of basement membrane and mesangial fluorescence were also seen, especially in patients with more severe dis- ease (Patients 10, 12, 14) or patients biopsied later in convalescence, but this-morphologic distribution was less frequent. In glomeruli from some pa- tients the fluorescent masses approached the size of erythrocytes and in some areas seemed to be fused with other masses in a local distribution sparing parts of the glomerulus. In tissue from other patients the nodules were smaller and gave a speckled appearance to the glomerulus at these magnifications. The deposits appear to follow the FIG. 4. TISSUE SHOWING A PORTION OF A GLOMERULUS course of the glomerular capillary walls at higher OBTAINED FROMN PATIENT 3, 8 DAYS AFTER ONSET OF ACUTE magnification, and in glomeruli from some patients GLOMERULONEPHRITIS, STAINED WITH FLUTORESCEIN-CON- the immune desposits clearly were present on the JUGATED RABBIT ANTIBODY AGAINST A GROUND-CELL PREPA- RATION OF A NEPHRITOGENIC TYPE 1, GROUP A, S-HEMO- epithelial surface of the glomerular basement mem- LYTIC . The discrete fluorescence cannot brane (Figure 2B). There was no evidence that be anatomically localized with respect to the basement the fluorescence represented nuclear, cytoplasmic, membrane, but appears to be present between cells or or cellular staining. Gamma G-globulin was not within the cytoplasm. (Fluorescent microscopy X 350.) present on the glomeruli in Patients 4, 9, 13, and 15. In these patients considerable amounts of 8,c- globulin were observed as discrete nodules. Beta1c-globulin was also demonstrated by an indirect technique in nine patients so studied, but when this method was used, the fluorescence was more linear in appearance than when the direct technique was used. In Patient 10, the amounts of yG- and /1c-glob- ulins were decreased at 112 days. At 272 days, only trace amounts of yG-globulin were noted on the glomeruli in focal distribution, but no flic- globulin could be detected. The tissues from Pa- tients 5 and 6, 193 and 184 days after onset, dem- onstrated neither Plic- nor yG-globulin. Slight local and focal deposition of both yG and f13c was observed in tissue obtained from Patient 12, 198 days after onset. FIG. 3. RENAL TISSUE FROM PATIENT 3, 8 DAYS AFTER ONSET, STAINED WITH FLUORESCEIN-LABELED RABBIT ANTI- The specificity of the fluorescence was confirmed HUMAN FIBRIN SERUM. The exact localization in the glo- by inhibition of fluorescence with unlabeled anti- merulus cannot be determined although the fibrin is prob- serum to -yG-globulin in 11 of 11 instances, by in- ably present within Bowman's space or along the base- hibition of fluorescence with antiserum to ment membrane; it is not deposited in the nodular form /,3c- seen with -yG- or 8,c-globulins (cf. Figure 2). (Fluo- globulin, and by negative fluorescence for human rescent microscopy X 350.) albumin in eight of eight preparations. No yM- 242 MICHAEL, DRUMMOND, GOOD, AND VERNIER

FIG. 5. ELECTRON MICROGRAPH OF A SMALL PORTION OF A GLOMERULUS FROM PATIENT 4, ILLUSTRAT- ING THE FREQUENCY AND VARIABILITY OF FORM OF DENSE IMMUNE DEPOSITS. ml (m = electron dense masses) appears to be attached to the basement membrane, which is more dense than normal in the area of attachment. A polymorphonuclear leukocyte (PMN) is adjacent to the endothelium at this site. m2 and m, appear to lie entirely within the epithelial cell although this is not proven. Note the dense halo of cytoplasmic material around M2. EP = epithelial cell; END = endothelial cell. (x 16,500.) globulin could be shown in the glomeruli of six cence was present in Patients 2 and 3 when either patients so studied. antiserum was used (Figure 4); this was inhibited Fibrin was observed in the glomeruli of initial by the prior application of unlabeled antisera. A biopsy specimens from five of eight patients; how- trace of fluorescence was also seen in the glomeruli ever, the distribution of the fluorescence differed of Patient 4, but none was seen in the other pa- from the nodular distribution observed with yG- tients. It was not possible to localize exactly the or /l3c-globulins (Figure 3). The pattern of fibrin fluorescence with respect to the glomerular base- deposition was often ribbon-like as though it were ment membrane, although no discrete nodules were present within Bowman's space, along the base- seen. Fluorescein-conjugated globulin against the ment membrane, or within the capillary lumen. extracellular streptococcal products failed to show In ten patients (No. 2 to 6, 10 to 12, 13. 