INFECTION AND IMMUNrrY, Nov. 1975, P. 1219-1221 Copyright C) 1975 American Society for Microbiology
Vol. 12, No. 5 Printed in U.SA.
Monocyte Activation in Horses Persistently Infected with Equine Infectious Anemia Virus KEITH L. BANKS1
Institute of Comparative Medicine and Veterinary Science, Washington State University, Pullman, Washington 99163
Received for publication 13 May 1975
The monocytes of horses infected with equine infectious anemia virus were shown by their failure to migrate from capillary tubes and their increased adherence to erythrocytes to be activated.
Equine infectious anemia (EIA) is a virusinduced disease, which may be fatal during acute infection or animals may become persistently viremic for years with an occasional recurrence of acute disease (reviewed in reference 7). The acute disease is characterized by fever, anemia, hepatitis, and increased titers of circulating virus. Horses with chronic disease become hypergammaglobulinemic with lower serum viral titers and develop immune complex glomerulonephritis. Antibody to virus antigens is produced throughout the infection. Several observations suggest that cells of the monocytemacrophage type may be important in the pathogenesis of EIA. The virus replicates in monocytes and macrophages both in vivo and in vitro, and the number of monocytes increases during infection. Macrophages of the spleen and liver often contain phagocytosed erythrocytes or erythrocyte breakdown products, including hemosiderin. These observations led to the study of the functional characteristics of monocytes of horses with EIA. The leukocytes studied were from Shetland ponies infected with the Texas or Wyoming strains of EIA virus and were maintained as previously described (10). Infection was confirmed on the basis of clinical signs and the development of serum antibody to EIA antigens detectable by an immuno-precipitation reaction (7). Horses having a rectal temperature over 101.5 F (ca. 38.6 C) were considered to have the acute disease, whereas afebrile periods were indications of chronic, quiescent disease. Anemia occurred with hematocrit readings of 26 + 6% (standard deviation) during acute infection and 33 + 7% (standard deviation) while in the chronic stages. Noninfected horses had hematocrit readings of 40 + 3% (standard deviation). Five of the horses had been infected for 1 to 3 years and none died during the study. Periph-
eral blood mononuclear cells and neutrophils were isolated by the Hypaque-Ficoll method as has been described for horses (1, 2). The cells were washed three times with Hanks balanced salt solution and counted. Mononuclear cell preparations isolated from normal horses were 1 to 9% neutrophils and 2 to 17% monocytes, and the remaining cells were lymphocytes. In
EIA-infected horses, occasionally more mono-
cytes were observed in the isolated band of cells. These proportions were 1 to 2% neutrophils and 1 to 29% monocytes, and the remaining cells were lymphocytes. Leukocyte preparations rich in neutrophils (98%) were obtained from the pellet of the Hypaque-Ficoll separation. The mononuclear cells were first examined for their ability to migrate in vitro. The isolated cells were packed into a short segment of a capillary tube, and the migration from the tube into a chamber filled with 80% Leibovitz-15 medium with 20% normnal horse serum was measured after 24 h. The details for this test using similar cell populations (3) or peritoneal exudate cells (4) are described elsewhere. Thirteen EIA horses and thirteen normal horses were studied, and each animal was examined one to seven times. A significant decrease in the migration of mononuclear cells (P < 0.001, Student's t test) from horses with EIA was observed when compared with the migration of cells from noninfected horses (Table 1). Neutrophils and monocytes were then tested for the ability to bind erythrocytes. Small chambers were prepared by attaching Lucite rings to cover glasses, and the cell preparations were added to the chambers. After the adherence of monocytes or neutrophils to the glass, nonadherent cells were removed by washing with Hanks balanced salt solution. Previous studies have shown that only monocytes and neutrophils bind to the glass (1). The leukocytes were ' Present address: International Laboratory for Research incubated at 37 C for 1 h with a 1% suspension on Animal Diseases, Nairobi, Kenya. 1219
1220
INFECT. IMMUN.
