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Effect of free radicals on lymphocyte response to mitogens and rosette formation
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
19, 319-324 (1981)
Effect of Free Radicals on Lymphocyte Response and Rosette Formation Y. Department
NISHIDA,
of Medicine
K. TANIMOTO,
to Mitogens
AND I. AKAOKA
and Physical Therapy, Faculty Bunkyo-ku 113, Japan
of Medicine,
University
of Tokyo,
Received August 11, 1980 When normal human lymphocytes were exposed to hypoxanthine plus xanthine oxidase system in vitro, the blastogenic response of lymphocytes to mitogens was markedly suppressed. The lymphocytes which respond to Con A were most sensitive to toxic effects of free radicals. In contrast, lymphocyte blastogenesis induced by PWM was less affected by free radicals. Superoxide dismutase was relatively ineffective in preventing the toxicity of free radicals on lymphocyte blastogenesis induced by PHA. In contrast, catalase showed a preventive effect on the oxidase-induced suppression of lymphocyte response to PHA. Uric acid plus uricase also suppressed blastogenic response of the lymphocytes to PHA. The suppressive effects of uric acid plus uricase on lymphocyte proliferation were reversed by catalase. Furthermore, hypoxanthine plus xanthine oxidase were demonstrated to have effects on the lymphocyte membrane receptors, by markedly inhibiting E rosettes; EAC rosettes were only slightly inhibited. These results suggest that H,O, suppresses lymphocyte function through damage to the cell membrane, and that T cells are more sensitive than B cells to the toxic effects of free radicals.
INTRODUCTION
The bacteriacidal effect of the superoxide radical (0;) in leukocytes is well established (l-4). Furthermore, several lines of evidence indicate that 0; and hydrogen peroxide (H202) are produced at the membrane surface of granulocytes and monocytes during phagocytosis and are released into the surrounding medium (5 12). Released 0; and or k,O, may participate in damage to surrounding tissues and other blood cells. The participation of the toxic effect of 0; in the inflammatory arthritis was suggested by Proctor ef al. (13). McCord has observed that 0; can produce degradation of purified hyaluronic acid and bovine synovial fluid (14) and also showed that synovial fluid contains only a negligible amount of the protective enzyme, superoxide dismutase. Handin et al. observed that 0; induced aggregation of human platelets and resulted in serotonine release (15). Because of these findings, it is reasonable to suggest that free radicals may also interfere with lymphocyte function in synovial fluid. In the present study we investigated the effect of free radicals on lymphocyte blastogenesis induced by plant mitogens in vitro. To clarify the mechanism of action of free radicals, the capacity of lymphocytes treated with hypoxanthine and xanthine oxidase to form rosettes with sheep red cells (E-rosette) and ox red cells treated with IgM antibody and complement (EAC-rosette) was studied. The results indicate that free radicals interfere with mitogen-induced lymphocyte proliferation and with binding of sheep red cells to T-lymphocyte membrane receptors. 319 009O-1229/81/060319-06$01.00/0 Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
320
NISHIDA,
TANIMOTO,
MATERIALS
AND
AKAOKA
AND METHODS
