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The effect of hydrocortisone on the immune response of mice treated with Corynebacterium parvum
The Effect of Hydrocortisone on the Immune Response of Mice Treated with Corynebacterium Parvum CHARLES
S. GREENBERG; .ANU NIKOLAY V.
Department of‘ Medicine, 19102. and Department
Hahnemann Medico/ C‘ollege. of Medicine. Mic,higun Starr Michigan 48824 Received
August
DIMITKOI
Philadelphia, Clni~~rrsity.
Penn.\~h~rnic~ East Lansing.
28. 1975
lntraperitoneal injection of Balbic mice with Co~nehocterium parvum 3 days prior to injection of sheep red blood cells produced a stimulation of direct (19s) and indirect (7s) plaque-forming cell response. Simultaneous intraperitoneal injection of C. parrum and hydrocortisone depressed the stimulatory effect of C. parvum on direct plaque cell formation in the spleen. Conversely, injection of c‘. l>crr\.l,nt and hydrocortisone did not significantly affect the indirect plaque cell response. The spleen weight was increased when C. parvum was injected alone or in combination with hydrocortisone. Administration of C. pcrrt’rrm alone decreased the weight of the thymus. This effect was blocked when the vaccine was combined with hydrocortisone.
INTRODUCTION
Administration of a killed vaccine of Corynrbacterium panwn has been shown to alter the immune response in mice to various antigens (l-3) and to inhibit the growth of various experimental tumors (3-6). During the last several years C. parvum has been used in the treatment of various human malignancies (7-10). The intravenous administration of the vaccine produced some side effects, most notably chills and fever, which occurred in 98% of the treated patients regardless of the injected dose (9, 10). In an attempt to reduce these side effects, simultaneous injections of corticosteroids and C. pan’um have been employed (8). Corticosteroids are known to affect the immune system which may interfere with the effect of C. parvum (11-13). It is well documented that corticosteroids have differential effects on the direct and indirect plaque-forming cells (14, 15). The direct plaque-forming cells are IgM-producing cells within the spleen, whereas the indirect are involved in IgG synthesis. It has been shown that a high dose of hydrocortisone selectively depressed the induction of both memory and synthesis in the IgG-forming cells (14). However, this effect appears to be a dose-dependent phenomenon (15). In this communication, we report the results from a study whose objective was to investigate the effect of simultaneous administration of C. partjurn and hydrocortisone on the immune response of mice to sheep red blood cells. MATERIALS AND METHODS Animals. Male Balb/c mice, 8-10 weeks old and weighing between 20 and 25 g
each, were obtained from the Jackson Laboratory. Bar Harbor, Me. Antigen. Fresh sheep red blood cells were washed twice in phosphate-buffered 264 CopyrIght All rights
ci_j 1976 by of reproduction
Academic in any
Prr\\. form
Inc recerved
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
saline (PBS) and then suspended to a concentration saline. Injection
schedule of C. parvum
and hydrocortisone.
PARVUM
265
of 1 x 10’ cells/O.25 ml in 0.9% Corynebacterium
parvum,
Batch No. PV365A, containing 7 mg/ml of formalin-killed organisms, was a gift from Wellcome Research Laboratory. The vaccine for use in these experiments was diluted to 500 pg/O.25 ml in normal saline. Hydrocortisone (Hydrocorton) produced by Merck, Sharp and Dohme Corporation was diluted to a concentration of 0.05 mg/0.25 ml in normal saline. Mice were separated into five groups which received intraperitoneal injections on 2 consecutive days of either 0.9% saline, 500 pg of C. parvum, 0.05 mg of hydrocortisone, or 500 pg of C. parvum plus 0.05 mg of hydrocortisone in volumes of 0.25 ml for each injection. Sheep red blood cells (SRBC) were injected ip 72 hr after the last pretreatment injection. Plaque assay for antibody-producing cells. Antibody-producing cells were assayed by the Jerne plaque technique as modified by Uyeki and Klassen (16). The assays were performed on days 5, 7, 10, 13 and 17 after the last injection of C. parl’um, hydrocortisone or normal saline. On each day three mice were sacrificed by cervical dislocation and weighed, and then each spleen was placed into a plastic petri dish containing 5 ml of Eagle’s medium. Dispersed cell suspensions were prepared by mechanically teasing the tissues. The individual spleen suspensions were decanted into a lo-ml test tube incubated in an ice bath for 3 min, and the cells were then decanted into fresh tubes. Spleen cells in suspension were counted in a hemacytometer. Tests of cell viability were done by the trypan blue exclusion technique. Assays were done in triplicate for each spleen cell dilution used. Each plate contained 2 ml of agar, 0.1 ml of a 10% sheep red blood cell suspension, and 0.1 ml of the spleen cell suspension containing from 1 x lo6 to 1 x lo7 viable cells. The petri dishes were incubated at 37°C for 1 hr, then a l-ml solution of a 1:20 dilution of guinea pig serum (Flow Labs) was added as a source of complement. For the development of the indirect plaques, rabbit anti-mouse IgG serum (Cappel Laboratories, Downingtown, Pa.) was added in the same dilution followed by a I-hr incubation at 37°C. The optimum concentration for development of plaques was found to be a 1% solution of anti-mouse IgG serum dissolved in PBS. A 1.5-ml aliquot of this solution was added to each plate and allowed to incubate for 45 min. After decanting the anti-mouse serum, 1 ml of the guinea pig serum (source of complement) was added and incubated for 45 min. Statistical analysis. All data were analyzed for significance by Student’s t test. Examination of spleen cell preparations. Spleen cells were stained with Wright’s stain for microscopic evaluation. RESULTS Effect of C. parvum and hydrocortisone on the direct plaque-forming cell (PFC) response. As presented in Fig. 1A the C. parvum group had a heightened response
on days 7, 10 and 13 after administration of the vaccine compared to the control and hydrocortisone groups (P < .OOl). This capacity of the vaccine to stimulate direct plaque cell production in the spleen was significantly reduced when the C. parvum injection was combined with hydrocortisone (P < 0.001) but was still in excess when compared to the control groups. The group of animals treated with
266
GREENBERG
AND
DIMITROV
El
c Parvunl Hydrocwlwme
70
plus
8.
