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Gammopathy with two M-components in a dog with IgA-type multiple myeloma
Veterinary
Immunology and Immunopathology 49 (1995) 161-168
Veterinay immunology and immunopathology
Short communication
Gammopathy with two M-components in a dog with &A-type multiple myeloma Hirotomo Kato a, Yasuyuki Momoi a, Kazuyuki Omori ‘, Hwa-Young Youn a, Takatsugu Yamada ‘, Naoaki Goto d, Kenichiro Ono ‘, Toshihiro Watari a, Hajime Tsujimoto a, Atsuhiko Hasegawa a9* ’ Department of Veterinary Internal Medicine, Faculty of Agriculture, Unitlersity of Tokyo. 1-l -I Yayoi. Bunkyo-ku, Tokyo 113, Japan ’ Department of Veterinary Clinical Pathoboiology, Faculty of Agriculture, Unit,ersih, of Tokyo. 1-l-1 Yayol. Bunkyo-ku, Tokyo 113, Japan ’ Ljepartment of Veterinary Internal Medicine, School of Veterinary Medicine, Azabu Unil~ersity.Sagamihara. Kanagawa 229, Japan ’ Department of Veterinary Pathology, Faculty of Agriculture. University of Tokyo, 1 -I -I Yayoi. Bunkyo-ku. Tokyo 113, Japan Accepted 20 March 1995
Abstract A 12-year-old neutered male mixed-breed dog was referred to hospital for evaluation of chronic diarrhea. Cellulose acetate electrophoresis of its serum revealed two monoclonal peaks in the gamma-globulin fraction. On immunoelectrophoretic analysis, the two monoclonal peaks in the gamma-globulin region were strongly precipitated with anti-dog IgA serum. On sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, the fractions corresponding to these two peaks were shown to be dimer and trimer or tetramer of immunoglobulin consisting of heavy and light chains. These results indicated that the studied dog had gammopathy with two M-components with dimer and trimer or tetramer of IgA. Accumulations of large amounts of these immunoglobulins with very high molecular weight in the serum were concluded to induce the hyperviscosity syndrome in this dog in the terminal stage. Keywords: Dog; Multiple myeloma; IgA
* Corresponding author. 0165.2427/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0165.2427(95)05462-6
162
H. Kato et al. / Veterinary Immunology
and Immunopathology
49 (1995) 161-168
1. Abbreviations kb, kilobase; sulfate;
PAGE,
polyacrylamide
gel electrophoresis;
SDS,
sodium
dodecyl
2. Introduction Multiple myeloma is an infrequently reported tumor in dogs and is a hematologic disorder characterized by neoplastic proliferation of immunoglobulin-producing plasma cells. Monoclonal gammopathy, neoplastic proliferation of plasma cells in bone marrow, Bence-Jones protein in urine and punched-out osteolytic lesions are characteristic features of multiple myeloma. IgG- and IgA-type myelomas have been reported in dogs (Osborne et al., 1968; Martinsson et al., 1973; Takahashi et al., 1980; Finnie and Wilks, 1982). Monoclonal gammopathy is shown by the presence of a single narrow band on cellulose acetate electrophoresis of the serum. On the other hand, a peculiar serum protein profile with two distinct monoclonal proteins, which is designated as gammopathy with two M-components or biclonal gammopathy, has been reported in humans (Vaerman et al., 1965a,b; Rudders et a1.,1973; Pruzanski et al., 1974; Kyle et al., 19811. Biclonal gammopathy was found to be very rare, occurring in about 1% of humans with myeloma (Kyle et al., 1981). The presence of two monoclonal proteins may be due to proliferation of two clones of plasma cells or production of two monoclonal proteins by a single clone of plasma cells. This paper describes a case of IgA-type multiple myeloma in a dog with gammopathy with two M-components, and the biochemical features of the two M-components from this case.
