Acta haemat. 62: 267-272 (1979)
Ferritin Deposits in Peripheral Blood Lymphocytes of Hodgkin’s Disease Patients Isaac Ben-Bassat, Bracha Ramol, Esther Aghai and Meir Djaldetti Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomer; Electron Microscopy Unit, Hasharon Hospital, Petah Tikva, and Sackler School of Medicine, Tel Aviv University, Tel Aviv
Key Words. Ferritin • Hodgkin’s disease • Lymphocytes Abstract. In view of the reported associations of Hodgkin's disease and ferritin, an elec tron microscopic study of the peripheral blood lymphocytes of these patients was done. In 5 out of the 6 patients studied intracellular ferritin deposits were seen. No such deposits were seen in the lymphocytes of healthy subjects or in those of a patient with ^-thalassemia. The lymphocyte ferritin accumulation in Hodgkin’s disease can arise either from increased synthesis or from phagocytosis.
these observations we undertook an electron microscopic study of peripheral blood lym phocytes of HD patients. Materials and Methods Peripheral blood mononuclear cells were sepa rated on a Ficoll-Hypaque gradient [4] and fixed in cold l"/o glutaraldehyde in phosphate buffer, pH 7.4. The cells were postfixed in osmium tetroxide, dehydrated in graded alcohols and embedded in Epon 812. Thin sections were cut with an LKB ul tratome III and examined with a Philips 300 elec tron microscope.
Patients 6 HD patients were studied: 1 was newly diag nosed and studied before treatment was started
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Several observations reported in recent years have associated Hodgkin’s disease (HD) with ferritin. Elevated serum concen trations of ferritin are consistently found in HD patients and it has been suggested that ferritin is a circulating tumor-associated an tigen [2, 3, 8], A HD-associated antigen, regularly demonstrable in high concentra tions in tumor tissue, has been subsequently identified as ferritin [6J. Recently, splenic tumor cells [14] and peripheral blood lym phocytes [15] have been shown to have an increased ferritin synthesis. We have shown that in HD there is a subpopulation of peripheral blood T lym phocytes that does not form E rosettes but can be unblocked by levamisole and that ferritin is shed from the surface of the treat ed lymphocytes [12, 13]. In view of all
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Fig. 2. A lymphocyte containing iron deposits with high (curved arrows) and lower (straight arrows) electron density. X 62,200.
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Fig. 1. Peripheral blood lym phocyte containing a few iron de posits. X 20,000.
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Ferritin in HI) Lymphocytes
Results No ferritin deposits were seen in the lymphocytes of 50 healthy subjects or in the thalassémie patient. In 5 out of the 6
Fig. 3. Higher magnification of ferritin cluster without limit ing membrane. X 155,400.
Fig. 4. Ferritin cluster with surrounding membrane. X227,125.
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and 5 were in remission, 1-4 years after the com pletion of therapy. At diagnosis 1 patient was in stage I, 4 in stage II, and 1 in stage III. As con trols the lymphocytes of 50 healthy subjects and a patient with homozygous ^-thalassemia with iron overload were studied.
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HD patients, intracellular ferritin deposits were seen in the peripheral lymphocytes. Figure 1 represents a lymphocyte containing a few iron deposits. The ultrastructure of the cell appears quite normal. In some cells (fig. 2) two types of deposits were noted: one with marked electron density, the sec ond with more pronounced granular appear ance. On higher magnification, the iron de posits represented ferritin clusters without (fig. 3), or with (fig. 4) a surrounding mem brane. In 1 patient some of the lymphocytes also contained phagocytized material (fig. 5).
Discussion In previous studies we have demonstrat ed that the immunomodulating drug levamisole raises the E rosette-forming cell num ber of HD patients [13]. By iodination of the lymphocyte membrane followed by levamisole treatment it has been shown that
ferritin is one of the proteins shed from the lymphocyte membrane [12J. It was there fore of interest to see whether ferritin could be detected on the surface of the lympho cyte membrane of these patients. Although membrane-bound ferritin was not detected, quite remarkable intracellular accumulation of ferritin was observed in the lymphocytes of 5 out of 6 HD patients. No ferritin depo sits were seen in the peripheral blood lym phocytes of normal subjects or in the lym phocytes of a homozygous /ï-thalassemia patient with systemic iron overload. As lym phocytes are not reticuloendothelial cells, this finding is not a nonspecific iron accu mulation as seen in chronic disorders. Du mont et al. [5] described marked deposition of iron in involved and uninvolved HD lymph nodes and particularly in those with nodular sclerosis type. The deposits seen by light microscopy were cellular, stromal and in the fibrous tissue. Sarcione et al. [15] have shown that HD peripheral blood lym phocytes synthesize ferritin 4.2 times faster
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Fig. 5. A lymphocyte with a phagocytized body. X 21,800.