r
13 Day
17
of Assay
FIG. I. (A), Distribution of direct PFC related to time and number of spleen cells. (B), Distribution of direct PFC related to time and the whole spleen. The results are expressed as means of three experiments -t standard error.
hydrocortisone alone showed a PFC response similar to the control groups. The group of animals treated with hydrocortisone alone showed a PFC response similar to the control group which was given 0.9% NaCl. By day 17 all the groups had similar values for plaque cell response when based on number of PFC/l@ spleen cells. When the values are based on the total number of PFClspleen, the C. parvum group shows an increased plaque formation throughout the experiment; the other groups returned to the control values by day 17 (Fig. 1B). Effect of C. parvum and hydrocortisone on the indirect plaque-forming cell response. The number of indirect PFC in the spleens of mice in all groups reached a peak at day 13. Indirect PFC in spleens of mice injected with C. parvum were
two to three times greater than those found in spleens of mice from the control
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
267
PARVUM
groups (P < 0.01). The combination of hydrocortisone with C. parvum did not influence this increase in indirect plaque cell response (Fig. 2A). Alone, hydrocortisone, as used in this experiment, had no significant effect on the indirect plaque cell response as compared to mice in the control group. When the results were expressed as total number of indirect PFUspleen (Fig. 2B), the group injected with C. parvum and hydrocortisone showed a significant decrease in plaque formation compared to the group of C. parvum on days 13 and 17 (P < 0.05 and P < 0.01, respectively). Effect of C. parvum and hydrocortisone on the spleen weight. As shown in Table 1, the C. parvum group responded with a progressive splenomegaly reaching a peak
13 days
after
the injection.
The
combination
of C. parvlrm with
hy-
drocortisone did not influence the increase in spleen weight up to day 13. At day 17. 14.0
rA
0
Control - 0.9%
@
Hydrocorlmne
NoCl
=” 12.0 s c f 10.0 -
q C.Parvum
w
IO 0 8.0
C Forvum
-
plus
Hydracortasone
25 k t 6.0 T! p 6 4.0 4 $
2.0
7
IO
13 Day
FIG. 2. (A), Distribution
of indirect
PFC related
17
of Assay
to time and number
of spleen cells.
(B), Distribution
of indirect PFC related to time and the whole spleen. The results are expressed as means of three experiments & standard error.
Days Treatment
5
Control mice (noninfected) Control mice (injected with SRBC) Hydrocortisone c. parL’lrrn C. prrr~~urn plus hydrocortisone ‘I Mean
spleen
weight
in mgil0
40.6 42.4 40.1 58.9 58.3 g of body
i -t r r +
after
7 4.5’1 3.7 6.1 6.9 8.2 weight
IO
59.6 t 4.0 56.3 -t 8.2 73.6 i 8.2 66.9 c 8.5 for three
treatment
63.3 62.0 X7.7 82.4
animals
t i < z
13
3.6 6.7 7.6 4.8
59.1 53.8 108.0 96.0
-t standard
z r t k
17
5.0 6.3 9.3 8.1
49.0 i45.4 * 79.5 f 54.3 :t
3.2 4.4 6.3 3.9
error.