NOTES
of autologous, unwashed erythrocytes that were collected the same day as the test was performed. Excess erythrocytes were removed by a gentle washing with Hanks balanced salt solution, and the degree of erythrocyte binding to the leukocytes was scored according to the following scale: + + + +, rosettes on 70 and 90% of cells; + + +, erythrocytes on almost every cell and rosettes on about 50% of the cells; + +, erythrocytes on 50% of the cells; and +, erythrocytes on some cells in every high-power field. In 64% of the observations with EIA monocytes, erythrocytes bound to the monocytes (Table 2). The number of erythrocytes bound was + + + or greater in 34% of the observations. With normal horse monocytes, only 14% of the observations were positive and on only one occasion was this above +. In 7% of the tests, erythrocytes bound to neutrophils of EIA horses whereas neutrophils of noninfected horses were always negative. The parameters affecting monocyte adherence to large numbers of erythrocytes were examined further. It was observed that rosette TABLE 1. Migration of mononuclear cells of EIAinfected and noninfected horses
horses
No. of observa-
tionsa
migrationb
6 7 13
15 10 21
0.289 ± 0.13c 0.301 + 0.10c 0.552 ± 0.19
No. of
Disease state
Acute EIA Chronic EIA Noninfected
Area of
a Each horse was examined one to seven times on different days. b The migration of mononuclear leukocytes is determined by projecting the cell pattern onto a screen and measuring the area by planimetry. The pattern was magnified six times by the projection, and the area is expressed in square inches plus or minus one standard deviation. c The area of migration from acute and chronic EIA-infected horses differs from the migration of noninfected horses with a P < 0.001 as determined by Student's t test.
formation was dependent upon incubation at 37 C, whereas at 4 C no adherence occurred. Adherence was not prevented by 50% serum, a procedure that blocks the binding of antibodycoated erythrocytes to the Fc receptor of monocytes (1, 9). Rosettes also were formed when washed erythrocytes of noninfected horses, sheep, or rabbits were incubated with the monocytes. Erythrocytes from horses with reactive monocytes did not bind to normal horse monocytes or neutrophils. Therefore, alterations of the monocytes, not changes in the erythrocytes, were responsible for the rosette formation. The greatest number of rosettes was formed when horses were in the acute stages of the disease. Monocytes from EIA horses had an increased binding to autologous erythrocytes or to erythrocytes from other horses, sheep, and rabbits. In addition, the monocytes migrated more slowly from a cell mass packed by centrifugation in capillary tubes. These findings suggest an increased "stickiness" of the monocytes, a property shown to be characteristic of activated macrophages and monocytes (4, 11, 15). Activation of EIA monocytes may be due to the continuous antigenic stimulation by the persistent virus infection. Macrophages can be activated by products of antigen-stimulated lymphocytes (13, 15) and during the immune response to bacterial (14) or chronic viral infections (5). The significance of activated monocytes in EIA was not determined, although these cells may be partially responsible for the increased extravascular hemolysis that occurs in this disease (10). The involvement of activated macrophages in the pathogenesis of anemia has been suggested by the observation that stimulated macrophages are cytotoxic to syngeneic erythrocytes in vitro (12), and anemia may accompany the stimulation of macrophages in vivo (6). Activated monocytes were present in EIA-infected horses when the anemia was most severe, a stage of the disease during which erythrophagocytosis by circulating monocytes has been observed (7). Although these findings suggest a
TABLE 2. Binding of autologous erythrocytes to monocytes and neutrophils of nornal and EIA-infected horses Leukocytes
Monocytes
Neutrophils
Disease state
EIA Normal EIA Normal
No. of
animals 14 22 14 22
% Observations with erythrocyte binding to leukocytes
No. of observationsa
92 40 74 37
0
+
++
+++
++++
36 86 93 100
16 12 1 0
14 0 0 0
16 2 3 0
18 0 3 0
a Each horse was examined one to eight times with each test done on a different day.
