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Desensitization of delayed-type hypersensitivity by antigen and specific antibody
CEIL~~LAR
IMMUNOLOGY
19, 129-136 (1975)
SHORT COMMUNICATIONS Desensitization
of Delayed-Type and Specific
Rrccizvd
Hypersensitivity Antibody
DCCCIJJ~XT
by Antigen
3, 19i4
The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP,,-BGG in Freund’s complete adjuvant (FCA) were desensitized 14 days later with various doses of DNPwBGG injected intravenously. It was found that only certain doses (100-500 rg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum ijt vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum sepanately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3-4 wk after desensitizing injection.
INTKODUCTION are currently under Mechanisms which suppress cell-mediated immunity much investigation. An understanding of these mechanisms is obviously of great and tumor immunology. The importance in transplantation, antimicrobial, in sensitized individuals by specific suppression of cell-mediated immunity enhancement” or “desensiantigen is variously referred to as “immunological tization” (1, 2). There is a large body of evidence indicating that the effector cells in cell-mediated immunity are thymus-derived (T) lymphocytes (3, 4). Classically, immunological enhancement refers to the situation in which sensitized T lymphocytes present in an individual are unable to mediate cellular immunity because of blocking by antibodies (5-7) or antigen-antibody complexes (8, 9), although the exact mechanism of action is not known. In contrast, desensitization of cell-mediated immunity, as exemplified by delayed-type hypersensitivity (DTH) can be achieved by injection of antigen in large or repeated doses (g-11). Again, the exact mechanism by which this desensitization is effected is not known (2, 12-14). It is believed by some that sensitized T lymphocytes are exhausted of their capacity to produce mediators and therefore are unable to respond to antigen encountered in the tissues ( 15). Thus the role of antibody in the desensitization of DTH is generally dismissed. In the present report, however, evidence will be presented suggesting that contrary to popular 129 Copyright All rights
1975 by Academic Press, Inc. oQ reproduction in any form reserved.
130
SHORT
COMMUNICATIONS
belief, antibocly plays an important role in the desensitization soluble antigen, DNP-BGG, in rats.
of DTH
to a
Female inbred AS strain rats 6-9 wk of age were used. Antigens Ovalbumin (British Drug House) was dissolved in phosphate-buffered saline (PBS) and stored frozen at a concentration of 20 mg/ml. Dinitrophenylated bovine gamma globulin (DNP-BGG) was prepared by reacting 1.0 g of dinitrobenzene sulphonic acid with 1.0 g of BGG (Sigma) in 50 ml distilled water containing 1.0 g of K,COJ. The mixture was stirred with a magnetic stirrer at room temperature for 18 hr. The reaction was stopped by rapid dialysis in the cold against normal saline. The degree of substitution was estimated photometrically to be 32 DNP group per BGG molecule (16). Antisera Rat anti-DNP-BGG hyperimmune serum was obtained from adult rats which were immunized subcutaneously with 1 mg/rat of DNP-BGG in Freund’s complete adjuvant (FCA) and boosted intraperitoneally 4 wk later with 1 mg of DNP-BGG in saline. The rats were bled out 1 wk after the booster injection. Serum was separated from the clot on the same day, pooled and stored at -10°C. Rat anti-OA hyperimmune serum was prepared similarly using ovalbumin as the immunizing antigen. Experiwaental
Procedures
