Immune complex disease associated with spontaneous murine leukemia: Incidence and pathogenesis of glomerulonephritis

CLINICAL

IMMUNOLOGY

AND

IMhfUNOP.4THOLOC;Y

1,

6-14

(1972)

Immune Complex Disease Associated with Spontaneous Murine leukemia: Incidence and Pathogenesis of Glomerulonephritis’

Glomerulonephritis accompanies a large proportion of murine leukemia viral infections. AKR mice, which spontaneously developed leukemia after natural infection by Gross murine leukemia virus showed progressive manifestations of immune complex glomerulonephritis. Hence, by 9 months of age, 68% of these mice had histologic evidence of nephritis, whereas 89% had granular deposits of viral antigen, host immunoglobulin, and complement in their glomeruli. Subepithelial and subendothelial deposits were seen by electron microscopy. Similarly, nearly all mice of strains ordinarily not subject to renal disease developed immune complex glomerulonephritis after experimental infection with Raucher leukemia virus.

Gross murine leukemia virus (GMuLV) is the etiologic agent of spontaneous leukemias of AKR mice (l-3). Such mice may develop glomerulonephritis (4), but neither the incidence nor pathogenic mechanism(s) have been described. The present study was undertaken to investigate these parameters. Our results showed that nephritis in these mice was common and associated with deposition of viral antigen(s), antibody to virus coded antigens and complement (C) presumably in immune complex form in the glomeruli. MATERIALS

AND

METHODS

Animuls AKR mice of both sexes were obtained from Jackson Laboratories, Bar Harbor, Maine. Random testing indicated that the mice were free of lymphocytic choriomeningitis (LCM), lactic dehydrogenase (LDV), and polyoma viral infections. Clinical study consisted of determinations of body weight, urinary protein, hematocrit, white blood count and differential, and antinuclear antibody (ANA) at 3,6,9, and 12 months of age. Proteinuria was de1 This is publication no. 563 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California, and was supported by United States Public Health Service Grants Al-09484, Al-07007 and CA-10596, and Atomic Energy Commission Contract AT(04-3)-410. * Recipient of Career Development Award I K04 AI-42580 AID from the United States Public Health Service. 3 Recipient of Fellowship from the National Kidney Foundation. Copyright All rights

@ 1972 by Academic Press, Inc. of reproduction in any form reserved.

6

IMMUNE

COMPLEXES,

GLOMERULONEPHIUTIS

AND

LEUKEMIA

7

termined by the sulfosalicylic acid method (5) on 24-hr collections; values over 5 mg/24 hr were considered abnormal. Heparinized blood was obtained via the retro-orbital venous plexus; the method for obtaining ANA has been reported (6). RFM and BALB/c mice were infected with 1000 ID,, of Raucher leukemia virus (RLV) by Dr. Arthur Upton and Dr. II. L. Tyndall of the Biology Division of Oak Ridge National Laboratories, Oak Ridge, Tennessee. RFM mice were infected when 8-10 weeks old, and BALB/c mice when 6-days old. At sacrifice, RFM mice were 4-6-months old, and BALB/c mice were 1-3months old. Kidney tissues from these animals snap-frozen in liquid nitrogen and formalin-fixed in paraffin as well as tissues from noninfected control RFM and BALB/c mice were sent to our laboratory.