15) localization in Patients 2, 3, 4, 5, and 10; the re- the tissue was stained with fluorescein-conju- mainder of the patients were not evaluated with gated globulin against two cellular preparations of this antiserum because of lack of tissue. Fluores- a nephritogenic type 1, group A, /8-hemolytic cent antibody against the ground-cell preparation streptococcus: a heat-killed and a ground-cell sus- pension." Definite discrete glomerular fluores- Armed Forces Epidemiological Board. The support of the offices of the Surgeon General, Department of the 11 These studies were done in collaboration with Dr. Army, Washington, D. C., and of the American Heart Lewis W. Wannamaker under the sponsorship of the Association (Dr. Wannamaker, Career Investigator) in Commission on Streptococcal and Staphylococcal Diseases, this collaborative effort is hereby acknowledged. ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS: IMMUNE DEPOSIT DISEASE 243 of type 1 streptococcus showed no glomerular staining in kidney tissue obtained from patients with the (2), chronic glo- merulonephritis (2), subacute glomerulonephritis (1), and mellitus (1), or in a normal individual (1). B) Ten years after acute glomerulonephritis. Renal biopsies from 14 patients who had had acute poststreptococcal glomerulonephritis 10 years previously were examined for the presence of yG- or 81c-globulins. All had normal renal function. The Addis counts in these patients were slightly abnormal, but the frequency of this abnormality was not significantly different from that of a con- trol Indian population with no history of glomeru- lonephritis (20). In 11 biopsies no glomerular deposition of yG- or,/1c-globulins was found; nine of these biopsies FIG. 6. TYPICAL EARLY CHANGES IN ENDOTHELIAL CELLS showed normal histology, but two showed mild AND BASEMENT MEMBRANE IN PATIENT 3. The lumen of this capillary is obliterated by blebs of endothelium. focal and local glomerular abnormalities. In three Densely stained material (arrows) lies within the base- biopsies, there was focal and local deposition of ment membrane and bulges outward toward the epi- -yG- and fl3c-globulins along the basement mem- thelium in one area. (X 25,700.) brane of approximately one-half of the glomeruli; the deposition was restricted to small loci and rep- crease in the number of polymorphonuclear leuko- resented a very small area of the glomerulus. His- cytes within the glomerular capillary lumina usu- tologically, two of these biopsies also showed mild ally adjacent to the dense deposits within the base- focal and local glomerular abnormalities, and one ment membrane or opposite nodules on the epi- was normal. No type of deposi- thelial side of the basement membrane; 4) tion resembling that irregu- found during acute glomerulo- lar circumscribed and linear areas nephritis was seen. of increased density within the basement membrane with oc- III) Electron microscopy casional zones of thinning of the lamina densa or irregular scalloped thickening (these were espe- Two to four glomeruli in each of 11 renal biopsy cially prominent in Patients 2, 3, and 12); and 5) specimens from 9 of the 16 patients included in this numerous dense, homogeneous, spherical deposits report were studied by electron microscopy. that projected from the epithelial side of the base- The ultrastructural changes within glomeruli ment membrane. The smaller masses often in- of the patients fell roughly into two patterns, each dented the lamina densa of the basement mem- related to the interval between onset of clinical brane (Figure 5) and appeared