Groups of five adult rats were immunized subcutaneously with 1 mg/rat of DNP-BGG in Freund’s complete adjuvant. Control rats were injected with FCA-saline. Fourteen days later, delayed type hypersensitivity (DTH) was elicited by the injection into the right hind footpads of 1Opg of DNP-BGG in 50 ~1 of saline. The left hind footpads was injected with 50 ~1 of saline. Desensitizing doses of DNP-BGG were injected intravenously at the same time as the eliciting antigen. DTH was determined 24 hr after the footpad injection. For adoptive transfer experiments, peritoneal cells (PC) from rats immunized 14 days previously with 1 mg/rat of DNP-BGG-FCA were used. The sensitized peritoneal cells were washed 4~ in cold PBS containing 5% normal rat serum. 2 x lo7 cells (one rat equivalent) were incubated in ice for half an hour with various doses of DNP-BGG or with DNP-BGG in the presence of either 0.5 ml of a rat anti-DNP-BGG hyperimmune serum or a rat anti-OA hyperimmune serum. The mixture was then washed three times in PBS containing 5% normal rat serum, resuspended in 1 ml of the medium, and injected intravenously into normal rats. Control rats were injected with the same number of the untreated cells. DTH was elicited in the recipients at the same time as the intravenous injection. DTH was determined 24 hr later. For the experiment on the time course of recovery from desensitization, rats were immunized with 1 mg/rat of DNP-BGG-FCA. Fourteen days later, half of the rats were desensitized by an intravenous injection of 100 pg/rat of
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DNP-BGG in 1.0 ml saline. Control rats were injected with 1.0 ml of saline only. The desensitized rats and the control rats were, respectively, divided into groups of five rats and elicited for DTH in the footpads at different times after the intravenous injection. Antibody
Estimation
Antibodies to DNP-BGG and OA were estimated by hemagglutination using sheep erythrocytes sensitized with DNP-EGG or OA, respectively, by a chromic chloride procedure (17). Assay for Delayed-Type
Hypersemitivity
Delayed-type hypersensitivity was determined by measuring the increase in footpad thickness after the injection of antigen in saline into the hind footpads. DNP-BGG specific reactions were elicited by injecting 10 pg of DNP-BGG in 50 ~1 saline into the right hind footpads and saline (50 ~1) alone into the left hind footpad. Footpad thickness was measured 24 hr after the injection and specific footpad swelling was determined by subtracting the thickness of the left hind footpad from the right. Footpad thickness was measured by a dial caliper (Mitutoyo, Japan) which had 0.01 mm graduations. The accuracy of the assay has been previously discussed (18). RESULTS Efect
of Antigen
Dose on the Desensitization
of Delayed-Type
Hypersensitivity
Adult female inbred AS strain rats were injected subcutaneously in the flank with 1 mg of dinitrophenylated bovine gamma globulin (DNP,g-BGG) in Freund’s complete adjuvant (FCA). These rats expressed high level of delayed-type hypersensitivity to DNP,,-BGG, as measured by the 24-hr footpad swelling method. The DTH response reached a plateau 12-14 days after sensitization and remained at the same level for at least 4 wk. Desensitizing doses of TABLE ANTIBODY
Group
1
2 3 4 5
Immune peritoneal cells
2 2 2 2 2
x x x x x
10’ 107 10’ 107 107
SPECIFICITY
1
IN THE
Antigen (100 rd
DNP-BGG
DNP-BGG DNP-BGG
* Groups of five adult female AS rats were injected 2 X lO’/rat of peritoneal washing cells (PC) from adult (1 mg/rat) 2 wk previously with DNP-BGG in FCA. in ice for half an hour with either antigen, antiserum, (group 1) were given the PC alone. Immediately after delayed hypersensitivity at the footpads. b P < 0.01 compared to group 1.
DESENSITIZATIONS
Antiserum (0.5 ml)
24 hr footpad swelling (l/lOth mm f SE)
-
2.3 f 0.2 2.4 i 0.3
Anti-DNP-BGG Anti-DNP-BGG Anti-OA
2.1 f 0.4 0.4 Yk O.lb
2.0 f 0.2
intravenously through the tail veins with female AS rats immunised subcutaneously Prior to injection, the cells were incubated or both, and then washed. Control rats the iv injection, the rats were elicited for
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OL b
0.01 0.05
0.1 Antigen
0.5 concentration
1.0
5.0
10.0
Img)
FIG. 1. Desensitization of DNP-BGG sensitized rats with various doses of Groups of five adult rats were immunized subcutaneously with 1 mgjrat FCA. Control rats (0) were injected with saline-FCA. All rats, except desensitized 14 days later by intravenous injection of the various doses
saline. All the animals were tested for DTH desensitizing injection. Vertical bars represent
DNP-BGG in V&O, of DNP,-BGG in the controls, were of DNPZS2-BGG in
(footpad swelling) at the same time as the standard errors of the arithmetic means.
