Acta med. scand. Vol. 197, pp. 265-269. 1975
MONOCLONAL IMMUNOGLOBULINAEMIA ASSOCIATED WITH GLOMERULOPATHY Herluf Jensen and Allan Wiik From Medical Department P . Division of Nephrology, Rigshospi!rrlet. the Immunological Laboratory, University Clinic f o r Infectious Diseases, Blegdamshospitale t . Copenhagen, and Medical Department B , Section of Nephrologv. Copenhagen Counw Hospital. Glostrup, Denmark
Abstract. Four patients with benign monoclonal immunoglobulinaemia and associated glomerulopathy are described. Immunohistochemical investigations of the immunoglobulin-containing - cells in the bone marrow revealed an unexpectedly pronounced predominance of monoclonal over .~ polvclonal cells as twicallv seen in .. macroglobulinaemia and multiple myeloma, but in conulasma cell ~roliferations the trast to the malignant percentage of immunoglobulin-containing cells only constitued 6 1 2 % of the nucleated cells. The possible pathogenetic mechanisms relating monoclonal immunoglobulinaemia and glomerulopathy are unknown. The sera did not contain antibodies to glomerular basement membrane, cryoglobulins. antinuclear factors or antiglobulins. The immunohistochemical technique certainly offers a clear advantage over conventional bone marrow cytology in the study of patients with monoclonal immunoglobulinaemia.
with essential monoclonal immunoglobulinaemia and glomer,,lopathy, ~mmuno~istoc~emica~ sis of the bone marrow was performed in each patient.
~
In 1%6 Meltzer et al. (10) described the association of essential cryoglobulinaemia and glomerulonephritis in three patients. Since then this combination has been reported by several investigators. In most cases the glomerulopathy has been poststreptococcal glomerulonephritis or rapidly progressive glomerulonephritis of unknown aetiology (1, 11). Monoclonal immunoglobulinaemia mostly witho u t circulating cryoglobulins is found in multiple myeloma, Waldenstrom's macroglobulinaemia and several other disorders, among which some are termed essential monoclonal immunoglobulinaemia because of lack of knowledge - about an underlying - cause. T h e latter disorder has only Once been described in combination with glomerulonephritis, by Kaplan and Kaplan (7). In the present paper we report o n four patients
M A T E R I A L AND METHODS Case histories Patient 1 . A 68-year-old male. The family history was not contributory. He had passed a renal stone 20 years ago. For the last 30 years he had suffered from muscle pain especially in cold weather. In March 1970 he was admitted to the Medical Department, Rigshospitalet, because of a nephrotic syndrome. The proteinuria was 4-6 g/24 h, serum creatinine 1.5 mg1100 ml, and endogenous creatinine clearance 79 ml/min. BP was 160/70 mmHg. Serum cholesterol and triglycerides were normal. A kidney biopsy showed membraneous glomerulonephritis. Serum contained an IgM-K M-component in a concentration of 2.9 g/l. determined by paper electrophoresis, but no rheumatoid factors, antinuclear factors or cryoglobulins (12). N o Bence Jones protein was found in the urine. The bone marrow contained I % normal plasma cells and 5% lymphoid cells with an irregular nucleus and a narrow rim of strongly basophilic cytoplasm. X-rays of the skull and pelvis were normal. Because of the nephrotic syndrome the patient was treated with prednisone, 60 mg a day, and azathioprine, 100 mg a day, for 4 weeks without any effect on proteinuria or the concentration of the M-component in serum. The patient was discharged on furosemide therapy. At the last control in March 1974 proteinuria was 3.4 g/24 h. serum creatinine 2.5 mg/lOO ml, and endogenous creatinine clearance 27 ml/min. BP was 200/100 mmHg on furosemide and methyldopa therapy. Serum concentration Of the M-component was 5.2 g/l. The concentration of polyclonal Ig in serum determined by the method of Weeke (14) was normal (Table I). No hyperviscosity syndrome or other signs of Waldenstrom's macroglobulinaemia were found Acta mt-d. scand. 197
266
H . Jensen and A . Wiik