Preparation of lymphocytes. Thirty milliliters of venous blood was collected from a healthy subject in a heparinized syringe. The lymphocytes were separated Ficoll-Hypaque density centrifugation (16) and washed three times in excess amounts of minimum essential medium (Grand Island Biological Company, Grand Island, N.Y.). The lymphocytes, suspended at 1 x lo6 cells/ml in RPM1 1640 medium (Gibco) or in RPM1 1640 medium containing 1 mM hypoxanthine were treated with from 0.25 to 5.0 ~1 of xanthine oxidase (8 U/m, P-L Biochemicals Inc., Milwaukee, Wise.) for 40 min at room temperature. Protective effects of scavengers of 0, and H,O, on the lymphocyte proliferation of activated oxygen toxicity were also studied. From 1 to 15 mg of superoxide dismutase (5500 U/mg, Mills Corp., South Africa), a scavenger of O;, and/or 25 pl(6.25 mg) of catalase (34000 U/mg protein, Sigma, St. Louis, MO.) was added to the lymphocyte suspension containing 1 mM hypoxanthine and 0.5 ~1 of xanthine oxidase. Other lymphocytes suspended in RPM1 1640 medium or RPM1 1640 medium containing 1 mM uric acid were treated with 20 or 30 ~1 of uricase (1.5 U/mg protein/O.5 ml, Seikagaku Kogyo Co., Tokyo, Japan) with or without 25 ~1 of catalase. Untreated lymphocytes were used as controls. After incubation, the lymphocytes were washed twice in RPM1 1640 medium and were resuspended at 1 x lo6 cells/ml in culture medium. Cell culture. Cultures were carried out in sterile microplates with round-bottom wells and were done in triplicate with 1 x 105 responding cells in 100 ~1 of RPM1 1640 containing 10% fetal calf serum, 100 units/ml penicillin, 100 &ml streptomycin, and 100 ~1 of mitogens, which were dissolved at appropriate concentrations in culture medium as follows: 4 pg/ml of phytohemagglutinin (PHA; Difco Laboratories, Detroit, Mich.), 5 pg/ml of concanavalin A (Con A: Pharmacia Fine Chemicals, Uppsala, Sweden), and 1:lOO diluted pokeweed mitogen (PWM: Grand Island Biological Company). The preparations were incubated for 96 hr at 37°C in a humidified atmosphere with 5% CO, and 95% air. Following 72 hr of culture, 1 PCi of [3H]thymidine (2 Ci/mmol: Radiochemical Centre, Amersham, England) was added to each well. Cells were harvested on glass fiber filter paper using a Millipore semiautomated harvester. Radioactivities of thymidine incorporated into lymphocyte DNA were measured in a liquid scintillation counter and they were expressed as counts per minute. Rosette formation. The binding of sheep red cells to lymphocyte membrane receptors was determined by rosette formation, as described by Jondal et al. (17). Lymphocytes (3.5 x lo6 cells/ml) treated with hypoxanthine-plus-xanthine oxidase system were suspended in 100 ~1 of fetal calf serum. The lymphocyte suspension was added to an equal volume of a 1% suspension of neuraminidase pretreated sheep red blood cells (E) or a suspension of ox red blood cells coated with antibody (IgM) and complement (EAC). The mixtures were incubated at 37°C for 30 min, centrifuged at 2OOg (5 min), and stored at 4°C for 1 hr. The cell pellet was gently resuspended, and the percentage of rosette-forming cells was measured microscopically by counting at least 200 lymphocytes. Lymphocytes surrounded with at least three erythrocytes were counted as positive rosette-forming cells. Each experiment was carried out in duplicate. Untreated lymphocytes were used as controls.
FREE RADICALS
321
AND LYMPHOCYTES
Viability studies. The viability of lymphocytes at several hours and 4 days after exposure to O;, H,Oo, xanthine oxidase, and uricase was examined by the trypan blue exclusion method. Cells were mixed with 1% solution of trypan blue and examined microscopically. The percentage of cells excluding the dye is represented as the percentage of viable cells. RESULTS