DNP,,-BGG (ranging from 10 pg to 10 mg in 1.0 ml of saline) were injected intravenously into groups of rats sensitized 14 days previously. Immediately after the intravenous injection the rats were tested for DTH in the footpads with 10 ,,.g of DNP-@GG in 50 ~1 of saline. Figure 1 shows that rats hypersensitive to DNP-BGG could be desensitized by optimum doses (100-500 pg P < 0.05) of DNP-BGG. Outside this dose range, the antigen either partially or completely failed to desensitize the delayed response in the rats. Desensitization
in the Adoptive
Transfer
System
Since rats sensitized with DNP-BGG-FCA contain high levels of antiDNP-BGG antibody on day 14 (av titre of 480), it could well be that the results reflected an in vivo optimum antigen/antibody ratio which effectively caused the desensitization of the delayed-type hypersensitivity. The following experiment was carried out to investigate this further. Peritoneal cells from rats sensitized with DNP-BGG-FCA 14 days previously were collected and washed four times by centrifugation in cold PBS containing 5% normal rat serum. The sensitized peritoneal cells were injected intravenously into normal recipient rats in the ratio of one donor to one recipient or 2 X lo7 cells per rat. Prior to injection, the cells were incubated in ice for half an hour with various doses of
DNP-BGG
with or without
rat anti-DNP-BGG
immune serum. The antiserum
(titre, 1000) was obtained from AS rats hyperimmunized with DNP-BGG and was added in the ratio of 0.5 ml per rat. After the incubation, cells were washed 3 x with cold PBS containing 5% normal rat seruni and resuspended Immediately after the cell transfer, the reto 2 x 10’ cells/ml in the medium. cipient
rats
were tested for DTH
reactions in the footpads. The 24-hr footpad
reactions are shown in Fig. 2. Normal rats receiving the sensitized peritoneal cells which were treated with the various doses of antigen alone expressed the
same level of delayed reaction as the control rats which were injected with the
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OL 0-
001
05
0.05 Artigen
concentration
5.0
imgl
FIG. 2. Desensitization of delayed type hypersensitivity (DTH) in adoptive transfer system. Peritoneal cells from adult female rats immunized 14 days previously with DNP-BGG-FCA were washed four times. 2 X 10’ cells were incubated in ice for half an hour with the various doses of DNP-BGG only (0), or with the antigen and 0.5 ml of a hyperimmune rat anti-DNP-BGG antiserum (0). The cells were washed again and injected intravenously into normal adult rats at the ratio of 2 X 10’ cells/recipient. Control rats (A) were not injected. DTH was elicited in the recipients at the same time as the cell transfer. Five rats per group. Vertical bars represent standard errors of the arithmetic means.
cells. In contrast, recipients injected with immune immune peritoneal peritoneal cells treated with the immune serum and 0.05 or 0.5 mg of DNPBGG showed little or no delayed type hypersensitivity to the antigen. However, when the cells were treated with 10 big or 5 mg of the antigen in the presence of the immune serum, their ability to transfer the DTH response was not diminished. Thus, it is clearly shown that, in this system, antigen alone is not effective in desensitizing the DTH response. What is essential for desensitization is that antigen is presented complexed to antibody at an optimum ratio. untreated
Specificity Antiserum.
of the Desensitization
of the Delayed
Response by Antigen
and
Peritoneal cells from rats sensitized 14 clays previously with 1 mg/rat of DNP-BGG-FCA were washed as described above. 2 X lo7 cells were incubated in ice for 30 min with 100 pg of DNP-BGG, 100 s of DNP-BGG with 0.5 ml of rat anti-DNP-BGG antiserum, or 100 H of DNP-BGG, with 0.5 ml of rat anti-OA antiserum. After the incubation the cells were washed 3~ and resuspended to 2 x 10’ cells/ml in medium. The cells were then injected into normal rats at the rate of 2 X 10’ cells/rat. Control rats were injected with either the sensitized peritoneal cells alone or with sensitized cells which had been incubated with rat anti-DNP-BGG antiserum. Immediately after cell transfer, the recipient rats were tested for delayed hypersensitivity in the footpads. The results are shown in Table 1. Normal rats receiving the sensitized peritoneal cells treated with 100 pg of DNP-BGG and 0.5 ml of a rat antiDNP-BGG antiserum showed little or no delayed type hypersensitivity (Group 4). In contrast, the sensitized peritoneal cells treated with 100 pug of DNP-BGG and an unrelated antiserum, rat anti-OA, transfered the same level of delayed reactivity (Group 5) as the control rats which received the untreated immune
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1
2
Tome after desensbtlmg
4
3 m,ecWm
Iweek:
FIG. 3. Time course of recovery from desensitization. Adult female AS rats were each immunized subcutaneously with 1 mg of DNP-BGG in FCA. Fmourteen days later, half of them were desensitized by an intravenous injection of 100 rg/rat of DNP-BGG in 1.0 ml saline (0). The desensitized rats and the control rats were respectively divided into groups of five rats and elicited for DTH in the footpads at different times after the intravenous injection. Footpad swelling was determined 24 hr after the eliciting injection. Vertical bars represent standard errors of the means.