3. Case history A 12-year-old neutered male mixed-breed dog was admitted to the Veterinary Medical Center, University of Tokyo, because of chronic diarrhea for 4 months. Hematologic examination revealed anemia (packed cell volume 25%, hemoglobin 9.9 g dl-‘1 and a markedly elevated serum protein level (12.8 g dll’l. Cellulose acetate electrophoresis showed two monoclonal peaks in the gamma-globulin fraction (Fig. 1). The major and minor monoclonal peaks corresponded to 6.0 and 3.1 g dl-’ gammaglobulin, respectively. Serum chemical analyses indicated moderate elevations of alkaline phosphatase (212 IU I-‘), alanine aminotransferase (226 IU 1-l) and aspartate aminotransferase (145 IU I-’ 1. No osteolytic lesion was found by radiographic examination. No Bence-Jones protein was detected in the urine. A bone marrow biopsy specimen from the femur appeared to be slightly hypocellular and contained a number of large amphophilic plasma cells, which showed morphologic features of ‘flame cells’ (Fig. 2). Mitoses were rare, but a few binucleated plasma cells were present. Diarrhea in this case could be controlled by fluid therapy and administration of digestives for 3 months. About 4 months after the first treatment, the dog showed recurrence of diarrhea. Furthermore, its plasma viscosity measured with an Ostwald
H. Kato et al. / Veterinary Immunology
and Immunopathology
r !-
:‘! / /
1
1 \
163
49 (1995) 161-168
r II
i
L
f
I
I
i
I/i
i
,:
‘I/ : \i \
[ :! I,’ ,
/i i ,r-..___L__Y
\I J Alb
Fig. 1. Cellulose acetate electrophoretic monoclonal peaks in the gamma-globulin
al pat,em zone.
a2
P
of the serum
11 . Y of the study
dog showing
two prominent
viscometer was markedly elevated (1101 s), which was more than ten times that of normal dogs (81-90 s>. Therefore, the dog was treated by chemotherapy with melphalan (1.5 mg m-‘) and prednisolone (40 mg m-* ). However, 5 days after the start of chemotherapy, the dog suffered circulatory depression and convulsions, and died, probably due to the hyperviscosity syndrome. Autopsy revealed splenomegaly and a number of small white nodules in the liver and spleen. Microscopic examination revealed proliferation of neoplastic plasma cells in the bone marrow. The growth and differentiation of normal hematopoietic cells were not severely impaired. Infiltration of neoplastic plasma cells was found in the portal triad and around the central vein in the liver. These neoplastic cells were also found in the cortex and medulla of the kidney.
Fig. 2. Neoplastic
plasma cells in the biopsy specimen of the bone marrow. Giemsa stain. X 1000.
164
H. Kato et al. / Veterinary Immunology and Immunopathology 49 (1995) I61 -168
4. Materials and methods 4. I. Immunoelectrophoresis Agarose gel (1.2%) prepared with Tris hydrochloric acid buffer (pH 8.6) of 0.025 ionic strength was used for immunoelectrophoresis of serum protein. After electrophoresis of serum samples from the study dog and a normal control dog, antiserum against dog whole serum (Organon Teknika, Durham, NC), dog IgG-Fc fragment (Bethyl Laboratories, Montgomery, TX), dog IgM (Bethyl Laboratories), or dog IgA (ICN ImmunoBiologicals, Lisle, IL) was pipetted into antiserum troughs cut into the agarose gel. 4.2. Gel filtration
of serum protein
A serum sample diluted with phosphate buffered saline was applied to a Superose 12 column (Pharmacia, Uppsala, Sweden) and fractionated with a fast performance liquid chromatography (FPLC) gel filtration system (Pharmacia). The prepared sample was collected in 32 fractions for further analysis. 4.3. Sodium dodecyl sulfate (SDS) polyacrylamide
gel electrophoresis
(PAGE)
SDS-PAGE under reduced conditions was performed using a 4-20% vertical slab gel (Daiichi Pure Chemicals, Tokyo, Japan) in an electrode buffer containing 0.025 M Tris-HCl (pH 7.61, 0.192 M glycine and 0.1% SDS. Samples dissolved in reducing loading buffer (0.0625 M Tris-HCI, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue (BPB)) were boiled for 3 min and electrophoresed for 1 h. For SDS-PAGE under non-reduced conditions, we used a 2-15% vertical slab gel (Daiichi Pure Chemicals) and samples diluted in the loading buffer without 2mercaptoethanol. After electrophoresis, the gels were stained with Coomassie Blue for 20 min and destained for 1 h. 4.4. Slot blot analysis For extraction of the RNA sample, a bone marrow biopsy specimen that had been stored at - 80°C was homogenized in liquid nitrogen and treated with RNAzol (Biotecx Houston, TX) containing guanidium thiocyanate, phenol and 2Laboratories, mercaptoethanol. The RNA sample was then extracted with chloroform and precipitated with isopropanol. The cellular RNA samples were spotted onto Biodyne B membranes (Pall Biosupport, East Hills, NY) and cross-linked by heating at 80°C for 15 min. The filters were prehybridized in a solution containing 50 mM Tris-HCl (pH 7.61, 5 X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% SDS, 5 X Denhardt solution and 100 mg ml-’ denatured salmon testis DNA for 3 h at 65°C. Hybridization was carried out with 3’P-labelled DNA probes for 18 h. As a lambda chain probe, we used a 0.6kilobase (kb) SacI-EcoRI fragment of clone S6-61 (kindly provided by Dr. S. Tokiyoshi, Chemo-Sero-Therapeutic Research Institute), which is a cDNA clone cover-
165
H. f&to er al. / Veterinaq Immunology and Immunopathology 49 119951161-I68 -
-
+ B
h
^ ^ _ _ . _I . ..
