Recombinant
Human
Erythropoietin,
Supplementation,
Improves
but
Anemia
Not
Iron
in Rats
with Ad j uvant Induced Arthritis Fumitoshi Biological
Research
Laboratories, Received
Asai
Sankyo October
Co.,
and Ltd.,
29, 1990
Takeshi 1-2-58
Oshima
Hiromachi,
Accepted
* Shinagawa-ku,
Tokyo
140, Japan
July 12, 1991
ABSTRACT-Pathophysiological and therapeutic properties of anemia in rats with adjuvant-induced arthritis (AA) were investigated. Both anemia and chronic in flammation were induced in rats by a single injection of Freund's complete adjuvant. This study confirmed other earlier data that these anemic rats with AA had reduced serum iron levels and that the anemia was characterized as mild, non-progressive, hypochromic, microcytic. In addition, our studies showed that these anemic rats had slightly but significantly enhanced erythropoietin titers, but not renal failure; there was no significant difference in blood urea nitrogen and creatinine levels in anemic and normal groups. The anemia in rats with AA was improved by recombinant hu man erythropoietin (r-HuEPO) at 30 and 100 U/kg/day, given i.v. for 5 days. In con trast, iron-chondroitinsulfate colloid (10 mg/kg/day, i.v. for 5 days) failed to improve the anemia and to enhance the effects of r-HuEPO. These data suggest that anemia in rats with adjuvant-induced arthritis is distinguished, pathophysiologically and thera peutically, from iron deficiency anemia, hemolytic anemia, and renal anemia.
Erythropoietin (EPO), the primary humoral regulator of erythropoiesis, is mainly produced in the kidney (1). Since the EPO gene was cloned in 1985 (2, 3), production of large quantities of recombinant human EPO (r HuEPO) has been possible. The main cause of anemia in patients with chronic renal failure involves reduced EPO production in the kid ney (4). The efficacy of r-HuEPO for correct ing this anemia has been established by recent human clinical studies (5-7). Mild to moderate anemia is frequently seen in chronic inflammation, including active rheumatoid arthritis (RA) (8, 9); the patho genesis or the response of this anemia to
r-HuEPO is a matter of controversy (10). Ex perimental adjuvant-induced polyarthritis in the rat shares certain features with human RA, morphologically and therapeutically (11, 12). Lukens et al. (13) first'showed that mild anemia also occurs in rats with adjuvant-in duced arthritis (AA). They also found that anemic rats with AA had reduced serum iron levels, although little is known about the pathogenesis or the response of this anemia to drugs. The purpose of this study was to further in vestigate the pathophysiological and thera peutical properties of anemia in rats with AA.
MATERIALS AND METHODS Animals and treatments Female Lewis rats from Charles River (Japan) that weighed approximately 170 g were used. All rats were maintained at 23 ± 1°C and humidity of 55 ± 5% in animal quar ters controlled to a 12-hr light-dark cycle; they were allowed normal rat chow and tap water ad libitum. The rats were injected in the right hind footpad with Freund's complete adjuvant containing 0.1 mg of heat-killed Mycobacter ium butyricum (Difco) in 0.05 ml paraffin, as described previously (14). Blood (0.5 ml) was collected from the tail vein at various intervals and analyzed with automated blood cell ana lyzers (ELT-8, Ortho, and HEG 120A, Omron). The development of inflammation was quantified by measuring the thickness of adjuvant-injected and non-injected footpads with a dial thickness gauge. The criteria for anemia and arthritis were a 10% reduction of the hemoglobin (Hb) level and a 2-fold in crease in footpad thickness, as compared with normal (adjuvant non-injected) rats. Experimental protocol On day 0, the rats were injected with ad juvant. On day 20, rats found to be anemic and arthritic were randomly divided into groups. r-HuEPO or vehicle was administered intravenously to these rats via the tail vein once a day for 5 days from day 21. On day 40, the rats were anesthetized with sodium pento barbital (35 mg/kg, i.p.), and blood was col lected from the abdominal aorta. Adjuvant non-injected rats were used as normal con trols. In other experiments, the effect of iron sup plementation alone or in combination with r HuEPO on the anemia was determined according to the protocol described above. The dose (10 mg/kg, i.v.) of iron-chondroitin sulfate colloid (Fe sulfate) given as iron sup plementation was chosen according to data on anemic rats with iron deficiency (15).