cells (Group 1). In addition, treatment with the antigen alone (Group 2) or with the antiserum alone (Group 3) had no effect on the ability of the immune cells to transfer delayed type hypensensitivity. Time Course of Recovery
from Desensitization
Figure 3 shows the time course of recovery of delayed-type hypersensitivity in rats primed with DNP-BGG and desensitized 14 days later with an intravenous injection of 100 e of DNP-BGG. Three weeks after the desensitizing injection, the delayed response was still significantly suppressed (P < 0.05) compared with the control animals. However, the delayed-type hypersensitivity had completely recovered by the fourth week after the desensitizing injection. DISCUSSION In this paper evidence is presented suggesting that antibody plays an important role in the desensitization of delayed-type hypersensitivity. Initially, evidence was obtained for this concept by the demonstration that with sensitized rats, only certain doses of soluble antigen could affectively desensitize delayed hypersensitivity. Subsequently, it was shown that when DTH was adoptively transfered, the sensitized cells were not desensitized by antigen alone, but were completely desensitized by the presence of a suitable ratio of antigen and specific antiserum. Consistent with these findings is the recent report (19) that delayed hypersensitivity to sheep erythrocytes in mice was not suppressed hy massive doses of the erythrocytes. However, these fiindings are seemingly in contrast with the results of Gel1 and Wolstencroft (10) and of Asherson and Stone (20). Gel1 and Wolstencroft failed to obtain consistent results in adoptive transfer experiments with cells from desensitized guinea pigs and with sensitized cells exposed to antigen in vitro. These experiments, however, are complicated by the
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fact that irradiated animals were used as recipients, and such animals are known to be deficient in accessory cells required for the expression of DTH. Asherson and Stone reported that the adoptive transfer of delayed hypersensitivity with guinea pig peritoneal exudate cells could be specifically diminished by exposure of the cells in vitro to 1 mg/ml of bovine gamma globulin. However, in their experiments the recipients were injected intravenously with 3 ml of immune serum a few hours before cell transfer and skin test, a situation not unlike the present system. A number of workers (9, 12) have investigated the specificity of desensitization of delayed hypersensitivity. For example, Uhr and Pappenheimer (9) demonstrated that the desensitization of egg albumin or tetanus toxoid DTH was specific if the guinea pigs were sensitized with albumin or toxoid separately. However, if the animals were sensitized with both antigens simultaneously, then desensitizing to one antigen affected the expression of DTH to the other antigen. On the other hand, Leskowitz and Jones (12) produced specific desensitization lasting 11 days with arsanyl-bovine serum albumin. The nonspecific desentization reported by Uhr and Pappenheimer might be due to a temporary decrease in the availability of one or more factors necessary for the expression of DTH, especially in view of the fact that the effect lasted only 24 hr. It is therefore likely that desensitization of delayed hypersensitivity is specific in its classical sense. The present study emphasizes the similarity between the mechanism of desensitization and that of immunological enhancement in that (a) both phenomena require the presence of specific antibody, (b) both effects are immunologically specific, and (c) the effects are only temporary. It may well be that the antigen-antibody complexes either directly or indirectly induce a specific temporary inactivation of the sensitized T cells. This concept that desensitization and enhancement represent a central effect, is consistent with observations by other workers (21-24). In subsequent publications a more comprehensive study of the interaction between antibody, antigen, and the cells involved will be presented. ACKNOWLEDGMENTS The author thanks Dr. C. R. Parish Mikin for technical assistance.
for
helpful
discussion
and advice
and Mr.
KEFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Feldman, J. D., Adzw. Znw~wzol. 15, 167, 1972. Asherson, G. L., Bra?. Med. Bull. 23, 24, 1967. Bloom, B. R., Adzm. Iwmnol. 13, 101, 1971. Cooper, M. G., and Ada, G. L., S~(zmf. J. 1~~1flxol. 1, 247, 1972. Voisin, G. A., Prog. Allergy 15, 328, 1971. Hellstrom, K. E., and Hellstrom, I., Adzwrr. Ctrrrrrr lies. 12, 169, 1069. Klein, W. J. Jr., J. Immunol. 109, 51, 1972. Jeckel, J. J., McKenzie, I. F. C., and Winn, H. J., J. I/llnm~tol. 108, 1017, lY72. Uhr, J. W., and Pappenheimer, A. M. Jr., J. Exp. Mrd. 108, 891, 1958. Gell, P. G. H., and Wilstenacroft, R. A., 13rit. Med. Bull. 23, 21, 1967. Dvorak, II. F., and Flax, M. H., J. I~znmnol. 96, 546, 1966. Leskowitz, S., and Jones, V. E., J. ImmrnoZ. 95, 331, 1965.