tl
.-_
Fig. 3. (A) Immunoektrophoresis of serum of the study dog against antisera to dog whole serum, IgG, IgM and IgA (1. 2, 3 and 4, respectiyely). Sera of the study dog and a normal dog were pipetted into antiserum troughs ‘a’ and ‘b’, respectively: (B) lmmunoelectrophoresis of Fractions 6 and 12 from serum of the studied dog against anti-dog IgA (Trough 4). Serum Fractions 6 and 12 were pipetted into antiserum troughs ‘c’ and ‘d’, respectively.
ing a constant region of the canine immunoglobulin lambda chain gene. A 2-kb PSI fragment of DEkSa (kindly provided by Dr. S. Tokiyoshil representing a constant region of canine immunoglobulin kappa chain cDNA was used as a kappa chain probe. After hybridization, the filter was washed three times with washing buffer containing 0.5 x SSC and 0.1% SDS at 65°C for 30 min and then exposed to X-ray film at - 80°C for 10 h.
5. Results Sera from the study dog and a normal dog were analyzed by immunoelectrophoresis using anti-dog whole serum, anti-dog IgG, anti-dog IgM and anti-dog IgA (Fig. 3(A)).
COD
280nm)
FlXliWl
+
Fig. 4. (A) Gel filtration of serum protein of the studied dog. Serum of the dog was collected in 32 fractions using a Superose 12 column in an FPLC system. (B) Electrophoresis of Fractions 6 (Lane a). 12 (Lane b). 25 (Lane c) and whole serum (Lane d) of the dog, on a cellulose acetate membrane.
166
H. Kato et al. / Veterinary Immunology
and Immunopathology
49 (1995) 161-168
A B a
h
c
d
(kd)
e
abed
e
669* 440) 232 ) 140)
Fig. 5. Analysis of serum protein of the dog by SDS-PAGE analysis under reduced (A) and non-reduced (B) conditions. Lanes a, b and c are Fractions 6, 12 and 25 of the dog’s serum, respectively; Lane d is the studied dog’s serum; Lane e is the normal dog serum.
Serum from the sick dog showed clear thickening and bowing of the arc in the gamma-globulin zone when antiserum against dog IgA was pipetted into antiserum troughs cut into the agarose gel. No obvious precipitate was found with anti-dog IgM or anti-dog IgG serum in this zone. This result indicated that the major monoclonal peak in the gamma-globulin zone was IgA. Serum of the dog was separated into 32 fractions by gel filtration (Fig. 4(A)), which were subjected to cellulose acetate electrophoresis. As shown in Fig. 4(B), Fractions 6 and 12 were found to correspond to major and minor peaks in the gamma-globulin zone, respectively. On immunoelectrophoresis both Fractions 6 and 12 gave distinct precipitates with anti-dog IgA (Fig. 3(B)). SDS-PAGE analysis of Fractions 6 and 12 under reduced conditions revealed bands of approximately 60 and 29 kDa (Fig. 5(A)). These bands corresponded in size to immunoglobulin heavy and light chains, respectively. On SDS-PAGE analysis under non-reduced conditions, Fractions 6 and 12 gave bands of approximately 520 and 350 kDa, corresponding in size to the trimer or tetramer and dimer of immunoglobulin, respectively (Fig. S(B)). These results indicated that the major and minor peaks in the gamma-globulin zone were those of the dimer and trimer or tetramer of IgA, respectively.
RNAW
5
1
0.2
b Fig. 6. Slot blot analysis of immunoglobulin light chains. were transferred to nylon membrane filters and hybridized chain probes.
RNA samples extracted from bone marrow cells with probes of canine lambda (a) and kappa (b)
H. Kato et al. / Veterinary Immunolo&y and Immunopathology
49 (1995) 161-l 68
lh7
Slot blot analysis using probes of canine immunoglobulin light chains revealed that the mRNA sample from bone marrow cells in this case hybridized with the canine immunoglobulin kappa chain probe, but much less with the canine immunoglobulin lambda chain probe (Fig. 6). A nucleic acid hybridization experiment indicated that the kappa chain was used as an immunoglobulin light chain in myeloma cells of this case.