Measurement Standard hematological techniques were used to analyze the blood, as described pre viously (14); blood cells (RBC, WBC, and platelet count), hematocrit (Ht), Hb, mean corpuscular volume (MCV), and mean cor puscular hemoglobin (MCH) were measured with an automated blood cell counter (ELT-8, Ortho Instrument). Reticulocytes were counted by an automated blood cell analyzer (HEG-120A, Omron) using a whole blood preparation stained with new methylene blue and Wright stain. Plasma and serum were obtained by centrifugation of the blood and stored until assay. Blood urea nitrogen (BUN) and creatinine were determined by an auto analyzer (Type-736, Hitachi). The serum levels of iron, ferritin, and transferrin were measured by respective assay kits (Wako Pure Chemicals). Total iron binding capacity (TIBC) was calculated by iron and transferrin data. Radioimmunoassay of serum EPO levels was conducted according to Egrie et al. (16). 125I-Labeled r-HuEPO was used as a tracer , and antibody was developed in rabbits. Serum from a rat injected with CoC12 (10 ,umol, s.c.) and exposed to hypoxia (0.45 atm) was assayed by exohypoxic mouse (17) to be 5.9 U/ml, and it was used as rat standard EPO. Reagents r-HuEPO used in the present study was pro duced by Chinese hamster ovarian cells in the laboratory of Kirin Brewery Co. (Tokyo), and its activity was assessed as being identical to natural EPO (7, 18). A working solution of r HuEPO was prepared daily and diluted in buf fer (20 mM citrate buffer containing 0.25% human serum albumin and 100 mM NaCl). Iron-chondroitinsulfate colloid for i.v. injec tion (Blutal, Dainippon Pharmaceut. Co.) was diluted with a saline solution. Statistical analysis Data were expressed as the mean ± S.E. Data were analyzed by Student's t-test for un paired observations, and analysis of variance
(ANOVA). A P-value less than 0.05 was con sidered to be statistically significant. RESULTS In the present study, all rats injected with adjuvant developed polyarthritis. Chronic in flammation, determined by the footpad thick ness of the adjuvant-non-injected side, appeared on day 15 and persisted until day 40 (Fig. 1). During this period, erythema and nodules were also observed in the ears, noses, and tails. In addition to this inflammatory re sponse, changes in Hb concentration, MCV, and MCH were observed, with a lag time of
15 days after adjuvant injection. In the first experiment, 27 of 35 rats in jected with adjuvant were found to be anemic on day 20 (Hb levels, 13.8 ± 0.1 g/dl, N = 27 for anemic rats with AA vs. 16.2 ± 0.1 g/dl, N = 13 for normal rats), and the anemia persisted until day 40 (Fig. 1). Table 1 summarizes hematologic data in normal rats and anemic rats with AA. Serum EPO titers and reticulocyte counts in anemic rats with AA were slightly but significantly (P < 0.05) increased compared with normal rats. Anemic rats with AA had a complicated iron state similar to human RA (19): significantly (P < 0.01) decreased serum iron concentration
Fig. 1. Time course of development of chronic inflammatory response and anemia induced by adjuvant in jection to rats. Data are expressed as the mean. 0, normal rats (N = 13); •, anemic rats with AA (N = 27). The standard error, not shown for clarity, was always less than 10% of the mean. * P < 0.05, * * P < 0.01 vs. normal rats. Hb, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglo bin.