Samai
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13. Kostiala, A. A. I., and Kosunen, T. U., Sra,ld. J. I~nwzwol. 1, 153, 1972. 14. Jokipii, L., Immunology 25, 283, 1973. 1.5. Bulletin WHO 48, 483, 1973. 16. Parish, C. R., J. Exp. Med. 134, 21, 1971. 17. Liew, F. Y., Immztnology 21, 1045, 1971. 18. Parish, C. R., and Liew, F. Y., J. Exp. Med. 135, 298, 1972. 19. Lagrange, P. H., MacKaness, G. B., and Miller, T. E., J. Exp. Med. 139, 528, 1974. 20. Asherson, G. L., and Stone, S. H., Immmzo1ogy 13, 469, 1967. 21. Dwyer, J. M., and Kantor, F. S., J. Exp. Med. 137, 32, 1972. 22. Radovich, J., and Talmage, D. W., Scirncc 158, 512, 1967. 23. Yoshida, T. O., and Anderson, B., Stand. J. Im~rz~~ol. 1, 401, 1972. N. A., and Tigelaar, R. E., Ia “Cellular Se24. Gorczynski, R., Kontiainen, S., Mitchison, lection and Regulation in the Immune Response.” (G. M. Edelman, Ed.), pp. 143-154. Raven Press, New York, 1974.
IMMUNOLOGY
19, 129-136 (1975)
SHORT COMMUNICATIONS Desensitization
of Delayed-Type and Specific
Rrccizvd
Hypersensitivity Antibody
DCCCIJJ~XT
by Antigen
3, 19i4
The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP,,-BGG in Freund’s complete adjuvant (FCA) were desensitized 14 days later with various doses of DNPwBGG injected intravenously. It was found that only certain doses (100-500 rg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum ijt vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum sepanately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3-4 wk after desensitizing injection.
INTKODUCTION are currently under Mechanisms which suppress cell-mediated immunity much investigation. An understanding of these mechanisms is obviously of great and tumor immunology. The importance in transplantation, antimicrobial, in sensitized individuals by specific suppression of cell-mediated immunity enhancement” or “desensiantigen is variously referred to as “immunological tization” (1, 2). There is a large body of evidence indicating that the effector cells in cell-mediated immunity are thymus-derived (T) lymphocytes (3, 4). Classically, immunological enhancement refers to the situation in which sensitized T lymphocytes present in an individual are unable to mediate cellular immunity because of blocking by antibodies (5-7) or antigen-antibody complexes (8, 9), although the exact mechanism of action is not known. In contrast, desensitization of cell-mediated immunity, as exemplified by delayed-type hypersensitivity (DTH) can be achieved by injection of antigen in large or repeated doses (g-11). Again, the exact mechanism by which this desensitization is effected is not known (2, 12-14). It is believed by some that sensitized T lymphocytes are exhausted of their capacity to produce mediators and therefore are unable to respond to antigen encountered in the tissues ( 15). Thus the role of antibody in the desensitization of DTH is generally dismissed. In the present report, however, evidence will be presented suggesting that contrary to popular 129 Copyright All rights
1975 by Academic Press, Inc. oQ reproduction in any form reserved.
130
SHORT
COMMUNICATIONS
belief, antibocly plays an important role in the desensitization soluble antigen, DNP-BGG, in rats.
of DTH
to a
Female inbred AS strain rats 6-9 wk of age were used. Antigens Ovalbumin (British Drug House) was dissolved in phosphate-buffered saline (PBS) and stored frozen at a concentration of 20 mg/ml. Dinitrophenylated bovine gamma globulin (DNP-BGG) was prepared by reacting 1.0 g of dinitrobenzene sulphonic acid with 1.0 g of BGG (Sigma) in 50 ml distilled water containing 1.0 g of K,COJ. The mixture was stirred with a magnetic stirrer at room temperature for 18 hr. The reaction was stopped by rapid dialysis in the cold against normal saline. The degree of substitution was estimated photometrically to be 32 DNP group per BGG molecule (16). Antisera Rat anti-DNP-BGG hyperimmune serum was obtained from adult rats which were immunized subcutaneously with 1 mg/rat of DNP-BGG in Freund’s complete adjuvant (FCA) and boosted intraperitoneally 4 wk later with 1 mg of DNP-BGG in saline. The rats were bled out 1 wk after the booster injection. Serum was separated from the clot on the same day, pooled and stored at -10°C. Rat anti-OA hyperimmune serum was prepared similarly using ovalbumin as the immunizing antigen. Experiwaental
Procedures
