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Changes in delayed hypersensitivity reaction in mice exposed to O3
ENVIRONMENTAL RESEARCH 43, 186-190 (1987)
Changes in Delayed Hypersensitivity Reaction in Mice Exposed to Oa H I D E K A Z U F U J I M A K I , F u J I O SHIRAISHI, TETSUO A S H I K A W A , * AND M A S A T A K A M U R A K A M I
Division of Basic Medical Sciences, The National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki 305, and *Department of Otorhinolaryngology, The Jikei University School of Medicine, Minato-ku, Tokyo 105, Japan Received March 6, 1986 BALB/c mice were continuously exposed to 0.8 ppm 0 3 for 1, 3, 7, and 14 days. Ozone exposure suppressed the delayed hypersensitivity (DH) reaction to sheep red blood cells (SRBC). The maximum effect was seen after 7 days of exposure. To estimate the suppression of the DH reaction by 03 exposure, the numbers of lymphocytes in thymus and blood of exposed mice were compared with those of control mice. A decrease in the numbers of lymphocytes in both thymus and blood was observed in ©3-exposed mice. The percentage of T and B lymphocytes in blood of exposed mice was the same as that in blood of control mice. These results suggest that 0.8 ppm 03 exposure affects the T lymphocytes required for OH reactions. © 1987AcademicPress, Inc.
INTRODUCTION
The cooperation of humoral and cell-mediated immunity protects the host from bacterial or viral infections, and defends against formation of tumors. The effects of 03 exposure on the humoral immune system have been investigated. Significantly suppressed anti-sheep red blood cell (SRBC) IgM antibody production was seen after a 1-hr exposure to 0.5 and 2.0 ppm 03 (Seto, 1975). Wolcott et al. (1982) reported no significant differences in magnitude of the virusneutralizing antibody response to 0.5 ppm 03 in exposed mice. Recently, we reported that the primary antibody response to SRBC (T-lymphocyte-dependent antigen) in the spleen of mice exposed to 0.8 ppm 03 was suppressed, but that no suppression was observed in antibody response to D N P - F i coll (T-lymphocyte-independent antigen) (Fujimaki et al., 1984). This study suggested that either the number or the function of T lymphocytes was affected by 03 exposure. However, effects of exposure to 03 on cellular immunity have not been examined adequately. The purpose of this study was to estimate the effects of 03 exposure on thymus and delayed hypersensitivity (DH) reaction. MATERIALS AND METHODS A n i m a l s . Male BALB/c mice (2 to 3 months old) were purchased from Charles River, Japan. 03 e x p o s u r e . Mice were continuously exposed to 0.8 -+ 0.02 ppm (SD) 03 for 1, 3, 7, and 14 days. 03 exposure was performed by the methods described previously (Fujimaki et al., 1984). In brief, 03 concentrations were continuously 186 0013-9351/87 $3.00 Copyright© i987by AcademicPress,Inc, All rightsof reproductionin any formreserved.
