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Subclinical immune complexes in the glomeruli of kidneys postmortem
Subclinical Immune Complexes in the Glomeruli of Kidneys Postmortem
JOHN C. SUTHERLAND,
M.D.*
ROY VANN MARKHAM,
Jr., M.D.7
MICHAEL R. MARDINEY,
Jr., M.D.
Baltimore, Maryland
From the Section of Immunology and Cell Biology, National Institutes of Health, National Cancer Institute, Baltimore Cancer Research Center, 3100 Wyman Park Drive, Baltimore, Maryland 21211 and from the Department of Pathology, United States Public Health Service Hospital, 3100 Wyman Park Drive, Baltimore, Maryland 2 12 11. Requests for reprints should be addressed to Dr. John C. Sutherland. Manuscript accepted January 17, 1974. Present address: University of Maryland, School of Medicine, Department of Pathology, B-1135 No. Hosp., 22 South Greene Street, Baltimore, Maryland 2 120 1. t Present address: Bavlor Universitv School . of Medicine, Houston, Texas 75246. l
536
October 1974
In a postmortem study of 303 kidneys examined by immunofluorescence, glomerular immune complex deposition was found in the glomeruli of 21. The incidence of immune complexes was highest in kidneys from patients with acute leukemta (14.3 per cent) and lowest in kidneys from patients with solid tumors (1.6 per cent). Immune complexes have also been found in kidneys from patients without tumors (11 per cent). The patients exhibited no clinical evidence of glomerulonephritis, and the kidneys were essentially normal by light microscopy although electrondense subendothelial deposits were seen in four of eight. Although previous studies have identified a mammalian oncornaviral antigen in the complexes of two kidneys, both from patients with acute leukemia, the identity of most of the antigens remains unknown. The detection and analyses of these complexes provide an important means of detecting host response to known and unknown antigens that are not now possible by routine serologic methods. The association of various bacterial [l-3], parasitic [4] and viral [5-81 infections with overt immune complex nephritis in man and animals has been well documented. The study in these situations of glomerular bound antigens and antibodies by staining kidney sections with specific antiserums or by elution of antibody from whole kidney homogenates has been useful in identifying the various antigens involved in these complexes. In addition to identification of suspected antigens, analysis of immune complexes in some situations has revealed host immune responsiveness when it was not expected. Thus, viral antigen and antibody complexes bound to glomerular basement membrane have been found in mice with lymphocytic choriomeningitis [9] and in AKR mice with spontaneous leukemia [ 10,l I], animals previously considered tolerant to virus. Because of the potential suggested by the latter two models of murine disease, we explored the possibility that subclinical immune complex disease could be present in the patient with neoplasia. Specifically, we examined kidneys obtained at autopsy by immunofluorescence to determine if immune complex deposition exists in the absence of clinical or histologic evidence of glomerulonephritis in man. A study of the kidneys of 303 patients obtained at autopsy revealed the presence of subclinical immune complex deposition in 2 1. On analysis of the complexes in the kidneys from two patients with acute leukemia, one antigen was found to be identical with or
The American Journal of Medicine
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SUBCLINICAL IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
related to the interspecies antigen of the mammalian oncornaviruses [ 121. We describe the immunofluorescence and light microscopic findings in these 21 kidneys and the ultrastructural findings in 8. Pertinent clinical details are
TABLE I
Percentages of Kidneys with Immune Complexes by Clinical Groups
Positivefor Kidneys Examined
discussed. Clinical Group
MATERIALS
AND METHODS
Kidneys were obtained postmortem from 303 patients. Half of these (146) were from patients with lymphoma-leukemia, 124 from patients with solid tumors and 33 from patients without tumors. The clinical classification of the patients and the numbers in each are given in Table I. The time after death before collection varied from 2 l/2 hours to 2 days. Blocks of cortical tissue were snap-frozen in an acetone-dry ice slurry and stored at -70°C. Cryostat-cut sections of renal cortex containing glomeruli were fixed in absolute acetone for 10 minutes, washed for 3 minutes and 10 minutes in phosphate-buffered saline solution at pH 7.2 and stained directly with fluorescein-labelled antihuman gamma globulin (immunoglobulins G (IgG), A (IgA) and M (IgM)) for 45 minutes at room temperature. Those positive for gamma globulin were further stained for human complement (C’3) and human fibrinogen. Sections were also stained with fluoresceinated antihuman albumin as a negative control. The commercial sources of these antiserums have been detailed [ 121. All antiserums were initially tested by immunoelectrophoresis for strength and specificity. Slides were evaluated with a Leitz microscope utilizing an Osram HB 200 light source and a UG 1 excitor filter in combination with a K460 barrier filter. For purposes of photography, either a BG 12 or KP 490 excitor filter in combination with a K 530 barrier filter was used. Positive fluorescence was recorded according to (1) distribution, whether capillary, mesangial or a combination of both; (2) pattern, whether linear or irregular; and (3) extent, whether all loops of all glomeruli were involved or a fraction thereof. For light microscopy formalin-fixed tissue was stained with hematoxylin and eosin, methenamine silver and by the Jones allochrome and periodic acid-Schiff methods. For electron microscopy tissue was fixed in glutaraldehyde, postfixed in osmium and stained with lead citrate. We examined tissue from three kidneys processed in this manner. Glutaraldehyde-fixed material from another five kidneys was further processed and examined by Dr. Ferman Tio of Georgetown University Hospital. Laboratory records of patients were examined for evidence of glomerulonephritis. It was also determined from the autopsy records whether the patients exhibited either clinical or histologic evidence of terminal viral infections.