Table 1. Hematologic data in normal rats and anemic rats with adjuvant-in duced arthritis
and TIBC, and significantly (P < 0.05) in creased serum ferritin concentration. There was no significant (P > 0.05) difference in BUN and creatinine values between the ane mic and normal groups. In the second experiment, the effect of r HuEPO, at various doses (3 to 100 U/kg, i.v.) administered once a day from day 21 for 5 days, to anemic rats with AA was examined. Figure 2A shows a clear dose-dependent in crease in Ht with r-HuEPO administration, and a statistically significant improvement in Ht was obtained with r-HuEPO at the doses of 30 and 100 U/kg, given i.v. A marked and dose-dependent increase in reticulocytes in
anemic rats treated with r-HuEPO was observed on day 25 (Fig. 2B). Hb, MCV, and MCH were normalized by r-HuEPO in a dose dependent manner (Table 2). Figure 2C shows the effects of iron-chon droitinsulfate colloid (10 mg/kg, i.v.) as iron supplementation, administered alone and in combination with r-HuEPO (30 U/kg, i.v.), on Ht in anemic rats. In contrast to r-HuEPO, iron injection alone failed to elevate Ht in anemic rats. In addition, the hemopoietic effect of r-HuEPO was not altered when administered in combination with iron (Fig. 2C and Table 2). Table 3 summarizes the influence of r
Fig. 2. The dose-response relationship of r-HuEPO with Ht (A) and reticulocytes (B), and the effects of iron supplementation on Ht (C). r-HuEPO (EI: 100, /: 30, eL: 10, A: 3 U/kg, i.v.), iron-chondro itinsulfate colloid (Fe sulfate, 10 mg/kg, i.v.) given alone (•) and in combination (K) with r-HuEPO (30 U/kg, i.v.). r-HuEPO and Fe sulfate were injected i.v. for 5 days from day 21 (arrowed). Data are expressed as the mean. The number of rats used for each group is similar to that in Table 2. The standard error, not shown for clarity, was always less than 10% of the mean. * P < 0.05, * * P < 0.01 vs. the control (0: vehi cle). r-HuEPO, recombinant human erythropoietin; Ht, hematocrit.
HuEPO on blood cells, inflammatory re sponse, and body weight, as determined on day 30, when the hemopoietic effect of r HuEPO was maximal (Fig. 2). Reduced Hb, MCV, and MCH in rats injected with ad juvant were normalized by r-HuEPO in a dose-dependent manner, whereas increased WBC and platelet counts were not affected by r-HuEPO at doses of up to 100 U/kg. In addi tion, neither inflammatory response nor body weight was altered in any group treated with r-HuEPO.
DISCUSSION The experimental model in this study was the rat with adjuvant-induced arthritis, which is commonly used for inflammatory studies be cause of its similarity to human RA (11, 12). In addition to inflammation, the anemia in rats with AA resembled the anemia in patients with chronic inflammation in the following aspects: it was mild, non-progressive, hypo chromic, and microcytic. These two latter characteristics and the low serum iron level, which are in agreement with the findings of
Table 2. Effects of r-HuEPO and iron supplementation induced arthritis
on the anemia in rats with adjuvant
Table 3. Effect of r-HuEPO on WBC, platelets, footpad thickness, and body weight of ane mic rats with adjuvant-induced arthritis
Lukens et al. (13), who first found anemia in rats with AA, may indicate that the anemia was due to iron deficiency. However, our pres ent data have also shown that changes in ferri tin and TIBC in anemic rats with AA were similar to those in human RA, but they were not similar to the findings in iron deficiency
anemia (19). Our therapeutic experiments showed that anemia in rats with AA can be improved by r HuEPO but not iron supplementation. The effects of r-HuEPO were selective on erythro cytes, and neither leukocyte nor platelet counts were affected. The marked and dose