The viability of the lymphocytes tested by trypan blue staining was more than 95% several hours after the exposure to O;, HzOz, xanthine oxidase, and uricase. In thymidine uptake assays, the viability of the cells which were exposed to hypoxanthine-plus-xanthine oxidase system and uric acid-plus-uricase system and were incubated 4 days in the culture medium were 12.2 to 29.3% and 9.4 to 33.6% respectively. The viability was dependent on the concentration of xanthine oxidase and uricase. The effects of xanthine oxidase alone or hypoxanthine plus xanthine oxidase on mitogen-induced blastogenesis are shown in Fig. 1. Large doses of xanthine oxidase suppressed the blastogenic responses of the lymphocytes to mitogens. Free radicals in high concentrations were found to have an inhibitory effect on the lymphocyte proliferative response to mitogens. Lymphocyte response to Con A was most markedly suppressed by free radicals. In contrast, cells stimulated with PWM seemed to be resistant to the suppressive effects of this system. The results of reversal of free radical toxicity on lymphocyte proliferation induced with PHA are shown in Fig. 2. Superoxide dismutase was relatively ineffective in reversing the toxicity of hypoxanthine-plus-xanthine oxidase system on lymphocyte blastogenesis-induced with PHA. In contrast, suppression of lymphocyte proliferation was reversed by catalase. Moreover, blastogenesis in response to PHA of the lymphocytes treated with uric acid plus uricase system was also suppressed. The suppressing effects of uric acid plus uricase system on lymphocyte blastogenesis was reversed by addition of catalase. The results of treatment with xanthine oxidase alone or hypoxanthine plus PHA
0. 25
u. z
2. 5
5.0
0.25
0.5
2. 5
5.0
0.25
CL5
2. 5
5.0
111
FIG. I. Effect of xanthine oxidase alone and hypoxanthine-plus-xanthine oxidase on lymphocyte blastogenesis induced with mitogens. Lymphocytes were pretreated with xanthine oxidase alone (0) and, xanthine oxidase plus 1 mkf hypoxanthine (0).
322
NISHIDA,
HypDxanthme + X.0
COlltrOl
TANIMOTO,
Hypoxanlhme + x.0 + 5.0.0. 2. 5 mg
AND
Hy,mxanthlne
urlcase
AKAOKA
mp1
“,I‘
arld
Uric
+ x.
0
Catalare
“mare
.md
Uric
+ 2op1
UrltaU
.olr,
dse
aid 2OPl
+ Catalare
25p,
25 PI
FIG. 2. Reversal of free radical toxicity on lymphocyte proliferation induced with PHA by superoxide dismutase and/or catalase. Lymphocytes were suspended for 45 min in the RPM1 1640 medium containing 1 m&f hypoxanthine plus 2.5 ~1 of xanthine oxidase and 1 mg of superoxide dismutase and/or 25 ~1 of catalase. Other lymphocytes were treated for 45 min with RPM1 1640 medium containing uric acid plus uricase with or without 25 ~1 of catalase. These lymphocytes were washed three times and used for cell cultures.
xanthine oxicase on rosette formation are shown in Fig. 3. The absolute percentages of rosette-forming cells in the untreated lymphocytes were E:EAC = 75.8: 14.7%. A marked reduction of E-rosette formation was seen when the lymphocytes were exposed to these systems. The percentage of EAC-rosette formation of lymphocytes treated with these systems was only slightly decreased. E rosette
0.25
0. 5
2. 5
EAC rosette
5. 0 Concentration
0.25
0.5
2. 5
of
xanthine
oxidase
5.0
UI
3. Effect of xanthine oxidase alone and hypoxanthine plus xanthine oxidase on E- and EACrosette formation. Lymphocytes were pretreated with xanthine oxidase alone (0) and xanthine oxidase plus 1 n&Z hypoxanthine (0). These lymphocytes were washed three times and used for rosette formation. FIG.