the C. purrturn group remained significantly elevated compared to the control group (P < O.OOl), whereas the group treated with C. parvum and hydrocortisone had a value similar to the control group and the group treated with hydrocortisone alone. The control group given 0.9% NaCl and the sheep red blood cells had a mild splenomegaly that reached a peak at day 10. The group injected with hydrocortisone alone had a pattern similar to that of the control group injected with SRBC. Effect qf C. parvum and hydrocortisone on the cell type recovered jkom the spfeen. The cell types defined by Wright’s staining showed that lymphocytes
comprised 8895% of the cells in all preparations, with 12-15% lymphoblastic cells. At day 5 after C. parvum was injected, there were 4-5% polymorphonuclear leukocytes (PMN). This cell population was also present in the spleens treated with C. parvum plus hydrocortisone. By day 7 after the C. parvum injection, the PMN could not be detected. At this time l-2%> eosinophils were present in the C. parlwm group and the group with C. par\wn and hydrocortisone. By day 10 after C. purlwm injection the cell population in all groups was rather uniform. containing mostly lymphocytes, 95-99%. Effect of C. purvum and hydrocortisonr on the thymus Mseight. When injected alone, C. parvum induced a significant decrease in the thymus size when compared to the control and hydrocortisone groups on days 7. 10 and 13 (I’ < 0.001). By day 17, thymuses in mice of the C. parvum group were comparable to the control values (Table 2). The injection of hydrocortisone alone had no significant effect on the thymus weight but when combined with C. parvum prevented the decrease in thymus weight produced by C. parvum alone. The thymus appeared to increase in size in the control groups injected with SRBC by day 7 after the injection of the erythrocytes (comparable to day 10 after treatment with C. parvum or hydrocortisone as indicated in the table). DISCUSSION
The immune response to SRBC is a multistep process requiring a series of cellular events. The complex phenomenon of antibody response includes three major steps: (a) cellular activity concerning recognition and processing of antigen, (b) generation of clones involved in synthesis and secretion of antibody and (c) development of memory cells. The effect of C. parvum and hydrocortisone, separately or in combination, may alter any of these three steps.
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
TABLE EFFECX
OF HYDROCORIXSONE
OS THYMUS
269
PARVUM
2 WF.IGH.I.OF C.
MI(:E
kmW7Z-TREA’I’k:D
Days after treatment Treatment
5
Control mice (noninjected) Control mice (injected with SRBC) Hydrocortisone
19.6 + 3.8” 19.9 + 3.6 20.2 2 6.1 14.1 f 4.6 19.3 2 3.3
C. parvum
C. parvum plus hydrocortisone
a Mean thymus weight in milligrams/l0
7
IO
22.3 19.2 7.1 20.1
13
2 2 -t 2
4.8 2.6
31.8 25.6
3.0 6.1
9.5 23.1
17
2 3.1 2 4.0 ? 2.6 -c 4.0
20.8 20.3 14.5 17.2
e + 2 t
4.6 4.9 2.8
21.4 19.1 18.9
6.0
18.8
k f ? k
4.0 2.2 4.2 3.2
grams of body weight for three animals k standard error.
The role of the macrophage in recognition and processing of antigen has been demonstrated (17). It has also been shown that C. parvum activates the macrophage and provides the added proliferative stimulus to the antigen-sensitive and, ultimately, antibody-producing B lymphocyte (3). On the other hand, hydrocortisone may inhibit phagocytosis (17) and may also interfere with the processing of the antigen through the stabilizing effect on lysozomal membrane (18). However, this inhibitory effect of hydrocortisone has been shown to be dose dependent (19). In addition, the increased nonspecific anti-tumor activity by C. parvum of peritoneal macrophages in mice has been inhibited by high doses of corticosteroids (20). In our experiments we have used lower doses of hydrocortisone, 2.5 mgikg, which is similar to the therapeutic dose used in humans for depression of the side effect of C. parvum (8). Simultaneous administration of C. parvum and hydrocortisone resulted in inhibition of the stimulatory effect of the vaccine when administered as a single agent (Fig. 1). Since C. parvum is an antigen which should be processed by the macrophages, its stimulatory effect on antibody production could be partially blocked by hydrocortisone at the site of the macrophage participation in the immune response. It is possible that the modified processing of the vaccine by the macrophage alters its functional capacity which may be responsible for the altered pattern of splenomegaly in the group of animals treated simultaneously with C. parvum and hydrocortisone (Table 1). Another role of the activated macrophage by C. parvum is considered to be that it may cause extended retention of lymphocytes within the spleen (2 1). Hydrocortisone may also alter this function of the stimulated by C. parvum macrophage, which may explain the reduction of the spleen size between day 13 and 17 (Table 1). The mechanisms involved in generation of cells producing antibody currently suggest cooperation between T and B cells and between macrophages and B cells in antibody production (22). The antibody response to SRBC is dependent on Tand B-cell function. However, the production of immunoglobulin classes requires different involvement of T and B cells. It is known that IgG responses usually require more T cells than do IgM responses (22). In agreement with the study of Howard et al. (3), our results show that C. parvum stimulates both IgG and IgM antibody-producing cells (Fig. 1 and 2). When C. parvum is injected simultane-