VOL. 12, 1975
NOTES
relationship of altered macrophages and anemia, direct proof of such a relationship remains to be measured. This investigation was supported by Public Health Service grant A1-07471 from the National Institute of Allergy and Infectious Diseases and by the Veterinary Science Research Division, Agricultural Research Service, U.S. Department of Agriculture. I wish to thank Colleen Wong and Kirby Smith for excellent technical assistance and Travis McGuire for discussions during these studies. LITERATURE CITED 1. Banks, K. L., and T. C. McGuire. 1975. Surface receptors on neutrophils and monocytes from immunodeficient and normal horses. Immunology 28:585-597. 2. Banks, K. L., and T. C. McGuire. 1975. Regulation by antibody of phytolectin induced lymphocyte proliferation. I. Evidence for two mechanisms of suppression. J. Immunol. 114:1307-1312. 3. Bull, D. M., J. R. Leibach, M. A. Williams, and R. A. Helms. 1973. Immunity to colon cancer assessed by antigen-induced inhibition of mixed mononuclear cell migration. Science 181:957-959. 4. David, J. R., S. Al-Askari, H. S. Lawrence, and L. Thomas. 1964. Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigen. J. Immunol. 93:264-273. 5. Gledhill, A. W., D. L. J. Bilbey, and J. S. F. Niven. 1965. Effect of certain murine pathogens on phago-
1221
cytic activity. Br. J. Exp. Pathol. 46:433-442. 6. Gorstein, F., and B. Benacerraf. 1960. Hyperactivity of the reticuloendothelial system and experimental anemia in mice. Am. J. Pathol. 37:569-578. 7. Henson, J. B., and T. C. McGuire. 1974. Equine infectious anemia. Prog. Med. Virol. 18:143-159. 8. Henson, J. B., T. C. McGuire, and J. R. Gorham. 1971. The detection of precipitating antibodies in equine infectious anemia and partial purification of the antigen. Arch. Gesamte Virusforsch. 35:385-391. 9. Huber, H., S. D. Douglas, and H. H. Fudenberg. 1969. The IgG receptor: an immunological marker for the characterization of mononuclear cells. Immunology 17:7-21. 10. McGuire, T. C., J. B. Henson, and S. E. Quist. 1969. Viral-induced hemolysis in equine infectious anemia. Am. J. Vet. Res. 30:2091-2097. 11. McLaughlin, J. F., N. H. Ruddle, and B. H. Waksman. 1972. Relationship between activation of peritoneal cells and their cytopathogenicity. RES J. Reticuloendothel. Soc. 12:293-304. 12. Melsom, H., and R. Seljelid. 1973. The cytotoxic effect of mouse macrophages on syngeneic and allogeneic erythrocytes. J. Exp. Med. 137:807-820. 13. Nathan, C. F., M. L. Karnovsky, and J. R. David. 1971. Alterations of macrophage functions by mediators from lymphocytes. J. Exp. Med. 133:1356-1376. 14. North, R. J. 1969. Cellular kinetics associated with the development of acquired cellular resistance. J. Exp. Med. 130:299-311. 15. Rocklin, R. E., C. T. Winston, and J. R. David, 1974. Activation of human blood monocytes by products of sensitized lymphocytes. J. Clin. Invest. 53:559-564.
Vol. 12, No. 5 Printed in U.SA.
Monocyte Activation in Horses Persistently Infected with Equine Infectious Anemia Virus KEITH L. BANKS1
Institute of Comparative Medicine and Veterinary Science, Washington State University, Pullman, Washington 99163
Received for publication 13 May 1975
The monocytes of horses infected with equine infectious anemia virus were shown by their failure to migrate from capillary tubes and their increased adherence to erythrocytes to be activated.
Equine infectious anemia (EIA) is a virusinduced disease, which may be fatal during acute infection or animals may become persistently viremic for years with an occasional recurrence of acute disease (reviewed in reference 7). The acute disease is characterized by fever, anemia, hepatitis, and increased titers of circulating virus. Horses with chronic disease become hypergammaglobulinemic with lower serum viral titers and develop immune complex glomerulonephritis. Antibody to virus antigens is produced throughout the infection. Several observations suggest that cells of the monocytemacrophage type may be important in the pathogenesis of EIA. The virus replicates in monocytes and macrophages both in vivo and in vitro, and the number of monocytes increases during infection. Macrophages of the spleen and liver often contain phagocytosed erythrocytes or erythrocyte breakdown products, including hemosiderin. These observations led to the study of the functional characteristics of monocytes of horses with EIA. The leukocytes studied were from Shetland ponies infected with the Texas or Wyoming strains of EIA virus and were