to lie wholly within disease and renal biopsy. the basement membrane. These ovoid bodies were A) Electron microscopic findings in six renal always more densely stained with lead citrate or biopsy specimens obtained 7 to 26 days after onset uranyl acetate than the lamina densa. The larger (Patients 1 to 4, 6, 12). The prominent abnor- masses were pedunculated and attached to the base- malities of ultrastructure in these biopsies included ment membrane by a narrow base that fused with (Figures 5 and 6) 1) an increase in volume of the lamina densa. Other larger spheres often ap- low density cytoplasmic material of luminal endo- peared to lie entirely within the epithelial cell al- thelial cells (endothelial swelling) ; 2) an increased though this is uncertain since serial sections would number of cells identified as either luminal endo- be needed to establish this point. Rarely, dense thelial cells (endothelial proliferation) or centro- granular material could be observed within endo- lobular cells (mesangial cells); 3) a striking in- thelial cytoplasmic vacuoles. 244 MICHAEL, DRUMMOND, GOOD, AND VERNIER B) Electron microscopic findings in five kidney dense material was decreased, and there were biopsy specimens obtained 1 month or more after fewer deposits within the basement membrane. onset (biopsy 2 from Patients 5, 6, and 14 and Nodular masses, where present, appeared to be biopsies 2 and 3 from Patient 10). The electron within enlarged epithelial cells that contained nu- microscopic findings in the group were similar to merous dilated cisternae and a decreased number those described above except that the amount of of mitochondria and lysosomes. A dense granular layer of material approximately 200 A in thickness surrounded the nodular deposits and appeared to blend into the epithelial cytoplasm. The base- ment membrane from later biopsy specimens showed focal areas of thickening alternating with areas of normal thickness and density. Glomeruli from two biopsy specimens (Patients 5 and 6), obtained 6 months after onset of the disease, dem- onstrated only an occasional nodular mass that appeared to be entirely within the epithelial cell; there was also an increase in membranous (stalk) material.

IV) Comparison of the immunofluorescent and ultramicroscopic morphology of acute glomeru- lonephritis with other renal diseases A nodular deposition of -yG- and flc-globulins has also been seen in tissue from certain patients with chronic glomerulonephritis and systemic lu- pus erythematosus (SLE). In the latter disease large amounts of immune-complex deposits may be present within or beneath the glomerular base- ment membrane or projecting from its epithelial surface (Figure 7A). Electron microscopy of a similarly involved glomerulus from the same pa- tient reveals numerous irregular dense masses within and projecting from the epithelial side of the basement membrane (Figure 8A). These masses are very different from the nodules de- FIG. 7. A) TISSUE OBTAINED FROM A 15-YEAR-OLD MALE scribed in acute glomerulonephritis in that the le- DURING THE COURSE OF SEVERE . Fluo- sions observed in SLE are less compact, are rescent microscopy of a segment of a glomerulus reveals rarely pedunculated, and have not been observed the presence of 7yG-globulin along the glomerular base- ment membrane. Deposits are primarily along the epi- as nodules entirely within the visceral epithelium. thelial side of the basement membrane (arrows), but are In other diffuse glomerular diseases such as also present within it. Betaic-globulin was present in a certain types of chronic glomerulonephritis and similar distribution. Compare with electron microscopic Goodpasture's disease, no nodules or masses are appearance in Figure 8. B) TISSUE OBTAINED FROM A 26-YEAR-OLD MAN WITH seen by light or fluorescent microscopy. Figure GOODPASTURE'S DISEASE. Gamma G-globulin is present 7B illustrates the fluorescent microscopic appear- within the glomerular basement membrane giving a dif- ance of the glomerulus in a patient with Good- fuse linear pattern. No isolated or discrete extramem- pasture's disease. The fine linear basement mem- branous deposits are seen. Betaic-globulin was present brane fluorescence is due to y-globulin deposition in a similar distribution. Compare with electron micro- scopic appearance in Figure 8. (Fluorescent microscopy in the basement membrane, which is uniformly x 875.) thickened and without nodules when viewed un- ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS: IMMUNE DEPOSIT DISEASE 245