Groups of five adult rats were immunized subcutaneously with 1 mg/rat of DNP-BGG in Freund’s complete adjuvant. Control rats were injected with FCA-saline. Fourteen days later, delayed type hypersensitivity (DTH) was elicited by the injection into the right hind footpads of 1Opg of DNP-BGG in 50 ~1 of saline. The left hind footpads was injected with 50 ~1 of saline. Desensitizing doses of DNP-BGG were injected intravenously at the same time as the eliciting antigen. DTH was determined 24 hr after the footpad injection. For adoptive transfer experiments, peritoneal cells (PC) from rats immunized 14 days previously with 1 mg/rat of DNP-BGG-FCA were used. The sensitized peritoneal cells were washed 4~ in cold PBS containing 5% normal rat serum. 2 x lo7 cells (one rat equivalent) were incubated in ice for half an hour with various doses of DNP-BGG or with DNP-BGG in the presence of either 0.5 ml of a rat anti-DNP-BGG hyperimmune serum or a rat anti-OA hyperimmune serum. The mixture was then washed three times in PBS containing 5% normal rat serum, resuspended in 1 ml of the medium, and injected intravenously into normal rats. Control rats were injected with the same number of the untreated cells. DTH was elicited in the recipients at the same time as the intravenous injection. DTH was determined 24 hr later. For the experiment on the time course of recovery from desensitization, rats were immunized with 1 mg/rat of DNP-BGG-FCA. Fourteen days later, half of the rats were desensitized by an intravenous injection of 100 pg/rat of
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DNP-BGG in 1.0 ml saline. Control rats were injected with 1.0 ml of saline only. The desensitized rats and the control rats were, respectively, divided into groups of five rats and elicited for DTH in the footpads at different times after the intravenous injection. Antibody
Estimation
Antibodies to DNP-BGG and OA were estimated by hemagglutination using sheep erythrocytes sensitized with DNP-EGG or OA, respectively, by a chromic chloride procedure (17). Assay for Delayed-Type
Hypersemitivity
Delayed-type hypersensitivity was determined by measuring the increase in footpad thickness after the injection of antigen in saline into the hind footpads. DNP-BGG specific reactions were elicited by injecting 10 pg of DNP-BGG in 50 ~1 saline into the right hind footpads and saline (50 ~1) alone into the left hind footpad. Footpad thickness was measured 24 hr after the injection and specific footpad swelling was determined by subtracting the thickness of the left hind footpad from the right. Footpad thickness was measured by a dial caliper (Mitutoyo, Japan) which had 0.01 mm graduations. The accuracy of the assay has been previously discussed (18). RESULTS Efect
of Antigen
Dose on the Desensitization
of Delayed-Type
Hypersensitivity
Adult female inbred AS strain rats were injected subcutaneously in the flank with 1 mg of dinitrophenylated bovine gamma globulin (DNP,g-BGG) in Freund’s complete adjuvant (FCA). These rats expressed high level of delayed-type hypersensitivity to DNP,,-BGG, as measured by the 24-hr footpad swelling method. The DTH response reached a plateau 12-14 days after sensitization and remained at the same level for at least 4 wk. Desensitizing doses of TABLE ANTIBODY
Group
1
2 3 4 5
Immune peritoneal cells
2 2 2 2 2
x x x x x
10’ 107 10’ 107 107
SPECIFICITY
1
IN THE
Antigen (100 rd
DNP-BGG
DNP-BGG DNP-BGG
* Groups of five adult female AS rats were injected 2 X lO’/rat of peritoneal washing cells (PC) from adult (1 mg/rat) 2 wk previously with DNP-BGG in FCA. in ice for half an hour with either antigen, antiserum, (group 1) were given the PC alone. Immediately after delayed hypersensitivity at the footpads. b P < 0.01 compared to group 1.
DESENSITIZATIONS
Antiserum (0.5 ml)
24 hr footpad swelling (l/lOth mm f SE)
-
2.3 f 0.2 2.4 i 0.3
Anti-DNP-BGG Anti-DNP-BGG Anti-OA
2.1 f 0.4 0.4 Yk O.lb
2.0 f 0.2
intravenously through the tail veins with female AS rats immunised subcutaneously Prior to injection, the cells were incubated or both, and then washed. Control rats the iv injection, the rats were elicited for
132
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COMMUNICATIONS
OL b
0.01 0.05
0.1 Antigen
0.5 concentration
1.0
5.0
10.0
Img)
FIG. 1. Desensitization of DNP-BGG sensitized rats with various doses of Groups of five adult rats were immunized subcutaneously with 1 mgjrat FCA. Control rats (0) were injected with saline-FCA. All rats, except desensitized 14 days later by intravenous injection of the various doses
saline. All the animals were tested for DTH desensitizing injection. Vertical bars represent
DNP-BGG in V&O, of DNP,-BGG in the controls, were of DNPZS2-BGG in
(footpad swelling) at the same time as the standard errors of the arithmetic means.