maintained as previously described (10). Infection was confirmed on the basis of clinical signs and the development of serum antibody to EIA antigens detectable by an immuno-precipitation reaction (7). Horses having a rectal temperature over 101.5 F (ca. 38.6 C) were considered to have the acute disease, whereas afebrile periods were indications of chronic, quiescent disease. Anemia occurred with hematocrit readings of 26 + 6% (standard deviation) during acute infection and 33 + 7% (standard deviation) while in the chronic stages. Noninfected horses had hematocrit readings of 40 + 3% (standard deviation). Five of the horses had been infected for 1 to 3 years and none died during the study. Periph-
eral blood mononuclear cells and neutrophils were isolated by the Hypaque-Ficoll method as has been described for horses (1, 2). The cells were washed three times with Hanks balanced salt solution and counted. Mononuclear cell preparations isolated from normal horses were 1 to 9% neutrophils and 2 to 17% monocytes, and the remaining cells were lymphocytes. In
EIA-infected horses, occasionally more mono-
cytes were observed in the isolated band of cells. These proportions were 1 to 2% neutrophils and 1 to 29% monocytes, and the remaining cells were lymphocytes. Leukocyte preparations rich in neutrophils (98%) were obtained from the pellet of the Hypaque-Ficoll separation. The mononuclear cells were first examined for their ability to migrate in vitro. The isolated cells were packed into a short segment of a capillary tube, and the migration from the tube into a chamber filled with 80% Leibovitz-15 medium with 20% normnal horse serum was measured after 24 h. The details for this test using similar cell populations (3) or peritoneal exudate cells (4) are described elsewhere. Thirteen EIA horses and thirteen normal horses were studied, and each animal was examined one to seven times. A significant decrease in the migration of mononuclear cells (P < 0.001, Student's t test) from horses with EIA was observed when compared with the migration of cells from noninfected horses (Table 1). Neutrophils and monocytes were then tested for the ability to bind erythrocytes. Small chambers were prepared by attaching Lucite rings to cover glasses, and the cell preparations were added to the chambers. After the adherence of monocytes or neutrophils to the glass, nonadherent cells were removed by washing with Hanks balanced salt solution. Previous studies have shown that only monocytes and neutrophils bind to the glass (1). The leukocytes were ' Present address: International Laboratory for Research incubated at 37 C for 1 h with a 1% suspension on Animal Diseases, Nairobi, Kenya. 1219
1220
INFECT. IMMUN.
NOTES
of autologous, unwashed erythrocytes that were collected the same day as the test was performed. Excess erythrocytes were removed by a gentle washing with Hanks balanced salt solution, and the degree of erythrocyte binding to the leukocytes was scored according to the following scale: + + + +, rosettes on 70 and 90% of cells; + + +, erythrocytes on almost every cell and rosettes on about 50% of the cells; + +, erythrocytes on 50% of the cells; and +, erythrocytes on some cells in every high-power field. In 64% of the observations with EIA monocytes, erythrocytes bound to the monocytes (Table 2). The number of erythrocytes bound was + + + or greater in 34% of the observations. With normal horse monocytes, only 14% of the observations were positive and on only one occasion was this above +. In 7% of the tests, erythrocytes bound to neutrophils of EIA horses whereas neutrophils of noninfected horses were always negative. The parameters affecting monocyte adherence to large numbers of erythrocytes were examined further. It was observed that rosette TABLE 1. Migration of mononuclear cells of EIAinfected and noninfected horses
horses
No. of observa-
tionsa
migrationb
6 7 13
15 10 21
0.289 ± 0.13c 0.301 + 0.10c 0.552 ± 0.19
No. of
Disease state
Acute EIA Chronic EIA Noninfected
Area of
a Each horse was examined one to seven times on different days. b The migration of mononuclear leukocytes is determined by projecting the cell pattern onto a screen and measuring the area by planimetry. The pattern was magnified six times by the projection, and the area is expressed in square inches plus or minus one standard deviation. c The area of migration from acute and chronic EIA-infected horses differs from the migration of noninfected horses with a P < 0.001 as determined by Student's t test.