ET AL.
Acute leukemia Chronic leukemia Lymphomas Solid tumors No tumor Total --I_
(no.> 83 9 54 124 33 303 _~
Positive for Both Gamma Globulin and Complement __.-. _ ~___ No. % 12 1 2 2 4 21
14.3 11 3.5 1.6 11 6.9
Gamma G~l~~~” (no.) 2 1 1 4 5 12
Figure 1. Fluorescence photomicrograph of a giomozlus stained for gamma globulin. Lumpy deposits may be seen chiefly along capillaries. This kidney was from a 21 year old man with acute myelogenous leukemia (Case 1, Table II). Protein and red blood cells were not detected in his urine by repeated single-specimen examinations; original magnification X 300, reduced by 15 per cent.
RESULTS Twenty-one kidneys stained positively for both gamma globulin (Figure 1) and complement (Figure 2) and an additional 12 stained positively for gamma
globulin alone. These positivities are grouped according to five broad clinical categories in Table I and are
Figure 2. C’3 in a glomerulus from the same kidney shown in Figure 1. The distribution of positivity is similar to that of gamma globulin. Original magnification X 750, reduced by 15 per cent
October 1974
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SUBCLINICAL
IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
TABLE II
Distribution
and Percentage
Involvement
ET AL.
of lmmunofluorescent
Positivities
GammaGlobulin Diagnosis
Case No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
NOTE: C = capillary;
C&M = capillary
Fibrinogen
Extent
Distribution
Extent
Distribution
C C C C C C C C C
1+ 4-F 1+ I+ 1+ 2+ 4+ 1+ 2+
2+ 4+ 1+ 1+ 2+ 2+ 4+ 1+ 1+
C C C C C C
2+ 3+ 1+ 2+ 4+ 1+ 0 0 0
C&M C C C C C C C C C C C
4+ 3+ 1+ 2+ 2+ 4+ 2+ 1+ 1+ 4+ 2+ 2+
C C C C C C C C C C&M C C C C C C C C C C C
4+ 2+ 1+ 1+ 2+ 4+ 2+ 2+ 3+ 2+ 2+ 2+
C C
4+ 4+ 0
C C C C
4+ 2+ 4+ 3+ 0
C
4+ 0
C C
1+ 1+
Distribution
Acute myelocytic leukemia Hodgkin’s disease Acute myelomonocytic leukemia Myocardial infarct Arteriosclerotic cardiovascular disease Acute myelocytic leukemia Acute myelomonocytic leukemia Acute myelocytic leukemia Cirrhosis Acute lymphocytic leukemia Acute myelocytic leukemia Carcinoma of esophagus Acute myelomonocytic leukemia Myeloid metaplasia Acute myelocytic leukemia Chronic myelocytic leukemia Acute myelomonocytic leukemia Carcinoma of colon Hodgkin’s disease Acute myelocytic leukemia Acute myelocytic leukemia
Complement
Extent
and mesangial.
listed according to individual diagnoses in Table II. The incidence of positivities (both gamma globulin and complement) was highest in kidneys from patients with acute leukemia (12 of 83 or 14.3 per cent), and lowest in kidneys from patients with solid tumors (2 of 124 or 1.6 per cent). Most positivities were capillary in distribution (Table II). Oily one kidney (Case 10, Table II) exhibited a mixed capillary-mesangial distribution. The pattern of positivity in each kidney was irregular or segmental. The extent of positivity was determined by calculating the average percentage involvement of
each of these proteins, however, II). In 14 kidneys the positivities
Figure 3. Two glomeruli stained with hematoxylin and eosin to show an essentially normal cellularity (Case 1, Table Ii). Original magnification X 120, reduced by 20 per cent.