6. Discussion Diagnosis of multiple myeloma in the present case was based on gammopathy with two M-components and proliferation of amphophilic plasma cells in the bone marrow. No osteolysis or Bence-Jones protein in the urine was observed, although these features are considered to be characteristic of multiple myeloma in dogs (Osborne et al., 1968). In a study on canine multiple myeloma, 56% of the cases were found to have osteolytic lesions and 31% to have Bence-Jones protein in the urine (Dorfman and Dimski, 1992). Conceivably, the presence of osteolytic lesions depends on the osteoclastic activating factor released from myeloma cells (Mundy et al., 1974), and detectable Bence-Jones protein may be due to overproduction of immunoglobulin light chains by myeloma cells (Osborne et al., 1968). The myeloma cells in the present case seemed to be devoid of these characteristics. Gammopathy with two M-components, which is rare in multiple myeloma, was found in the present case. Biochemical analysis indicated that the two M-proteins in this case were dimer and trimer or tetramer of IgA. Only one previous case of gammopathy with two M-components in the dog with multiple myeloma has been reported (Jacobs et al., 19861, but detailed biochemical analysis on this reported case was not shown. In humans, gammopathy with two M-components is reported to occur in approximately 1% of all cases of multiple myeloma (Kyle et al., 1981). The presence of biclonal M-protein may be due to proliferation of two clones of plasma cells, each producing an unrelated monoclonal immunoglobulin, or may result from production of biclonal M-protein by a single clone of plasma cells. Switching of the heavy chain class of immunoglobulin occurs during normal development and differentiation of B lymphocytes (Dreyer and Bennett, 1965; Seidman et al., 1978). In spite of class switching of the heavy chain, the antibody molecules retain the same antigenic specificity. Of human cases of multiple myeloma with biclonal gammopathy, many were shown to have IgG and IgA components, while a few were found to have two IgA proteins (Vaerman et al., 1965a.b; Kyle et al., 1981). Therefore, the biclonal gammopathy with dimer and trimer or tetramer of IgA demonstrated in the present case was considered to be very rare in multiple myeloma. Analysis of serum protein by SDS-PAGE under reduced conditions indicated that both Fractions 6 and 12 gave bands of approximately 60 and 29 kDa, corresponding to the sizes of immunoglobulin heavy and light chains, respectively. On the other hand, heavy and light chains in normal dog serum were shown to be 50 and 25 kDa, respectively, in size. The size differences of heavy and light chains in the serum of the present case from those in normal dog serum were thought to be due to the difference of immunoglobulin class; that is to say, the immunoglobulin class of the present case was
168
H. Kato et ai./ Veterinary Immunology and Immunopathotogy 49 (1995) 161-168
IgA, whereas that of the normal dog was mainly IgG. Two bands of approximately 60 kDa might be due to some difference in glycosylation of these proteins. Analyzed by SDS-PAGE under non-reduced conditions, Fraction 12 clearly contained two bands, and it might be also due to the difference of glycosylation. The hyperviscosity syndrome is not always observed in cases of multiple myeloma, but is closely associated with serious symptoms in the circulation and hemostasis. The hyperviscosity syndrome is frequently observed in cases of myeloma with high molecular weight M-proteins such as IgM and polymeric IgA. The IgG-associated hyperviscosity syndrome is reported to be rare because IgG has a low molecular weight and is a monomer (Dorfman and Dimski, 1992). In the present case, sudden death was considered to be due to the hyperviscosity syndrome because severe circulation insufficiency was observed before death. The clinical signs in this case were considered to be closely related to large amounts of polymeric IgA with high molecular weights.
Acknowledgments We thank Dr. S. Tokiyoshi (Chemo-Sero-Therapeutic Research Institute) for canine lambda and kappa chain gene probes. This study was supported in part by grants from the Ministry of Education, Science and Culture in Japan.