03 A N D D E L A Y E D H Y P E R S E N S I T I V I T Y R E A C T I O N
187
checked with an 03 monitor (Kimoto Co., Ltd., Japan). In the chamber condition, the temperature was maintained between 24 and 26°C and humidity between 50 and 60%. During 03 exposure, food was exchanged daily for fresh sterilized food, and water was also replaced every 2 days. Assay of D H reaction. By the method of Mitsuoka et al. (1978) the delayed hypersensitivity reaction was assayed. Immediately before or after 03 exposure, mice were injected intravenously with 1 x 106 SRBC (Toyo Serum Co., Ltd.). Four days later, SRBC (1 x 108/0.02 ml) were injected into the right footpad, and the same volume of saline was injected into the left footpad as control. The thickness between instep and sole of the foot was measured 24 hr after SRBC injection using a dial thickness gauge with 0.01-mm graduations (Peacock G. Ozaki Seisakusho, Tokyo) (Tamura et al., 1973). The difference in the thickness between the test and the control footpads was the indication of intensity of the DH reaction. The differences were evaluated statistically by Student's t test. Separation of lymphocytes. Lymphocytes in thymus were prepared by teasing the thymus through a stainless-steel mesh and the tissue suspension was washed with balanced salt solution (BSS). Lymphocytes from heart blood were separated with Ficoll-Paque (Pharmacia) by centrifugation (400g for 30 min) and washed with BSS. The numbers of Thy 1.2 antigen positive (T) and surface immunoglobulin positive (B) cells were counted with an incident light immunofluorescence microscope (Olympus Model BH-REF) by the methods described in our previous paper (Fujimaki and Shimizu, 1981). RESULTS
Effects of O.8 p p m
0 3 Exposure
on D H Reaction in Mice
Mice were continuously exposed to 0.8 ppm 03 for 1, 3, 7, or 14 days, and then SRBC were injected intravenously. Four days after immunization, SRBC were reinjected into the footpad, and 24 hr later the footpad swelling was measured (Table 1). Compared with control, the DH reactions decreased gradually from 1 day to 7 days exposure. Particularly 03 exposure for 7 days significantly suppressed the DH reaction (63% of the control value). No suppression of the DH reaction was observed after 14 days of exposure. TABLE 1 EFFECTS OF 0.8 ppm 0 3 EXPOSURE ON DH REACTION IN MICE Footpad swelling ( × 0.01 mm) a Duration of exposure (days) 1 3 7 14
Control 40.3 48.0 41.8 31.0
_+ 3.8 _+ 4.9 _+ 3.7 _+ 2.3
Exposed 35.2 35.8 26.5 30.2
_ -_+ _+
2.8 2.8 5.0* 2.4
* P < 0.05 (Student's t test). Each value is expressed as the mean _+ SE of 7 to 10 mice. b Percentage of control value. a
(87) b (75) (63) (97)
No. of mice per treatment group 8 7 7 10
188
FUJIMAKI
ET AL.
Effects of Timing of 03 Exposure on DH Reaction To investigate the effect of timing between 03 exposure and SRBC injection, mice were exposed to 0.8 ppm 03 for 3 days immediately before (group B) or after (group D) intravenous SRBC injection (Table 2). Both group A (the control for group B) and group C (the control for group D) were also injected with SRBC without 03 exposure. In the group D, the DH reaction was significantly suppressed compared with that in group C (P < 0.01). But no significant suppression of DH reaction in group B was observed compared with that in group A.
Effects of O.8 ppm 03 Exposure on Thymus in Mice To estimate the suppression of DH reaction described above (Tables 1 and 2), the effects of 0.8 ppm 03 exposure for 3 and 7 days on thymus were studied (Fig. I). 03 exposure for both days markedly decreased the cell numbers and the weights of the thymus. The percentage of Thy 1.2 positive cells in the thymus of exposed mice was decreased by 7 days 03 exposure (74%), but was not changed by 3 days exposure (96%) compared with that of control mice. It seems that 03 exposure for 7 days impaired the thymus cells more severely than that for 3 days. Effects of 03 Exposure on Lymphocytes in Blood of Mice To investigate the effect of 03 exposure on lymphocytes in blood, mice were exposed to 0.8 ppm 03 for 3 or 7 days, and then blood was drawn from the heart. The lymphocytes were separated from the blood of the O3-exposed and control mice by the use of Ficoll-Paque. The recovered numbers of lymphocytes were significantly decreased in the mice exposed for 3 days (Table 3). The number was still decreased after 7 days of exposure but the change was not significant. No significant difference in the percentage of T and B lymphocytes was observed in the blood of mice exposed for 3 or 7 days. DISCUSSION
This paper studied the effects of 03 exposure on cellular immunity and showed that 0.8 ppm 03 exposure not only suppressed the DH reaction but also decreased the numbers of lymphocytes in thymus and blood. Both T lymphocytes and macTABLE 2 EFFECT OF TIMING OF 0 3 EXPOSURE ON D H REACTION
Group
SRBC (iv) a
0.8 p p m 0 3 exposure b
SRBC (iv)
A B C D
+ +
+ +
+ + -
Footpad swelling ( x 0.01 r a m ) 26.9 23.3 34.4 19.3
+ _+ _+ -+
No. of mice
2.0 c 2.7 2.4 1.1"*
Note. Group A was the control for Group B, and Group C was the control for Group D. ** P < 0.01 ( S t u d e n t ' s t test). a Intravenous injection. b Duration of exposure was 3 days. c E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S E .