formation was dependent upon incubation at 37 C, whereas at 4 C no adherence occurred. Adherence was not prevented by 50% serum, a procedure that blocks the binding of antibodycoated erythrocytes to the Fc receptor of monocytes (1, 9). Rosettes also were formed when washed erythrocytes of noninfected horses, sheep, or rabbits were incubated with the monocytes. Erythrocytes from horses with reactive monocytes did not bind to normal horse monocytes or neutrophils. Therefore, alterations of the monocytes, not changes in the erythrocytes, were responsible for the rosette formation. The greatest number of rosettes was formed when horses were in the acute stages of the disease. Monocytes from EIA horses had an increased binding to autologous erythrocytes or to erythrocytes from other horses, sheep, and rabbits. In addition, the monocytes migrated more slowly from a cell mass packed by centrifugation in capillary tubes. These findings suggest an increased "stickiness" of the monocytes, a property shown to be characteristic of activated macrophages and monocytes (4, 11, 15). Activation of EIA monocytes may be due to the continuous antigenic stimulation by the persistent virus infection. Macrophages can be activated by products of antigen-stimulated lymphocytes (13, 15) and during the immune response to bacterial (14) or chronic viral infections (5). The significance of activated monocytes in EIA was not determined, although these cells may be partially responsible for the increased extravascular hemolysis that occurs in this disease (10). The involvement of activated macrophages in the pathogenesis of anemia has been suggested by the observation that stimulated macrophages are cytotoxic to syngeneic erythrocytes in vitro (12), and anemia may accompany the stimulation of macrophages in vivo (6). Activated monocytes were present in EIA-infected horses when the anemia was most severe, a stage of the disease during which erythrophagocytosis by circulating monocytes has been observed (7). Although these findings suggest a
TABLE 2. Binding of autologous erythrocytes to monocytes and neutrophils of nornal and EIA-infected horses Leukocytes
Monocytes
Neutrophils
Disease state
EIA Normal EIA Normal
No. of
animals 14 22 14 22
% Observations with erythrocyte binding to leukocytes
No. of observationsa
92 40 74 37
0
+
++
+++
++++
36 86 93 100
16 12 1 0
14 0 0 0
16 2 3 0
18 0 3 0
a Each horse was examined one to eight times with each test done on a different day.
VOL. 12, 1975
NOTES
relationship of altered macrophages and anemia, direct proof of such a relationship remains to be measured. This investigation was supported by Public Health Service grant A1-07471 from the National Institute of Allergy and Infectious Diseases and by the Veterinary Science Research Division, Agricultural Research Service, U.S. Department of Agriculture. I wish to thank Colleen Wong and Kirby Smith for excellent technical assistance and Travis McGuire for discussions during these studies. LITERATURE CITED 1. Banks, K. L., and T. C. McGuire. 1975. Surface receptors on neutrophils and monocytes from immunodeficient and normal horses. Immunology 28:585-597. 2. Banks, K. L., and T. C. McGuire. 1975. Regulation by antibody of phytolectin induced lymphocyte proliferation. I. Evidence for two mechanisms of suppression. J. Immunol. 114:1307-1312. 3. Bull, D. M., J. R. Leibach, M. A. Williams, and R. A. Helms. 1973. Immunity to colon cancer assessed by antigen-induced inhibition of mixed mononuclear cell migration. Science 181:957-959. 4. David, J. R., S. Al-Askari, H. S. Lawrence, and L. Thomas. 1964. Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigen. J. Immunol. 93:264-273. 5. Gledhill, A. W., D. L. J. Bilbey, and J. S. F. Niven. 1965. Effect of certain murine pathogens on phago-
1221
cytic activity. Br. J. Exp. Pathol. 46:433-442. 6. Gorstein, F., and B. Benacerraf. 1960. Hyperactivity of the reticuloendothelial system and experimental anemia in mice. Am. J. Pathol. 37:569-578. 7. Henson, J. B., and T. C. McGuire. 1974. Equine infectious anemia. Prog. Med. Virol. 18:143-159. 8. Henson, J. B., T. C. McGuire, and J. R. Gorham. 1971. The detection of precipitating antibodies in equine infectious anemia and partial purification of the antigen. Arch. Gesamte Virusforsch. 35:385-391. 9. Huber, H., S. D. Douglas, and H. H. Fudenberg. 1969. The IgG receptor: an immunological marker for the characterization of mononuclear cells. Immunology 17:7-21. 10. McGuire, T. C., J. B. Henson, and S. E. Quist. 1969. Viral-induced hemolysis in equine infectious anemia. Am. J. Vet. Res. 30:2091-2097. 11. McLaughlin, J. F., N. H. Ruddle, and B. H. Waksman. 1972. Relationship between activation of peritoneal cells and their cytopathogenicity. RES J. Reticuloendothel. Soc. 12:293-304. 12. Melsom, H., and R. Seljelid. 1973. The cytotoxic effect of mouse macrophages on syngeneic and allogeneic erythrocytes. J. Exp. Med. 137:807-820. 13. Nathan, C. F., M. L. Karnovsky, and J. R. David. 1971. Alterations of macrophage functions by mediators from lymphocytes. J. Exp. Med. 133:1356-1376. 14. North, R. J. 1969. Cellular kinetics associated with the development of acquired cellular resistance. J. Exp. Med. 130:299-311. 15. Rocklin, R. E., C. T. Winston, and J. R. David, 1974. Activation of human blood monocytes by products of sensitized lymphocytes. J. Clin. Invest. 53:559-564.