Patient 2. A 53-year-old male. The patient’s mother had had diabetes at the age of 60.At the age of 24 a diagnosis of diabetes mellitus was suspected in the patient but no therapy was started. In 1%3 proteinuria was found. In 1%6 the patient passed 3 renal stones. In March 1%7 he was admitted to the Medical Department, Rigshospitalet, because of headache and fatigue. H e was obese. BP 180/120 mmHg, serum creatinine 2.7 mg/lOO ml, and endogenous creatinine clearance 49 ml/min. A proteinuria of 5-10 g/24 h was found. Urinary excretion of glucose &2 g/24 h. Fasting plasma glucose was normal. A percutaneous kidney biopsy showed lobular glomerulonephritis. Serum contained an M-component of type IgG-K in a concentration of 13.4 g/l. The same globulin (19% of total urine protein) plus Bence Jones protein was found in the urine. No rheumatoid factors, antinuclear factors or cryoproteins were found in serum. The bone marrow contained only 2% normal plasma cells. X-rays of the skull, pelvis and vertebral spine were normal. The patient was treated with thiazides and methyldopa, and was followed in the Outpatient Department. The kidney function deteriorated and in Nov. 1973 chronic intermittent peritoneal dialysis was started. The concentration of the M-component in serum was at that time unaltered. Serum concentration of polyclonal Ig was normal (Table I) and there were still no signs of multiple myeloma. Patient 3. A 56-year-old male. No family history of renal disease, diabetes mellitus or hypertension. At the age of 20 the patient was successfully treated with gold salts for rheumatoid arthritis. At the age of 40 he was treated for a period with glucocorticoids for the same disease. During the last 20 years the patient had consumed large amounts of analgesics because of joint pain. In 1966 proteinuria (2-3 g/24 h) was discovered. Serum creatinine was normal. In Dec. 1%9 the patient was admitted to the Medical Department, Rigshospitalet, in terminal uraemia. Serum creatinine was 10 mg/100 ml and endogenous creatinine clearance was about 8 ml/min. Proteinuria was 4-5 g/24 h. BP was normal. N o subjective or objective signs from the joints were found. A kidney biopsy was performed. A histological diagnosis was difficult to establish because of scarring of the kidney tissue, but compatible with chronic poliferative glomerulonephritis. No signs of myelomatous kidney alterations or amyloidosis were found. A biopsy of the rectal mucosa was normal. Serum contained an
M-component of the type IgG-K in a concentration of 12 g/l. N o rheumatoid factors, antinuclear factors or circulating cryoglobulins were found in the serum. The urine contained the same M-component in an amount of 1 I % of the total protein excretion. No Bence Jones protein was found in the urine. Examination of the bone marrow demonstrated 9% plasma cells, some of which were atypical with vacuolated nuclei and several nuclei per cell. X-rays of the skeleton were without signs of multiple myeloma. The patient was treated with intermittent dialysis until March 1971, when he received a cadaveric kidney. At the time of transplantation the concentration of M-component in serum was 6.2 g/l. Prednisone (initially 150 mg/day) and azathioprine (100-200 mg/day) in combination with extracorporeal irradiation of the blood were given as immunosuppressive treatment. The course was uneventful and at the last follow-up in April 1974, the patient was doing well with an endogenous creatinine clearance of 75 ml/min. From the time of transplantation until Oct. 1971 (i.e. 7 months) the urine contained no protein, but from this date increased urine protein was seen, thus at the last examination 7.1 g/24 h. The concentration of the M-component in serum was at that time 9.6 g/l. The same M-component was found in the urine. The bone marrow contained 8 % plasma cells and as in the first bone marrow specimen some of these were atypical. X-rays of the skeleton showed no signs of multiple myeloma. The concentration of polyclonal Ig was normal (Table I). Patient 4 . A 71-year-old female. Her daughter has chronic kidney disease. For several years the patient had suffered from nocturnal dyspnoea and ECG had shown a WPW block. In Aug. 1971 the patient was admitted to the Medical Department, Copenhagen County Hospital, with a nephrotic syndrome. Proteinuria was 8-12 g/24 h, serum creatinine 2.0 mg/lOO ml, and endogenous creatinine clearance 38 mllmin. Serum contained an IgM-K M-component in a concentration of 9 g/l but no rheumatoid factors, antinuclear factors or cryoglobulins. The urine contained no Bence Jones protein. The bone marrow was normal with 2 % plasma cells. X-rays of the skull, vertebral spine and extremities were normal. No hyperviscosity syndrome or other signs of Waldenstrom’s macroglobulinaemia were present. A kidney biopsy showed “minimal changes”. The patient was treated with prednisone and had a complete remission of the nephrotic syndrome, and pred-
Table I. Monoclonal and polyclonal immunoglobulins in serum ( g l l ) and histological findings in bone marrow Pat. no.
M-comp.O
IgGb
1
2.9
7.6
1.2
0.85
2 3
13.4 12.0
11.2 17.6
1 .o 0.61
0.67 0.88
4
10.0
8.6
0.62
1.32
Determined electrophoretically. Acra med. scand. 197
IgA
IgM
Histological findings in bone marrow 1% normal plasma cells and 5 % lymphoid cells 2% normal plasma cells 9 % plasma cells, some with vacuolated nuclei 2% normal plasma cells
Determined immunochemically.