FREE
RADICALS
AND
LYMPHOCYTES
323
DISCUSSION
In the present study, xanthine oxidase was shown to have various effects on lymphocyte responsiveness to plant mitogens. E- and EAC-rosette formation was also inhibited by pretreatment with large doses of xanthine oxidase. The mechanisms of the effects of xanthine oxidase on lymphocyte function remains unclear. However, the differences between the actions of xanthine oxidase and those of xanthine oxidase plus hypoxanthine on lymphocyte function may be explained by the specific effect of free radicals. Similarly, the differences between the action of uricase and those of uricase plus uric acid may represent the specific effect of HA Our results showed that free radicals inhibited the lymphocyte response to some mitogens. The lymphocytes which respond to Con A were most sensitive to toxic effects of free radicals. On the other hand PWM-stimulated lymphocytes were less affected by free radicals. Furthermore, free radicals demonstrated effects on lymphocyte membranes, markedly inhibiting E- and slightly inhibiting EAC-rosette formation. The viability of the lymphocytes was not reduced shortly after exposure to free radicals, but dropped markedly within a few days of cell cultures. Superoxide dismutase had relatively little effect on the toxicity of these systems to lymphocyte blastogenesis. H,O, is also suppressive of lymphocyte proliferation produced by the uric acid plus uricase system. The preventive effects of catalase on the free radical-induced suppression of lymphocyte proliferation with mitogens suggests the toxic effects are due to H,O,. We are in agreement with the previous findings of Sagone ef al. (18) concerning the toxic effects of H& on lymphocyte blastogenesis. We assume further that Hz02 suppresses mainly T-cell function and that one of the sites of the action of activated oxygen is the outer surface of the lymphocyte membrane. Many biologic factors have been described that are capable of affecting lymphocyte function. Among many factors possibly present in the synovial fluids, immune complexes, activated complement, and lysosomal enzymes from polymorphonuclear leukocytes are known to alter the lymphocyte function (19-21). Lymphocytes obtained from the synovial fluids of patients with rheumatoid arthritis have been found to have diminished response to plant mitogens (22). It is possible that free radicals also interfere with lymphocyte function in the synovial fluids in such patients. The biological importance of these free radicals on lymphocyte function should be further investigated. REFERENCES I. Fridovich, I., N. Engl. J. Med. 290, 624, 1974. 2. Johnston, R. B. Jr.. Keele, B. B., Jr., Misra, H. P., Lehmeyer, J. E., Webb, L. S., Baehner, R. L.. and Rajagopalan, K. V., J. C/in. Invest. 55, 1357, 1975. 3. Klebanoff, S. J., J. Biol. Chem. 249, 3724, 1974. 4. Babior, B. M., Cumutte, J. T., and Kipnes, R. S., J. Lab. Clin. Med. 85, 235, 1975. 5. Curnutte, J. T., Whitten, D. M., and Babior, B. M., N. Engl. J. Med. 290, 593, 1974. 6. Babior, B. M., Kipnes, R. S.; and Curnutte, J. T., J. Clin. Invest. 52, 741, 1973. 7. Sahn, M. L., and McCord, J. M., J. Clin. Invest. 54, 1005, 1974. 8. Goldstein, I. M., Cerqueira, M., Lind, S., and Kaplan, H. B., J. Clin. Invest. 59, 249, 1977. 9. Weening, R. S., Wever, R., and Roos, D., J. Lab. Clin. Med. 85, 245, 1975. 10. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissmann, G., J. Clin. Invest. 56, 1155, 1975.
324 11. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22.
NISHIDA,
TANIMOTO,
AND
AKAOKA
Root, R. K., Metcalf, J., Oshino, N., and Chance, B., .I. Clin. Invest. 55, 945, 1975. Salin, M. L., and McCord, J. M., .I. C/in. Znvesr. 56, 1319, 1975. Proctor, P. H., Kirkpatrick, D. S., and McGinness, J. E., Lancer 2, 95. 1978. McCord, J. M., Science 185, 529, 1974. Handin, R. I., Karabin, R., and Boxer, G. J., .I. Clin. Invest. 59, 959, 1977. Boyum, A., Stand. J. Clin. Lab. Invest. 21, (Suppl. 97), 1968. Jondal, M., Holm, G., and Wigzell, H., 1. Exp. Med. 136, 207, 1972. Sagone, A. L., Kamps, S., and Campbell, R., Photo&m. Photobiol. 28, 909, 1978. Bloch-Shtacher, N., Hirschhorn, K., and Uhr, J. W., C(in. Exp. Zmmunot. 3, 889, 1968 Vischer, T. L., Bretz, U., and Baggiolini, M., .I. Exp. Med. 144, 863, 1976. Yamasaki, K., and Ziff, M., Clin. Exp. Immunol,. 27, 254, 1977. Sheldon, P. J., Papamichail, M., and Halborow, E. J., Ann. Rheum. Dis. 33, 509, 1974.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
19, 319-324 (1981)