and release it 2.4 times faster than normal lymphocytes. No relationship was observed between relative rates of lymphocyte ferritin synthesis and sex, age or pathologic stage of the disease. These data suggest that the iron deposits seen by electron microscopy could be the result of increased synthesis. Howev er, in view of the phagocytic activity of some of these lymphocytes, (fig. 5), pinocytosis or phagocytosis of ferritin by the lym phocytes cannot be excluded. Phagocytic activity by normal peripheral blood lympho cytes [9], acute leukemia lymphoblasts |7| and hairy-cell leukemia cells [16] has been previously described but has not, to the best of our knowledge, been reported in HD lymphocytes. Increased ferritin levels are present in several human tumor cells like hepatoma [1], carcinoma of the breast and pancreas 110], The interesting point in our observa tion is that the iron deposits are found in cells not considered to be malignant and their presence is unrelated to the disease ac tivity. The significance of these morphologic findings to the immunologic abberations in HD is unknown and should be the subject for further study. It should be noted, how ever, that ferritin can significantly suppress the mitogen-induced blastic transformation of normal lymphocytes [ 111.
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References 1 Alpert, E.; Coston, R. L., anti Drysdale, J. W.: Carcino-foelal human liver ferritins. Nature, Lontl. 242: 194-195 (1973). 2 Aungst, C. W.: Ferritin in body fluids. J. Lab. clin. Med. 71: 517-522 (1968). 3 Bieber, C. P. and Bieber, M. M.: Detection of ferritin as a circulating tumor-associated anti-
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gen in Hodgkin's disease. Natn. Cancer Inst. Monogr. 36: 145-157 (1973). Boyurn, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21: suppl. 97, pp. 77-89 (1968). Dumont, A. E.; Ford, R. J., and Becker, F. F.: Siderosis of lymph nodes in patients with Hodgkin’s disease. Cancer 3H: 1247-1252 (1976). Eshhar, Z.; Order, S. E., and Katz. D. H.: Fer ritin, a Hodgkin's disease associated antigen. Proc. natn. Acad. Sci. USA 71: 3956-3960 (1974). Foadi, M. D.; Slater, A. M., and Pegrum, G. D.: Erythrophagocytosis by acute lymphoblas tic leukaemic cells. Scand. J. Haematol. 20: 85-88 (1978). lones, P. A. E.; Miller, F. M.; Worwood, M., and Jacobs, A.: Ferritinaemia in leukaemia and Hodgkin's disease. Br. J. Cancer 27: 212-217 (1973). Koszewski, B. L; Emerick. C. W., and Dicus, D. R.: Studies of phagocytic activity of lym phocytes. III. Phagocytosis of intravenous In dia ink in human subjects. Blood 12: 559-566 (1957). Marcus, D. M. and Zinberg, N.: Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents. Archs Biochem. Biophys. 162: 493-501 (1974). Matzner, Y.; Hershko, C.; Polliack, A.; Konijn, A. M., and Izak, G.: Suppressive effect of fer ritin on in vitro lymphocyte function. Br. J. Haemat. (in press, 1979). Moroz, C.; Lahat, N.; Biniaminov, M., and Ramot, B.: Ferritin on the surface of lympho cytes in Hodgkin's disease patients. A possible blocking substance removed by levamisole. Clin. exp. Immunol. 29: 30-36 (1977). Ramot, B.; Biniaminov, M.; Shoham, C., and Rosenthal, E.: Effect of levamisole on E-rosette forming cells in vivo and in vitro in Hodgkin’s disease. New Engl. J. Med. 294: 809-811 (1976). Sarcione, E. J.; Stutzman, L., and Mittelman, A.: Ferritin synthesis by splenic tumor tissue of Hodgkin’s disease. Experientia 31: 1334-1335 (1975). Sarcione, E. J.; Smalley, J. R.; Lema, M. J., and Stutzman, L.: Increased ferritin synthesis
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Ferritin in HD Lymphocytes
and release by Hodgkin’s disease peripheral blood lymphocytes. Int. J. Cancer 20: 339-346 (1977). 16 Utsinger, P. D.; Jount, W. J.; Fuller, C. R.; Logue, M. J., and Orringer, E. P.: Hairy cell leukemia. B-lymphocyte and phagocytic prop erties. Blood 49: 19-27 (1977).
