Mixed IgG-IgM cryoglobulinemia with glomerulonephritis

Mixed IgG-IgM Cryoglobulinemia with Glomerulonephritis Immunochemical,

Fluorescent

and Ultrastructural

Study of Kidney and

In Vitro Cryoprecipitate

DANIEL CORDONNIER,M.D. HUGUETTEMARTIN, M.D. PAULE GROSLAMBERT, M.D. CLAUDE MICOUIN, M.D. Grenoble, France

FRANCOISE CHENAIS, M.D.’ San Francisco, California

PIERRE STOEBNER, M.D. Grenoble. France

From the Service de Reanimation et Nephrologie and the following laboratories: Anatomie Pathologique, Biochimie Pathologique, Immunopathologie, Centre de Transfusion Sanguine; Microscopie Electronique, Centre Hospitalier Universitaire et Faculte de Medecine de Grenoble, La Tronche, France. This work was supported in part by a grant from the lnstitut National de La Sante et de La Recherche Medicale (ATP 72 3 466 10). This report was presented in part at the Fiih International Congress of Nephrology. Mexico City, October 12, 1972. Requests for reprints should be addressed to Dr. Cordonnier, Service de Reanimation et Ntiphrologie. Centre Hospltalier Universfteire de Grenoble, 38700 La Tronche. France. Manuscript accepted March 28, 1975. l Present address: Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, 80 Barre Street, Charleston. South Carolina 29407.

Comparative studies of renal blopsy specimens and in vitro cryoprecipitate were carried out in a patient with mixed immunoglobulin G (IgG)-immunoglobulin M (IgM) cryogiobulinemia associated with glomerulonephritis. The IgM isolated from the cryoprecipitate was an antlbody with anti-IgG activity. Proliferative endocapillary glomerulonephritis was found in the kidney, with large amorphous deposits in the capillary walls. On immunofluorescent examination, these deposits contained IgG and C3. Ultrastructural studies of both cryoprecipltate and glomerular deposits revealed unusual structures designated as “cylindrical or annular bodies.” The morphologic characteristics of these bodies were exactly the same in the kidney and in the cryoprecipitate. These findings suggest an identity between the glomerular deposits and the circulating cryoglobulln, supporting the hypothesis that the glomerulonephritis reported here Is an immune-complex disease. Study of mixed essential immunoglobulin G (IgG)-immunoglobulin M (IgM) cryoglobulinemia is clinically useful because in this disorder there is an abundance of cryoprecipitate that can be separated into its constituents, and because the possible association of mixed cryoglobulinemia with glomerulonephritis strongly suggests a disease similar to experimental serum sickness. We studied by immunochemical, immunofluorescence and electron microscopic methods a patient with mixed essential cryoglobulinemia associated with glomerulonephritis. We found unusual deposits, formed of cylindrical or annular bodies, in the glomerular walls; the same bodies appeared in the cryoprecipitate studied in vitro. This suggests, as proposed by Melzer et al. [I], that the mixed cryoglobulins may be immune complexes that are deposited in the glomeruli and that induce glomerulonephritis. If confirmed, this finding may allow a new approach to the pathophysiology of glomerulonephritis. CASE REPORT The patient, a white woman born in 1906, had a family history including three relatives with coronary disease. She had had vertebral degenerative arthritis since 1960. In 1968 arterial hypertension developed; shortly thereafter, mild proteinuria was found, always less than 1 g/24 hours and with no abnormality of the urinary sediment. An intravenous urogram appeared normal and her creatinine clearance was 80 ml/min. Arteriolar ne-