Monocloncil immir nog lo hi4 linaem iri u nd g lorn eridopa thy nisone was withdrawn in Nov. 1971. In May 1972 the patient was admitted to a surgical department and a cholecystectomy was performed. During this admission Albustix" was positive but no further examinations were done. The patient was discharged and readmitted in July 1972 with a relapse of the nephrotic syndrome. Prednisone treatment was resumed, this time without success. Proteinuria persisted in an amount of 2-3 g/24 h. Therefore in May 1973 the treatment was supplemented with cyclophosphamide (I50mg a day). In June the same year the urine became free of protein and predisone was withdrawn in Dec., as was cyclophosphamide in March 1974. At this last follow-up serum creatinine was 1.1 mg/100 ml, endogenous creatinine clearance 41 mlimin. BP was 150/70 mmHg. The urine contained no protein and the urinary sediment was normal. Polyclonal Ig were normal (Table I). Special investigations Immunohistochemical studies of bone marrow. Bone marrow specimens were studied by direct immunofluorescence technique according to the method of Hijmans et al. ( 5 ) . Unlabelled and fluoresceinisothiocyanatc (F1TC)-labelled rabbit IgG specific for human a, p, y, K and A chains were purchased from Dakopatts A/S, Copenhagen. The unlabelled rabbit antibodies were labelled with tetramethylrhodamineisothiocyanate (TMRITC) as described by Cebra and Goldstein (2). Before use all conjugates were tested for specificity by crossed immunoelectrophoresis (8) and by performance tests using monoclonal bone marrow cells from patients with multiple myeloma and rnacroglobulinaemia (15). In all patients double staining experiments were done to determine in the same preparation whether the cells containing one particular type of heavy chain also contained only one type of light chain, the minimal criterion for calling the plasmocytic proliferation monoclonal. All cells containing a particular heavy chain type were counted in each cytocentrifuge slide preparation and subsequently the relative percentages were calculated. The K I A ratio was always counted in one slide preparation stained with FITC-anti+ and TMRITC-anti-A. Antiglomerular basement membrane antibodies. Cryostate sections of normal human kidney tissue (4-6 /A thick) sewed as substrate for detection of antiglomerular basement membrane antibodies by indirect immuno-
267
fluorescence technique. Sera were screened at dilutions 1 : I , 1 : 4 and 1 : 16 using a 1 : 50 diluted FITClabelled rabbit antiserum specific for human a, p , y . K and A chains (Dakopatts A/S, Copenhagen).
RESULTS In patients 1 and 4 with an IgM-K M-component in serum, 83% of the monoclonal bone marrow cells contained p chains and 96 and 7296, respectively, contained K-chains. In patients 2 and 3 with an IgG-K M-component in serum, the corresponding figures were 98,97 and 98%, respectively. Ig-containing cells as a percentage of nucleated cells were determined in 3 patients and varied from 6.3 to 12.2. In patients 1 and 4 the Ig-containing cells were predominantly lymphoid, and in patients 2 and 3 mainly plasmocytoid (Table 11). N o antiglomerular basement membrane antibodies were found in any of the patients.
DISCUSSION
I n addition to patients with multiple myeloma and Waldenstrom's macroglobulinaemia, monoclonal immunoglobulins are encountered among patients with cancer, amyloidosis, and leukaemias. These diseases were excluded on clinical or histological grounds in the present patients. As monoclonal Ig in plasma can be found in apparently healthy persons, especially in old age (6, 13), there could have been a chance association between benign monoclonal immunoglobulinaemia and kidney disease in our patients. However, it is remarkable that all the patients suffered from a glomerulopathy and not from different renal disorders. Furthermore, such an association would be rare considering the low incidence of both diseases. Therefore it is tempting to postulate some kind of
Table 11. Immunohistochemically determined relative percentages of bone marrow cells containing y , cr and p heavy chains and K and A light chains. Morphological features Pat.
Cells containing heavy and light chains (%)
Ig-conQining c & ~ 100 nucleated cells
no.