related increase in reticulocyte counts would indicate that the correction of anemia by r HuEPO was due to its erythropoietic action. Masunaga et al. (20) found that neither ane mic rats with iron deficiency nor rats with hemolytic anemia respond to exogenously administered natural EPO. Thus, from a thera peutical view-point, anemia in rats with AA is clearly distinguished from iron deficiency ane mia and hemolytic anemia in rats. Although the anemia in RA is characterized by a slightly increased rate of erythrocyte pro duction (21), results measuring serum EPO titers in RA have been conflicting (8, 9, 22, 23). The serum EPO level measured by RIA in rats with AA was found to be significantly increased in accordance with the increased re ticulocyte count (Table 1). However, the de gree of elevation of EPO titers in anemic rats with AA is modest when compared with the 10 to 1000-fold increases seen in anemic rats with iron deficiency and chemically induced hemolytic anemic rats (20). Renal anemia is characterized by a low EPO titer, which may be due to renal failure, and by improvement with r-HuEPO treatment (5-7, 24). BUN and creatinine, which reflect renal function, were not different between anemic rats with AA and normal rats (Table 1). Therefore, the main cause of the present anemia would seem to be different from that of renal anemia, whereas anemia in rats with AA was improved with r-HuEPO. Improvement of the decreased Ht values in anemic rats by treatment with r-HuEPO was obtained at 30 U/kg/day, given i.v. for 5 days. Hemopoietic effects of r-HuEPO at a similar dose were also demonstrated in normal rats (24). Thus, it is unlikely that reduced sensitiv ity for endogenous EPO or inhibitors of erythropoiesis is a cause of the present ane mia. The hemopoietic effect of r-HuEPO persists for 2 to 3 weeks in normal rats (24), which contrasts with our data in anemic rats with AA (less than 10 days, Fig. 2). The shortened duration of the hemopoietic effects of r HuEPO appears to reflect the shortened life
span of erythrocytes in the present anemia model. We recently have found that anemic rats with AA have reduced erythrocyte defor mability and swelling of the spleen (14). It has been shown that reduced erythrocyte deforma bility results in a shortened half-life span of human erythrocytes (25, 26). Therefore, the present data would be consistent with our pre vious hypothesis that enhanced sequestration of less-deformable erythrocytes by the spleen would play a causative role in the anemia of rats with AA. In summary, the present studies confirmed that a single injection of Freund's complete adjuvant causes anemia and reduced serum iron levels in rats. Our data also provided additional evidence that 1) anemic rats with AA had increased EPO titers, but no renal failure, 2) r-HuEPO, but not iron supple mentation, improves this anemia. These data suggest that anemia in rats with adjuvant-in duced arthritis is distinguished, pathophysio logically and therapeutically, from iron defi ciency anemia, hemolytic anemia, and renal anemia. Acknowledgments The authors wish to thank Dr. E.R. McCluskey for reviewing the manuscript, Dr. N. Nagano for carrying out RIA for serum EPO, and Mrs. T. Shimoji for her excellent technical assistance. REFERENCES
1
2
3
4
Jacobson, L.O., Goldwasser, E., Fried, W. and Plazak, L.: Role of the kidney in erythropoiesis. Nature 179, 633-634 (1957) Lin, F-K., Suggs, S., Lin, C.-H., Browne, J.K., Smalling, R., Egrie, J.C., Chen, K.K., Fox, G., Martin, F., Stabinsky, Z., Badrawe, S.M., Lai, P H. and Goldwasser, E.: Cloning and expression of the human erythropoietin gene. Proc. Natl. Acad. Sci. U.S.A. 82, 7580-7584 (1985) Jacob, K., Shoemaker, C., Rudesdorf, R., Neil, S.D., Kaufman, R.J., Mufson, A., Seehra, J., Jones, S.S., Hewick, R., Frithsh, E.F., Kawakita, M., Shimizu, T. and Miyake, T.: Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313, 806-810 (1985) Adamson, J.W., Eschbach, J.W. and Finch, C.A.:
The kidney and erythropoiesis. Am. J. Med. 44, 725 733 (1968) 5 Winerls, C.G., Oliver, D.O., Pippard, M.J., Reid, C., Downing, M.R. and Cotes, P.M.: Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic hemodialysis. Lancet 2, 1175-1178 (1986) 6 Eschbach, J.W., Egrie, J.C., Downing, M.R., Brone, J.K. and Adamson, J.W.: Correction of the anemia of endo-stage renal disease with re combinant human erythropoietin. N. Engl. J. Med. 316, 73-78 (1987) 7 Urabe, U., Takaku, F., Mizoguchi, H., Kubo, K., Ota, K., Shimizu, N., Tanaka, K., Miura, N. and Nihei, H.: Effect of recombinant human erythro poietin on the anemia of chronic renal failure. Int. J. Cell Cloning 6, 179 -191 (1988) 8 Mowat, A.G.: Hematologic abnormalities in rheumatoid arthritis. Semin. Arthritis Rheum. 1, 195-219 (1971) 9 Cartwright, G.E. and Lee, G. R.: The anemia of chronic disorders. Br. J. Haematol. 