References Dorfman, M. and Dimski, D.S., 1992. Paraproteinemias in small animal medicine. Compend. Contin. Educ. Pratt. Vet., 14: 621-631. Dreyer, W.J. and Bennett, J.C., 196.5. The molecular basis of antibody formation: a paradox. Proc. Nat]. Acad. Sci. USA, 54: 864-869. Finnie, J.W. and Wilks, C.R., 1982. Two cases of multiple myeloma in the dog. J. Small Anim. Pratt., 23: 19-27. Jacobs, R.M., Couto, C.G. and Wellman, M.L., 1986. Biclonal gammopathy in a dog with myeloma and cutaneous lymphoma. Vet. Pathol., 23: 211-213. Kyle, R.A., Robinson, R.A. and Katzmann, J.A., 1981. The clinical aspects of biclonal gammopathies. Am. J. Med., 71: 999-1008. Martinsson, K., Jonsson, L. and Johansson, H.-E., 1973. Multiple myeloma of IgA type in a dog. Zbl. Vet. Med. A, 20: 826-835. Mundy, G.R., Raisz, L.G., Cooper, R.A., Schechter, G.P. and Salmon, SE., 1974. Evidence for the secretion of an osteoclast stimulating factor in myeloma. N. Engl. J. Med., 291: 1041-1046. Osborne, CA., Sautter, J.H., Stevens, J.B. and Hanlon, G.F., 1968. Multiple myeloma in the dog. J. Am. Vet. Med. Assoc., 153: 1300-1319. Pruzanski, W., Underdown, B., Silver, E.H. and Katz, A., 1974. Macroglobulinemia-myeloma double gammopathy. Am. J. Med., 57: 259-266. Rudders, R.A., Yakulis, V. and Heller, P., 1973. Double myeloma. Am. J. Med., 55: 215-221. Seidman, J.G., Leder, A., Nau, M., Norman, B. and Leder, P., 1978. Antibody diversity. Science, 202: 11-17. Takahashi, K., Katami, K., Nakamura, K., Tomoda, I. and Fujiwara, K., 1980. IgG type myeloma in a dog. Jpn. J. Vet. Sci., 42: 271-275. of the Vaerman, J.P., Fudenberg, H.H., Vaerman, C. and Mandy, W.J., 1965a. On the significance heterogeneity in molecular size of human serum yA-globulins. Immunochemistry, 2: 263-272. Vaerman, J.P., Johnson, L.B., Mandy, W. and Fudenberg, H.H., 1965b. Multiple myeloma with two paraprotein peaks: an instructive case. J. Lab. Clin. Med., 65: 18-25.
Immunology and Immunopathology 49 (1995) 161-168
Veterinay immunology and immunopathology
Short communication
Gammopathy with two M-components in a dog with &A-type multiple myeloma Hirotomo Kato a, Yasuyuki Momoi a, Kazuyuki Omori ‘, Hwa-Young Youn a, Takatsugu Yamada ‘, Naoaki Goto d, Kenichiro Ono ‘, Toshihiro Watari a, Hajime Tsujimoto a, Atsuhiko Hasegawa a9* ’ Department of Veterinary Internal Medicine, Faculty of Agriculture, Unitlersity of Tokyo. 1-l -I Yayoi. Bunkyo-ku, Tokyo 113, Japan ’ Department of Veterinary Clinical Pathoboiology, Faculty of Agriculture, Unit,ersih, of Tokyo. 1-l-1 Yayol. Bunkyo-ku, Tokyo 113, Japan ’ Ljepartment of Veterinary Internal Medicine, School of Veterinary Medicine, Azabu Unil~ersity.Sagamihara. Kanagawa 229, Japan ’ Department of Veterinary Pathology, Faculty of Agriculture. University of Tokyo, 1 -I -I Yayoi. Bunkyo-ku. Tokyo 113, Japan Accepted 20 March 1995
Abstract A 12-year-old neutered male mixed-breed dog was referred to hospital for evaluation of chronic diarrhea. Cellulose acetate electrophoresis of its serum revealed two monoclonal peaks in the gamma-globulin fraction. On immunoelectrophoretic analysis, the two monoclonal peaks in the gamma-globulin region were strongly precipitated with anti-dog IgA serum. On sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, the fractions corresponding to these two peaks were shown to be dimer and trimer or tetramer of immunoglobulin consisting of heavy and light chains. These results indicated that the studied dog had gammopathy with two M-components with dimer and trimer or tetramer of IgA. Accumulations of large amounts of these immunoglobulins with very high molecular weight in the serum were concluded to induce the hyperviscosity syndrome in this dog in the terminal stage. Keywords: Dog; Multiple myeloma; IgA
* Corresponding author. 0165.2427/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0165.2427(95)05462-6
162
H. Kato et al. / Veterinary Immunology
and Immunopathology
49 (1995) 161-168
1. Abbreviations kb, kilobase; sulfate;
PAGE,
polyacrylamide
gel electrophoresis;
SDS,
sodium
dodecyl
2. Introduction Multiple myeloma is an infrequently reported tumor in dogs and is a hematologic disorder characterized by neoplastic proliferation of immunoglobulin-producing plasma cells. Monoclonal gammopathy, neoplastic proliferation of plasma cells in bone marrow, Bence-Jones protein in urine and punched-out osteolytic lesions are characteristic features of multiple myeloma. IgG- and IgA-type myelomas have been reported in dogs (Osborne et al., 1968; Martinsson et al., 1973; Takahashi et al., 1980; Finnie and Wilks, 1982). Monoclonal gammopathy is shown by the presence of a single narrow band on cellulose acetate electrophoresis of the serum. On the other hand, a peculiar serum protein profile with two distinct monoclonal proteins, which is designated as gammopathy with two M-components or biclonal gammopathy, has been reported in humans (Vaerman et al., 1965a,b; Rudders et a1.,1973; Pruzanski et al., 1974; Kyle et al., 19811. Biclonal gammopathy was found to be very rare, occurring in about 1% of humans with myeloma (Kyle et al., 1981). The presence of two monoclonal proteins may be due to proliferation of two clones of plasma cells or production of two monoclonal proteins by a single clone of plasma cells. This paper describes a case of IgA-type multiple myeloma in a dog with gammopathy with two M-components, and the biochemical features of the two M-components from this case.