y
a
/A
K
A
1
I5
2
83
96
4
12.2
2 3 4
98 97 12
1 2 5
1
98 98 72
2 2 28
-
1 83
9.0 6.3
Characteristics of monoclonal cells Mainly small lymphoid cells with narrow rim of cytoplasm Mainly plasmocytoid cells Mainly plasmocytoid cells Both plasmocytoid and lymphoid cells Acto med. scand. 197
268
H . Jensen a n d A . Wiik
causal relationship between the monoclonal immunoglobulinaemia and the glomerulopathy. At our present state of knowledge the two pathogenetic mechanisms for the induction of glomerulonephritis should be considered, one involving type I1 reaction between antiglomerular basement membrane antibodies. glomerular basement membrane and complement leading to damage of the membrane, the other involving type 111 reaction due to immune complex deposition in the glomeruli. The latter mechanism is thought to be the more common. We cannot say which of the two mechanisms is the most likely in our patients, since no immunohistochemical studies were performed on the renal biopsies. The only earlier report of monoclonal immunoglobulinaemia accompanied by glomerulopathy (7) did not include immunohistochemical studies either. The lack of antiglomerular basement membrane antibody activity in the sera does not exclude type I1 reactions as such antibodies may be completely trapped in the glomeruli (9). Serum electrophoresis did not indicate aggregate formation of the M-components, but this does not exclude that the M-component might participate in immune complexes. Monoclonal immunoglobulinaemia in combination with rheumatoid arthritis has been described by several authors (3, 16). The M-component often showed antiglobulin activity. In our rheumatoid arthritis patient (no. 3) such activity of the M-component was not detected. However the possibility remains that the M-component might have antibody activity against circulating autologous antigens. Finally the M-component might itself act as foreign antigen. triggering antibody production and immune complex formation. As none of the patients had circulating cryoglobulins, the pathogenetic mechanism may be different from that hypothesized by Skrifvars et al. ( I 1). In the immunohistochemical investigations of the bone marrow the percentage of nucleated cells containing Ig was increased compared to normal persons, but not to an extent found in malignant proliferation of plasma cells o r lymphoid cells as seen in multiple myeloma or macroglobulinaemia. However, the monoclonal population constituted a very high percentage of the lg-containing cells, quite similar t o that found as a rule in malignant cell proliferation (4). The last finding compared to the relatively low quantities of Arta rned. wand. 197
M-component in the corresponding sera suggests that either I ) the monoclonal cells secrete less Ig than polyclonal cells, 2) the M-component is hypercatabolized or excreted very fast, or 3) most of the polyclonal Ig is produced in lymphoid organs outside the bone marrow. At present none of these possibilities can be excluded. although the last one seems the most likely. The results of conventional histological and immunohistochemical investigation of the bone marrow in this study shows that only the latter investigation gives a true picture of the activity of the bone marrow in producing M-components in monoclonal immunoglobulinaemias. The experience gained from this study seems to indicate that both the percentage of Ig-containing cells among the nucleated cells and the relative percentage of the Ig-containing cells constituted by M-component-containing cells are relevant in attempting to differentiate between benign and malignant plasma cell proliferations. Obviously, longitudinal studies of these parameters are even more important for this distinction.
REFERENCES I . Barnett, E. V.: Cryoglobulins and disease. Ann. in-
tern. Med. 73:95. 1970. 2. Cebra, J. & Goldstein, G.: Chromatographic purification of tetramethylrhodamine-immuneglobulin conjugates and their use in the cellular localization of rabbit y-globulin polypeptide chains. J. Immunol. 95: 230. 1965.
3. Dryll, A , , Ryckewaert. A. & Kahn, M. F.: Rheumatismes inflammatoires et paraprottine a propos de neuf observations personelles. Rev. Rhum. 34: 155, 1967. 4. Hijmans, W . , Schuit, H. R. E. & Hulsing-Hesselnik. E.: An imrnunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann. N.Y. Acad. Sci. 177: 290, 1971. 5 . Hijmans, W . , Schuit, H. R. E. & Klein, F.: An im-
munofluorescence procedure for the detection of intracellular immunoglobulins. Clin. exp. Immunol. 4:457. 1969. 6. Halltn, J.: Discrete gammaglobulin (M-)components in serum. Acta med. scand., Suppl. 462, 1%6. 7. Kaplan, N . G. & Kaplan, K. C.: Monoclonal gam-
mopathy, glomerulonephritis. and the nephrotic syndrome. Arch. intern. Med. 125: 696, 1970. 8. Laurell, C.-B.: Antigen-antibody crossed electrophoresis. Anal. Biochem. 10: 358, 1965. 9. McPhaul, J. J. & Dixon, F. J.: The presence of antiglomerular basement membrane antibodies in peripheral blood. J. Immunol. 103: 1168, 1%9.
Monoclonal irnmunoglubilinaemia und glomerulopathy 10. Meltzer, M.. Franklin, E. C . , Elias. K . , McCluskey, R. T. & Cooper, N . : Cryoglobulinemia-A clinical and laboratory study. 11. Cryoglobulins with rheumatoid factor activit). Amel-. J Med. 40:837, 1966. 11. Skrifvars, B., Tallquist. (i. gL Tornroth, T.: Renal involvement in essential cryoglohulinaemia. Acta med. scand. 194: 229, 1973. 12. Wager, O., Mustakallio, K . K . & Rislinen, J . A.: Mixed IgA-lgG cryoglobuhnemia. lmmunologkal studies and case reports of three patients. Amer. J . Med. 44: 179, 1%8. 13. Waldenstrom, J . G.: Benign monoclonal gammapathies. In: Multiple myelomaand related disordersted.
269
H. A. Azar & M . Potter), vol. I , pp. 247-286. Harper & Row, New York, Evonston, San Francisco and London 1973. 14. Weeke, B.: Quantitative estimation of human immunoglobulins following carbamylation by electrophoresis i n antibody-containing agarose gel. Scand. J . d i n . Lab. Invest. 22: 107, 1968. IS. Wiik, A . & Munthe. E.: Restrictions among heavy and light chain determinants of granulocyte-specific antinuclear factors. Immunology 23: 53, 1972. 16. Zawadski, Z. A. & Benedek, I. G.: Rheumatoid arthritis, dysproteinemic arthropathy and paraproteinemia. Arthr. and Rheum. 12: 5 5 5 . 1969.