Effect of Free Radicals on Lymphocyte Response and Rosette Formation Y. Department
NISHIDA,
of Medicine
K. TANIMOTO,
to Mitogens
AND I. AKAOKA
and Physical Therapy, Faculty Bunkyo-ku 113, Japan
of Medicine,
University
of Tokyo,
Received August 11, 1980 When normal human lymphocytes were exposed to hypoxanthine plus xanthine oxidase system in vitro, the blastogenic response of lymphocytes to mitogens was markedly suppressed. The lymphocytes which respond to Con A were most sensitive to toxic effects of free radicals. In contrast, lymphocyte blastogenesis induced by PWM was less affected by free radicals. Superoxide dismutase was relatively ineffective in preventing the toxicity of free radicals on lymphocyte blastogenesis induced by PHA. In contrast, catalase showed a preventive effect on the oxidase-induced suppression of lymphocyte response to PHA. Uric acid plus uricase also suppressed blastogenic response of the lymphocytes to PHA. The suppressive effects of uric acid plus uricase on lymphocyte proliferation were reversed by catalase. Furthermore, hypoxanthine plus xanthine oxidase were demonstrated to have effects on the lymphocyte membrane receptors, by markedly inhibiting E rosettes; EAC rosettes were only slightly inhibited. These results suggest that H,O, suppresses lymphocyte function through damage to the cell membrane, and that T cells are more sensitive than B cells to the toxic effects of free radicals.
INTRODUCTION
The bacteriacidal effect of the superoxide radical (0;) in leukocytes is well established (l-4). Furthermore, several lines of evidence indicate that 0; and hydrogen peroxide (H202) are produced at the membrane surface of granulocytes and monocytes during phagocytosis and are released into the surrounding medium (5 12). Released 0; and or k,O, may participate in damage to surrounding tissues and other blood cells. The participation of the toxic effect of 0; in the inflammatory arthritis was suggested by Proctor ef al. (13). McCord has observed that 0; can produce degradation of purified hyaluronic acid and bovine synovial fluid (14) and also showed that synovial fluid contains only a negligible amount of the protective enzyme, superoxide dismutase. Handin et al. observed that 0; induced aggregation of human platelets and resulted in serotonine release (15). Because of these findings, it is reasonable to suggest that free radicals may also interfere with lymphocyte function in synovial fluid. In the present study we investigated the effect of free radicals on lymphocyte blastogenesis induced by plant mitogens in vitro. To clarify the mechanism of action of free radicals, the capacity of lymphocytes treated with hypoxanthine and xanthine oxidase to form rosettes with sheep red cells (E-rosette) and ox red cells treated with IgM antibody and complement (EAC-rosette) was studied. The results indicate that free radicals interfere with mitogen-induced lymphocyte proliferation and with binding of sheep red cells to T-lymphocyte membrane receptors. 319 009O-1229/81/060319-06$01.00/0 Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
320
NISHIDA,
TANIMOTO,
MATERIALS
AND
AKAOKA
AND METHODS
Preparation of lymphocytes. Thirty milliliters of venous blood was collected from a healthy subject in a heparinized syringe. The lymphocytes were separated Ficoll-Hypaque density centrifugation (16) and washed three times in excess amounts of minimum essential medium (Grand Island Biological Company, Grand Island, N.Y.). The lymphocytes, suspended at 1 x lo6 cells/ml in RPM1 1640 medium (Gibco) or in RPM1 1640 medium containing 1 mM hypoxanthine were treated with from 0.25 to 5.0 ~1 of xanthine oxidase (8 U/m, P-L Biochemicals Inc., Milwaukee, Wise.) for 40 min at room temperature. Protective effects of scavengers of 0, and H,O, on the lymphocyte proliferation of activated oxygen toxicity were also studied. From 1 to 15 mg of superoxide dismutase (5500 U/mg, Mills Corp., South Africa), a scavenger of O;, and/or 25 pl(6.25 mg) of catalase (34000 U/mg protein, Sigma, St. Louis, MO.) was added to the lymphocyte suspension containing 1 mM hypoxanthine and 0.5 ~1 of xanthine oxidase. Other lymphocytes suspended in RPM1 1640 medium or RPM1 1640 medium containing 1 mM uric acid were treated with 20 or 30 ~1 of uricase (1.5 U/mg protein/O.5 ml, Seikagaku Kogyo Co., Tokyo, Japan) with or without 25 ~1 of catalase. Untreated lymphocytes were used as controls. After incubation, the lymphocytes were washed twice in RPM1 