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phrosclerosis was diagnosed and, since 1968, she has received alpha methyl dopa to control her hypertension. In April 1971, arthritis of the joints of the fingers developed. A few days later, she complained of violent intrathoracic pain, but there were no signs of coronary insufficiency and the pain disappeared spontaneously. Her blood pressure was 2501120 mm Hg; her proteinuria had increased to 17 g/24 hours. Six weeks later, she was hospitalized in the Service de N&phrologie du Centre Hospitalier Universitaire de Grenoble with edema of the ankles and slight back pain. Her blood pressure was 3101170 mm Hg, but her cardiorespiratory system appeared to be undamaged. There was no splenomegaly or palpable lymph nodes, and she had no fever. Urinary protein content was 17 g/24 hours, hematuria 30,00O/min (Addis count), creatinine clearance 35 ml/ min, erythrocytes 4,000,000/mm3, serum proteins 6.3 g/100 ml, with 43 per cent albumin and 3 per cent alpha* globulin: IgA 0.10 g/100 ml (normal, 0.22 f 0.08 g/100 ml); IgG 0.18 g/100 ml (normal, 1.17 f 0.27 g/100 ml); IgM 1.7 g/100 ml (normal, 0.12 f 0.05 g/100 ml); and C3 0.05 g/100 ml (normal, 0.14 f 0.02 g/100 ml). There was abundant mixed IgGlgM cryoglobulinemia. A latex test was positive (3-t) as was a Waaler-Rose test at 1:1,024. Two myelograms were obtAined; there were signs of inflammation, but no lymphocytic abnormalities. Her skeletal films showed no abnormalities other than her arthritis. A renal biopsy was performed. She received furosemide (80 mg/day), clonidine (450 mg/day) and chlorambucil (10 mg/day). In October 1971, her proteinuria was 0.17 g/24 hours and hematuria had disappeared; the cryoglobulinemia and hypocomplementemia were still present, but the blood immunoglobulins were back to normal. Chlorambucil therapy was discontinued in February 1972 because of medullary hypoplasia. In July 1972, her blood pressure was 150190 mm Hg; there had been no proteinuria for six months; complement was 0.04 g/100 ml; and cryoglobulin was still present. A second biopsy was performed. Routine follow-up examinations since that time have revealed no change. At no time did the patient have any symptoms while exposed to the cold. MATERIALS

AND METHODS

Isolation and Identification of Cryoglobulln. Blood drawn with a warmed syringe was allowed to clot at 37’C for 1 to 2 hours. Serum was kept at 4’C for 48 hours, then centrifuged at 3,000 rpm for 30 minutes. The supernatant was removed, and the cryoprecipitate was washed in normal saline solution and dissolved in a glycine-hydrochloride buffer, 0.1 M. pH 3.7. Fractions were separated at 10°C in a Sephadexm G 200 superfine column in phosphate-sodium chloride buffer at 0.05 M and 0.01 M, pH 7.5. ldentification of fractions was made by immunoelectrophoresis, and quantitation by radial /mmunodiffusion, at 4OC. Search for Immunologic Actlvlty In the Products of Dlssoclatlon of the Complex. Two trials were made: search for ihe autologous cryoprecipitate phenomenon in capillary tubes incubated at 37’C and 4’C, control tubes being obtained with normal IgG and IgM, and search for antigamma

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Part of a glomerular fiocculus, little modified by the endocapillary cellular proliferation. Two homogeneous nodular deposits thicken the capillary loops (arrows). Periodic aci&Schiff stain; original magnification X 500, reduced by 2 7 per cent.

globulin activity by passive agglutination of latex particles sensitized with human IgG (Institut Pasteur, Paris, France) oi rabbit IgG (Burroughs Wellcome Products, Research Triangle Park, North Carolina). Morphologic Study. For light microscopy, a fragment of tissue obtained during renal biopsy was fixed in Bouin’s solution, cut at 4 k and stained by standard histologic methods. For immunofluorescence, a specimen was placed in OCT Compound@ (Ames Company, Division, Miles Laboratories, Inc., Elkhart, Indiana) and frozen in liquid nitrogen. It was cut at 3 to 5 ~1in a cryostat at -2O’C. The sections were fixed in acetone and stained with fluorescein-labeled antiserums directed against human IgA, IgG, IgM, C3 and fibrinogen (Hyland Laboratories, Division of Travenol Laboratories, Inc., Costa Mesa, California). Ultrastructure. A fragment of the renal tissue obtained at the time of the first biopsy was cut into small cubes, fixed at 4’C in 3.8 per cent glutaraldehyde at pH 7, and postfixed in Palade’s liquid. After alcoholic dehydration, it was immersed in Araldite@ and Epon@. Half-thin-sections were stained with toluidine blue. Ultrathin sections were made on a Reichert OMU microtome and examined with a Philips EM 300 microscope. Fixing solutions were added to cryoprecipitates as described to determine the effect, if any, of fixation on the morphologic appearance of the cryoprecipitates. The following combinations were examined: 3.8 per cent glutaraldehyde with postfixation in Palade’s liquid, glutaraldehyde only, paraformaldehyde and osmic acid. Five cryoprecipitates, obtained at different times, were so treated. Other Studles. Enzymatic digestions using the technics of Monneron and Bernhard [2] were attempted on ultrathin sections. No positive results were obtained. RESULTS Immunochemistry of Cryoprecipitates. The immunochemical studies revealed the presence of IgG and