Acta med. scand. 197
MONOCLONAL IMMUNOGLOBULINAEMIA ASSOCIATED WITH GLOMERULOPATHY Herluf Jensen and Allan Wiik From Medical Department P . Division of Nephrology, Rigshospi!rrlet. the Immunological Laboratory, University Clinic f o r Infectious Diseases, Blegdamshospitale t . Copenhagen, and Medical Department B , Section of Nephrologv. Copenhagen Counw Hospital. Glostrup, Denmark
Abstract. Four patients with benign monoclonal immunoglobulinaemia and associated glomerulopathy are described. Immunohistochemical investigations of the immunoglobulin-containing - cells in the bone marrow revealed an unexpectedly pronounced predominance of monoclonal over .~ polvclonal cells as twicallv seen in .. macroglobulinaemia and multiple myeloma, but in conulasma cell ~roliferations the trast to the malignant percentage of immunoglobulin-containing cells only constitued 6 1 2 % of the nucleated cells. The possible pathogenetic mechanisms relating monoclonal immunoglobulinaemia and glomerulopathy are unknown. The sera did not contain antibodies to glomerular basement membrane, cryoglobulins. antinuclear factors or antiglobulins. The immunohistochemical technique certainly offers a clear advantage over conventional bone marrow cytology in the study of patients with monoclonal immunoglobulinaemia.
with essential monoclonal immunoglobulinaemia and glomer,,lopathy, ~mmuno~istoc~emica~ sis of the bone marrow was performed in each patient.
~
In 1%6 Meltzer et al. (10) described the association of essential cryoglobulinaemia and glomerulonephritis in three patients. Since then this combination has been reported by several investigators. In most cases the glomerulopathy has been poststreptococcal glomerulonephritis or rapidly progressive glomerulonephritis of unknown aetiology (1, 11). Monoclonal immunoglobulinaemia mostly witho u t circulating cryoglobulins is found in multiple myeloma, Waldenstrom's macroglobulinaemia and several other disorders, among which some are termed essential monoclonal immunoglobulinaemia because of lack of knowledge - about an underlying - cause. T h e latter disorder has only Once been described in combination with glomerulonephritis, by Kaplan and Kaplan (7). In the present paper we report o n four patients
M A T E R I A L AND METHODS Case histories Patient 1 . A 68-year-old male. The family history was not contributory. He had passed a renal stone 20 years ago. For the last 30 years he had suffered from muscle pain especially in cold weather. In March 1970 he was admitted to the Medical Department, Rigshospitalet, because of a nephrotic syndrome. The proteinuria was 4-6 g/24 h, serum creatinine 1.5 mg1100 ml, and endogenous creatinine clearance 79 ml/min. BP was 160/70 mmHg. Serum cholesterol and triglycerides were normal. A kidney biopsy showed membraneous glomerulonephritis. Serum contained an IgM-K M-component in a concentration of 2.9 g/l. determined by paper electrophoresis, but no rheumatoid factors, antinuclear factors or cryoglobulins (12). N o Bence Jones protein was found in the urine. The bone marrow contained I % normal plasma cells and 5% lymphoid cells with an irregular nucleus and a narrow rim of strongly basophilic cytoplasm. X-rays of the skull and pelvis were normal. Because of the nephrotic syndrome the patient was treated with prednisone, 60 mg a day, and azathioprine, 100 mg a day, for 4 weeks without any effect on proteinuria or the concentration of the M-component in serum. The patient was discharged on furosemide therapy. At the last control in March 1974 proteinuria was 3.4 g/24 h. serum creatinine 2.5 mg/lOO ml, and endogenous creatinine clearance 27 ml/min. BP was 200/100 mmHg on furosemide and methyldopa therapy. Serum concentration Of the M-component was 5.2 g/l. The concentration of polyclonal Ig in serum determined by the method of Weeke (14) was normal (Table I). No hyperviscosity syndrome or other signs of Waldenstrom's macroglobulinaemia were found Acta mt-d. scand. 197
266
H . Jensen and A . Wiik
Patient 2. A 53-year-old male. The patient’s mother had had diabetes at the age of 60.At the age of 24 a diagnosis of diabetes mellitus was suspected in the patient but no therapy was started. In 1%3 proteinuria was found. In 1%6 the patient passed 3 renal stones. In March 1%7 he was admitted to the Medical Department, Rigshospitalet, because of headache and fatigue. H e was obese. BP 180/120 mmHg, serum creatinine 2.7 mg/lOO ml, and endogenous creatinine clearance 49 ml/min. A proteinuria of 5-10 g/24 h was found. Urinary excretion of glucose &2 g/24 h. Fasting plasma glucose was normal. A percutaneous kidney biopsy showed lobular glomerulonephritis. Serum contained an M-component of type IgG-K in a concentration