270
GREENBERG
AND
DIMITROV
ously with hydrocortisone this effect is significantly inhibited for IgM-producing cells (Fig. 1). Hydrocortisone alone has been shown to have no effect on the capacity of T cells to cooperate in antibody response to SRBC (23), which is in agreement with our results when hydrocortisone alone was used. Recently the role of a subpopulation of thymus-derived cells has been demonstrated which can inhibit antibody production by their effect on B cells (24). These suppressor cells appear to be produced by the thymus and home to the spleen where they suppress the antibody response to SRBC (25). One may speculate that the production of suppressor T cells is diminished by C. parvum or that these cells are destroyed by C. parvum-activated spleen which may augment the antibody response. Scott (26) has shown that a similar mechanism exists for destruction of delayed type hypersensitivity-reactive T cells. The interpretation of changes in thymus size must consider the fact that the thymus is a dynamic organ involved with lymphocytopoiesis in the newborn and young animal. Recent studies have shown that cell proliferation occurs in both the thymic cortex and medulla, with substantial cell emigration, and turnover (27). In an attempt to explain the reduction of the thymus weight in the C. purvum group, one could suggest that there was either an increase in intrathymic cell death, a decrease in cell proliferation or an increase in cell emigration. This experiment can not differentiate between these possibilities. Hydrocortisone capacity to block the ability of C. parvum to cause a decrease in thymic weight might be explained by modifying C. parvum processing, resulting in less C. purvum going to the thymus and exciting a toxic effect, or reducing the level of stress evoked by the vaccine, so fewer endogenous corticosteroids are formed which would have the same effect as high doses of exogenous corticosteroids to suppress thymus weight. Further studies are necessary to define the mechanism of the decrease in thymus weight by C. purvum and hydrocortisone’s protective action. Smears of cells isolated from the spleens of each group of animals revealed that combining C. purvum with hydrocortisone did not alter the population of cells recovered from the spleens. But it was apparent from the results on a plaqueforming production that the cells in the C. purvum and hydrocortisone group had modified functional capacity when compared to the C. parvum-treated group. The role of C, in cell cooperation is just beginning to be investigated but it remains interesting to note that C. parvum treatment in patients alters C, levels, and presumably in animal models this effect would occur and could modify the immune response to SRBC (28). The final event in the antibody response is the development of memory cells which was not investigated in this study and should be the subject of further investigations. ACKNOWLEDGMENTS The technical assistance of Mr. Thomas Denny and Mrs. Donna thank Dr. B. Landau for his help in reviewing the manuscript.
West is highly
appreciated.
REFERENCES I. Scott, 2. Neveu,
M. T., Semis. T., Branellec.
One-ol. 1, 367, 1974. A.. and Biozzi, G.. Ann.
Insr.
Pasteur
Pwi.s
106, 771, 1%4.
We also
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
PARVUM
271
3. Howard, J. G., Scott, M. T., and Christie, G. H., In “Immunopotentiation,” Ciba Found. Symp. 19, 101, Little, Brown, Boston, 1973. 4. Halpern, B. N., Biozzi, G., Stiffel, C., and Mouton, D., Nature (London) 212, 853, 1966. 5. Paslin, D., Dimitrov, N. V., Heaton, C., J. Nat. Cancer Inst. 52, 571, 1974. 6. Castro, J. E., Eur. .I. Cancer 10, 121, 1974. 7. Israel, L., and Edelstein, R., In “26th Annual M.D. Anderson Symposium on Fundamental Cancer Research, 1973,” in press. 8. Fischer, B., Rubin, H., Sartiano, G., Ennis, L., and Walmark, H., Cancer, in press. 9. Reed, R. C., Gutterman, J. U., Mauligit, G. M., and Hersh, E. M., Proc. AACR ASCO 16, 228, 1975. 10. Band, P. R., Jao-King, C., Urtasun, R., and Haraphonope, M., Proc. AACR ASCO 16,9, 1975. 11. Baxter, J. D., and Harris, A. W., Transplant. Proc. 7, 55, 1975. 12. Claman, H. N., J. Allergy C/in. Immunol. 55, 145, 1975. 13. Butler, W. T., Transplant. hoc. 7, 49, 1975. 14. Petranyi, Gy., Benezur, M., and Alfoldy, P., immunology 21, 151, 1971. 15. Janah, S., Hussain, Q. Z., Chaudhuri, S. N., fndian J. Med. Res. 58, 1206, 1970. 16. Uyeki, E. H., and Klassen, R. S., J. Immune/. 101, 271, 1968. 17. Heller, J. H., Endocrinology 56, 80, 1955. 18. de Duve, C., In “Injury, Inflammation and Immunity” (L. Thomas et al., Eds.), pp. 288-311, Williams and Wilkins, Baltimore, 1%4. 19. Gotjamanos, T., J. Reticuloendothel. Sot. 8, 421, 1970. 20. Scott, M. T.,J. Narl. Cancer Inst. 54, 789, 1975. 21. Frost, P., and Lance, E. M., In “Immunopotentiation,” Ciba Found. Symp. 18, 29, Little, Brown, Boston, 1973. 22. Feldmann, M., Ser. Haematol. 7, 593, 1974. 23. Cohen, J. J., and Claman, H., 1. Exp. Med. 133, 1026, 1971. 24. Gershon, R. K., Cohen, P., Henein, R., and Liebhaber, S. A., J. Immunol. 108, 586, 1972. 25. WU, C-Y., and Lance, E. M., Cell. Immunol. 13, 1, 1974. 26. Scott, M. T., Cell. Immunol. 13, 251, 1974. 27. Joel, D. D., Chanana, A. D., and Cronkite, E. P., Ser. Haemato/. 7, 464, 1974. 28. Dimitrov, N. V., Israel, L., and Peltier, A. J., submitted for publication.