41~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i

FIG. 8. SECTIONS OF TYPICAL GLOMERULAR CAPILLARIES FROM A) A PATIENT WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND B) A PATIENT WITH PULMONARY HEMORRHAGE AND GLOMERULONEPHRITIS (GOODPASTURE'S SYNDROME). Mottled density within the basement membrane and loosely packed nodular masses (arrows) on the epithelial side of the basement membrane are features of the ultrastructural pa- thology in SLE. Compare with Figure 7. In Goodpasture's syndrome the basement membrane is dif- fusely and rather uniformly infiltrated with electron dense material (,y-globulin). Compare with Figure 7. L = capillary lumen. (A, X 26,000; B, X 18,000.) der the electron microscope (Figure 8B). This evidence of preceding streptococcal , and picture stands in sharp contrast to the morpho- characteristic renal morphology. The salient find- logic appearance of the glomerulus in acute post- ing was the presence of discrete nodular deposits streptococcal glomerulonephritis. of either 8,c- or yG-globulin, or both, in the glo- meruli of all 16 patients with acute disease and Discussion the correlation of this type of deposition with den- Strict criteria for the diagnosis of acute post- sities, similar in conformation and location, in elec- streptococcal glomerulonephritis in 16 of our pa- tron microscopy and thin section microscopy of tients included a characteristic clinical picture, tissue from the same biopsies. These deposits are 246 MICHAEL, DRUMMOND, GOOD, AND VERNIER characteristic of the disease. In a few patients tron-dense material adjacent to and within the glo- with severe disease, a prominent diffuse linear merular basement membrane (42-44). Ferritin- basement membrane and mesangial fluorescence labeled studies have shown that these deposits was also observed. During convalescence, the contain bovine serum albumin (antigen) and rab- immune-complex deposits were either not present bit y-globulin (antibody) (48). The discrete at all or in minimal quantity by fluorescence mi- nodular fluorescent and electron microscopic ap- croscopy and seemed to be resolving in the few pearance of these nodular masses on the epithelial patients studied by electron microscopy. In side of the basement membrane in acute serum these late specimens the densities seemed to be sickness in rabbits is indistinguishable from the entirely within the epithelial cells and showed renal lesion of acute poststreptococcal glomerulo- changes interpretable of ongoing lysis. nephritis in children (49). This lesion is morphologically distinct from the Although the exact mechanism of production of diffuse linear distribution of y-globulin and com- acute glomerulonephritis is speculative, the se- plement observed by fluorescent microscopy quence of events may be similar to that occurring within glomerular basement membrane in Good- in experimental antigen-antibody complex disease. pasture's disease and in some patients with chronic The presence of streptococcal antigen in the glo- glomerulonephritis, in which no discrete deposits meruli of some patients with this disease tends to are present. Nodular basement membrane fluo- support but does not prove the antigen-antibody rescence has also been observed in disseminated complex hypothesis. lupus erythematosus and in some cases of chronic Although the deposits of acute glomerulo- glomerulonephritis (39); however, the yG and nephritis bear similarities to those of lupus nephri- Pic are not as discretely deposited. tis and some forms of chronic glomerulonephritis, Beta1c-globulin was demonstrated