3. Case history A 12-year-old neutered male mixed-breed dog was admitted to the Veterinary Medical Center, University of Tokyo, because of chronic diarrhea for 4 months. Hematologic examination revealed anemia (packed cell volume 25%, hemoglobin 9.9 g dl-‘1 and a markedly elevated serum protein level (12.8 g dll’l. Cellulose acetate electrophoresis showed two monoclonal peaks in the gamma-globulin fraction (Fig. 1). The major and minor monoclonal peaks corresponded to 6.0 and 3.1 g dl-’ gammaglobulin, respectively. Serum chemical analyses indicated moderate elevations of alkaline phosphatase (212 IU I-‘), alanine aminotransferase (226 IU 1-l) and aspartate aminotransferase (145 IU I-’ 1. No osteolytic lesion was found by radiographic examination. No Bence-Jones protein was detected in the urine. A bone marrow biopsy specimen from the femur appeared to be slightly hypocellular and contained a number of large amphophilic plasma cells, which showed morphologic features of ‘flame cells’ (Fig. 2). Mitoses were rare, but a few binucleated plasma cells were present. Diarrhea in this case could be controlled by fluid therapy and administration of digestives for 3 months. About 4 months after the first treatment, the dog showed recurrence of diarrhea. Furthermore, its plasma viscosity measured with an Ostwald
H. Kato et al. / Veterinary Immunology
and Immunopathology
r !-
:‘! / /
1
1 \
163
49 (1995) 161-168
r II
i
L
f
I
I
i
I/i
i
,:
‘I/ : \i \
[ :! I,’ ,
/i i ,r-..___L__Y
\I J Alb
Fig. 1. Cellulose acetate electrophoretic monoclonal peaks in the gamma-globulin
al pat,em zone.
a2
P
of the serum
11 . Y of the study
dog showing
two prominent
viscometer was markedly elevated (1101 s), which was more than ten times that of normal dogs (81-90 s>. Therefore, the dog was treated by chemotherapy with melphalan (1.5 mg m-‘) and prednisolone (40 mg m-* ). However, 5 days after the start of chemotherapy, the dog suffered circulatory depression and convulsions, and died, probably due to the hyperviscosity syndrome. Autopsy revealed splenomegaly and a number of small white nodules in the liver and spleen. Microscopic examination revealed proliferation of neoplastic plasma cells in the bone marrow. The growth and differentiation of normal hematopoietic cells were not severely impaired. Infiltration of neoplastic plasma cells was found in the portal triad and around the central vein in the liver. These neoplastic cells were also found in the cortex and medulla of the kidney.
Fig. 2. Neoplastic
plasma cells in the biopsy specimen of the bone marrow. Giemsa stain. X 1000.
164
H. Kato et al. / Veterinary Immunology and Immunopathology 49 (1995) I61 -168
4. Materials and methods 4. I. Immunoelectrophoresis Agarose gel (1.2%) prepared with Tris hydrochloric acid buffer (pH 8.6) of 0.025 ionic strength was used for immunoelectrophoresis of serum protein. After electrophoresis of serum samples from the study dog and a normal control dog, antiserum against dog whole serum (Organon Teknika, Durham, NC), dog IgG-Fc fragment (Bethyl Laboratories, Montgomery, TX), dog IgM (Bethyl Laboratories), or dog IgA (ICN ImmunoBiologicals, Lisle, IL) was pipetted into antiserum troughs cut into the agarose gel. 4.2. Gel filtration
of serum protein
A serum sample diluted with phosphate buffered saline was applied to a Superose 12 column (Pharmacia, Uppsala, Sweden) and fractionated with a fast performance liquid chromatography (FPLC) gel filtration system (Pharmacia). The prepared sample was collected in 32 fractions for further analysis. 4.3. Sodium dodecyl sulfate (SDS) polyacrylamide
gel electrophoresis
(PAGE)