S. GREENBERG; .ANU NIKOLAY V.
Department of‘ Medicine, 19102. and Department
Hahnemann Medico/ C‘ollege. of Medicine. Mic,higun Starr Michigan 48824 Received
August
DIMITKOI
Philadelphia, Clni~~rrsity.
Penn.\~h~rnic~ East Lansing.
28. 1975
lntraperitoneal injection of Balbic mice with Co~nehocterium parvum 3 days prior to injection of sheep red blood cells produced a stimulation of direct (19s) and indirect (7s) plaque-forming cell response. Simultaneous intraperitoneal injection of C. parrum and hydrocortisone depressed the stimulatory effect of C. parvum on direct plaque cell formation in the spleen. Conversely, injection of c‘. l>crr\.l,nt and hydrocortisone did not significantly affect the indirect plaque cell response. The spleen weight was increased when C. parvum was injected alone or in combination with hydrocortisone. Administration of C. pcrrt’rrm alone decreased the weight of the thymus. This effect was blocked when the vaccine was combined with hydrocortisone.
INTRODUCTION
Administration of a killed vaccine of Corynrbacterium panwn has been shown to alter the immune response in mice to various antigens (l-3) and to inhibit the growth of various experimental tumors (3-6). During the last several years C. parvum has been used in the treatment of various human malignancies (7-10). The intravenous administration of the vaccine produced some side effects, most notably chills and fever, which occurred in 98% of the treated patients regardless of the injected dose (9, 10). In an attempt to reduce these side effects, simultaneous injections of corticosteroids and C. pan’um have been employed (8). Corticosteroids are known to affect the immune system which may interfere with the effect of C. parvum (11-13). It is well documented that corticosteroids have differential effects on the direct and indirect plaque-forming cells (14, 15). The direct plaque-forming cells are IgM-producing cells within the spleen, whereas the indirect are involved in IgG synthesis. It has been shown that a high dose of hydrocortisone selectively depressed the induction of both memory and synthesis in the IgG-forming cells (14). However, this effect appears to be a dose-dependent phenomenon (15). In this communication, we report the results from a study whose objective was to investigate the effect of simultaneous administration of C. partjurn and hydrocortisone on the immune response of mice to sheep red blood cells. MATERIALS AND METHODS Animals. Male Balb/c mice, 8-10 weeks old and weighing between 20 and 25 g
each, were obtained from the Jackson Laboratory. Bar Harbor, Me. Antigen. Fresh sheep red blood cells were washed twice in phosphate-buffered 264 CopyrIght All rights
ci_j 1976 by of reproduction
Academic in any
Prr\\. form
Inc recerved
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
saline (PBS) and then suspended to a concentration saline. Injection
schedule of C. parvum
and hydrocortisone.
PARVUM
265
of 1 x 10’ cells/O.25 ml in 0.9% Corynebacterium
parvum,
Batch No. PV365A, containing 7 mg/ml of formalin-killed organisms, was a gift from Wellcome Research Laboratory. The vaccine for use in these experiments was diluted to 500 pg/O.25 ml in normal saline. Hydrocortisone (Hydrocorton) produced by Merck, Sharp and Dohme Corporation was diluted to a concentration of 0.05 mg/0.25 ml in normal saline. Mice were separated into five groups which received intraperitoneal injections on 2 consecutive days of either 0.9% saline, 500 pg of C. parvum, 0.05 mg of hydrocortisone, or 500 pg of C. parvum plus 0.05 mg of hydrocortisone in volumes of 0.25 ml for each injection. Sheep red blood cells (SRBC) were injected ip 72 hr after the last pretreatment injection. Plaque assay for antibody-producing cells. Antibody-producing cells were assayed by the Jerne plaque technique as modified by Uyeki and Klassen (16). The assays were performed on days 5, 7, 10, 13 and 17 after the last injection of C. parl’um, hydrocortisone or normal saline. On each day three mice were sacrificed by cervical dislocation and weighed, and then each spleen was placed into a plastic petri dish containing 5 ml of Eagle’s medium. Dispersed cell suspensions were prepared by mechanically teasing the tissues. The individual spleen suspensions were decanted into a lo-ml test tube incubated in an ice bath for 3 min, and the cells were then decanted into fresh tubes. Spleen cells in suspension were counted in a hemacytometer. Tests of cell viability were done by the trypan blue exclusion technique. Assays were done in triplicate for each spleen cell dilution used. Each plate contained 2 ml of agar, 0.1 ml of a 10% sheep red blood cell suspension, and 0.1 ml of the spleen cell suspension containing from 1 x lo6 to 1 x lo7 viable cells. The petri dishes were incubated at 37°C for 1 hr, then a l-ml solution of a 1:20 dilution of guinea pig serum (Flow Labs) was added as a source of complement. For the development of the indirect plaques, rabbit anti-mouse IgG serum (Cappel Laboratories, Downingtown, Pa.) was added in the same dilution followed by a I-hr incubation at 37°C. The optimum concentration for development of plaques was found to be a 1% solution of anti-mouse IgG serum dissolved in PBS. A 1.5-ml aliquot of this solution was added to each plate and allowed to incubate for 45 min. After decanting the anti-mouse serum, 1 ml of the guinea pig serum (source of complement) was added and incubated for 45 min. Statistical analysis. All data were analyzed for significance by Student’s t test. Examination of spleen cell preparations. Spleen cells were stained with Wright’s stain for microscopic evaluation. RESULTS Effect of C. parvum and hydrocortisone on the direct plaque-forming cell (PFC) response. As presented in Fig. 1A the C. parvum group had a heightened response
on days 7, 10 and 13 after administration of the vaccine compared to the control and hydrocortisone groups (P < .OOl). This capacity of the vaccine to stimulate direct plaque cell production in the spleen was significantly reduced when the C. parvum injection was combined with hydrocortisone (P < 0.001) but was still in excess when compared to the control groups. The group of animals treated with
266
GREENBERG
AND
DIMITROV
El
c Parvunl Hydrocwlwme
70
plus
8.