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Capillary loop modified by proliferation of mesangial cells (M), by deposits of membranoid material (d) and by denser irregular deposits (arrows) which may be nodular (D) and can be considered homologous to the homogeneous deposits of Figure 1. All these deposits are situated inside the lamina densa. Original magnification X 26,700, reduced by 24 per cent.

Figure 3.

Figure 2. Marble-like glomerular fluorescence serum to IgG. Magnification X 320.

with anti-

IgM with a two-zone morphology for IgM. IgA and C3 were not seen. We did not search for fibrinogen or for fibrin degradation products. Chromatography allowed the isolation of IgM in fraction I and IgG in fraction II (confirmed by immunochemical identification). The IgM in the cryoprecipitate had antibody activity directed against the IgG. If the two fractions were kept separately at 4’C there was no change in the tubes after 48 hours. A mixture of the two, kept at the same temperature, revealed a distinct precipitate after a few hours. Stored at 40°C, the mixture showed no change after 48 hours. Fraction I (IgM) very clearly agglutinated latex particles sensitized with IgG. The agglutination reaction was positive at l:l,OOO with human IgG, and at 1500 with rabbit IgG. Fraction II (IgG) did not agglutinate the same particles. Antibodies to human IgG were detected at levels up to 1:40,000 in whole serum heated at 4OOC; levels of 1:2,000 were detected in the cryoprecipitate after washing and mixing at 40°C in a volume of phosphate-buffered saline solution equal to the original volume. In cryoprecipitate-free serum heated at 40°C, antibodies were detected at a dilution of 1: 20,000.

Light Microscopy and lmmunofluorescence Studies of Renal Biopsy Specimens. Light microscopic study of the biopsy specimens taken in June 197 1 suggested proliferative endocapillary glomerulonephritis. The walls of some capillary loops were thickened by large, amorphous, homogeneous deposits that were eosinophilic, periodic acid-Schiff positive and nonmetachromatic with toluidine blue (Figure 1).

One year later, proliferative endocapillary glomerulonephritis was still seen, but the large deposits were no longer visible. The deposits contained IgG and C3. In specimens from both biopsies, the anti-IgG and anti-C3 serums gave a positive granular fluorescence along the glomerular capillary walls. The deposits also glowed (Figure 2). There was no fluorescence with antiserum to IgM.

Electron

Microscopy

of Renal Biopsy Specimen.

Several findings were unimportant from a clinical standpoint: foot processes were slightly spread out, and some capillary walls were thickened by a membranoid material that occupied the lamina densa of the subendothelial region (Figure 3). The same membranoid material was found in the intercapillary areas, and was sometimes responsible for stenosis of the capillary lumen. Some of the deposits on the capillary walls consisted of unusual structures which, depending on the orientation of the microscopic section, could be described as “cylindrical or annular bodies.” The cylindrical bodies (Figure 4) appeared as clear spaces limited by two straight parallel membrane-like walls no more than 20 nm apart. The outer side of each wall was covered by a membranoid curd-like material 10 to 25 nm in thickness. The minimal length of these cylinders was 100 nm; the maximum was 1 CL. The distance between adjacent structures was rarely less than 40 nm. The annular bodies (Figures 4 and 5) appeared to be cross sections of the cylindrical structures. They

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Figure 5.