10 10 7 7
189
0 3 AND DELAYED HYPERSENSITIVITY REACTION
100
¢ 5C 0 U
|
!
|
Control
7 days
3 days
Duration
of 0 3 e x p o s u r e
FIG. 1. Effects of 0.8 ppm 03 exposure on thymus in mice. Each bar represents the standard error of six mice. O, weight of thymus; A, No. of thymus cells. Control mice showed 49.2 + 1.6 (mg) of thymus weights and 127.2 ± 10.6 ( x 106) of the numbers of thymus cells. The thymus weights and numbers of thymus cells at both 3 and 7 days of exposure were decreased significantly (P < 0.01).
rophages play an important role in the DH reaction. Therefore, 0.8 ppm 03 exposure may affect the numbers and the function of T lymphocytes or macrophages. There are many papers concerning the effects of 03 exposure on the functions of macrophages. The depression of phagocytic activity of alveolar macrophages and the bactericidal activity by 03 exposure was reported by Coffin et al. (1968) and Goldstein et al. (1971). On the contrary, Christman and Schwartz (1982) and Aranyi et al. (1983) recently observed enhanced phagocytic and bactericidal activities after 03 exposure. These discrepancies may be due to the differences in the doses and the exposure periods of 03. However, as shown in Table 2, the DH reaction in mice exposed to 0.8 ppm 03 after SRBC injection was significantly TABLE 3 CHANGES OF LYMPHOCYTE SUBPOPULATIONS IN BLOOD OF 0.8 ppm 0 3 EXPOSED MICE a
Duration of 03 exposure 3 days
No. T/B T B
of lymphoyctes ( x 104/ml of blood) lymphocyte proportion cells (%) cells (%)
7 days
Control
Exposed
Control
Exposed
76.7 ± 4.3
26.3 ± 3.4**
77.0 ± 3.5
46.0 ± 8.0
70 ± 2 30 ± 2
63 ± 10 37 ± 10
75 ± 3 25 ± 3
** P < 0.01 (Student's t test). Each value is expressed as the mean ± SE of six mice.
80 ± 3 20 ± 3
190
FUJIMAKI ET AL.
suppressed, but no significant suppression of DH reaction in mice exposed to 03 before SRBC injection was observed. This result suggests that 03 exposure induced the effects on growth and differentiation of T lymphocytes activated by SRBC antigen rather than those on the functions of macrophages required for DH reaction. Previously, we have observed that the primary antibody response to SRBC as T-lymphocyte-dependent antigen was suppressed by 03 exposure, but that primary antibody response to D N P - F i c o l l as T-lymphocyte-independent antigen showed no suppression (Fujimaki et al., 1984). Recently Ozawa et al. (1985) studied the effect of 03 on the IgE antibody production and showed that the suppression of IgE antibody production was caused in part by the delay of helper T-cell function. From the present result and these previous studies, it seems that the impairment of T lymphocytes was induced by a short-term exposure to 0.8 ppm 03. The mechanisms of the impairment or depressed functions of lymphocytes induced by 03 exposure are unclear. But Kraut and Sagone (1981) studied the effect of oxidant stress on the lymphocyte membrane and lymphocyte functions, and showed a marked reduction in lymphocytes' ability to bind SRBC and to form caps for concanavalin A stimulation after in vitro oxidant injury. Further investigation of the effects of 03 exposure on the cell membrane or cellular components in T lymphocytes or macrophages is needed. REFERENCES Aranyi, C., Vana, S. C., Thomas, P. T., Bradof, J. N., and Fenters, J. D. (1983). Effects of subchronic exposure to a mixture of 03, SOz, and (NH4)2SO 4 on host defenses of mice. J. Toxicol. Environ. Health 12, 55-71. Christman, C. A., and Schwartz, L. W. (1982). Enhanced phagocytosis by alveolar macrophages induced by short-term ozone insult. Environ. Res. 28, 241-250. Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. (1968). Influence of ozone on pulmonary cells. Arch. Environ. Health 16, 633-636. Fujimaki, H., Ozawa, M., Imai, T., and Shimizu, F. (1984). Effect of short-term exposure to 03 on antibody response in mice. Environ. Res. 35,490-496. Fujimaki, H., and Shimizu, F. (1981). Effects of acute exposure to nitrogen dioxide on pulmonary antibody response. Arch. Environ. Health 36, 114-119. Goldstein, E., Tyler, W. S., Hoeprich, R D., and Eagle, C. (1971). Ozone and the antibacterial defense mechanisms of the murine lung. Arch. Intern. Med. 127, 1099-1102. Kraut, E. H., and Sagone, A. L. (1981). The effect of oxidant injury on the lymphocyte membrane and functions. J. Lab. Clin. Mad. 98, 697-703. Mitsuoka, A., Teramatsu, T., Baba, M., Morikawa, S., and Yasuhira, K. (1978). Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: Evidence for tuberculin type delayed hypersensitivity of the reaction. Immunology 34, 363-370. Ozawa, M., Fujimaki, H., Imai, T., Honda, Y., and Watanabe, N. (1985). Suppression of IgE antibody production after exposure to ozone in mice. Int. Arch. Allergy Appl. Immunol. 76, 16-19. Seto, H. (1975). Effect of ozone on humoral immunity. Igakunoayumi 94, 250-251 (in Japanese). Tamura, S., Kurata, T., Sugimoto, M., and Egashira, Y. (1973). Cellular and humoral immune responses in mice. I. Development of delayed-type footpad swelling against sheep erythrocytes and its suppression by intraperitoneal administration of the antigen. Japan J. Med. Sci. Biol. 26, 161-168. Wolcott, J. A., Zee, Y. C., and Osebold, J. W. (1982). Exposure to ozone reduces influenza severity and alters distribution of influenza viral antigens in murine lungs. Appl. Environ. Microsc. 44, 723-731.
Changes in Delayed Hypersensitivity Reaction in Mice Exposed to Oa H I D E K A Z U F U J I M A K I , F u J I O SHIRAISHI, TETSUO A S H I K A W A , * AND M A S A T A K A M U R A K A M I
Division of Basic Medical Sciences, The National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki 305, and *Department of Otorhinolaryngology, The Jikei University School of Medicine, Minato-ku, Tokyo 105, Japan Received March 6, 1986 BALB/c mice were continuously exposed to 0.8 ppm 0 3 for 1, 3, 7, and 14 days. Ozone exposure suppressed the delayed hypersensitivity (DH) reaction to sheep red blood cells (SRBC). The maximum effect was seen after 7 days of exposure. To estimate the suppression of the DH reaction by 03 exposure, the numbers of lymphocytes in thymus and blood of exposed mice were compared with those of control mice. A decrease in the numbers of lymphocytes in both thymus and blood was observed in ©3-exposed mice. The percentage of T and B lymphocytes in blood of exposed mice was the same as that in blood of control mice. These results suggest that 0.8 ppm 03 exposure affects the T lymphocytes required for OH reactions. © 1987AcademicPress, Inc.