r
13 Day
17
of Assay
FIG. I. (A), Distribution of direct PFC related to time and number of spleen cells. (B), Distribution of direct PFC related to time and the whole spleen. The results are expressed as means of three experiments -t standard error.
hydrocortisone alone showed a PFC response similar to the control groups. The group of animals treated with hydrocortisone alone showed a PFC response similar to the control group which was given 0.9% NaCl. By day 17 all the groups had similar values for plaque cell response when based on number of PFC/l@ spleen cells. When the values are based on the total number of PFClspleen, the C. parvum group shows an increased plaque formation throughout the experiment; the other groups returned to the control values by day 17 (Fig. 1B). Effect of C. parvum and hydrocortisone on the indirect plaque-forming cell response. The number of indirect PFC in the spleens of mice in all groups reached a peak at day 13. Indirect PFC in spleens of mice injected with C. parvum were
two to three times greater than those found in spleens of mice from the control
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
267
PARVUM
groups (P < 0.01). The combination of hydrocortisone with C. parvum did not influence this increase in indirect plaque cell response (Fig. 2A). Alone, hydrocortisone, as used in this experiment, had no significant effect on the indirect plaque cell response as compared to mice in the control group. When the results were expressed as total number of indirect PFUspleen (Fig. 2B), the group injected with C. parvum and hydrocortisone showed a significant decrease in plaque formation compared to the group of C. parvum on days 13 and 17 (P < 0.05 and P < 0.01, respectively). Effect of C. parvum and hydrocortisone on the spleen weight. As shown in Table 1, the C. parvum group responded with a progressive splenomegaly reaching a peak
13 days
after
the injection.
The
combination
of C. parvlrm with
hy-
drocortisone did not influence the increase in spleen weight up to day 13. At day 17. 14.0
rA
0
Control - 0.9%
@
Hydrocorlmne
NoCl
=” 12.0 s c f 10.0 -
q C.Parvum
w
IO 0 8.0
C Forvum
-
plus
Hydracortasone
25 k t 6.0 T! p 6 4.0 4 $
2.0
7
IO
13 Day
FIG. 2. (A), Distribution
of indirect
PFC related
17
of Assay
to time and number
of spleen cells.
(B), Distribution
of indirect PFC related to time and the whole spleen. The results are expressed as means of three experiments & standard error.
Days Treatment
5
Control mice (noninfected) Control mice (injected with SRBC) Hydrocortisone c. parL’lrrn C. prrr~~urn plus hydrocortisone ‘I Mean
spleen
weight
in mgil0
40.6 42.4 40.1 58.9 58.3 g of body
i -t r r +
after
7 4.5’1 3.7 6.1 6.9 8.2 weight
IO
59.6 t 4.0 56.3 -t 8.2 73.6 i 8.2 66.9 c 8.5 for three
treatment
63.3 62.0 X7.7 82.4
animals
t i < z
13
3.6 6.7 7.6 4.8
59.1 53.8 108.0 96.0
-t standard
z r t k
17
5.0 6.3 9.3 8.1
49.0 i45.4 * 79.5 f 54.3 :t
3.2 4.4 6.3 3.9
error.