Structure of deposits of “annular or cylindrical bodies” in the kidney. Here the annular bodies are few in number and are in contact with the endothelial cell inside the lamina densa. They are well defined (arrows): central clear zone, dense median wall, irregularly condensed membranoid covering. Original magnification X 84,000, reduced by 24 per cent.

Electron Microscopy of Cryoprecipitates.

Figure 4. Structure of deposits of “‘annular or cylindrical bodies” in the kidney. The irregular dense deposits are made up of annular structures (annular bodies) in transverse sections and cylindrical structures (cylindrical bodies) in longitudinal sections (arrows). Magnification X 40,000.

had an inner clear central area 18 to 20 nm in diameter, limited by an external. curd-like membranoid material about 20 nm in width. Five to eight globular condensations were seen in this material, oblique or perpendicular to the central ring. The total diameter of the annular bodies was about 60 nm. The membranoid covering was often poorly organized, and it was not always possible to see these structures clearly. However, the annular bodies were always independent one from the other. When not numerous, the cyclindrical or annular bodies were seen on the endocapillary side in intimate contact with the endothelial cells. They were sometimes loose, but more often these bodies were conglomerated. They were always separated from the foot processes by intact, unthickened basal material. They lay close to the endocapillary cells and seemed occasionally to enter in contact with their membranes. Twice we observed the cylindrical or annular bodies in polymorphonuclear neutrophils. As far as could be judged on the limited material studied, only the glomeruli contained these cylindrical or annular bodies. They were never seen on the lamina rara externa or in the interstitial vessels.

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Whatever fixation technic was used, and whenever the cryoprecipitate was obtained, there was no variation in its morphologic appearance: it was constituted of cylindrical or annular bodies, identical to those found in the glomeruli. The central clear area did not exceed 20 nm in diameter, and the total diameter was about 75 nm. The width of the wall varied in accordance to its structure, which appeared to be complex on certain pictures. This applied also to the membranoid covering. However, there was clearly morphologic identity between

the elements

pitate and the elements

of the cryopreci-

of the capillary walls (Figure

6). COMMENTS Glomerulonephritis associated with mixed IgG-IgM cryoglobulinemia is now a recognized fact. MorelMaroger and associates [3,4] located 12 cases in the literature, and added 10 personal cases. Brouet et al. [6] found its incidence to be 35 per cent in 19 patients with mixed IgG-IgM cryoglobulinemia. Others have found cylindrical or annular bodies in tissues and in cryoprecipitates [6,7]; nevertheless, they represent an exceptionally rare finding. To our knowledge, only two such observations have been made: in the kidney of a patient with lupus glomerulopathy [6] and in the lymph nodes and cryoprecipitates of a patient with Waldenstrom’s macroglobulinemia [ 71, In the first instance, cryoglobulins were not sought for; in the second, the kidney was not examined despite the presence of nephropathy.

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ies (arrows) are seen similar to ihose.in glomeruli. Magnification X 59,400.

Cylindrical or annular bodies appear to differ from other entities described in the literature. For example, the spherical microparticles described by Burkholder et al. [8] were smaller in size (500 to 580 A),and had a dense core with a trilaminar outer limiting membrane. The bodies we found do not resemble viroid inclusion bodies such as those observed in systemic lupus erythematosus [ 9, lo]. These inclusion bodies are tubular, but curving and interwoven. They measure 20 to 25 nm in diameter and are always situated in the ergastoplasm of cells, especially endothelial cells. Further, their simple membrane limit differs from the complex structure of the bodies described. The existence of similar observations by others and the consistent presence of identical structures in cryoprecipitates obtained at various times excludes, from our point of view, the possibility of an artifact. Metallic and mineral precipitates, for example, do not display the characteristics described here. The structures obtained after cooling the patient’s serum, observed under the electron microscope, ap-

ET AL.