Figure 4. Glomerulus stained by the Jones allochrome technic to show normal capillary loops. There is possibly a slight increase in mesangial matrix (Case 1, Table II). Original magnification X 300, reduced by 20 per cent.
538
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The American Journal of Medicine
each glomerulus in a section and is expressed on a scale of from 1-F to 4+. Thus 4-I indicates positive fluorescence of all lobules in all glomeruli whereas 1-F indicates involvement of 25 per cent or less of all lobules. In the latter instance, the involvement might vary considerably in extent from glomerulus to glomerulus and 1-I represents an average. Usually the distribution and pattern of positivities in a given kidney were similar for gamma globulin, complement and fibrinogen. The extent of positivity for
Volume 57
often varied (Table for gamma globulin
SUBCLINICAL IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
were the same as those for complement, in 4 they were greater and in 3 they were less. Fibrinogen equaled in extent the positivities for gamma globulin and complement in five kidneys, exceeded them in six, was less in four and could not be detected in six. Kidneys examined by light microscopy were evaluated for increased cellularity and basement membrane and mesangial thickening. Except for an occasional thickened capillary loop or mesangial stalk, all were essentially normal (Figures 3 and 4). Eight kidneys were examined by electron microscopy. Electron-dense, finely granular, subendothelial deposits were found in the glomeruli of four (Figure 5) and intramembranous deposits in one. Two of these kidneys were from patients with acute myelocytic leukemia, one was from a patient with acute myelomonocytic leukemia and one was from a patient with myeloid metaplasia (Cases 1,3,14 and 17, Table II). The intramembranous deposits were found in a kidney from a patient with acute myelomonocytic leukemia (Case 7, Table II). Laboratory findings that might be attributable to immune complex deposition were minimal. A few urine specimens obtained on admission contained small quantities of red blood cells and protein but in no greater amounts or frequencies than were seen in urine from patients without immune complexes. On admission all blood urea nitrogen determinations in patients with immune complexes were within normal
ET AL.
limits. Terminally, microscopic hematuria, proteinuria and azotemia occurred no more frequently in patients with immune
complexes
than in those without.
The occurrence of these complexes appears to be equally as frequent in patients with and without clinical or histologic evidence of terminal viral infections. Of the 282 patients without immune complexes 13 (4.7 per cent) had clinical or histologic evidence of terminal viral infection. Six had viral hepatitis, five either herpes zoster or varicella and two disseminated cytomegaloviral infections. Two of the 21 patients with immune complexes (9.5 per cent) had clinical or histologic evidence of a terminal viral infection. One had a herpes stomatitis and the other an Australia antigen-associated viral hepatitis. The difference is not significant. Further, homogenates of whole kidney from the patient with the AU+ hepatitis prepared as described in [ 121 were negative for Au antigen and antibody when tested by counterimmunoelectrophoresis. COMMENTS These studies demonstrate that subclinical immune complex deposition can be found in otherwise normal kidneys at autopsy. The criteria for assuming immune complex deposition include the finding of both gamma globulin and complement in a segmental or irregular distribution in the same locus on basement
A portion of a capillary loop revealing irregularity and focal thickening of baseFigure 5. ment membrane (i3M) and the presence of dense subendothelial deposits (0). Encendothelial cell (Case 1, Table II). Original magnification X 10,000, reduced by 4 per cent.