21, 147-152 (1971) 10 Birgegard, G.: Erythropoietin and inflammation. In Erythropoietin; From Molecular Structure to Clinical Application, Edited by Baldamus, C.A., Scigalla, P., Wieczoorek, L.W. and Koch, K.M., p. 330-341, Karger, Basel (1989) 11 Billingham, M.E.J.: Models and the search for antiarthritic drugs. Pharmacol. Ther. 21, 389-428 (1983) 12 Rainford, K.D.: Adjuvant polyarthritis in rats: Is this a satisfactory model for screening antiarthritis drugs? Agents Actions 12, 452-458 (1982) 13 Lukens, J.N., Cartwright, G.E. and Winthrobe, M.M.: Anemia of adjuvant-induced inflammation in rats. Proc. Soc. Exp. Biol. Med. 126, 346-349 (1967) 14 Asai, F. and Oshima, T.: Reduced erythrocyte de formability in anemic rats with adjuvant arthritis. Japan. J. Pharmacol. 53, 510-514 (1990) 15 Kurabayashi, M.: Studies on iron metabolism and iron deficiency anemia in pregnant women; theo retical discussion on the relation between serum
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17
18
19 20
21 22
23
24
25
26
iron level and iron dose to be administrated, and clinical result after iron therapy. Acta Obst. Gynaecol. Japon. 32, 833-841 (1980) Egrie, J.C., Cotes, P.M., Lane, J., Das, R.E.G. and Tam, R.C.: Development of radioimmunoas says for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J. Im munol. Methods 99, 235-241 (1987) Adamson, J.W.: The erythropoietin/hematocrit relationship in normal and polycythemic man: implications for marrow regulation. Blood 4, 597 609 (1968) Satake, R., Kubota, M., Wada, T., Watanabe, T., Ukai, S., Inoue, N. and Asano, K.: Structure and physicochemical properties of KRN 5702. Clin. Rep. 22, 5173-5181 (1988) Mowat, A.G.: Connective tissue diseases. Clin. Haematol. 1, 573-594 (1972) Masunaga, H., Murakami, A., Goto, M. and Ueda, M.: Effects of erythropoietin injection on the anemic rats with different serum erythropoietin titer. Japan. J. Vet. Sci. 49, 1-6 (1986) Cartwright, G.E.: The anemia of chronic dis orders. Semin. Hematol. 3, 351-375 (1966) Birgegard, G., Hallgren, R. and Caro, J.: Serum erythropoietin in rheumatoid arthritis and other inflammatory arthritis: relationship to anemia and the effect of anti-inflammatory treatment. Br. J. Haematol. 65, 479-483 (1987) Erslev, A.J., Wilson, J. and Caro, J.: Erythro poietin titers in anemic, nonuremic patients. J. Lab. Clin. Med. 109, 429-433 (1987) Arai, H., Kabatani, K., Nagano, N., Kawagishi, M. and Kusaka, M.: Effects of KRN 5702 on erythropoiesis in normal rats and anemic model rats with renal failure. Clin. Rep. 22, 5531-5546 (1988) Allard, C., Mohandas, N. and Bessis, M.: Red cell deformability changes in hemolytic anemias estimated by diffractometric methods (ektacy tometry). Blood Cells 3, 209-228 (1977) LaCelle, P.L. and Weed, R.I.: The contribution of normal and pathologic erythrocytes to blood rheol ogy. Semin. Hematol. 7, 1-32 (1970)
Human
Erythropoietin,
Supplementation,
Improves
but
Anemia
Not
Iron
in Rats
with Ad j uvant Induced Arthritis Fumitoshi Biological
Research
Laboratories, Received
Asai
Sankyo October
Co.,
and Ltd.,
29, 1990
Takeshi 1-2-58
Oshima
Hiromachi,
Accepted
* Shinagawa-ku,
Tokyo
140, Japan
July 12, 1991
ABSTRACT-Pathophysiological and therapeutic properties of anemia in rats with adjuvant-induced arthritis (AA) were investigated. Both anemia and chronic in flammation were induced in rats by a single injection of Freund's complete adjuvant. This study confirmed other earlier data that these anemic rats with AA had reduced serum iron levels and that the anemia was characterized as mild, non-progressive, hypochromic, microcytic. In addition, our studies showed that these anemic rats had slightly but significantly enhanced erythropoietin titers, but not renal failure; there was no significant difference in blood urea nitrogen and creatinine levels in anemic and normal groups. The anemia in rats with AA was improved by recombinant hu man erythropoietin (r-HuEPO) at 30 and 100 U/kg/day, given i.v. for 5 days. In con trast, iron-chondroitinsulfate colloid (10 mg/kg/day, i.v. for 5 days) failed to improve the anemia and to enhance the effects of r-HuEPO. These data suggest that anemia in rats with adjuvant-induced arthritis is distinguished, pathophysiologically and thera peutically, from iron deficiency anemia, hemolytic anemia, and renal anemia.