INTRODUCTION
The cooperation of humoral and cell-mediated immunity protects the host from bacterial or viral infections, and defends against formation of tumors. The effects of 03 exposure on the humoral immune system have been investigated. Significantly suppressed anti-sheep red blood cell (SRBC) IgM antibody production was seen after a 1-hr exposure to 0.5 and 2.0 ppm 03 (Seto, 1975). Wolcott et al. (1982) reported no significant differences in magnitude of the virusneutralizing antibody response to 0.5 ppm 03 in exposed mice. Recently, we reported that the primary antibody response to SRBC (T-lymphocyte-dependent antigen) in the spleen of mice exposed to 0.8 ppm 03 was suppressed, but that no suppression was observed in antibody response to D N P - F i coll (T-lymphocyte-independent antigen) (Fujimaki et al., 1984). This study suggested that either the number or the function of T lymphocytes was affected by 03 exposure. However, effects of exposure to 03 on cellular immunity have not been examined adequately. The purpose of this study was to estimate the effects of 03 exposure on thymus and delayed hypersensitivity (DH) reaction. MATERIALS AND METHODS A n i m a l s . Male BALB/c mice (2 to 3 months old) were purchased from Charles River, Japan. 03 e x p o s u r e . Mice were continuously exposed to 0.8 -+ 0.02 ppm (SD) 03 for 1, 3, 7, and 14 days. 03 exposure was performed by the methods described previously (Fujimaki et al., 1984). In brief, 03 concentrations were continuously 186 0013-9351/87 $3.00 Copyright© i987by AcademicPress,Inc, All rightsof reproductionin any formreserved.
03 A N D D E L A Y E D H Y P E R S E N S I T I V I T Y R E A C T I O N
187
checked with an 03 monitor (Kimoto Co., Ltd., Japan). In the chamber condition, the temperature was maintained between 24 and 26°C and humidity between 50 and 60%. During 03 exposure, food was exchanged daily for fresh sterilized food, and water was also replaced every 2 days. Assay of D H reaction. By the method of Mitsuoka et al. (1978) the delayed hypersensitivity reaction was assayed. Immediately before or after 03 exposure, mice were injected intravenously with 1 x 106 SRBC (Toyo Serum Co., Ltd.). Four days later, SRBC (1 x 108/0.02 ml) were injected into the right footpad, and the same volume of saline was injected into the left footpad as control. The thickness between instep and sole of the foot was measured 24 hr after SRBC injection using a dial thickness gauge with 0.01-mm graduations (Peacock G. Ozaki Seisakusho, Tokyo) (Tamura et al., 1973). The difference in the thickness between the test and the control footpads was the indication of intensity of the DH reaction. The differences were evaluated statistically by Student's t test. Separation of lymphocytes. Lymphocytes in thymus were prepared by teasing the thymus through a stainless-steel mesh and the tissue suspension was washed with balanced salt solution (BSS). Lymphocytes from heart blood were separated with Ficoll-Paque (Pharmacia) by centrifugation (400g for 30 min) and washed with BSS. The numbers of Thy 1.2 antigen positive (T) and surface immunoglobulin positive (B) cells were counted with an incident light immunofluorescence microscope (Olympus Model BH-REF) by the methods described in our previous paper (Fujimaki and Shimizu, 1981). RESULTS
Effects of O.8 p p m
0 3 Exposure
on D H Reaction in Mice
Mice were continuously exposed to 0.8 ppm 03 for 1, 3, 7, or 14 days, and then SRBC were injected intravenously. Four days after immunization, SRBC were reinjected into the footpad, and 24 hr later the footpad swelling was measured (Table 1). Compared with control, the DH reactions decreased gradually from 1 day to 7 days exposure. Particularly 03 exposure for 7 days significantly suppressed the DH reaction (63% of the control value). No suppression of the DH reaction was observed after 14 days of exposure. TABLE 1 EFFECTS OF 0.8 ppm 0 3 EXPOSURE ON DH REACTION IN MICE Footpad swelling ( × 0.01 mm) a Duration of exposure (days) 1 3 7 14
Control 40.3 48.0 41.8 31.0
_+ 3.8 _+ 4.9 _+ 3.7 _+ 2.3
Exposed 35.2 35.8 26.5 30.2
_ -_+ _+
2.8 2.8 5.0* 2.4
* P < 0.05 (Student's t test). Each value is expressed as the mean _+ SE of 7 to 10 mice. b Percentage of control value. a
(87) b (75) (63) (97)
No. of mice per treatment group 8 7 7 10
188
FUJIMAKI
ET AL.
Effects of Timing of 03 Exposure on DH Reaction To investigate the effect of timing between 03 exposure and SRBC injection, mice were exposed to 0.8 ppm 03 for 3 days immediately before (group B) or after (group D) intravenous SRBC injection (Table 2). Both group A (the control for group B) and group C (the control for group D) were also injected with SRBC without 03 exposure. In the group D, the DH reaction was significantly suppressed compared with that in group C (P < 0.01). But no significant suppression of DH reaction in group B was observed compared with that in group A.