lmmuno~uorescent,

Histologic,

and Electron

Microscopic

Techniques

Tissue blocks were snap-frozen in liquid nitrogen, and sections 4 micron thick were cut, fixed in ether alcohol, and then examined by direct immunofluorescence for host IgG, C, and fibrinogen. Details as to handling of tissues, preparation of monospecific antisera, and its conjugation to fluorescein isothiocyanate (FITC) have been reported (7). Rat antiserum to Gross agent, kindly supplied by Dr. Wallace Rowe of the National Institute of Allergy and Infectious Disease, Bethesda, MD, and rabbit anti-rat IgG FITC were used in the indirect staining procedure. Both reagents were absorbed with mouse Ig. Such absorbed rabbit antiserum to rat IgG FITC did not stain mouse IgG deposited in a known positive tissue section. Procedure for determining both in tiiuo and in vitro fixation of guinea pig C3 in heterologous glomeruli has been published (8). Monospecific rabbit antiserum to guinea pig C3 was prepared according to the method of Linscott and Cochrane (9). In addition, kidney tissue from AKR mice was processed for electron microscopy. After fixation in glutaraldehyde and osmium tetraoxide, the tissue was dehydrated in alcohol, embedded in vestopal, sectioned, and stained. At least ten glomeruli from each mouse were examined, and a total of ten mice were studied. Formalin-fixed paraffin sections of tissues were stained by hematoxylin and eosin, and periodic acid-Schiff (PAS) methods.

IgG and Anti-Gross

Antibody

in the Kidneys

of Leukemic

Mice

Initially, the sediments from washed kidney homogenates were suspended in 0.02 M glycine-HCI buffer, pH 3.2,20 ml/g of kidney and incubated at 37°C for 60 min with constant shaking. After low-speed centrifugation, the supematants were removed, concentrated, and spun in a swinging bucket rotor at 105,500g for 2 hr. The upper half of fluid was carefully removed, and molarity and pH corrected to 0.15 M, pH 7. The glycine-HCI-treated sediments were then incubated with 3 M NaCl, 20 ml/g of kidney, at 37°C for 30 min and the resultant 105,500g supematant processed as above. Supematants obtained by both procedures were pooled and concentrated by lyophilization. Elutions were done on (a) pools of AKR kidneys having heavy glomerular deposits of IgG, (b) pools of AKR kidneys with no or minimal IgG deposits, (c) pools of

8

OLDSTONE

ET

AL.

Age of mice

Proteinuria (>5 mg/24 hr) Histologic nephritis Deposition of host Mortality ” Number

of mice

IgG

and C3

showing

positive

3

(( ; )

6

(‘F !

2138”

(5)

l/3,5

(1 1)

4122 (18) 12122 (54) o/39 (0) findings

18124 (75) 20/24 (83) 7138 118) over

total

number

(months) 9

(‘il

12

I’;:

10138 (26 )

1 ‘i/35

3 1145 (68) 40/45 (89) 111.38 (28)

20/24 (83) 24/24 iI 00) 15138 (39)

of mice

(483

observrtl.

kidneys from RFM adult mice infected with RLV, (d) pools of kidneys from BALB/c adult mice infected with RLV, (e) and (f) pools from control RFM and BALB/c mice. The amount of IgG eluted per kidney was quantitated by radioimmunodifliision employing antibody agar plates (10,ll). Renal eluates at a concentration of 200 pg IgG/ml were assayed for the presence of fluorescent binding and C-fixing antibodies to GMuLV antigens, antibody to Gross cell surface antigen (GCSA), ANA and anti-glomerular basement membrane antibodies by previously reported techniques (6,7,12). RESULTS

Incidence c?fr~ephritis. AKR mice were followed for 1 yr to study the development of glomerulonephritis. Table 1 indicates that by 3 months of age, 5% of these mice had proteinuria, and 18% histologic evidence of nephritis. By 9 months of age, 26% had proteinuria and 68% histologic evidence of nephritis, while at 1 yr of age 48% had proteinuria and 83% nephritis. Histologic study. Histologically, the earliest manifestations of renal disease were endothelial and mesangial cell proliferation. Later, various gradations of basement membrane thickening, intracapillary hyalinization, and occlusion occurred (Fig. 1A and B). There appeared to be no relationship between interstitial leukemic infiltrates in the kidney (Fig. 1C) and the development of nephritis. 11721)111)IOflual.~sL’e)lt study. In the kidneys, both IgG and C3 were seen deposited in a granular pattern in the glomeruli along the basement membranes of capillaries and in the mesangia (Fig. 2A and B) in over 80% of the 6 to 9 -month-old AKR mice. In 25% of the glomeruli examined there were corresponding deposits of mouse fibrinogen. These glomeruli had the heaviest deposits of host IgG and C3, and were histologically the most severely damFIG. month-old erating mouse month-old normal