of 13.4 g/l. The same globulin (19% of total urine protein) plus Bence Jones protein was found in the urine. No rheumatoid factors, antinuclear factors or cryoproteins were found in serum. The bone marrow contained only 2% normal plasma cells. X-rays of the skull, pelvis and vertebral spine were normal. The patient was treated with thiazides and methyldopa, and was followed in the Outpatient Department. The kidney function deteriorated and in Nov. 1973 chronic intermittent peritoneal dialysis was started. The concentration of the M-component in serum was at that time unaltered. Serum concentration of polyclonal Ig was normal (Table I) and there were still no signs of multiple myeloma. Patient 3. A 56-year-old male. No family history of renal disease, diabetes mellitus or hypertension. At the age of 20 the patient was successfully treated with gold salts for rheumatoid arthritis. At the age of 40 he was treated for a period with glucocorticoids for the same disease. During the last 20 years the patient had consumed large amounts of analgesics because of joint pain. In 1966 proteinuria (2-3 g/24 h) was discovered. Serum creatinine was normal. In Dec. 1%9 the patient was admitted to the Medical Department, Rigshospitalet, in terminal uraemia. Serum creatinine was 10 mg/100 ml and endogenous creatinine clearance was about 8 ml/min. Proteinuria was 4-5 g/24 h. BP was normal. N o subjective or objective signs from the joints were found. A kidney biopsy was performed. A histological diagnosis was difficult to establish because of scarring of the kidney tissue, but compatible with chronic poliferative glomerulonephritis. No signs of myelomatous kidney alterations or amyloidosis were found. A biopsy of the rectal mucosa was normal. Serum contained an
M-component of the type IgG-K in a concentration of 12 g/l. N o rheumatoid factors, antinuclear factors or circulating cryoglobulins were found in the serum. The urine contained the same M-component in an amount of 1 I % of the total protein excretion. No Bence Jones protein was found in the urine. Examination of the bone marrow demonstrated 9% plasma cells, some of which were atypical with vacuolated nuclei and several nuclei per cell. X-rays of the skeleton were without signs of multiple myeloma. The patient was treated with intermittent dialysis until March 1971, when he received a cadaveric kidney. At the time of transplantation the concentration of M-component in serum was 6.2 g/l. Prednisone (initially 150 mg/day) and azathioprine (100-200 mg/day) in combination with extracorporeal irradiation of the blood were given as immunosuppressive treatment. The course was uneventful and at the last follow-up in April 1974, the patient was doing well with an endogenous creatinine clearance of 75 ml/min. From the time of transplantation until Oct. 1971 (i.e. 7 months) the urine contained no protein, but from this date increased urine protein was seen, thus at the last examination 7.1 g/24 h. The concentration of the M-component in serum was at that time 9.6 g/l. The same M-component was found in the urine. The bone marrow contained 8 % plasma cells and as in the first bone marrow specimen some of these were atypical. X-rays of the skeleton showed no signs of multiple myeloma. The concentration of polyclonal Ig was normal (Table I). Patient 4 . A 71-year-old female. Her daughter has chronic kidney disease. For several years the patient had suffered from nocturnal dyspnoea and ECG had shown a WPW block. In Aug. 1971 the patient was admitted to the Medical Department, Copenhagen County Hospital, with a nephrotic syndrome. Proteinuria was 8-12 g/24 h, serum creatinine 2.0 mg/lOO ml, and endogenous creatinine clearance 38 mllmin. Serum contained an IgM-K M-component in a concentration of 9 g/l but no rheumatoid factors, antinuclear factors or cryoglobulins. The urine contained no Bence Jones protein. The bone marrow was normal with 2 % plasma cells. X-rays of the skull, vertebral spine and extremities were normal. No hyperviscosity syndrome or other signs of Waldenstrom’s macroglobulinaemia were present. A kidney biopsy showed “minimal changes”. The patient was treated with prednisone and had a complete remission of the nephrotic syndrome, and pred-
Table I. Monoclonal and polyclonal immunoglobulins in serum ( g l l ) and histological findings in bone marrow Pat. no.
M-comp.O
IgGb
1
2.9
7.6
1.2
0.85
2 3
13.4 12.0
11.2 17.6
1 .o 0.61
0.67 0.88
4
10.0
8.6
0.62
1.32
Determined electrophoretically. Acra med. scand. 197
IgA
IgM
Histological findings in bone marrow 1% normal plasma cells and 5 % lymphoid cells 2% normal plasma cells 9 % plasma cells, some with vacuolated nuclei 2% normal plasma cells
Determined immunochemically.