the C. purrturn group remained significantly elevated compared to the control group (P < O.OOl), whereas the group treated with C. parvum and hydrocortisone had a value similar to the control group and the group treated with hydrocortisone alone. The control group given 0.9% NaCl and the sheep red blood cells had a mild splenomegaly that reached a peak at day 10. The group injected with hydrocortisone alone had a pattern similar to that of the control group injected with SRBC. Effect qf C. parvum and hydrocortisone on the cell type recovered jkom the spfeen. The cell types defined by Wright’s staining showed that lymphocytes
comprised 8895% of the cells in all preparations, with 12-15% lymphoblastic cells. At day 5 after C. parvum was injected, there were 4-5% polymorphonuclear leukocytes (PMN). This cell population was also present in the spleens treated with C. parvum plus hydrocortisone. By day 7 after the C. parvum injection, the PMN could not be detected. At this time l-2%> eosinophils were present in the C. parlwm group and the group with C. par\wn and hydrocortisone. By day 10 after C. purlwm injection the cell population in all groups was rather uniform. containing mostly lymphocytes, 95-99%. Effect of C. purvum and hydrocortisonr on the thymus Mseight. When injected alone, C. parvum induced a significant decrease in the thymus size when compared to the control and hydrocortisone groups on days 7. 10 and 13 (I’ < 0.001). By day 17, thymuses in mice of the C. parvum group were comparable to the control values (Table 2). The injection of hydrocortisone alone had no significant effect on the thymus weight but when combined with C. parvum prevented the decrease in thymus weight produced by C. parvum alone. The thymus appeared to increase in size in the control groups injected with SRBC by day 7 after the injection of the erythrocytes (comparable to day 10 after treatment with C. parvum or hydrocortisone as indicated in the table). DISCUSSION
The immune response to SRBC is a multistep process requiring a series of cellular events. The complex phenomenon of antibody response includes three major steps: (a) cellular activity concerning recognition and processing of antigen, (b) generation of clones involved in synthesis and secretion of antibody and (c) development of memory cells. The effect of C. parvum and hydrocortisone, separately or in combination, may alter any of these three steps.
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
TABLE EFFECX
OF HYDROCORIXSONE
OS THYMUS
269
PARVUM
2 WF.IGH.I.OF C.
MI(:E
kmW7Z-TREA’I’k:D
Days after treatment Treatment
5
Control mice (noninjected) Control mice (injected with SRBC) Hydrocortisone
19.6 + 3.8” 19.9 + 3.6 20.2 2 6.1 14.1 f 4.6 19.3 2 3.3
C. parvum
C. parvum plus hydrocortisone
a Mean thymus weight in milligrams/l0
7
IO
22.3 19.2 7.1 20.1
13
2 2 -t 2
4.8 2.6
31.8 25.6
3.0 6.1
9.5 23.1
17
2 3.1 2 4.0 ? 2.6 -c 4.0
20.8 20.3 14.5 17.2
e + 2 t
4.6 4.9 2.8
21.4 19.1 18.9
6.0
18.8
k f ? k
4.0 2.2 4.2 3.2
grams of body weight for three animals k standard error.
The role of the macrophage in recognition and processing of antigen has been demonstrated (17). It has also been shown that C. parvum activates the macrophage and provides the added proliferative stimulus to the antigen-sensitive and, ultimately, antibody-producing B lymphocyte (3). On the other hand, hydrocortisone may inhibit phagocytosis (17) and may also interfere with the processing of the antigen through the stabilizing effect on lysozomal membrane (18). However, this inhibitory effect of hydrocortisone has been shown to be dose dependent (19). In addition, the increased nonspecific anti-tumor activity by C. parvum of peritoneal macrophages in mice has been inhibited by high doses of corticosteroids (20). In our experiments we have used lower doses of hydrocortisone, 2.5 mgikg, which is similar to the therapeutic dose used in humans for depression of the side effect of C. parvum (8). Simultaneous administration of C. parvum and hydrocortisone resulted in inhibition of the stimulatory effect of the vaccine when administered as a single agent (Fig. 1). Since C. parvum is an antigen which should be processed by the macrophages, its stimulatory effect on antibody production could be partially blocked by hydrocortisone at the site of the macrophage participation in the immune response. It is possible that the modified processing of the vaccine by the macrophage alters its functional capacity which may be responsible for the altered pattern of splenomegaly in the group of animals treated simultaneously with C. parvum and hydrocortisone (Table 1). Another role of the activated macrophage by C. parvum is considered to be that it may cause extended retention of lymphocytes within the spleen (2 1). Hydrocortisone may also alter this function of the stimulated by C. parvum macrophage, which may explain the reduction of the spleen size between day 13 and 17 (Table 1). The mechanisms involved in generation of cells producing antibody currently suggest cooperation between T and B cells and between macrophages and B cells in antibody production (22). The antibody response to SRBC is dependent on Tand B-cell function. However, the production of immunoglobulin classes requires different involvement of T and B cells. It is known that IgG responses usually require more T cells than do IgM responses (22). In agreement with the study of Howard et al. (3), our results show that C. parvum stimulates both IgG and IgM antibody-producing cells (Fig. 1 and 2). When C. parvum is injected simultane-