more consis- there are differences. In acute nephritis the de- tently in the glomeruli of patients than was yG- posits tend to be discrete and appear to be present globulin, which was not demonstrable even in wholly within the epithelial cell; in the other dis- trace amounts in four children. This may be eases, the nodular deposits may be located entirely analogous to the non-y-globulin Coombs' test in within the basement membrane, as in some cases certain hemolytic anemias where complement or its of chronic glomerulonephritis (39) ; the electron- components, and not y-globulin, may be found on dense material or yG- and fl1c-globulins may also the red cell surface (40, 41). Leddy, Hill, appear as a linear density within the basement Swisher, and Vaughan (41) have suggested that membrane without extramembranous deposits as the y-globulin is present, but sterically hindered in Goodpasture's disease. The appearance of the from reacting with its homologous antiserum, or glomeruli on immunofluorescent staining of the that y-globulin has in fact dissociated from the biopsies in the latter disease is identical to that cell leaving complement behind. It may also be previously shown to be characteristic of nephro- that the location of the deposit in some way deter- toxic serum nephritis (3). In acute nephritis mines detectability of the y-globulin. there is little distortion or disruption of the base- The closest experimental analogue to the lesions ment membrane, and the masses are not usually in acute poststreptococcal glomerulonephritis is trapped or incorporated within the basement mem- antigen-antibody complex nephritis, as observed brane; in the other diseases the basement mem- by Dixon, Feldman, and co-workers (42-45). brane is extensively involved and becomes thick- Our own studies (3) and those of Germuth and ened and distorted. McKinnon (46, 47) support the concept that the Summary lesions of serum sickness reflect the toxicity of these complexes, which are also complement fix- 1. A coordinate study of the light, immunofluor- ing. Light microscopy of the kidney in rabbits escent, and electron microscopy of the kidney was with experimental serum sickness shows acute carried out in 16 children with acute poststrepto- proliferative glomerulonephritis and , and coccal glomerulonephritis. electron microscopy reveals swelling and prolifera- 2. Discrete deposits of fic-globulin or yG-globu- tion of the endothelial cells and deposition of elec- lin or both were present within and along the epi- ACUTE POSTSTREPTOCOCCAL GLOMERULONEPHRITIS: IMMUNE DEPOSIT DISEASE 247 thelial surface of the glomerular basement mem- in vivo bound complement in tissue sections. J. brane during the acute phase of the disease. By exp. Med. 1962, 115, 63. 9. Koffler, D., and F. Paronetto. Immunofluorescent electron and thin section microscopy, these im- localization of immunoglobulin, complement, and mune deposits were shown as dense masses lying fibrinogen in human disease. II. Acute, subacute, on the epithelial surface of the basement mem- and chronic glomerulonephritis. J. dlin. Invest. brane, and rarely within the endothelial cell. 1965, 44, 1665. This discrete deposition of yG- and 10. Seegal, B. C., G. A. Andres, K. C. Hsu, and J. B. /l3c-globulins Zabriskie. Studies on the pathogenesis of acute is morphologically distinct from other glomerular and progressive glomerulonephritis in man by im- diseases. Correlative experimental evidence indi- munofluorescein and immunoferritin techniques. cates that these deposits represent immune aggre- Fed. Proc. 1965, 24, 100. gates, probably antigen-antibody complexes. In 11. Steiner, J. W., and R. Volpe. Autoimmunization-a three of ten patients streptococcal cell antigen was possible mechanism of tissue injury. III. Clinical aspects of . Canad. med. Ass. J. also present in the ilomerulus. 1961, 84, 1297. 3. The amount of yG- and /31c-globulin in the 12. Movat, H. Z., J. W. Steiner, and D. Huhn. The glomeruli decreased markedly or disappeared in fine structure of the glomerulus in acute glomeru- four patients studied up to 9 months after onset. lonephritis. Lab. Invest. 