Monocloncil immir nog lo hi4 linaem iri u nd g lorn eridopa thy nisone was withdrawn in Nov. 1971. In May 1972 the patient was admitted to a surgical department and a cholecystectomy was performed. During this admission Albustix" was positive but no further examinations were done. The patient was discharged and readmitted in July 1972 with a relapse of the nephrotic syndrome. Prednisone treatment was resumed, this time without success. Proteinuria persisted in an amount of 2-3 g/24 h. Therefore in May 1973 the treatment was supplemented with cyclophosphamide (I50mg a day). In June the same year the urine became free of protein and predisone was withdrawn in Dec., as was cyclophosphamide in March 1974. At this last follow-up serum creatinine was 1.1 mg/100 ml, endogenous creatinine clearance 41 mlimin. BP was 150/70 mmHg. The urine contained no protein and the urinary sediment was normal. Polyclonal Ig were normal (Table I). Special investigations Immunohistochemical studies of bone marrow. Bone marrow specimens were studied by direct immunofluorescence technique according to the method of Hijmans et al. ( 5 ) . Unlabelled and fluoresceinisothiocyanatc (F1TC)-labelled rabbit IgG specific for human a, p, y, K and A chains were purchased from Dakopatts A/S, Copenhagen. The unlabelled rabbit antibodies were labelled with tetramethylrhodamineisothiocyanate (TMRITC) as described by Cebra and Goldstein (2). Before use all conjugates were tested for specificity by crossed immunoelectrophoresis (8) and by performance tests using monoclonal bone marrow cells from patients with multiple myeloma and rnacroglobulinaemia (15). In all patients double staining experiments were done to determine in the same preparation whether the cells containing one particular type of heavy chain also contained only one type of light chain, the minimal criterion for calling the plasmocytic proliferation monoclonal. All cells containing a particular heavy chain type were counted in each cytocentrifuge slide preparation and subsequently the relative percentages were calculated. The K I A ratio was always counted in one slide preparation stained with FITC-anti+ and TMRITC-anti-A. Antiglomerular basement membrane antibodies. Cryostate sections of normal human kidney tissue (4-6 /A thick) sewed as substrate for detection of antiglomerular basement membrane antibodies by indirect immuno-
267
fluorescence technique. Sera were screened at dilutions 1 : I , 1 : 4 and 1 : 16 using a 1 : 50 diluted FITClabelled rabbit antiserum specific for human a, p , y . K and A chains (Dakopatts A/S, Copenhagen).
RESULTS In patients 1 and 4 with an IgM-K M-component in serum, 83% of the monoclonal bone marrow cells contained p chains and 96 and 7296, respectively, contained K-chains. In patients 2 and 3 with an IgG-K M-component in serum, the corresponding figures were 98,97 and 98%, respectively. Ig-containing cells as a percentage of nucleated cells were determined in 3 patients and varied from 6.3 to 12.2. In patients 1 and 4 the Ig-containing cells were predominantly lymphoid, and in patients 2 and 3 mainly plasmocytoid (Table 11). N o antiglomerular basement membrane antibodies were found in any of the patients.
DISCUSSION
I n addition to patients with multiple myeloma and Waldenstrom's macroglobulinaemia, monoclonal immunoglobulins are encountered among patients with cancer, amyloidosis, and leukaemias. These diseases were excluded on clinical or histological grounds in the present patients. As monoclonal Ig in plasma can be found in apparently healthy persons, especially in old age (6, 13), there could have been a chance association between benign monoclonal immunoglobulinaemia and kidney disease in our patients. However, it is remarkable that all the patients suffered from a glomerulopathy and not from different renal disorders. Furthermore, such an association would be rare considering the low incidence of both diseases. Therefore it is tempting to postulate some kind of
Table 11. Immunohistochemically determined relative percentages of bone marrow cells containing y , cr and p heavy chains and K and A light chains. Morphological features Pat.
Cells containing heavy and light chains (%)
Ig-conQining c & ~ 100 nucleated cells
no.