Erythropoietin (EPO), the primary humoral regulator of erythropoiesis, is mainly produced in the kidney (1). Since the EPO gene was cloned in 1985 (2, 3), production of large quantities of recombinant human EPO (r HuEPO) has been possible. The main cause of anemia in patients with chronic renal failure involves reduced EPO production in the kid ney (4). The efficacy of r-HuEPO for correct ing this anemia has been established by recent human clinical studies (5-7). Mild to moderate anemia is frequently seen in chronic inflammation, including active rheumatoid arthritis (RA) (8, 9); the patho genesis or the response of this anemia to
r-HuEPO is a matter of controversy (10). Ex perimental adjuvant-induced polyarthritis in the rat shares certain features with human RA, morphologically and therapeutically (11, 12). Lukens et al. (13) first'showed that mild anemia also occurs in rats with adjuvant-in duced arthritis (AA). They also found that anemic rats with AA had reduced serum iron levels, although little is known about the pathogenesis or the response of this anemia to drugs. The purpose of this study was to further in vestigate the pathophysiological and thera peutical properties of anemia in rats with AA.
MATERIALS AND METHODS Animals and treatments Female Lewis rats from Charles River (Japan) that weighed approximately 170 g were used. All rats were maintained at 23 ± 1°C and humidity of 55 ± 5% in animal quar ters controlled to a 12-hr light-dark cycle; they were allowed normal rat chow and tap water ad libitum. The rats were injected in the right hind footpad with Freund's complete adjuvant containing 0.1 mg of heat-killed Mycobacter ium butyricum (Difco) in 0.05 ml paraffin, as described previously (14). Blood (0.5 ml) was collected from the tail vein at various intervals and analyzed with automated blood cell ana lyzers (ELT-8, Ortho, and HEG 120A, Omron). The development of inflammation was quantified by measuring the thickness of adjuvant-injected and non-injected footpads with a dial thickness gauge. The criteria for anemia and arthritis were a 10% reduction of the hemoglobin (Hb) level and a 2-fold in crease in footpad thickness, as compared with normal (adjuvant non-injected) rats. Experimental protocol On day 0, the rats were injected with ad juvant. On day 20, rats found to be anemic and arthritic were randomly divided into groups. r-HuEPO or vehicle was administered intravenously to these rats via the tail vein once a day for 5 days from day 21. On day 40, the rats were anesthetized with sodium pento barbital (35 mg/kg, i.p.), and blood was col lected from the abdominal aorta. Adjuvant non-injected rats were used as normal con trols. In other experiments, the effect of iron sup plementation alone or in combination with r HuEPO on the anemia was determined according to the protocol described above. The dose (10 mg/kg, i.v.) of iron-chondroitin sulfate colloid (Fe sulfate) given as iron sup plementation was chosen according to data on anemic rats with iron deficiency (15).
Measurement Standard hematological techniques were used to analyze the blood, as described pre viously (14); blood cells (RBC, WBC, and platelet count), hematocrit (Ht), Hb, mean corpuscular volume (MCV), and mean cor puscular hemoglobin (MCH) were measured with an automated blood cell counter (ELT-8, Ortho Instrument). Reticulocytes were counted by an automated blood cell analyzer (HEG-120A, Omron) using a whole blood preparation stained with new methylene blue and Wright stain. Plasma and serum were obtained by centrifugation of the blood and stored until assay. Blood urea nitrogen (BUN) and creatinine were determined by an auto analyzer (Type-736, Hitachi). The serum levels of iron, ferritin, and transferrin were measured by respective assay kits (Wako Pure Chemicals). Total iron binding capacity (TIBC) was calculated by iron and transferrin data. Radioimmunoassay of serum EPO levels was conducted according to Egrie et al. (16). 125I-Labeled r-HuEPO was used as a tracer , and antibody was developed in rabbits. Serum from a rat injected with CoC12 (10 ,umol, s.c.) and exposed to hypoxia (0.45 atm) was assayed by exohypoxic mouse (17) to be 5.9 U/ml, and it was used as rat standard EPO. Reagents r-HuEPO used in the present study was pro duced by Chinese hamster ovarian cells in the laboratory of Kirin Brewery Co. (Tokyo), and its activity was assessed as being identical to natural EPO (7, 18). A working solution of r HuEPO was prepared daily and diluted in buf fer (20 mM citrate buffer containing 0.25% human serum albumin and 100 mM NaCl). Iron-chondroitinsulfate colloid for i.v. injec tion (Blutal, Dainippon Pharmaceut. Co.) was diluted with a saline solution. Statistical analysis Data were expressed as the mean ± S.E. Data were analyzed by Student's t-test for un paired observations, and analysis of variance