DNP,,-BGG (ranging from 10 pg to 10 mg in 1.0 ml of saline) were injected intravenously into groups of rats sensitized 14 days previously. Immediately after the intravenous injection the rats were tested for DTH in the footpads with 10 ,,.g of DNP-@GG in 50 ~1 of saline. Figure 1 shows that rats hypersensitive to DNP-BGG could be desensitized by optimum doses (100-500 pg P < 0.05) of DNP-BGG. Outside this dose range, the antigen either partially or completely failed to desensitize the delayed response in the rats. Desensitization
in the Adoptive
Transfer
System
Since rats sensitized with DNP-BGG-FCA contain high levels of antiDNP-BGG antibody on day 14 (av titre of 480), it could well be that the results reflected an in vivo optimum antigen/antibody ratio which effectively caused the desensitization of the delayed-type hypersensitivity. The following experiment was carried out to investigate this further. Peritoneal cells from rats sensitized with DNP-BGG-FCA 14 days previously were collected and washed four times by centrifugation in cold PBS containing 5% normal rat serum. The sensitized peritoneal cells were injected intravenously into normal recipient rats in the ratio of one donor to one recipient or 2 X lo7 cells per rat. Prior to injection, the cells were incubated in ice for half an hour with various doses of
DNP-BGG
with or without
rat anti-DNP-BGG
immune serum. The antiserum
(titre, 1000) was obtained from AS rats hyperimmunized with DNP-BGG and was added in the ratio of 0.5 ml per rat. After the incubation, cells were washed 3 x with cold PBS containing 5% normal rat seruni and resuspended Immediately after the cell transfer, the reto 2 x 10’ cells/ml in the medium. cipient
rats
were tested for DTH
reactions in the footpads. The 24-hr footpad
reactions are shown in Fig. 2. Normal rats receiving the sensitized peritoneal cells which were treated with the various doses of antigen alone expressed the
same level of delayed reaction as the control rats which were injected with the
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OL 0-
001
05
0.05 Artigen
concentration
5.0
imgl
FIG. 2. Desensitization of delayed type hypersensitivity (DTH) in adoptive transfer system. Peritoneal cells from adult female rats immunized 14 days previously with DNP-BGG-FCA were washed four times. 2 X 10’ cells were incubated in ice for half an hour with the various doses of DNP-BGG only (0), or with the antigen and 0.5 ml of a hyperimmune rat anti-DNP-BGG antiserum (0). The cells were washed again and injected intravenously into normal adult rats at the ratio of 2 X 10’ cells/recipient. Control rats (A) were not injected. DTH was elicited in the recipients at the same time as the cell transfer. Five rats per group. Vertical bars represent standard errors of the arithmetic means.
cells. In contrast, recipients injected with immune immune peritoneal peritoneal cells treated with the immune serum and 0.05 or 0.5 mg of DNPBGG showed little or no delayed type hypersensitivity to the antigen. However, when the cells were treated with 10 big or 5 mg of the antigen in the presence of the immune serum, their ability to transfer the DTH response was not diminished. Thus, it is clearly shown that, in this system, antigen alone is not effective in desensitizing the DTH response. What is essential for desensitization is that antigen is presented complexed to antibody at an optimum ratio. untreated
Specificity Antiserum.
of the Desensitization
of the Delayed
Response by Antigen
and
Peritoneal cells from rats sensitized 14 clays previously with 1 mg/rat of DNP-BGG-FCA were washed as described above. 2 X lo7 cells were incubated in ice for 30 min with 100 pg of DNP-BGG, 100 s of DNP-BGG with 0.5 ml of rat anti-DNP-BGG antiserum, or 100 H of DNP-BGG, with 0.5 ml of rat anti-OA antiserum. After the incubation the cells were washed 3~ and resuspended to 2 x 10’ cells/ml in medium. The cells were then injected into normal rats at the rate of 2 X 10’ cells/rat. Control rats were injected with either the sensitized peritoneal cells alone or with sensitized cells which had been incubated with rat anti-DNP-BGG antiserum. Immediately after cell transfer, the recipient rats were tested for delayed hypersensitivity in the footpads. The results are shown in Table 1. Normal rats receiving the sensitized peritoneal cells treated with 100 pg of DNP-BGG and 0.5 ml of a rat antiDNP-BGG antiserum showed little or no delayed type hypersensitivity (Group 4). In contrast, the sensitized peritoneal cells treated with 100 pug of DNP-BGG and an unrelated antiserum, rat anti-OA, transfered the same level of delayed reactivity (Group 5) as the control rats which received the untreated immune
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1
2
Tome after desensbtlmg
4
3 m,ecWm
Iweek:
FIG. 3. Time course of recovery from desensitization. Adult female AS rats were each immunized subcutaneously with 1 mg of DNP-BGG in FCA. Fmourteen days later, half of them were desensitized by an intravenous injection of 100 rg/rat of DNP-BGG in 1.0 ml saline (0). The desensitized rats and the control rats were respectively divided into groups of five rats and elicited for DTH in the footpads at different times after the intravenous injection. Footpad swelling was determined 24 hr after the eliciting injection. Vertical bars represent standard errors of the means.