1962, 11, 117. 13. Kimmelstiel, P., 0. J. Kim, and J. Beres. Studies 4. Immunofluorescent studies on kidney tissue on renal biopsy specimens, with the aid of the obtained from 14 patients 10 years after epidemic electron microscope. II. Glomerulonephritis and acute poststreptococcal glomerulonephritis reveals . Amer. J. dlin. Path. 1962, no deposition in 11 and minimal abnormalities in 3. 38, 280. 14. Trump, B. F., and E. P. Benditt. Electron micro- scopic studies of human renal disease. Observa- Acknowledgments tions of normal visceral glomerular epithelium We appreciate the thoughtful editorial assistance of and its modification in disease. Lab. Invest. 1962, Miss Ann Gabrielsen. 11, 753. 15. Churg, J., W. Mautner, E. Grishman, and G. M. Eisner. Structure of glomerular capillaries in References . Arch. intern. Med. 1962, 109, 97. 1. Michael, A. M., K. N. Drummond, R. A. Good, and 16. Movat, H. Z., J. W. Steiner, and R. J. Slater. The R. L. Vernier. Immunoglobulins: clarification of fine structure of the glomerulus in Bright's dis- their significance in renal disease and demonstra- ease: a clinico-pathological study in Ciba Founda- tion of response to immunosuppressive therapy tion Symposium on Renal Biopsy, G. E. W. Wol- (abstract). J. clin. Invest. 1964, 43, 1291. stenholme and M. P. Cameron, Eds. London, J. 2. Schick, B. Die Nachkrankheiten des Scharlach. Jb. and A. Churchill, 1961, p. 103. Kinderheilk. 1907, 65, 132. 17. Strunk, S. W., W. S. Hammond, and E. P. Benditt. 3. Michael, A. F., K. N. Drummond, R. L. Vernier, and The resolution of acute glomerulonephritis. An R. A. Good. Immunologic basis of renal disease. electron microscopic study of four sequential biop- Pediat. Clin. N. Amer. 1964, 11, 685. sies. Lab. Invest. 1964, 13, 401. 4. Taft, L. I., J. K. Dineen, and I. R. Mackay. The 18. Farquhar, M. G., R. L. Vernier, and R. A. Good. localization and binding of serum proteins in the An electron microscope study of the glomerulus glomeruli of kidney biopsies in disseminated lupus in , glomerulonephritis, and lupus erythe- erythematosus and glomerulonephritis. Aust. Ann. matosus. J. exp. Med. 1957, 106, 649. Med. 1958, 7, 5. 19. Vernier, R. L. Kidney biopsy in the study of renal 5. Freedman, P., J. H. Peters, and R. M. Kark. Lo- disease. Pediat. Clin. N. Amer. 1960, 7, 353. calization of gamma-globulin in the diseased kid- 20. Perlman, L. V., R. C. Herdman, H. Kleinman, and ney. Arch. intern. Med. 1960, 105, 524. R. L. Vernier. Poststreptococcal glomerulonephri- 6. Freedman, P., and A. S. Markowitz. Gamma globu- tis: a ten year follow-up of an epidemic. J. Amer. lin and complement in the diseased kidney. J. dlin. med. Ass. 1965, 194, 63. Invest. 1962, 41, 328. 21. Condie, R. M., R. Y. Tousignant, and R. A. Good. 7. Seegal, B. Fluorescent antibody studies of human Rapid staining of osmic acid-fixed methacrylate- glomerulonephritis in Hereditary, Developmental, embedded tissue sections for light microscopy. and Immunologic Aspects of , J. Anat. Rec. 1958, 130, 399. Metcoff, Ed. Evanston, Northwestern University 22. Chandra, S., and F. R. Skelton. Staining juxtaglo- Press, 1962, p. 148. merular cell granules with toluidine blue or with 8. Lachmann, P. J., H. J. Miller-Eberhard, H. G. basic fuchsin for light microscopy after Epon em- Kunkel, and F. Paronetto. The localization of bedding. Stain Technol. 1964, 39, 107. 248 MICHAEL, DRUMMOND, GOOD, AND VERNIER 23. Watson, M. L. Staining of tissue sections for elec- 38. Frommhagen, L. H., and M. J. Martins. A com- tron microscopy with heavy metals. J. biophys. parison of fluorescein-labeled 'y-globulins purified biochem. Cytol. 1958, 4, 475. by rivanol and DEAE chromatography. J. Im- 24. Reynolds, E. S. The use of lead citrate of high pH munol. 1963, 90, 116. as an electron-opaque stain in electron microscopy. 39. Michael, A. F. Unpublished observations. J. Cell Biol. 1963, 17, 208. 