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1974
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membranes [13,14J. Conclusive evidence of immune complex deposition would also include identification of the antigen involved. None of the patients whose kidneys contained immune complexes was considered clinically to have glomerulonephritis, and all kidneys essentially were normal histologically. Although proteinuria and microscopic hematuria were found in some of the urines, these are not unexpected findings in cancer patients treated intensively with irradiation and chemotherapy. Further, they were found as frequently in patients whose kidneys were negative for immune complexes. We have also found subclinical immune complexes in renal biopsy specimens taken from patients with Hodgkin’s disease while they were undergoing staging laparotomies early in the course of their disease and before the commencement of intensive therapy [ 151. In addition, Markham et al. [ 161 have shown that immune complex deposition is ubiquitous in the normal laboratory mouse. In the latter study, the assay involved is somewhat more sensitive in that optimal material for study was consistently utilized. The unique handicap of the present study is that the kidneys obtained at autopsy were at least 2 hours old and, in the worst situation, 2 days old. Such a study is limited in that background fluorescence increases, therefore, making those positivities considered absolute more difficult to obtain. At this time, our hypothesis is that during the natural history of all or a majority of antibody responses in vivo a select population of immune complexes are deposited in glomerular basement membranes. There may of course be a wide spectrum in the extent of such deposition, and it is clear that certain situations allow for the greater deposition of such complexes. The full significance of the complexes observed in the present study will not be known until all the antigens are defined. If they are the result of viral infections, the most likely candidates are those characterized by chronicity of infection and minimal host responsiveness [ 171. The lack of correlation between the presence of these immune complexes and terminal viral infection supports this conclusion. Other
ET AL
possibilities besides chronic viral infection can account for these glomerular immune complexes. They may represent host response to tumor specific antigens (Lewis et al. [ 181 described the occurrence of the nephrotic syndrome and bronchial carcinoma) or a response to antigens associated with bacterial infections (i.e., staphylococcal septicemia [ 1,2]). Multiple complexes may be present in a single kidney as suggested by analysis of immune complex nephritis in NZB mice [ 191. Forty-five per cent of the antibody eluted from these kidneys were antinuclear antibodies, 2 1 per cent were Gross viral antibody and the remainder were not identifiable. Whatever their source, it is clear that in most situations, the antigens eliciting significant immune complex deposition have been those present in the host for some period of time. The presence of immune complexes in 11 per cent (4 of 33) of the kidneys of patients without tumor, although of unknown origin, suggests that future studies may demonstrate that the ubiquitous occurrence of immune complex deposition among laboratory mice [ 161 is also true for man. Currently, we believe that the kidney represents a focal point of study for those disease entities in which host responsiveness to antigen may be weak and in which the antigen may be unknown. This is specifically relevant to the cancer problem in which etiology is still in part debatable. Our own studies indicate that oncornaviral-related antigens can be observed in immune complexes present in patients with acute myelogenous leukemia. Continued work in this area allowing for increased sensitivity of the assays involved will allow for a more definitive analysis of the complexes both as to antibody specificity and antigen. We believe this approach is of extreme importance, and the present study is further evidence that efforts in this direction will bear fruit as to incidence of subclinical immune complex deposition and, finally, possible pathogenesis of disease. ACKNOWLEDGMENT We are indebted to Mrs. Jean Hargest for excellent technical assistance.
REFERENCES 1. KaufmanDB, McIntosh R: The pathogenesis of a renal le-
2.
3. 4.
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sion in a patient with streptococcal disease, infected ventriculoatrial shunt, cryoglobulinemia and nephritis. Am J Med 50: 262, 1971. Gutman RA, Striker GE, Gilllland BC, Cutler RE: The immune complex glomerulonephritis of bacterial endocarditis. Medicine 51: 1, 1972. Levy RL, Hong R: The immune nature of subacute bacterial endocardltls (SBE) nephritis. Am J Med 54: 645, 1973. Alllson AC, Hendrickse RG, Edington GM, Houba V, de Petris S, Adeniyi A: Immune complexes in the nephrotic syndrome of African children. Lancet 1: 1232, 1969.
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5.
6.
7.
8.
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Henson JF, Gorham JR, Padgett GA, Davis EC: Pathogenesis of the glomerular lesions in Aleutian disease of mink: immunofluorescent studies. Arch Pathol 87: 21, 1969. Chevllle NF, Mengeling WL, Zinober MR: Ultrastructural and immunofluorescent studies of glomerulonephritis in chronic hog cholera. Lab Invest 22: 458, 1970. Combes B, Shorey J, Barrera A, Stastny P, Eigenbrodt EH, Hull AR, Carter NW: Glomerulonephritis with depositlon of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2: 234, 1971. Banks KL, Henson JB, McGuire TC: Immunologically mediated glomerulitis of horses. I. Pathogenesis in persis-
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9.
10.
11.
12.
13.
IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
tent infection by equine infectious anemia virus. Lab Invest 26: 701, 1972. Oldstone MBA, Dixon FJ: Lymphocytic choriomeningitis. Production of antibody by “tolerant” infected mice. Science 158: 1193, 1967. Oldstone MBA, Aoki T, Dixon FJ: The antibody response of mice to murine leukemia virus in spontaneous infection. Absence of classical immunologic tolerance. Proc Natl Acad Sci USA 69: 134, 1972. Markham RV Jr, Sutherland JC, Cimino EF, Drake WP, Mardiney MR Jr: Immune complexes localized in the renal glomeruli of AKR mice. The presence of MuLV_gs1 and C-type RNA virus gs-3 determinants. Rev Eur Etud Clin Biol 17: 11, 1972. Sutherland JC, Mardiney MR Jr: Immune complex disease in the kidneys of lymphoma-leukemia patients. The presence of an oncornavirus-related antigen. J Nat1 Cancer lnst 50: 633, 1973. Feldman JD, Mardiney MR Jr, Shuler SE: Immunology and
October
14. 15.