1640 medium and were resuspended at 1 x lo6 cells/ml in culture medium. Cell culture. Cultures were carried out in sterile microplates with round-bottom wells and were done in triplicate with 1 x 105 responding cells in 100 ~1 of RPM1 1640 containing 10% fetal calf serum, 100 units/ml penicillin, 100 &ml streptomycin, and 100 ~1 of mitogens, which were dissolved at appropriate concentrations in culture medium as follows: 4 pg/ml of phytohemagglutinin (PHA; Difco Laboratories, Detroit, Mich.), 5 pg/ml of concanavalin A (Con A: Pharmacia Fine Chemicals, Uppsala, Sweden), and 1:lOO diluted pokeweed mitogen (PWM: Grand Island Biological Company). The preparations were incubated for 96 hr at 37°C in a humidified atmosphere with 5% CO, and 95% air. Following 72 hr of culture, 1 PCi of [3H]thymidine (2 Ci/mmol: Radiochemical Centre, Amersham, England) was added to each well. Cells were harvested on glass fiber filter paper using a Millipore semiautomated harvester. Radioactivities of thymidine incorporated into lymphocyte DNA were measured in a liquid scintillation counter and they were expressed as counts per minute. Rosette formation. The binding of sheep red cells to lymphocyte membrane receptors was determined by rosette formation, as described by Jondal et al. (17). Lymphocytes (3.5 x lo6 cells/ml) treated with hypoxanthine-plus-xanthine oxidase system were suspended in 100 ~1 of fetal calf serum. The lymphocyte suspension was added to an equal volume of a 1% suspension of neuraminidase pretreated sheep red blood cells (E) or a suspension of ox red blood cells coated with antibody (IgM) and complement (EAC). The mixtures were incubated at 37°C for 30 min, centrifuged at 2OOg (5 min), and stored at 4°C for 1 hr. The cell pellet was gently resuspended, and the percentage of rosette-forming cells was measured microscopically by counting at least 200 lymphocytes. Lymphocytes surrounded with at least three erythrocytes were counted as positive rosette-forming cells. Each experiment was carried out in duplicate. Untreated lymphocytes were used as controls.
FREE RADICALS
321
AND LYMPHOCYTES
Viability studies. The viability of lymphocytes at several hours and 4 days after exposure to O;, H,Oo, xanthine oxidase, and uricase was examined by the trypan blue exclusion method. Cells were mixed with 1% solution of trypan blue and examined microscopically. The percentage of cells excluding the dye is represented as the percentage of viable cells. RESULTS
The viability of the lymphocytes tested by trypan blue staining was more than 95% several hours after the exposure to O;, HzOz, xanthine oxidase, and uricase. In thymidine uptake assays, the viability of the cells which were exposed to hypoxanthine-plus-xanthine oxidase system and uric acid-plus-uricase system and were incubated 4 days in the culture medium were 12.2 to 29.3% and 9.4 to 33.6% respectively. The viability was dependent on the concentration of xanthine oxidase and uricase. The effects of xanthine oxidase alone or hypoxanthine plus xanthine oxidase on mitogen-induced blastogenesis are shown in Fig. 1. Large doses of xanthine oxidase suppressed the blastogenic responses of the lymphocytes to mitogens. Free radicals in high concentrations were found to have an inhibitory effect on the lymphocyte proliferative response to mitogens. Lymphocyte response to Con A was most markedly suppressed by free radicals. In contrast, cells stimulated with PWM seemed to be resistant to the suppressive effects of this system. The results of reversal of free radical toxicity on lymphocyte proliferation induced with PHA are shown in Fig. 2. Superoxide dismutase was relatively ineffective in reversing the toxicity of hypoxanthine-plus-xanthine oxidase system on lymphocyte blastogenesis-induced with PHA. In contrast, suppression of lymphocyte proliferation was reversed by catalase. Moreover, blastogenesis in response to PHA of the lymphocytes treated with uric acid plus uricase system was also suppressed. The suppressing effects of uric acid plus uricase system on lymphocyte blastogenesis was reversed by addition of catalase. The results of treatment with xanthine oxidase alone or hypoxanthine plus PHA
0. 25
u. z
2. 5
5.0
0.25
0.5
2. 5
5.0
0.25
CL5
2. 5
5.0
111
FIG. I. Effect of xanthine oxidase alone and hypoxanthine-plus-xanthine oxidase on lymphocyte blastogenesis induced with mitogens. Lymphocytes were pretreated with xanthine oxidase alone (0) and, xanthine oxidase plus 1 mkf hypoxanthine (0).