40. Muiller-Eberhard, H. J. Identification of complement 25. Coons, A. H., and M. H. Kaplan. Localization of antigens coating red cells in acquired hemolytic antigen in tissue cells. II. Improvements in a anemia in Immunopathology, Third International method for the detection of antigen by means of Symposium, P. Grabar and P. A. Miescher, Eds. fluorescent antibody. J. exp. Med. 1950, 91, 1. Basel, Schwabe, 1963, p. 332. 26. Coons, A. H. Fluorescent antibody methods in Gen- 41. Leddy, J. P., R. W. Hill, S. N. Swisher, and J. H. eral Cytochemical Methods, J. F. Danielli, Ed. Vaughan. Observations on the immunochemical New York, Academic Press, 1958, vol. 1, p. 399. nature of red cell autosensitization in Immuno- 27. Mellors, R. C. Fluorescent antibody-method in Ana- pathology, Third International Symposium, P. lytical Cytology, R. C. Mellors, Ed. New York, Grabar and P. A. Miescher, Eds. Basel, Schwabe, McGraw-Hill, 1959, p. 1. 1963, p. 318. 28. Ortega, L. G., and R. C. Mellors. Analytical pa- 42. Dixon, F. J., J. D. Feldman, and J. J. Vazquez. Ex- thology. IV. The role of localized antibodies in perimental glomerulonephritis. The pathogenesis the pathogenesis of nephrotoxic nephritis in the of a laboratory model resembling the spectrum of rat. J. exp. Med. 1956, 104, 151. human glomerulonephritis. J. exp. Med. 1961, 113, 29. Muller-Eberhard, H. J., U. Nilsson, and T. Aronsson. 899. Isolation and characterization of two 6,-glycopro- teins of human serum. J. exp. Med. 1960, 111, 201. 43. Feldman, J. D. Electron microscopy of serum sick- 30. Laki, K. The polymerization of proteins: the action ness nephritis. J. exp. Med. 1958, 108, 957. of thrombin on fibrinogen. Arch. Biochem. 1951, 44. Feldman, J. D. Pathogenesis of ultrastructural 32, 317. glomerular changes induced by immunologic means 31. Mills, D. A., R. Coyne, B. Pollara, and R. W. von in Immunopathology, Third International Sym- Korff. The action of plasmin on fibrinogen and posium, P. Grabar and P. Miescher, Eds. Basel, fibrin. I. Changes in the N-terminal residues. Schwabe, 1963, p. 263. Biochim. biophys. Acta (Amst.) 1964, 86, 527. 45. Dixon, F. J., J. J. Vazquez, W. 0. Weigle, and C. G. 32. Wannamaker, L. W. Electrophoretic studies of the Cochrane. Pathogenesis of serum sickness. Arch. extracellular products of group A streptococci. Path. 1958, 65, 18. J. exp. Med. 1958, 107, 783. 46. Germuth, F. G., Jr. A comparative histologic and 33. Marshall, J. D., W. C. Eveland, and C. W. Smith. immunologic study in rabbits of induced hyper- Superiority of fluorescein isothiocyanate (Riggs) sensitivity of the serum sickness type. J. exp. for fluorescent-antibody technic with a modifica- Med. 1953, 97, 257. tion of its application. Proc. Soc. exp. Biol. 47. Germuth, F. G., Jr., and G. E. McKinnon. Studies (N. Y.) 1958, 98, 898. on the biological properties of antigen-antibody 34. Riggs, J. L., R. J. Seiwald, J. H. Burckhalter, C. M. complexes. I. Anaphylactic shock induced by Downs, and T. G. Metcalf. Isothiocyanate com- soluble antigen-antibody complexes in unsensitized pounds as fluorescent labeling agents for immune normal guinea pigs. Bull. Johns Hopk. Hosp. serum. Amer. J. Path. 1958, 34, 1081. 1957, 101, 13. 35. Fothergill, J. E., and R. C. Nairn. Purification of 48. Andres, G. A., B. C. Seegal, K. C. Hsu, M. S. fluorescent protein conjugates: comparison of Rothenberg, and M. L. Chapeau. Electron micro- charcoal and "Sephadex." Nature (Lond.) 1961, scopic studies of experimental nephritis with ferritin- 192, 1073. conjugated antibody. J. exp. Med. 1963, 117, 691. 36. George, W., and K. W. Walton. Purification and concentration of dye-protein conjugates by gel fil- 49. Fish, A., A. F. Michael, R. L. Vernier, and R. A. tration. Nature (Lond.) 1961, 192, 1188. Good. Correlation of experimental serum sick- 37. Goldstein, G., I. S. Slizys, and M. W. Chase. Stud- ness nephritis with acute post-streptococcal glo- ies on fluorescent antibody staining. I. Non- merulonephritis in man, Seventeenth Annual Con- specific fluorescence with fluorescein-coupled sheep ference on the Kidney, J. Metcoff, Ed. Warrenton, antirabbit globulins. J. exp. Med. 1961, 114, 89. Va., 1965, in press.