1. Light AKR glomerular showing AKR glomeruli.

photomicrographs of renal glomeruli from AKR mice stained with PAS. (A) A 6mouse showing extensive glomerular injury with PAS positive deposits oblitcapillary loops associated with crescent formation: (B) g-month-old AKB extensive intravascular and mesangial deposits of PAS positive material; (C) 9mouse which despite interstitial infiltration of leukemic cells has relatively-

”

9

10

OLDSTONE

ET

AL.

FIG. 2. Fluorescent photomicrograph ofrenal glomeruli from AKR mice. The preparations wen’ stained with fluorescein-conjugated rabbit antiserum to mouse IgC. (A) A 9-montli-old AKR mouse with IgG deposited along the peripheral walls of glomerular capillaries anti in thr mesangial areas in a granular pattern. A similar pattern was observed when adjaceiit scctirjns were stained with rabbit antiserum to mouse C3 or rat antiserum to Gross viral antigens; (II) 6month-old AKR mouse with IgC deposited along the glomerular basement membranes alid xccumulated heavily in the mesangial area. Neither anti-glomerular basement membrane antibodies nor ANA were recovered from these kidneys, while antibodies to both GM&\ ant1 GCSA were.

aged. IgG and C3 deposits were limited to the glomeruli. In contrast, GMuLV antigens were observed in the cells of the convoluted tubules, connective tissue, arterial endothelium, and glomeruli. In the glomeruli, GMuLV antigens were found deposited in a granular pattern along the basement membranes of capillaries and in the mesangia.

IMMUNE

COMPLEXES,

GLOMERULONEPHRITIS

AND

11

LEUKEMIA

Nutzlre und concentration of IgG eluted from the kidney. The IgG eluted from AKR kidneys contained immunofluorescent binding and C-fixing antibodies to GMuLV antigens as well as antibodies to GCSA (12). Neither antibody was detected in the circulation by using any of the above assays. Conversely, ANA in low titer (highest dilution-i) was found in the plasmas of 14% of 6-12-month-old AKR mice but not in the renal eluates. Antiglomerular basement membrane antibody was not detected in renal eluates. The amount of IgG deposited in the glomerulus increased as the animals aged. Table 2 shows that 6.8, 18.2, and 40.4 pg IgG were deposited per AKR mouse kidney at 2-4, 5-8, and 9-12 months of age, respectively.

QUANTITY

OF IgC

ELUTED

TABLE 2 FROM THE KIDNEYS

Age Strain __AKR AKR AKR BALB/c BALB/c RFM RFM ” None

(months) 2-4 5-8 9-12 l-2 1-2 4-6 4-6

Leukemia GMuLV GMuLV GMuLV RLV not infected RLV not infected

OF

LEUKEMIC

MICE pg of IgG per mouse

eluted kidney

6.8 18.2 40.4 36.4 ND” 48.5 ND

detectable.

Time of uppeurunce of glomerulur bound 1gG. By direct immunofluorescence, kidneys from ten AKR male and female mice were studied monthly for the presence of mouse IgG. Faint mesangial deposition of IgG was found in the glomeruli of an occasional mouse at 4 weeks of age, while moderate glomerular deposits of IgG occurred in over 50% of mice by 3 months of age. By 9 months of age 89% of AKR mice showed both IgG and C3 deposits in the glomeruli with 70% of these having heavy mesangial and capillary wall deposits (Fig. 2A). Fixation of guineu pig C3 irz glomeruli of AKR mice. Using direct immunofluorescence, we detected guinea pig C3 in both mesangia and capillary glomerular walls of g-month-old AKR mice 6 hr after iv injection of 0.5 ml fresh guinea pig serum, yet injection of guinea pig serum decomplemented with zymosan did not result in C3 fixation. In vitro, guinea pig C3 fixed to AKR glomeruli. Electron microscopic study. Glomeruli from AKR mice showed varying degrees of injury. Nonhomogeneous, dense, subepithelial deposits were prominent in most of the glomeruli, while about $ of glomeruli had both subepithelial and subendothelial deposits (Fig. 3). Associated with the appearance of these deposits were the loss of foot processes of epithelial cells, and increase of mesangial cells.