pear completely identical to those found in the capillary walls. Each new cryoprecipitate obtained from this patient had these structures, and no others. Because the immunochemical analysis of the cryoprecipitates showed only two immunoglobulins, IgG and IgM, we believe it reasonable to conclude that these two immunoglobulins correspond to the cylindrical and annular bodies seen in the cryoprecipitates. It is true that the immunoglobulins described by Almeida et al. [ 111, Valentine and Green [ 121, Chesebro et al. [13] and Bogaars et al. [14] did not look like the cylindrical or annular bodies seen by us: however, these studies were made on isolated human or animal immunoglobulins. It is obvious that the morphologic similarity between the renal specimens and those from cryoprecipitates cannot allow us to reach a firm conclusion about their qualitative similarity. In order to support such a conclusion, it is necessary to demonstrate that the antigen and antibody in the kidney are strictly the same as those in the serum. However, in the absence of eluates we could not study and compare the detailed chemical structure of the two IgG’s. As IgM could not be detected in the kidney, we can also imagine that the antibody from the two sources is not exactly the same. Elution studies of the renal biopsy specimen might have determined whether IgM was the antibody but was engulfed by IgG, or if the antibody was not IgM. The evidence summarized suggests the probability that this patient has an “immune complex disease.” The fact that C3 remained fixed on the glomerular capillaries in a granular manner, and that its level in serum remained very low throughout these chemical studies, seems to support this hypothesis. ACKNOWLEDGMENT We are indebted to Professor Andre Cruchaud (Geneva, Switzerland) for helpful discussion of the manuscript. We gratefully acknowledge the technical assistance of Mrs. M. C. Barioz, C. Dussert, S. Lossardo and C. Robert.

REFERENCES 1. Meitzer M, Franklin EC, Elias K, McCluskeyRT, Cooper N:

2. 3.

Cryoglobulinemia-A clinical and laboratory study. II. Cryloglobulins with rheumatoid factor actlvll. Am J Med 40: 837, 1966. Monneron A, Bernhard W: Action de certaines enzymes sur des tissus inclus en epon. J Microscopic 5: 697. 1966. Verroust P, M&y J-P, Morel-Maroger L, Cfauvel J-P, Richet G Glomerular lesions in monocfonal gammopathfes and mixed essential cryoglobulinemias IgGIgM. Adv Nephrol 1: 161. 1971.

4.

Morel-MarogerL, M&y J-Ph: Renal lesionsin mixed IgG-lgM

5.

essential cryoglobulinemia. Proceedings of the Fifth International Congress of Nephrology, Mexico, 1972. Vol 1. Morphology and Pathology (Villarreal H, ed), Basel. S. Karger, 1974, p 173. Brouet J-C, Clauvel J-P, Danon F. Klein M, Seiiimann M: Biologic and clinical significance of cryogfobulins: a report of 88 cases. Am J Med 57: 775. 1974. Fresco R: Virus-like particles in systemic lupus erythematosus. N Engl J Med 283: 1231, 1970.

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7.

8.

9.

10.

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Cruchaud A, Wigren S, Nicod I, Vassali P: Monoclonal IgM globulin with anti IgG activity: an immunofluorescent and electron microscopic study. Protides of the Biological Fluids, 20th Colloquium, Bruges, Belgium, 1972. Proteins and Related Subjects, vol 20 (Peeters H, ed), New York, Pergamon Press, 1973, p 39. Burkholder PM, Hyman LB, Barber TA: Extracellular clusters of spherical microparticies in glomeruli in human renal glomerular diseases. Lab Invest 28: 416, 1973. Gyorkey F, Min K-W. Sincovics JO. Gyiorkey P: Systemic lupus erythematosus and myxovirus. N Engl J Med 28: 333, 1989. Norton WL: Endotheiiil inclusions in active lesions of sys-

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ET AL.

11. 12.

13.’

14.

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temic lupus erythematosus. J Lab Clin Med 74: 369. 1989. Almeida JD, Brown F, Waterson AP: The morphologic characteristics of 19s antibody. J lmmuno198: 186, 1967. Valentine RC. Green NM: Electron microscopy of an antibody-hapten complex [letter to the editor]. J Molec Biol 27: 615, 1967. Chesebro B, Bloth B. Svehag S-E: The ultrastructure of normal and pathological IgM immunoglobulins. J Exp Med 127: 399. 1968. Bogaars HA, Kakleron AE, Cummings FJ, Kaplan S, Melnicoff I, Park C, Diamond I, Calabresi P: Human IgG cryoglobulin with tubular crystal structure. Nature (New Biol) 245: 117, 1973.