16.
17. 18.
19.
1974
ET AL.
morphology of acute post-streptococcal glomerulonephritis. Lab Invest 15: 283, 1966. Dixon FJ: The pathogenesis of glomerulonephritis. Am J Med 44: 493, 1968. Sutherland JC, Markham RV Jr, Ramsey HE, Mardiney MR Jr: Subclinical immune complex nephritis in patients with Hodgkin’s disease. Cancer Res 34: 1179, 1974. Markham RV Jr, Sutherland JC, Mardiney MR Jr: The ubiquitous occurrence of immune complex localization in the renal glomeruli of normal mice. Lab Invest 29: 111, 1973. Oldstone MBA, Dixon FJ: Immune complex disease in chronic viral infections. J Exp Med 134: 32s. 1971. Lewis MB, Loughridge LW, Phillips TM: Immunological studies in nephrotic syndrome associated with extrarenal malignant disease. Lancet 2: 134, 197 1. Tonietti GJ. Oldstone MBA, Dixon FJ: The effect of induced chronic viral infections on the immunologic diseases of New Zealand mice. J Exp Med 132: 89, 1970.
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JOHN C. SUTHERLAND,
M.D.*
ROY VANN MARKHAM,
Jr., M.D.7
MICHAEL R. MARDINEY,
Jr., M.D.
Baltimore, Maryland
From the Section of Immunology and Cell Biology, National Institutes of Health, National Cancer Institute, Baltimore Cancer Research Center, 3100 Wyman Park Drive, Baltimore, Maryland 21211 and from the Department of Pathology, United States Public Health Service Hospital, 3100 Wyman Park Drive, Baltimore, Maryland 2 12 11. Requests for reprints should be addressed to Dr. John C. Sutherland. Manuscript accepted January 17, 1974. Present address: University of Maryland, School of Medicine, Department of Pathology, B-1135 No. Hosp., 22 South Greene Street, Baltimore, Maryland 2 120 1. t Present address: Bavlor Universitv School . of Medicine, Houston, Texas 75246. l
536
October 1974
In a postmortem study of 303 kidneys examined by immunofluorescence, glomerular immune complex deposition was found in the glomeruli of 21. The incidence of immune complexes was highest in kidneys from patients with acute leukemta (14.3 per cent) and lowest in kidneys from patients with solid tumors (1.6 per cent). Immune complexes have also been found in kidneys from patients without tumors (11 per cent). The patients exhibited no clinical evidence of glomerulonephritis, and the kidneys were essentially normal by light microscopy although electrondense subendothelial deposits were seen in four of eight. Although previous studies have identified a mammalian oncornaviral antigen in the complexes of two kidneys, both from patients with acute leukemia, the identity of most of the antigens remains unknown. The detection and analyses of these complexes provide an important means of detecting host response to known and unknown antigens that are not now possible by routine serologic methods. The association of various bacterial [l-3], parasitic [4] and viral [5-81 infections with overt immune complex nephritis in man and animals has been well documented. The study in these situations of glomerular bound antigens and antibodies by staining kidney sections with specific antiserums or by elution of antibody from whole kidney homogenates has been useful in identifying the various antigens involved in these complexes. In addition to identification of suspected antigens, analysis of immune complexes in some situations has revealed host immune responsiveness when it was not expected. Thus, viral antigen and antibody complexes bound to glomerular basement membrane have been found in mice with lymphocytic choriomeningitis [9] and in AKR mice with spontaneous leukemia [ 10,l I], animals previously considered tolerant to virus. Because of the potential suggested by the latter two models of murine disease, we explored the possibility that subclinical immune complex disease could be present in the patient with neoplasia. Specifically, we examined kidneys obtained at autopsy by immunofluorescence to determine if immune complex deposition exists in the absence of clinical or histologic evidence of glomerulonephritis in man. A study of the kidneys of 303 patients obtained at autopsy revealed the presence of subclinical immune complex deposition in 2 1. On analysis of the complexes in the kidneys from two patients with acute leukemia, one antigen was found to be identical with or