SDS-PAGE under reduced conditions was performed using a 4-20% vertical slab gel (Daiichi Pure Chemicals, Tokyo, Japan) in an electrode buffer containing 0.025 M Tris-HCl (pH 7.61, 0.192 M glycine and 0.1% SDS. Samples dissolved in reducing loading buffer (0.0625 M Tris-HCI, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue (BPB)) were boiled for 3 min and electrophoresed for 1 h. For SDS-PAGE under non-reduced conditions, we used a 2-15% vertical slab gel (Daiichi Pure Chemicals) and samples diluted in the loading buffer without 2mercaptoethanol. After electrophoresis, the gels were stained with Coomassie Blue for 20 min and destained for 1 h. 4.4. Slot blot analysis For extraction of the RNA sample, a bone marrow biopsy specimen that had been stored at - 80°C was homogenized in liquid nitrogen and treated with RNAzol (Biotecx Houston, TX) containing guanidium thiocyanate, phenol and 2Laboratories, mercaptoethanol. The RNA sample was then extracted with chloroform and precipitated with isopropanol. The cellular RNA samples were spotted onto Biodyne B membranes (Pall Biosupport, East Hills, NY) and cross-linked by heating at 80°C for 15 min. The filters were prehybridized in a solution containing 50 mM Tris-HCl (pH 7.61, 5 X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% SDS, 5 X Denhardt solution and 100 mg ml-’ denatured salmon testis DNA for 3 h at 65°C. Hybridization was carried out with 3’P-labelled DNA probes for 18 h. As a lambda chain probe, we used a 0.6kilobase (kb) SacI-EcoRI fragment of clone S6-61 (kindly provided by Dr. S. Tokiyoshi, Chemo-Sero-Therapeutic Research Institute), which is a cDNA clone cover-
165
H. f&to er al. / Veterinaq Immunology and Immunopathology 49 119951161-I68 -
-
+ B
h
^ ^ _ _ . _I . ..
tl
.-_
Fig. 3. (A) Immunoektrophoresis of serum of the study dog against antisera to dog whole serum, IgG, IgM and IgA (1. 2, 3 and 4, respectiyely). Sera of the study dog and a normal dog were pipetted into antiserum troughs ‘a’ and ‘b’, respectively: (B) lmmunoelectrophoresis of Fractions 6 and 12 from serum of the studied dog against anti-dog IgA (Trough 4). Serum Fractions 6 and 12 were pipetted into antiserum troughs ‘c’ and ‘d’, respectively.
ing a constant region of the canine immunoglobulin lambda chain gene. A 2-kb PSI fragment of DEkSa (kindly provided by Dr. S. Tokiyoshil representing a constant region of canine immunoglobulin kappa chain cDNA was used as a kappa chain probe. After hybridization, the filter was washed three times with washing buffer containing 0.5 x SSC and 0.1% SDS at 65°C for 30 min and then exposed to X-ray film at - 80°C for 10 h.
5. Results Sera from the study dog and a normal dog were analyzed by immunoelectrophoresis using anti-dog whole serum, anti-dog IgG, anti-dog IgM and anti-dog IgA (Fig. 3(A)).
COD
280nm)
FlXliWl
+
Fig. 4. (A) Gel filtration of serum protein of the studied dog. Serum of the dog was collected in 32 fractions using a Superose 12 column in an FPLC system. (B) Electrophoresis of Fractions 6 (Lane a). 12 (Lane b). 25 (Lane c) and whole serum (Lane d) of the dog, on a cellulose acetate membrane.
166
H. Kato et al. / Veterinary Immunology
and Immunopathology
49 (1995) 161-168
A B a
h
c
d
(kd)
e
abed
e
669* 440) 232 ) 140)
Fig. 5. Analysis of serum protein of the dog by SDS-PAGE analysis under reduced (A) and non-reduced (B) conditions. Lanes a, b and c are Fractions 6, 12 and 25 of the dog’s serum, respectively; Lane d is the studied dog’s serum; Lane e is the normal dog serum.
Serum from the sick dog showed clear thickening and bowing of the arc in the gamma-globulin zone when antiserum against dog IgA was pipetted into antiserum troughs cut into the agarose gel. No obvious precipitate was found with anti-dog IgM or anti-dog IgG serum in this zone. This result indicated that the major monoclonal peak in the gamma-globulin zone was IgA. Serum of the dog was separated into 32 fractions by gel filtration (Fig. 4(A)), which were subjected to cellulose acetate electrophoresis. As shown in Fig. 4(B), Fractions 6 and 12 were found to correspond to major and minor peaks in the gamma-globulin zone, respectively. On immunoelectrophoresis both Fractions 6 and 12 gave distinct precipitates with anti-dog IgA (Fig. 3(B)). SDS-PAGE analysis of Fractions 6 and 12 under reduced conditions revealed bands of approximately 60 and 29 kDa (Fig. 5(A)). These bands corresponded in size to immunoglobulin heavy and light chains, respectively. On SDS-PAGE analysis under non-reduced conditions, Fractions 6 and 12 gave bands of approximately 520 and 350 kDa, corresponding in size to the trimer or tetramer and dimer of immunoglobulin, respectively (Fig. S(B)). These results indicated that the major and minor peaks in the gamma-globulin zone were those of the dimer and trimer or tetramer of IgA, respectively.