Ben-Bassat/Ramot/Aghai/Djaldetti
Received: March 13, 1979 Accepted: March 23, 1979
Isaac Ben-Bassat, MD, Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomcr (Israel)
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
272
Ferritin Deposits in Peripheral Blood Lymphocytes of Hodgkin’s Disease Patients Isaac Ben-Bassat, Bracha Ramol, Esther Aghai and Meir Djaldetti Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomer; Electron Microscopy Unit, Hasharon Hospital, Petah Tikva, and Sackler School of Medicine, Tel Aviv University, Tel Aviv
Key Words. Ferritin • Hodgkin’s disease • Lymphocytes Abstract. In view of the reported associations of Hodgkin's disease and ferritin, an elec tron microscopic study of the peripheral blood lymphocytes of these patients was done. In 5 out of the 6 patients studied intracellular ferritin deposits were seen. No such deposits were seen in the lymphocytes of healthy subjects or in those of a patient with ^-thalassemia. The lymphocyte ferritin accumulation in Hodgkin’s disease can arise either from increased synthesis or from phagocytosis.
these observations we undertook an electron microscopic study of peripheral blood lym phocytes of HD patients. Materials and Methods Peripheral blood mononuclear cells were sepa rated on a Ficoll-Hypaque gradient [4] and fixed in cold l"/o glutaraldehyde in phosphate buffer, pH 7.4. The cells were postfixed in osmium tetroxide, dehydrated in graded alcohols and embedded in Epon 812. Thin sections were cut with an LKB ul tratome III and examined with a Philips 300 elec tron microscope.
Patients 6 HD patients were studied: 1 was newly diag nosed and studied before treatment was started
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Several observations reported in recent years have associated Hodgkin’s disease (HD) with ferritin. Elevated serum concen trations of ferritin are consistently found in HD patients and it has been suggested that ferritin is a circulating tumor-associated an tigen [2, 3, 8], A HD-associated antigen, regularly demonstrable in high concentra tions in tumor tissue, has been subsequently identified as ferritin [6J. Recently, splenic tumor cells [14] and peripheral blood lym phocytes [15] have been shown to have an increased ferritin synthesis. We have shown that in HD there is a subpopulation of peripheral blood T lym phocytes that does not form E rosettes but can be unblocked by levamisole and that ferritin is shed from the surface of the treat ed lymphocytes [12, 13]. In view of all
268
Ben-Bassat Ramot/Aghai/Djaldetti
Fig. 2. A lymphocyte containing iron deposits with high (curved arrows) and lower (straight arrows) electron density. X 62,200.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Fig. 1. Peripheral blood lym phocyte containing a few iron de posits. X 20,000.
269
Ferritin in HI) Lymphocytes
Results No ferritin deposits were seen in the lymphocytes of 50 healthy subjects or in the thalassémie patient. In 5 out of the 6
Fig. 3. Higher magnification of ferritin cluster without limit ing membrane. X 155,400.
Fig. 4. Ferritin cluster with surrounding membrane. X227,125.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
and 5 were in remission, 1-4 years after the com pletion of therapy. At diagnosis 1 patient was in stage I, 4 in stage II, and 1 in stage III. As con trols the lymphocytes of 50 healthy subjects and a patient with homozygous ^-thalassemia with iron overload were studied.
270
Ben-Bassat/Ramot/Aghai/Djaldetti
HD patients, intracellular ferritin deposits were seen in the peripheral lymphocytes. Figure 1 represents a lymphocyte containing a few iron deposits. The ultrastructure of the cell appears quite normal. In some cells (fig. 2) two types of deposits were noted: one with marked electron density, the sec ond with more pronounced granular appear ance. On higher magnification, the iron de posits represented ferritin clusters without (fig. 3), or with (fig. 4) a surrounding mem brane. In 1 patient some of the lymphocytes also contained phagocytized material (fig. 5).
Discussion In previous studies we have demonstrat ed that the immunomodulating drug levamisole raises the E rosette-forming cell num ber of HD patients [13]. By iodination of the lymphocyte membrane followed by levamisole treatment it has been shown that
ferritin is one of the proteins shed from the lymphocyte membrane [12J. It was there fore of interest to see whether ferritin could be detected on the surface of the lympho cyte membrane of these patients. Although membrane-bound ferritin was not detected, quite remarkable intracellular accumulation of ferritin was observed in the lymphocytes of 5 out of 6 HD patients. No ferritin depo sits were seen in the peripheral blood lym phocytes of normal subjects or in the lym phocytes of a homozygous /ï-thalassemia patient with systemic iron overload. As lym phocytes are not reticuloendothelial cells, this finding is not a nonspecific iron accu mulation as seen in chronic disorders. Du mont et al. [5] described marked deposition of iron in involved and uninvolved HD lymph nodes and particularly in those with nodular sclerosis type. The deposits seen by light microscopy were cellular, stromal and in the fibrous tissue. Sarcione et al. [15] have shown that HD peripheral blood lym phocytes synthesize ferritin 4.2 times faster
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Fig. 5. A lymphocyte with a phagocytized body. X 21,800.