The American Journal of Medicine
Volume 57
SUBCLINICAL IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
related to the interspecies antigen of the mammalian oncornaviruses [ 121. We describe the immunofluorescence and light microscopic findings in these 21 kidneys and the ultrastructural findings in 8. Pertinent clinical details are
TABLE I
Percentages of Kidneys with Immune Complexes by Clinical Groups
Positivefor Kidneys Examined
discussed. Clinical Group
MATERIALS
AND METHODS
Kidneys were obtained postmortem from 303 patients. Half of these (146) were from patients with lymphoma-leukemia, 124 from patients with solid tumors and 33 from patients without tumors. The clinical classification of the patients and the numbers in each are given in Table I. The time after death before collection varied from 2 l/2 hours to 2 days. Blocks of cortical tissue were snap-frozen in an acetone-dry ice slurry and stored at -70°C. Cryostat-cut sections of renal cortex containing glomeruli were fixed in absolute acetone for 10 minutes, washed for 3 minutes and 10 minutes in phosphate-buffered saline solution at pH 7.2 and stained directly with fluorescein-labelled antihuman gamma globulin (immunoglobulins G (IgG), A (IgA) and M (IgM)) for 45 minutes at room temperature. Those positive for gamma globulin were further stained for human complement (C’3) and human fibrinogen. Sections were also stained with fluoresceinated antihuman albumin as a negative control. The commercial sources of these antiserums have been detailed [ 121. All antiserums were initially tested by immunoelectrophoresis for strength and specificity. Slides were evaluated with a Leitz microscope utilizing an Osram HB 200 light source and a UG 1 excitor filter in combination with a K460 barrier filter. For purposes of photography, either a BG 12 or KP 490 excitor filter in combination with a K 530 barrier filter was used. Positive fluorescence was recorded according to (1) distribution, whether capillary, mesangial or a combination of both; (2) pattern, whether linear or irregular; and (3) extent, whether all loops of all glomeruli were involved or a fraction thereof. For light microscopy formalin-fixed tissue was stained with hematoxylin and eosin, methenamine silver and by the Jones allochrome and periodic acid-Schiff methods. For electron microscopy tissue was fixed in glutaraldehyde, postfixed in osmium and stained with lead citrate. We examined tissue from three kidneys processed in this manner. Glutaraldehyde-fixed material from another five kidneys was further processed and examined by Dr. Ferman Tio of Georgetown University Hospital. Laboratory records of patients were examined for evidence of glomerulonephritis. It was also determined from the autopsy records whether the patients exhibited either clinical or histologic evidence of terminal viral infections.
ET AL.
Acute leukemia Chronic leukemia Lymphomas Solid tumors No tumor Total --I_
(no.> 83 9 54 124 33 303 _~
Positive for Both Gamma Globulin and Complement __.-. _ ~___ No. % 12 1 2 2 4 21
14.3 11 3.5 1.6 11 6.9
Gamma G~l~~~” (no.) 2 1 1 4 5 12
Figure 1. Fluorescence photomicrograph of a giomozlus stained for gamma globulin. Lumpy deposits may be seen chiefly along capillaries. This kidney was from a 21 year old man with acute myelogenous leukemia (Case 1, Table II). Protein and red blood cells were not detected in his urine by repeated single-specimen examinations; original magnification X 300, reduced by 15 per cent.
RESULTS Twenty-one kidneys stained positively for both gamma globulin (Figure 1) and complement (Figure 2) and an additional 12 stained positively for gamma
globulin alone. These positivities are grouped according to five broad clinical categories in Table I and are
Figure 2. C’3 in a glomerulus from the same kidney shown in Figure 1. The distribution of positivity is similar to that of gamma globulin. Original magnification X 750, reduced by 15 per cent
October 1974
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SUBCLINICAL
IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
TABLE II
Distribution
and Percentage
Involvement
ET AL.