Effects of O.8 ppm 03 Exposure on Thymus in Mice To estimate the suppression of DH reaction described above (Tables 1 and 2), the effects of 0.8 ppm 03 exposure for 3 and 7 days on thymus were studied (Fig. I). 03 exposure for both days markedly decreased the cell numbers and the weights of the thymus. The percentage of Thy 1.2 positive cells in the thymus of exposed mice was decreased by 7 days 03 exposure (74%), but was not changed by 3 days exposure (96%) compared with that of control mice. It seems that 03 exposure for 7 days impaired the thymus cells more severely than that for 3 days. Effects of 03 Exposure on Lymphocytes in Blood of Mice To investigate the effect of 03 exposure on lymphocytes in blood, mice were exposed to 0.8 ppm 03 for 3 or 7 days, and then blood was drawn from the heart. The lymphocytes were separated from the blood of the O3-exposed and control mice by the use of Ficoll-Paque. The recovered numbers of lymphocytes were significantly decreased in the mice exposed for 3 days (Table 3). The number was still decreased after 7 days of exposure but the change was not significant. No significant difference in the percentage of T and B lymphocytes was observed in the blood of mice exposed for 3 or 7 days. DISCUSSION
This paper studied the effects of 03 exposure on cellular immunity and showed that 0.8 ppm 03 exposure not only suppressed the DH reaction but also decreased the numbers of lymphocytes in thymus and blood. Both T lymphocytes and macTABLE 2 EFFECT OF TIMING OF 0 3 EXPOSURE ON D H REACTION
Group
SRBC (iv) a
0.8 p p m 0 3 exposure b
SRBC (iv)
A B C D
+ +
+ +
+ + -
Footpad swelling ( x 0.01 r a m ) 26.9 23.3 34.4 19.3
+ _+ _+ -+
No. of mice
2.0 c 2.7 2.4 1.1"*
Note. Group A was the control for Group B, and Group C was the control for Group D. ** P < 0.01 ( S t u d e n t ' s t test). a Intravenous injection. b Duration of exposure was 3 days. c E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S E .
10 10 7 7
189
0 3 AND DELAYED HYPERSENSITIVITY REACTION
100
¢ 5C 0 U
|
!
|
Control
7 days
3 days
Duration
of 0 3 e x p o s u r e
FIG. 1. Effects of 0.8 ppm 03 exposure on thymus in mice. Each bar represents the standard error of six mice. O, weight of thymus; A, No. of thymus cells. Control mice showed 49.2 + 1.6 (mg) of thymus weights and 127.2 ± 10.6 ( x 106) of the numbers of thymus cells. The thymus weights and numbers of thymus cells at both 3 and 7 days of exposure were decreased significantly (P < 0.01).
rophages play an important role in the DH reaction. Therefore, 0.8 ppm 03 exposure may affect the numbers and the function of T lymphocytes or macrophages. There are many papers concerning the effects of 03 exposure on the functions of macrophages. The depression of phagocytic activity of alveolar macrophages and the bactericidal activity by 03 exposure was reported by Coffin et al. (1968) and Goldstein et al. (1971). On the contrary, Christman and Schwartz (1982) and Aranyi et al. (1983) recently observed enhanced phagocytic and bactericidal activities after 03 exposure. These discrepancies may be due to the differences in the doses and the exposure periods of 03. However, as shown in Table 2, the DH reaction in mice exposed to 0.8 ppm 03 after SRBC injection was significantly TABLE 3 CHANGES OF LYMPHOCYTE SUBPOPULATIONS IN BLOOD OF 0.8 ppm 0 3 EXPOSED MICE a
Duration of 03 exposure 3 days
No. T/B T B
of lymphoyctes ( x 104/ml of blood) lymphocyte proportion cells (%) cells (%)
7 days
Control
Exposed
Control
Exposed
76.7 ± 4.3
26.3 ± 3.4**
77.0 ± 3.5
46.0 ± 8.0
70 ± 2 30 ± 2
63 ± 10 37 ± 10
75 ± 3 25 ± 3
** P < 0.01 (Student's t test). Each value is expressed as the mean ± SE of six mice.
80 ± 3 20 ± 3
190
FUJIMAKI ET AL.