and release it 2.4 times faster than normal lymphocytes. No relationship was observed between relative rates of lymphocyte ferritin synthesis and sex, age or pathologic stage of the disease. These data suggest that the iron deposits seen by electron microscopy could be the result of increased synthesis. Howev er, in view of the phagocytic activity of some of these lymphocytes, (fig. 5), pinocytosis or phagocytosis of ferritin by the lym phocytes cannot be excluded. Phagocytic activity by normal peripheral blood lympho cytes [9], acute leukemia lymphoblasts |7| and hairy-cell leukemia cells [16] has been previously described but has not, to the best of our knowledge, been reported in HD lymphocytes. Increased ferritin levels are present in several human tumor cells like hepatoma [1], carcinoma of the breast and pancreas 110], The interesting point in our observa tion is that the iron deposits are found in cells not considered to be malignant and their presence is unrelated to the disease ac tivity. The significance of these morphologic findings to the immunologic abberations in HD is unknown and should be the subject for further study. It should be noted, how ever, that ferritin can significantly suppress the mitogen-induced blastic transformation of normal lymphocytes [ 111.
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References 1 Alpert, E.; Coston, R. L., anti Drysdale, J. W.: Carcino-foelal human liver ferritins. Nature, Lontl. 242: 194-195 (1973). 2 Aungst, C. W.: Ferritin in body fluids. J. Lab. clin. Med. 71: 517-522 (1968). 3 Bieber, C. P. and Bieber, M. M.: Detection of ferritin as a circulating tumor-associated anti-
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gen in Hodgkin's disease. Natn. Cancer Inst. Monogr. 36: 145-157 (1973). Boyurn, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21: suppl. 97, pp. 77-89 (1968). Dumont, A. E.; Ford, R. J., and Becker, F. F.: Siderosis of lymph nodes in patients with Hodgkin’s disease. Cancer 3H: 1247-1252 (1976). Eshhar, Z.; Order, S. E., and Katz. D. H.: Fer ritin, a Hodgkin's disease associated antigen. Proc. natn. Acad. Sci. USA 71: 3956-3960 (1974). Foadi, M. D.; Slater, A. M., and Pegrum, G. D.: Erythrophagocytosis by acute lymphoblas tic leukaemic cells. Scand. J. Haematol. 20: 85-88 (1978). lones, P. A. E.; Miller, F. M.; Worwood, M., and Jacobs, A.: Ferritinaemia in leukaemia and Hodgkin's disease. Br. J. Cancer 27: 212-217 (1973). Koszewski, B. L; Emerick. C. W., and Dicus, D. R.: Studies of phagocytic activity of lym phocytes. III. Phagocytosis of intravenous In dia ink in human subjects. Blood 12: 559-566 (1957). Marcus, D. M. and Zinberg, N.: Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents. Archs Biochem. Biophys. 162: 493-501 (1974). Matzner, Y.; Hershko, C.; Polliack, A.; Konijn, A. M., and Izak, G.: Suppressive effect of fer ritin on in vitro lymphocyte function. Br. J. Haemat. (in press, 1979). Moroz, C.; Lahat, N.; Biniaminov, M., and Ramot, B.: Ferritin on the surface of lympho cytes in Hodgkin's disease patients. A possible blocking substance removed by levamisole. Clin. exp. Immunol. 29: 30-36 (1977). Ramot, B.; Biniaminov, M.; Shoham, C., and Rosenthal, E.: Effect of levamisole on E-rosette forming cells in vivo and in vitro in Hodgkin’s disease. New Engl. J. Med. 294: 809-811 (1976). Sarcione, E. J.; Stutzman, L., and Mittelman, A.: Ferritin synthesis by splenic tumor tissue of Hodgkin’s disease. Experientia 31: 1334-1335 (1975). Sarcione, E. J.; Smalley, J. R.; Lema, M. J., and Stutzman, L.: Increased ferritin synthesis
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
Ferritin in HD Lymphocytes
and release by Hodgkin’s disease peripheral blood lymphocytes. Int. J. Cancer 20: 339-346 (1977). 16 Utsinger, P. D.; Jount, W. J.; Fuller, C. R.; Logue, M. J., and Orringer, E. P.: Hairy cell leukemia. B-lymphocyte and phagocytic prop erties. Blood 49: 19-27 (1977).
Ben-Bassat/Ramot/Aghai/Djaldetti
Received: March 13, 1979 Accepted: March 23, 1979
Isaac Ben-Bassat, MD, Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomcr (Israel)
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/13/2019 9:26:51 AM
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