of lmmunofluorescent
Positivities
GammaGlobulin Diagnosis
Case No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
NOTE: C = capillary;
C&M = capillary
Fibrinogen
Extent
Distribution
Extent
Distribution
C C C C C C C C C
1+ 4-F 1+ I+ 1+ 2+ 4+ 1+ 2+
2+ 4+ 1+ 1+ 2+ 2+ 4+ 1+ 1+
C C C C C C
2+ 3+ 1+ 2+ 4+ 1+ 0 0 0
C&M C C C C C C C C C C C
4+ 3+ 1+ 2+ 2+ 4+ 2+ 1+ 1+ 4+ 2+ 2+
C C C C C C C C C C&M C C C C C C C C C C C
4+ 2+ 1+ 1+ 2+ 4+ 2+ 2+ 3+ 2+ 2+ 2+
C C
4+ 4+ 0
C C C C
4+ 2+ 4+ 3+ 0
C
4+ 0
C C
1+ 1+
Distribution
Acute myelocytic leukemia Hodgkin’s disease Acute myelomonocytic leukemia Myocardial infarct Arteriosclerotic cardiovascular disease Acute myelocytic leukemia Acute myelomonocytic leukemia Acute myelocytic leukemia Cirrhosis Acute lymphocytic leukemia Acute myelocytic leukemia Carcinoma of esophagus Acute myelomonocytic leukemia Myeloid metaplasia Acute myelocytic leukemia Chronic myelocytic leukemia Acute myelomonocytic leukemia Carcinoma of colon Hodgkin’s disease Acute myelocytic leukemia Acute myelocytic leukemia
Complement
Extent
and mesangial.
listed according to individual diagnoses in Table II. The incidence of positivities (both gamma globulin and complement) was highest in kidneys from patients with acute leukemia (12 of 83 or 14.3 per cent), and lowest in kidneys from patients with solid tumors (2 of 124 or 1.6 per cent). Most positivities were capillary in distribution (Table II). Oily one kidney (Case 10, Table II) exhibited a mixed capillary-mesangial distribution. The pattern of positivity in each kidney was irregular or segmental. The extent of positivity was determined by calculating the average percentage involvement of
each of these proteins, however, II). In 14 kidneys the positivities
Figure 3. Two glomeruli stained with hematoxylin and eosin to show an essentially normal cellularity (Case 1, Table Ii). Original magnification X 120, reduced by 20 per cent.
Figure 4. Glomerulus stained by the Jones allochrome technic to show normal capillary loops. There is possibly a slight increase in mesangial matrix (Case 1, Table II). Original magnification X 300, reduced by 20 per cent.
538
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The American Journal of Medicine
each glomerulus in a section and is expressed on a scale of from 1-F to 4+. Thus 4-I indicates positive fluorescence of all lobules in all glomeruli whereas 1-F indicates involvement of 25 per cent or less of all lobules. In the latter instance, the involvement might vary considerably in extent from glomerulus to glomerulus and 1-I represents an average. Usually the distribution and pattern of positivities in a given kidney were similar for gamma globulin, complement and fibrinogen. The extent of positivity for
Volume 57
often varied (Table for gamma globulin
SUBCLINICAL IMMUNE COMPLEXES IN KIDNEYS POSTMORTEM-SUTHERLAND
were the same as those for complement, in 4 they were greater and in 3 they were less. Fibrinogen equaled in extent the positivities for gamma globulin and complement in five kidneys, exceeded them in six, was less in four and could not be detected in six. Kidneys examined by light microscopy were evaluated for increased cellularity and basement membrane and mesangial thickening. Except for an occasional thickened capillary loop or mesangial stalk, all were essentially normal (Figures 3 and 4). Eight kidneys were examined by electron microscopy. Electron-dense, finely granular, subendothelial deposits were found in the glomeruli of four (Figure 5) and intramembranous deposits in one. Two of these kidneys were from patients with acute myelocytic leukemia, one was from a patient with acute myelomonocytic leukemia and one was from a patient with myeloid metaplasia (Cases 1,3,14 and 17, Table II). The intramembranous deposits were found in a kidney from a patient with acute myelomonocytic leukemia (Case 7, Table II). Laboratory findings that might be attributable to immune complex deposition were minimal. A few urine specimens obtained on admission contained small quantities of red blood cells and protein but in no greater amounts or frequencies than were seen in urine from patients without immune complexes. On admission all blood urea nitrogen determinations in patients with immune complexes were within normal
ET AL.
limits. Terminally, microscopic hematuria, proteinuria and azotemia occurred no more frequently in patients with immune
complexes
than in those without.
The occurrence of these complexes appears to be equally as frequent in patients with and without clinical or histologic evidence of terminal viral infections. Of the 282 patients without immune complexes 13 (4.7 per cent) had clinical or histologic evidence of terminal viral infection. Six had viral hepatitis, five either herpes zoster or varicella and two disseminated cytomegaloviral infections. Two of the 21 patients with immune complexes (9.5 per cent) had clinical or histologic evidence of a terminal viral infection. One had a herpes stomatitis and the other an Australia antigen-associated viral hepatitis. The difference is not significant. Further, homogenates of whole kidney from the patient with the AU+ hepatitis prepared as described in [ 121 were negative for Au antigen and antibody when tested by counterimmunoelectrophoresis. COMMENTS These studies demonstrate that subclinical immune complex deposition can be found in otherwise normal kidneys at autopsy. The criteria for assuming immune complex deposition include the finding of both gamma globulin and complement in a segmental or irregular distribution in the same locus on basement
A portion of a capillary loop revealing irregularity and focal thickening of baseFigure 5. ment membrane (i3M) and the presence of dense subendothelial deposits (0). Encendothelial cell (Case 1, Table II). Original magnification X 10,000, reduced by 4 per cent.