RNAW
5
1
0.2
b Fig. 6. Slot blot analysis of immunoglobulin light chains. were transferred to nylon membrane filters and hybridized chain probes.
RNA samples extracted from bone marrow cells with probes of canine lambda (a) and kappa (b)
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Slot blot analysis using probes of canine immunoglobulin light chains revealed that the mRNA sample from bone marrow cells in this case hybridized with the canine immunoglobulin kappa chain probe, but much less with the canine immunoglobulin lambda chain probe (Fig. 6). A nucleic acid hybridization experiment indicated that the kappa chain was used as an immunoglobulin light chain in myeloma cells of this case.
6. Discussion Diagnosis of multiple myeloma in the present case was based on gammopathy with two M-components and proliferation of amphophilic plasma cells in the bone marrow. No osteolysis or Bence-Jones protein in the urine was observed, although these features are considered to be characteristic of multiple myeloma in dogs (Osborne et al., 1968). In a study on canine multiple myeloma, 56% of the cases were found to have osteolytic lesions and 31% to have Bence-Jones protein in the urine (Dorfman and Dimski, 1992). Conceivably, the presence of osteolytic lesions depends on the osteoclastic activating factor released from myeloma cells (Mundy et al., 1974), and detectable Bence-Jones protein may be due to overproduction of immunoglobulin light chains by myeloma cells (Osborne et al., 1968). The myeloma cells in the present case seemed to be devoid of these characteristics. Gammopathy with two M-components, which is rare in multiple myeloma, was found in the present case. Biochemical analysis indicated that the two M-proteins in this case were dimer and trimer or tetramer of IgA. Only one previous case of gammopathy with two M-components in the dog with multiple myeloma has been reported (Jacobs et al., 19861, but detailed biochemical analysis on this reported case was not shown. In humans, gammopathy with two M-components is reported to occur in approximately 1% of all cases of multiple myeloma (Kyle et al., 1981). The presence of biclonal M-protein may be due to proliferation of two clones of plasma cells, each producing an unrelated monoclonal immunoglobulin, or may result from production of biclonal M-protein by a single clone of plasma cells. Switching of the heavy chain class of immunoglobulin occurs during normal development and differentiation of B lymphocytes (Dreyer and Bennett, 1965; Seidman et al., 1978). In spite of class switching of the heavy chain, the antibody molecules retain the same antigenic specificity. Of human cases of multiple myeloma with biclonal gammopathy, many were shown to have IgG and IgA components, while a few were found to have two IgA proteins (Vaerman et al., 1965a.b; Kyle et al., 1981). Therefore, the biclonal gammopathy with dimer and trimer or tetramer of IgA demonstrated in the present case was considered to be very rare in multiple myeloma. Analysis of serum protein by SDS-PAGE under reduced conditions indicated that both Fractions 6 and 12 gave bands of approximately 60 and 29 kDa, corresponding to the sizes of immunoglobulin heavy and light chains, respectively. On the other hand, heavy and light chains in normal dog serum were shown to be 50 and 25 kDa, respectively, in size. The size differences of heavy and light chains in the serum of the present case from those in normal dog serum were thought to be due to the difference of immunoglobulin class; that is to say, the immunoglobulin class of the present case was
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IgA, whereas that of the normal dog was mainly IgG. Two bands of approximately 60 kDa might be due to some difference in glycosylation of these proteins. Analyzed by SDS-PAGE under non-reduced conditions, Fraction 12 clearly contained two bands, and it might be also due to the difference of glycosylation. The hyperviscosity syndrome is not always observed in cases of multiple myeloma, but is closely associated with serious symptoms in the circulation and hemostasis. The hyperviscosity syndrome is frequently observed in cases of myeloma with high molecular weight M-proteins such as IgM and polymeric IgA. The IgG-associated hyperviscosity syndrome is reported to be rare because IgG has a low molecular weight and is a monomer (Dorfman and Dimski, 1992). In the present case, sudden death was considered to be due to the hyperviscosity syndrome because severe circulation insufficiency was observed before death. The clinical signs in this case were considered to be closely related to large amounts of polymeric IgA with high molecular weights.
Acknowledgments We thank Dr. S. Tokiyoshi (Chemo-Sero-Therapeutic Research Institute) for canine lambda and kappa chain gene probes. This study was supported in part by grants from the Ministry of Education, Science and Culture in Japan.
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