12

OLUSTONE

ET

AL.

FIG. 3. Electron micrograph of a glomerular capillary wall from a Y-month-old AKK mouse. .A very large electron-dense deposit is visible on the endothelial aspect of the glomerular basement membrane. Smaller, more electron-dense deposits are visible on the epithelial aspect of the glomendar basement membrane (arrows). CL-capillary lumen; END-endothelial cell cytoplasm; DEP-deposit; BM-glomerular basement membrane: EP-epithelial cell cytoplasm.

Nearly all RFM (4-6 months old) or BALB/c (l-2 months old) mice infected with RLV had severe glomerulonephritis. There was, however, no correlation between the degree of nephritis and leukemic infiltrates in the kidney. In BALB/c and RFM mice infected with RLV both IgG and C3 were found in the mesangia and peripheral capillary walls in irregular deposits (Fig. 4). Histologic examination of tissue from 20 control BALB/c and 20 control RFM mice sacrificed at 5 months revealed no evidence of nephritis, although with immunofluorescence faint mesangial deposits of IgG and C3 were seen in three control BALB/c and five control RFM mice. From the kidneys of a BALB/c and RFM mouse infected with RLV, 36.4 and 48.5 ,ug IgG were eluted, respectively (Table 2). IgG was not detected in renal eluates from nonleukemia-infected mice of either strain. 111citro fixation of guinea pig C on tissue sections from both BALB/c and RFM mice infected with RLV occured.

IMMUNE

COMPLEXES.

GLOMERULONEPHRITIS

AND

LEUKEMIA

13

FIG. 4. Flourescent photomicrograph of a renal glomerulus from a 4-month-old RFM mouse infected with RLV. The preparation was stained with fluorescein-conjugated rabbit antiserum to mouse C3. Heavy C3 deposits in a granular pattern are seen along the glomerular basement membranes and in the mesangia. Similar deposits of IgG occurred when the adjacent section was stained with fluorescein-conjugated rabbit antiserum to mouse IgG.

DISCUSSION AKR mice naturally infected with GMuLV and BALB/c or RFM mice artificially infected with RLV developed a high incidence of glomerulonephritis. On the basis of immunofluorescent and immunologic study it would appear that trapping of virus-antibody complexes in glomerular capillary walls and mesangia was the pathogenic mechanism of these nephritides. In AKR mice it was shown that there were (1) GMuLV antigen(s) deposited in association with host IgG and C3 along the glomerular basement membrane in a granular pattern, (2) specific concentrations of antibodies to GMuLV or GCSA in the injured glomeruli, (3) fixation of C to the injured glomeruli in oitro and in ~iuo, and (4) dense deposits in the glomeruli seen by electron microscopy. Although leukemia viruses appear essential to development of nephritis, the viruses themselves are not primarily injurious to the glomerulus. Hence, this is not a true “viral nephritis” but rather an immune complex nephritis similar to that developing in man with serum sickness (13,14), and lupus erythematosus (15,16), or in animals with persistent LCM (7), LDV (17,18) and Moloney sarcoma infections (19), in Aleutian disease of mink (20,21), and in equine infectious anemia (22). Previously Recher et al. (4) postulated that

14

OLDSTONE

ET

AL.