(ANOVA). A P-value less than 0.05 was con sidered to be statistically significant. RESULTS In the present study, all rats injected with adjuvant developed polyarthritis. Chronic in flammation, determined by the footpad thick ness of the adjuvant-non-injected side, appeared on day 15 and persisted until day 40 (Fig. 1). During this period, erythema and nodules were also observed in the ears, noses, and tails. In addition to this inflammatory re sponse, changes in Hb concentration, MCV, and MCH were observed, with a lag time of
15 days after adjuvant injection. In the first experiment, 27 of 35 rats in jected with adjuvant were found to be anemic on day 20 (Hb levels, 13.8 ± 0.1 g/dl, N = 27 for anemic rats with AA vs. 16.2 ± 0.1 g/dl, N = 13 for normal rats), and the anemia persisted until day 40 (Fig. 1). Table 1 summarizes hematologic data in normal rats and anemic rats with AA. Serum EPO titers and reticulocyte counts in anemic rats with AA were slightly but significantly (P < 0.05) increased compared with normal rats. Anemic rats with AA had a complicated iron state similar to human RA (19): significantly (P < 0.01) decreased serum iron concentration
Fig. 1. Time course of development of chronic inflammatory response and anemia induced by adjuvant in jection to rats. Data are expressed as the mean. 0, normal rats (N = 13); •, anemic rats with AA (N = 27). The standard error, not shown for clarity, was always less than 10% of the mean. * P < 0.05, * * P < 0.01 vs. normal rats. Hb, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglo bin.
Table 1. Hematologic data in normal rats and anemic rats with adjuvant-in duced arthritis
and TIBC, and significantly (P < 0.05) in creased serum ferritin concentration. There was no significant (P > 0.05) difference in BUN and creatinine values between the ane mic and normal groups. In the second experiment, the effect of r HuEPO, at various doses (3 to 100 U/kg, i.v.) administered once a day from day 21 for 5 days, to anemic rats with AA was examined. Figure 2A shows a clear dose-dependent in crease in Ht with r-HuEPO administration, and a statistically significant improvement in Ht was obtained with r-HuEPO at the doses of 30 and 100 U/kg, given i.v. A marked and dose-dependent increase in reticulocytes in
anemic rats treated with r-HuEPO was observed on day 25 (Fig. 2B). Hb, MCV, and MCH were normalized by r-HuEPO in a dose dependent manner (Table 2). Figure 2C shows the effects of iron-chon droitinsulfate colloid (10 mg/kg, i.v.) as iron supplementation, administered alone and in combination with r-HuEPO (30 U/kg, i.v.), on Ht in anemic rats. In contrast to r-HuEPO, iron injection alone failed to elevate Ht in anemic rats. In addition, the hemopoietic effect of r-HuEPO was not altered when administered in combination with iron (Fig. 2C and Table 2). Table 3 summarizes the influence of r
Fig. 2. The dose-response relationship of r-HuEPO with Ht (A) and reticulocytes (B), and the effects of iron supplementation on Ht (C). r-HuEPO (EI: 100, /: 30, eL: 10, A: 3 U/kg, i.v.), iron-chondro itinsulfate colloid (Fe sulfate, 10 mg/kg, i.v.) given alone (•) and in combination (K) with r-HuEPO (30 U/kg, i.v.). r-HuEPO and Fe sulfate were injected i.v. for 5 days from day 21 (arrowed). Data are expressed as the mean. The number of rats used for each group is similar to that in Table 2. The standard error, not shown for clarity, was always less than 10% of the mean. * P < 0.05, * * P < 0.01 vs. the control (0: vehi cle). r-HuEPO, recombinant human erythropoietin; Ht, hematocrit.
HuEPO on blood cells, inflammatory re sponse, and body weight, as determined on day 30, when the hemopoietic effect of r HuEPO was maximal (Fig. 2). Reduced Hb, MCV, and MCH in rats injected with ad juvant were normalized by r-HuEPO in a dose-dependent manner, whereas increased WBC and platelet counts were not affected by r-HuEPO at doses of up to 100 U/kg. In addi tion, neither inflammatory response nor body weight was altered in any group treated with r-HuEPO.