suppressed, but no significant suppression of DH reaction in mice exposed to 03 before SRBC injection was observed. This result suggests that 03 exposure induced the effects on growth and differentiation of T lymphocytes activated by SRBC antigen rather than those on the functions of macrophages required for DH reaction. Previously, we have observed that the primary antibody response to SRBC as T-lymphocyte-dependent antigen was suppressed by 03 exposure, but that primary antibody response to D N P - F i c o l l as T-lymphocyte-independent antigen showed no suppression (Fujimaki et al., 1984). Recently Ozawa et al. (1985) studied the effect of 03 on the IgE antibody production and showed that the suppression of IgE antibody production was caused in part by the delay of helper T-cell function. From the present result and these previous studies, it seems that the impairment of T lymphocytes was induced by a short-term exposure to 0.8 ppm 03. The mechanisms of the impairment or depressed functions of lymphocytes induced by 03 exposure are unclear. But Kraut and Sagone (1981) studied the effect of oxidant stress on the lymphocyte membrane and lymphocyte functions, and showed a marked reduction in lymphocytes' ability to bind SRBC and to form caps for concanavalin A stimulation after in vitro oxidant injury. Further investigation of the effects of 03 exposure on the cell membrane or cellular components in T lymphocytes or macrophages is needed. REFERENCES Aranyi, C., Vana, S. C., Thomas, P. T., Bradof, J. N., and Fenters, J. D. (1983). Effects of subchronic exposure to a mixture of 03, SOz, and (NH4)2SO 4 on host defenses of mice. J. Toxicol. Environ. Health 12, 55-71. Christman, C. A., and Schwartz, L. W. (1982). Enhanced phagocytosis by alveolar macrophages induced by short-term ozone insult. Environ. Res. 28, 241-250. Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. (1968). Influence of ozone on pulmonary cells. Arch. Environ. Health 16, 633-636. Fujimaki, H., Ozawa, M., Imai, T., and Shimizu, F. (1984). Effect of short-term exposure to 03 on antibody response in mice. Environ. Res. 35,490-496. Fujimaki, H., and Shimizu, F. (1981). Effects of acute exposure to nitrogen dioxide on pulmonary antibody response. Arch. Environ. Health 36, 114-119. Goldstein, E., Tyler, W. S., Hoeprich, R D., and Eagle, C. (1971). Ozone and the antibacterial defense mechanisms of the murine lung. Arch. Intern. Med. 127, 1099-1102. Kraut, E. H., and Sagone, A. L. (1981). The effect of oxidant injury on the lymphocyte membrane and functions. J. Lab. Clin. Mad. 98, 697-703. Mitsuoka, A., Teramatsu, T., Baba, M., Morikawa, S., and Yasuhira, K. (1978). Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: Evidence for tuberculin type delayed hypersensitivity of the reaction. Immunology 34, 363-370. Ozawa, M., Fujimaki, H., Imai, T., Honda, Y., and Watanabe, N. (1985). Suppression of IgE antibody production after exposure to ozone in mice. Int. Arch. Allergy Appl. Immunol. 76, 16-19. Seto, H. (1975). Effect of ozone on humoral immunity. Igakunoayumi 94, 250-251 (in Japanese). Tamura, S., Kurata, T., Sugimoto, M., and Egashira, Y. (1973). Cellular and humoral immune responses in mice. I. Development of delayed-type footpad swelling against sheep erythrocytes and its suppression by intraperitoneal administration of the antigen. Japan J. Med. Sci. Biol. 26, 161-168. Wolcott, J. A., Zee, Y. C., and Osebold, J. W. (1982). Exposure to ozone reduces influenza severity and alters distribution of influenza viral antigens in murine lungs. Appl. Environ. Microsc. 44, 723-731.