322
NISHIDA,
HypDxanthme + X.0
COlltrOl
TANIMOTO,
Hypoxanlhme + x.0 + 5.0.0. 2. 5 mg
AND
Hy,mxanthlne
urlcase
AKAOKA
mp1
“,I‘
arld
Uric
+ x.
0
Catalare
“mare
.md
Uric
+ 2op1
UrltaU
.olr,
dse
aid 2OPl
+ Catalare
25p,
25 PI
FIG. 2. Reversal of free radical toxicity on lymphocyte proliferation induced with PHA by superoxide dismutase and/or catalase. Lymphocytes were suspended for 45 min in the RPM1 1640 medium containing 1 m&f hypoxanthine plus 2.5 ~1 of xanthine oxidase and 1 mg of superoxide dismutase and/or 25 ~1 of catalase. Other lymphocytes were treated for 45 min with RPM1 1640 medium containing uric acid plus uricase with or without 25 ~1 of catalase. These lymphocytes were washed three times and used for cell cultures.
xanthine oxicase on rosette formation are shown in Fig. 3. The absolute percentages of rosette-forming cells in the untreated lymphocytes were E:EAC = 75.8: 14.7%. A marked reduction of E-rosette formation was seen when the lymphocytes were exposed to these systems. The percentage of EAC-rosette formation of lymphocytes treated with these systems was only slightly decreased. E rosette
0.25
0. 5
2. 5
EAC rosette
5. 0 Concentration
0.25
0.5
2. 5
of
xanthine
oxidase
5.0
UI
3. Effect of xanthine oxidase alone and hypoxanthine plus xanthine oxidase on E- and EACrosette formation. Lymphocytes were pretreated with xanthine oxidase alone (0) and xanthine oxidase plus 1 n&Z hypoxanthine (0). These lymphocytes were washed three times and used for rosette formation. FIG.