DISCUSSION The experimental model in this study was the rat with adjuvant-induced arthritis, which is commonly used for inflammatory studies be cause of its similarity to human RA (11, 12). In addition to inflammation, the anemia in rats with AA resembled the anemia in patients with chronic inflammation in the following aspects: it was mild, non-progressive, hypo chromic, and microcytic. These two latter characteristics and the low serum iron level, which are in agreement with the findings of
Table 2. Effects of r-HuEPO and iron supplementation induced arthritis
on the anemia in rats with adjuvant
Table 3. Effect of r-HuEPO on WBC, platelets, footpad thickness, and body weight of ane mic rats with adjuvant-induced arthritis
Lukens et al. (13), who first found anemia in rats with AA, may indicate that the anemia was due to iron deficiency. However, our pres ent data have also shown that changes in ferri tin and TIBC in anemic rats with AA were similar to those in human RA, but they were not similar to the findings in iron deficiency
anemia (19). Our therapeutic experiments showed that anemia in rats with AA can be improved by r HuEPO but not iron supplementation. The effects of r-HuEPO were selective on erythro cytes, and neither leukocyte nor platelet counts were affected. The marked and dose
related increase in reticulocyte counts would indicate that the correction of anemia by r HuEPO was due to its erythropoietic action. Masunaga et al. (20) found that neither ane mic rats with iron deficiency nor rats with hemolytic anemia respond to exogenously administered natural EPO. Thus, from a thera peutical view-point, anemia in rats with AA is clearly distinguished from iron deficiency ane mia and hemolytic anemia in rats. Although the anemia in RA is characterized by a slightly increased rate of erythrocyte pro duction (21), results measuring serum EPO titers in RA have been conflicting (8, 9, 22, 23). The serum EPO level measured by RIA in rats with AA was found to be significantly increased in accordance with the increased re ticulocyte count (Table 1). However, the de gree of elevation of EPO titers in anemic rats with AA is modest when compared with the 10 to 1000-fold increases seen in anemic rats with iron deficiency and chemically induced hemolytic anemic rats (20). Renal anemia is characterized by a low EPO titer, which may be due to renal failure, and by improvement with r-HuEPO treatment (5-7, 24). BUN and creatinine, which reflect renal function, were not different between anemic rats with AA and normal rats (Table 1). Therefore, the main cause of the present anemia would seem to be different from that of renal anemia, whereas anemia in rats with AA was improved with r-HuEPO. Improvement of the decreased Ht values in anemic rats by treatment with r-HuEPO was obtained at 30 U/kg/day, given i.v. for 5 days. Hemopoietic effects of r-HuEPO at a similar dose were also demonstrated in normal rats (24). Thus, it is unlikely that reduced sensitiv ity for endogenous EPO or inhibitors of erythropoiesis is a cause of the present ane mia. The hemopoietic effect of r-HuEPO persists for 2 to 3 weeks in normal rats (24), which contrasts with our data in anemic rats with AA (less than 10 days, Fig. 2). The shortened duration of the hemopoietic effects of r HuEPO appears to reflect the shortened life
span of erythrocytes in the present anemia model. We recently have found that anemic rats with AA have reduced erythrocyte defor mability and swelling of the spleen (14). It has been shown that reduced erythrocyte deforma bility results in a shortened half-life span of human erythrocytes (25, 26). Therefore, the present data would be consistent with our pre vious hypothesis that enhanced sequestration of less-deformable erythrocytes by the spleen would play a causative role in the anemia of rats with AA. In summary, the present studies confirmed that a single injection of Freund's complete adjuvant causes anemia and reduced serum iron levels in rats. Our data also provided additional evidence that 1) anemic rats with AA had increased EPO titers, but no renal failure, 2) r-HuEPO, but not iron supple mentation, improves this anemia. These data suggest that anemia in rats with adjuvant-in duced arthritis is distinguished, pathophysio logically and therapeutically, from iron defi ciency anemia, hemolytic anemia, and renal anemia. Acknowledgments The authors wish to thank Dr. E.R. McCluskey for reviewing the manuscript, Dr. N. Nagano for carrying out RIA for serum EPO, and Mrs. T. Shimoji for her excellent technical assistance. REFERENCES
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