the glomerulonephritis of AKR mice was caused by either a direct viral nephritogenic effect or an autoimmune reaction secondary to alteration of glomerular tissue. From the present study it is apparent that neither pathogenic mechanism occurred. Despite the persistence of leukemia virus, a host anti-viral immune response occurred, and interaction of virus with the products of this immune response led to progressive glomerular tissue injue. Upon recovery of antibodies to both GMuLV and GCSA from the injured kidneys of AKR mice the Gross virus was identifed as an etiologic immunogen in the associated nephritis. Whether other etiologic agents are involved in this immune complex disease is not known, although the absence of both ANA and anti-glomerular basement membrane antibodies in the diseased kidney excludes at least these antigens as playing a significant role. While elution studies have several technical limitations, they nevertheless provide the only direct identification of antibodies actually present in diseased tissue. However, until the concentration of antibodies to GMuLV and GCSA in the total IgG eluate is known, the possibility of additional etiologic agents exists. Finally, glomeruli may be a promising source of antibodies in those diseases suspected of arising from persistent infection in which the infectious agent circulates and elicits a low antibody response. ACKNOWLEDGMENTS The authors tissues.

thank

Dr. Tyndall

and Dr.

Upton

for supplying

RLV-infected

REFERENCES 1. OLD.

I,. J,, BOYS&

2. WAHHEN, 3. GEEHIM:,

B.. Nntvw (:., OLD.

E. A., AXI)

STOCKEHT,

(Lw&l~) L. J,. ANI)

205, 409, BouSE,

E., C~III~I.

!?es.

E. A., .I. EF:z/‘. .\fc,t/.

4. RECHER, I,.. TANAKA, T., SYKES, J,. Yuiw~o, T., &MAN, Xc/f. CU~ICW Itut. Mowgr. 22, 459, 1966. 5. KINCSBUHT, F., CLMK;, C.. WILLIAMS, C:.. ANI) Pas-I-. 6. LAMBERT,

P.. AND

7. OLDSTONE,

DIXON,

F. J., J. I:‘x)J.

Merl.

127,

41. B. .4., AND E.. ,\ND DIXON,

9. 10.

LINSCOTT. MANCINI,

W.. MD COCHRANE, C.. /. I~?~??lrrrwl. c;., CAHBONAHA. A.. AND HEHEMAAX

11. 12.

FAHEY, J., .&ND OLDSTONE, hl.

13.

DIXON, New DIXON,

14. 15. 16.

PORTER, HIRSCII, PORTER, HENSON, BANKS,

E., 1. Inlwrrrtol. T., AND Drxo;\r,

F. J., 111 “Harvey Lectures, 1962-1963” York, 1963. F. J., Amer. J, Med. 44, 493, 1968.

KOFFLER, KHISHNAN. 17. OLDSTONE,

18. 19. 20. 21. 22.

MCKELVEY, B. A., AoKI,

813,

1965.

507,

753,

124,

1966.

C.. Yowl,

I,.. AXI)

A.. ,I. /,trh. 1968.

(‘lirt.

DIXON, F. J., J. Es/). Med. 129, 483, F. J., J. Es!,. M&. 119, 965, 1964.

UNANUE.

8.

25.

1965.

H., J. Iwr,trrr~o/. AND HARVEY. J,AND PORTER, H.. PADGETT, G.. AXD J., Fed. Pwc,. 28,

MM/.

11, Ml,

93, 972, 1964. J.? I,nr,rrr1lo(,ltr,r2i,\.l~!l

2, 235,

Nufurc

1264,

1967.

1971.

(Lodm)

./. E’:s~I. Men. DAVIS. W.. 752, 1969.

223, 739, 130, .I&.

1926.

1965.

94, 84, 1965. F. J., PM,. Sot. ;htl. Qi. I’. S. 69, 134, Academic (R. D. All en. Ed.), pp. 31-52,

106,

I .

1969.

D.. SCMJR, P., .&ND KUNKEL. H. C., J. Ex~J, Met/. 126, 607, C., ANT) %&PLAN, M., J. Clirr. Ztlcwf. 46, 569, 1967. ICI. B. A., -\ND DIXXX, F. J,, 1. Z,rtrt,rrtro/. 106, 1260, lQ71. D., ASD POHTER. hl., ALLZON, A., D., LARSEN. A., J., COHHAM. J., K., AND HENSON,

DI\IM:HO\VS~I.

1969. 575, 1969. P&M. 87, 21,

1969.

1972. Press,