270
GREENBERG
AND
DIMITROV
ously with hydrocortisone this effect is significantly inhibited for IgM-producing cells (Fig. 1). Hydrocortisone alone has been shown to have no effect on the capacity of T cells to cooperate in antibody response to SRBC (23), which is in agreement with our results when hydrocortisone alone was used. Recently the role of a subpopulation of thymus-derived cells has been demonstrated which can inhibit antibody production by their effect on B cells (24). These suppressor cells appear to be produced by the thymus and home to the spleen where they suppress the antibody response to SRBC (25). One may speculate that the production of suppressor T cells is diminished by C. parvum or that these cells are destroyed by C. parvum-activated spleen which may augment the antibody response. Scott (26) has shown that a similar mechanism exists for destruction of delayed type hypersensitivity-reactive T cells. The interpretation of changes in thymus size must consider the fact that the thymus is a dynamic organ involved with lymphocytopoiesis in the newborn and young animal. Recent studies have shown that cell proliferation occurs in both the thymic cortex and medulla, with substantial cell emigration, and turnover (27). In an attempt to explain the reduction of the thymus weight in the C. purvum group, one could suggest that there was either an increase in intrathymic cell death, a decrease in cell proliferation or an increase in cell emigration. This experiment can not differentiate between these possibilities. Hydrocortisone capacity to block the ability of C. parvum to cause a decrease in thymic weight might be explained by modifying C. parvum processing, resulting in less C. purvum going to the thymus and exciting a toxic effect, or reducing the level of stress evoked by the vaccine, so fewer endogenous corticosteroids are formed which would have the same effect as high doses of exogenous corticosteroids to suppress thymus weight. Further studies are necessary to define the mechanism of the decrease in thymus weight by C. purvum and hydrocortisone’s protective action. Smears of cells isolated from the spleens of each group of animals revealed that combining C. purvum with hydrocortisone did not alter the population of cells recovered from the spleens. But it was apparent from the results on a plaqueforming production that the cells in the C. purvum and hydrocortisone group had modified functional capacity when compared to the C. parvum-treated group. The role of C, in cell cooperation is just beginning to be investigated but it remains interesting to note that C. parvum treatment in patients alters C, levels, and presumably in animal models this effect would occur and could modify the immune response to SRBC (28). The final event in the antibody response is the development of memory cells which was not investigated in this study and should be the subject of further investigations. ACKNOWLEDGMENTS The technical assistance of Mr. Thomas Denny and Mrs. Donna thank Dr. B. Landau for his help in reviewing the manuscript.
West is highly
appreciated.
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M. T., Semis. T., Branellec.
One-ol. 1, 367, 1974. A.. and Biozzi, G.. Ann.
Insr.
Pasteur
Pwi.s
106, 771, 1%4.
We also
CORTICOSTEROIDS
AND
CORYNEBACTERIUM
PARVUM
271
3. Howard, J. G., Scott, M. T., and Christie, G. H., In “Immunopotentiation,” Ciba Found. Symp. 19, 101, Little, Brown, Boston, 1973. 4. Halpern, B. N., Biozzi, G., Stiffel, C., and Mouton, D., Nature (London) 212, 853, 1966. 5. Paslin, D., Dimitrov, N. V., Heaton, C., J. Nat. Cancer Inst. 52, 571, 1974. 6. Castro, J. E., Eur. .I. Cancer 10, 121, 1974. 7. Israel, L., and Edelstein, R., In “26th Annual M.D. Anderson Symposium on Fundamental Cancer Research, 1973,” in press. 8. Fischer, B., Rubin, H., Sartiano, G., Ennis, L., and Walmark, H., Cancer, in press. 9. Reed, R. C., Gutterman, J. U., Mauligit, G. M., and Hersh, E. M., Proc. AACR ASCO 16, 228, 1975. 10. Band, P. R., Jao-King, C., Urtasun, R., and Haraphonope, M., Proc. AACR ASCO 16,9, 1975. 11. Baxter, J. D., and Harris, A. W., Transplant. Proc. 7, 55, 1975. 12. Claman, H. N., J. Allergy C/in. Immunol. 55, 145, 1975. 13. Butler, W. T., Transplant. hoc. 7, 49, 1975. 14. Petranyi, Gy., Benezur, M., and Alfoldy, P., immunology 21, 151, 1971. 15. Janah, S., Hussain, Q. Z., Chaudhuri, S. N., fndian J. Med. Res. 58, 1206, 1970. 16. Uyeki, E. H., and Klassen, R. S., J. Immune/. 101, 271, 1968. 17. Heller, J. H., Endocrinology 56, 80, 1955. 18. de Duve, C., In “Injury, Inflammation and Immunity” (L. Thomas et al., Eds.), pp. 288-311, Williams and Wilkins, Baltimore, 1%4. 19. Gotjamanos, T., J. Reticuloendothel. Sot. 8, 421, 1970. 20. Scott, M. T.,J. Narl. Cancer Inst. 54, 789, 1975. 21. Frost, P., and Lance, E. M., In “Immunopotentiation,” Ciba Found. Symp. 18, 29, Little, Brown, Boston, 1973. 22. Feldmann, M., Ser. Haematol. 7, 593, 1974. 23. Cohen, J. J., and Claman, H., 1. Exp. Med. 133, 1026, 1971. 24. Gershon, R. K., Cohen, P., Henein, R., and Liebhaber, S. A., J. Immunol. 108, 586, 1972. 25. WU, C-Y., and Lance, E. M., Cell. Immunol. 13, 1, 1974. 26. Scott, M. T., Cell. Immunol. 13, 251, 1974. 27. Joel, D. D., Chanana, A. D., and Cronkite, E. P., Ser. Haemato/. 7, 464, 1974. 28. Dimitrov, N. V., Israel, L., and Peltier, A. J., submitted for publication.