FREE
RADICALS
AND
LYMPHOCYTES
323
DISCUSSION
In the present study, xanthine oxidase was shown to have various effects on lymphocyte responsiveness to plant mitogens. E- and EAC-rosette formation was also inhibited by pretreatment with large doses of xanthine oxidase. The mechanisms of the effects of xanthine oxidase on lymphocyte function remains unclear. However, the differences between the actions of xanthine oxidase and those of xanthine oxidase plus hypoxanthine on lymphocyte function may be explained by the specific effect of free radicals. Similarly, the differences between the action of uricase and those of uricase plus uric acid may represent the specific effect of HA Our results showed that free radicals inhibited the lymphocyte response to some mitogens. The lymphocytes which respond to Con A were most sensitive to toxic effects of free radicals. On the other hand PWM-stimulated lymphocytes were less affected by free radicals. Furthermore, free radicals demonstrated effects on lymphocyte membranes, markedly inhibiting E- and slightly inhibiting EAC-rosette formation. The viability of the lymphocytes was not reduced shortly after exposure to free radicals, but dropped markedly within a few days of cell cultures. Superoxide dismutase had relatively little effect on the toxicity of these systems to lymphocyte blastogenesis. H,O, is also suppressive of lymphocyte proliferation produced by the uric acid plus uricase system. The preventive effects of catalase on the free radical-induced suppression of lymphocyte proliferation with mitogens suggests the toxic effects are due to H,O,. We are in agreement with the previous findings of Sagone ef al. (18) concerning the toxic effects of H& on lymphocyte blastogenesis. We assume further that Hz02 suppresses mainly T-cell function and that one of the sites of the action of activated oxygen is the outer surface of the lymphocyte membrane. Many biologic factors have been described that are capable of affecting lymphocyte function. Among many factors possibly present in the synovial fluids, immune complexes, activated complement, and lysosomal enzymes from polymorphonuclear leukocytes are known to alter the lymphocyte function (19-21). Lymphocytes obtained from the synovial fluids of patients with rheumatoid arthritis have been found to have diminished response to plant mitogens (22). It is possible that free radicals also interfere with lymphocyte function in the synovial fluids in such patients. The biological importance of these free radicals on lymphocyte function should be further investigated. REFERENCES I. Fridovich, I., N. Engl. J. Med. 290, 624, 1974. 2. Johnston, R. B. Jr.. Keele, B. B., Jr., Misra, H. P., Lehmeyer, J. E., Webb, L. S., Baehner, R. L.. and Rajagopalan, K. V., J. C/in. Invest. 55, 1357, 1975. 3. Klebanoff, S. J., J. Biol. Chem. 249, 3724, 1974. 4. Babior, B. M., Cumutte, J. T., and Kipnes, R. S., J. Lab. Clin. Med. 85, 235, 1975. 5. Curnutte, J. T., Whitten, D. M., and Babior, B. M., N. Engl. J. Med. 290, 593, 1974. 6. Babior, B. M., Kipnes, R. S.; and Curnutte, J. T., J. Clin. Invest. 52, 741, 1973. 7. Sahn, M. L., and McCord, J. M., J. Clin. Invest. 54, 1005, 1974. 8. Goldstein, I. M., Cerqueira, M., Lind, S., and Kaplan, H. B., J. Clin. Invest. 59, 249, 1977. 9. Weening, R. S., Wever, R., and Roos, D., J. Lab. Clin. Med. 85, 245, 1975. 10. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissmann, G., J. Clin. Invest. 56, 1155, 1975.
324 11. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22.
NISHIDA,
TANIMOTO,
AND
AKAOKA
Root, R. K., Metcalf, J., Oshino, N., and Chance, B., .I. Clin. Invest. 55, 945, 1975. Salin, M. L., and McCord, J. M., .I. C/in. Znvesr. 56, 1319, 1975. Proctor, P. H., Kirkpatrick, D. S., and McGinness, J. E., Lancer 2, 95. 1978. McCord, J. M., Science 185, 529, 1974. Handin, R. I., Karabin, R., and Boxer, G. J., .I. Clin. Invest. 59, 959, 1977. Boyum, A., Stand. J. Clin. Lab. Invest. 21, (Suppl. 97), 1968. Jondal, M., Holm, G., and Wigzell, H., 1. Exp. Med. 136, 207, 1972. Sagone, A. L., Kamps, S., and Campbell, R., Photo&m. Photobiol. 28, 909, 1978. Bloch-Shtacher, N., Hirschhorn, K., and Uhr, J. W., C(in. Exp. Zmmunot. 3, 889, 1968 Vischer, T. L., Bretz, U., and Baggiolini, M., .I. Exp. Med. 144, 863, 1976. Yamasaki, K., and Ziff, M., Clin. Exp. Immunol,. 27, 254, 1977. Sheldon, P. J., Papamichail, M., and Halborow, E. J., Ann. Rheum. Dis. 33, 509, 1974.