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1974
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Journal
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membranes [13,14J. Conclusive evidence of immune complex deposition would also include identification of the antigen involved. None of the patients whose kidneys contained immune complexes was considered clinically to have glomerulonephritis, and all kidneys essentially were normal histologically. Although proteinuria and microscopic hematuria were found in some of the urines, these are not unexpected findings in cancer patients treated intensively with irradiation and chemotherapy. Further, they were found as frequently in patients whose kidneys were negative for immune complexes. We have also found subclinical immune complexes in renal biopsy specimens taken from patients with Hodgkin’s disease while they were undergoing staging laparotomies early in the course of their disease and before the commencement of intensive therapy [ 151. In addition, Markham et al. [ 161 have shown that immune complex deposition is ubiquitous in the normal laboratory mouse. In the latter study, the assay involved is somewhat more sensitive in that optimal material for study was consistently utilized. The unique handicap of the present study is that the kidneys obtained at autopsy were at least 2 hours old and, in the worst situation, 2 days old. Such a study is limited in that background fluorescence increases, therefore, making those positivities considered absolute more difficult to obtain. At this time, our hypothesis is that during the natural history of all or a majority of antibody responses in vivo a select population of immune complexes are deposited in glomerular basement membranes. There may of course be a wide spectrum in the extent of such deposition, and it is clear that certain situations allow for the greater deposition of such complexes. The full significance of the complexes observed in the present study will not be known until all the antigens are defined. If they are the result of viral infections, the most likely candidates are those characterized by chronicity of infection and minimal host responsiveness [ 171. The lack of correlation between the presence of these immune complexes and terminal viral infection supports this conclusion. Other
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possibilities besides chronic viral infection can account for these glomerular immune complexes. They may represent host response to tumor specific antigens (Lewis et al. [ 181 described the occurrence of the nephrotic syndrome and bronchial carcinoma) or a response to antigens associated with bacterial infections (i.e., staphylococcal septicemia [ 1,2]). Multiple complexes may be present in a single kidney as suggested by analysis of immune complex nephritis in NZB mice [ 191. Forty-five per cent of the antibody eluted from these kidneys were antinuclear antibodies, 2 1 per cent were Gross viral antibody and the remainder were not identifiable. Whatever their source, it is clear that in most situations, the antigens eliciting significant immune complex deposition have been those present in the host for some period of time. The presence of immune complexes in 11 per cent (4 of 33) of the kidneys of patients without tumor, although of unknown origin, suggests that future studies may demonstrate that the ubiquitous occurrence of immune complex deposition among laboratory mice [ 161 is also true for man. Currently, we believe that the kidney represents a focal point of study for those disease entities in which host responsiveness to antigen may be weak and in which the antigen may be unknown. This is specifically relevant to the cancer problem in which etiology is still in part debatable. Our own studies indicate that oncornaviral-related antigens can be observed in immune complexes present in patients with acute myelogenous leukemia. Continued work in this area allowing for increased sensitivity of the assays involved will allow for a more definitive analysis of the complexes both as to antibody specificity and antigen. We believe this approach is of extreme importance, and the present study is further evidence that efforts in this direction will bear fruit as to incidence of subclinical immune complex deposition and, finally, possible pathogenesis of disease. ACKNOWLEDGMENT We are indebted to Mrs. Jean Hargest for excellent technical assistance.
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Henson JF, Gorham JR, Padgett GA, Davis EC: Pathogenesis of the glomerular lesions in Aleutian disease of mink: immunofluorescent studies. Arch Pathol 87: 21, 1969. Chevllle NF, Mengeling WL, Zinober MR: Ultrastructural and immunofluorescent studies of glomerulonephritis in chronic hog cholera. Lab Invest 22: 458, 1970. Combes B, Shorey J, Barrera A, Stastny P, Eigenbrodt EH, Hull AR, Carter NW: Glomerulonephritis with depositlon of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2: 234, 1971. Banks KL, Henson JB, McGuire TC: Immunologically mediated glomerulitis of horses. I. Pathogenesis in persis-
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