y
a
/A
K
A
1
I5
2
83
96
4
12.2
2 3 4
98 97 12
1 2 5
1
98 98 72
2 2 28
-
1 83
9.0 6.3
Characteristics of monoclonal cells Mainly small lymphoid cells with narrow rim of cytoplasm Mainly plasmocytoid cells Mainly plasmocytoid cells Both plasmocytoid and lymphoid cells Acto med. scand. 197
268
H . Jensen a n d A . Wiik
causal relationship between the monoclonal immunoglobulinaemia and the glomerulopathy. At our present state of knowledge the two pathogenetic mechanisms for the induction of glomerulonephritis should be considered, one involving type I1 reaction between antiglomerular basement membrane antibodies. glomerular basement membrane and complement leading to damage of the membrane, the other involving type 111 reaction due to immune complex deposition in the glomeruli. The latter mechanism is thought to be the more common. We cannot say which of the two mechanisms is the most likely in our patients, since no immunohistochemical studies were performed on the renal biopsies. The only earlier report of monoclonal immunoglobulinaemia accompanied by glomerulopathy (7) did not include immunohistochemical studies either. The lack of antiglomerular basement membrane antibody activity in the sera does not exclude type I1 reactions as such antibodies may be completely trapped in the glomeruli (9). Serum electrophoresis did not indicate aggregate formation of the M-components, but this does not exclude that the M-component might participate in immune complexes. Monoclonal immunoglobulinaemia in combination with rheumatoid arthritis has been described by several authors (3, 16). The M-component often showed antiglobulin activity. In our rheumatoid arthritis patient (no. 3) such activity of the M-component was not detected. However the possibility remains that the M-component might have antibody activity against circulating autologous antigens. Finally the M-component might itself act as foreign antigen. triggering antibody production and immune complex formation. As none of the patients had circulating cryoglobulins, the pathogenetic mechanism may be different from that hypothesized by Skrifvars et al. ( I 1). In the immunohistochemical investigations of the bone marrow the percentage of nucleated cells containing Ig was increased compared to normal persons, but not to an extent found in malignant proliferation of plasma cells o r lymphoid cells as seen in multiple myeloma or macroglobulinaemia. However, the monoclonal population constituted a very high percentage of the lg-containing cells, quite similar t o that found as a rule in malignant cell proliferation (4). The last finding compared to the relatively low quantities of Arta rned. wand. 197
M-component in the corresponding sera suggests that either I ) the monoclonal cells secrete less Ig than polyclonal cells, 2) the M-component is hypercatabolized or excreted very fast, or 3) most of the polyclonal Ig is produced in lymphoid organs outside the bone marrow. At present none of these possibilities can be excluded. although the last one seems the most likely. The results of conventional histological and immunohistochemical investigation of the bone marrow in this study shows that only the latter investigation gives a true picture of the activity of the bone marrow in producing M-components in monoclonal immunoglobulinaemias. The experience gained from this study seems to indicate that both the percentage of Ig-containing cells among the nucleated cells and the relative percentage of the Ig-containing cells constituted by M-component-containing cells are relevant in attempting to differentiate between benign and malignant plasma cell proliferations. Obviously, longitudinal studies of these parameters are even more important for this distinction.
REFERENCES I . Barnett, E. V.: Cryoglobulins and disease. Ann. in-
tern. Med. 73:95. 1970. 2. Cebra, J. & Goldstein, G.: Chromatographic purification of tetramethylrhodamine-immuneglobulin conjugates and their use in the cellular localization of rabbit y-globulin polypeptide chains. J. Immunol. 95: 230. 1965.
3. Dryll, A , , Ryckewaert. A. & Kahn, M. F.: Rheumatismes inflammatoires et paraprottine a propos de neuf observations personelles. Rev. Rhum. 34: 155, 1967. 4. Hijmans, W . , Schuit, H. R. E. & Hulsing-Hesselnik. E.: An imrnunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann. N.Y. Acad. Sci. 177: 290, 1971. 5 . Hijmans, W . , Schuit, H. R. E. & Klein, F.: An im-
munofluorescence procedure for the detection of intracellular immunoglobulins. Clin. exp. Immunol. 4:457. 1969. 6. Halltn, J.: Discrete gammaglobulin (M-)components in serum. Acta med. scand., Suppl. 462, 1%6. 7. Kaplan, N . G. & Kaplan, K. C.: Monoclonal gam-
mopathy, glomerulonephritis. and the nephrotic syndrome. Arch. intern. Med. 125: 696, 1970. 8. Laurell, C.-B.: Antigen-antibody crossed electrophoresis. Anal. Biochem. 10: 358, 1965. 9. McPhaul, J. J. & Dixon, F. J.: The presence of antiglomerular basement membrane antibodies in peripheral blood. J. Immunol. 103: 1168, 1%9.
Monoclonal irnmunoglubilinaemia und glomerulopathy 10. Meltzer, M.. Franklin, E. C . , Elias. K . , McCluskey, R. T. & Cooper, N . : Cryoglobulinemia-A clinical and laboratory study. 11. Cryoglobulins with rheumatoid factor activit). Amel-. J Med. 40:837, 1966. 11. Skrifvars, B., Tallquist. (i. gL Tornroth, T.: Renal involvement in essential cryoglohulinaemia. Acta med. scand. 194: 229, 1973. 12. Wager, O., Mustakallio, K . K . & Rislinen, J . A.: Mixed IgA-lgG cryoglobuhnemia. lmmunologkal studies and case reports of three patients. Amer. J . Med. 44: 179, 1%8. 13. Waldenstrom, J . G.: Benign monoclonal gammapathies. In: Multiple myelomaand related disordersted.
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H. A. Azar & M . Potter), vol. I , pp. 247-286. Harper & Row, New York, Evonston, San Francisco and London 1973. 14. Weeke, B.: Quantitative estimation of human immunoglobulins following carbamylation by electrophoresis i n antibody-containing agarose gel. Scand. J . d i n . Lab. Invest. 22: 107, 1968. IS. Wiik, A . & Munthe. E.: Restrictions among heavy and light chain determinants of granulocyte-specific antinuclear factors. Immunology 23: 53, 1972. 16. Zawadski, Z. A. & Benedek, I. G.: Rheumatoid arthritis, dysproteinemic arthropathy and paraproteinemia. Arthr. and Rheum. 12: 5 5 5 . 1969.
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