cells (Group 1). In addition, treatment with the antigen alone (Group 2) or with the antiserum alone (Group 3) had no effect on the ability of the immune cells to transfer delayed type hypensensitivity. Time Course of Recovery
from Desensitization
Figure 3 shows the time course of recovery of delayed-type hypersensitivity in rats primed with DNP-BGG and desensitized 14 days later with an intravenous injection of 100 e of DNP-BGG. Three weeks after the desensitizing injection, the delayed response was still significantly suppressed (P < 0.05) compared with the control animals. However, the delayed-type hypersensitivity had completely recovered by the fourth week after the desensitizing injection. DISCUSSION In this paper evidence is presented suggesting that antibody plays an important role in the desensitization of delayed-type hypersensitivity. Initially, evidence was obtained for this concept by the demonstration that with sensitized rats, only certain doses of soluble antigen could affectively desensitize delayed hypersensitivity. Subsequently, it was shown that when DTH was adoptively transfered, the sensitized cells were not desensitized by antigen alone, but were completely desensitized by the presence of a suitable ratio of antigen and specific antiserum. Consistent with these findings is the recent report (19) that delayed hypersensitivity to sheep erythrocytes in mice was not suppressed hy massive doses of the erythrocytes. However, these fiindings are seemingly in contrast with the results of Gel1 and Wolstencroft (10) and of Asherson and Stone (20). Gel1 and Wolstencroft failed to obtain consistent results in adoptive transfer experiments with cells from desensitized guinea pigs and with sensitized cells exposed to antigen in vitro. These experiments, however, are complicated by the
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fact that irradiated animals were used as recipients, and such animals are known to be deficient in accessory cells required for the expression of DTH. Asherson and Stone reported that the adoptive transfer of delayed hypersensitivity with guinea pig peritoneal exudate cells could be specifically diminished by exposure of the cells in vitro to 1 mg/ml of bovine gamma globulin. However, in their experiments the recipients were injected intravenously with 3 ml of immune serum a few hours before cell transfer and skin test, a situation not unlike the present system. A number of workers (9, 12) have investigated the specificity of desensitization of delayed hypersensitivity. For example, Uhr and Pappenheimer (9) demonstrated that the desensitization of egg albumin or tetanus toxoid DTH was specific if the guinea pigs were sensitized with albumin or toxoid separately. However, if the animals were sensitized with both antigens simultaneously, then desensitizing to one antigen affected the expression of DTH to the other antigen. On the other hand, Leskowitz and Jones (12) produced specific desensitization lasting 11 days with arsanyl-bovine serum albumin. The nonspecific desentization reported by Uhr and Pappenheimer might be due to a temporary decrease in the availability of one or more factors necessary for the expression of DTH, especially in view of the fact that the effect lasted only 24 hr. It is therefore likely that desensitization of delayed hypersensitivity is specific in its classical sense. The present study emphasizes the similarity between the mechanism of desensitization and that of immunological enhancement in that (a) both phenomena require the presence of specific antibody, (b) both effects are immunologically specific, and (c) the effects are only temporary. It may well be that the antigen-antibody complexes either directly or indirectly induce a specific temporary inactivation of the sensitized T cells. This concept that desensitization and enhancement represent a central effect, is consistent with observations by other workers (21-24). In subsequent publications a more comprehensive study of the interaction between antibody, antigen, and the cells involved will be presented. ACKNOWLEDGMENTS The author thanks Dr. C. R. Parish Mikin for technical assistance.
for
helpful
discussion
and advice
and Mr.
KEFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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13. Kostiala, A. A. I., and Kosunen, T. U., Sra,ld. J. I~nwzwol. 1, 153, 1972. 14. Jokipii, L., Immunology 25, 283, 1973. 1.5. Bulletin WHO 48, 483, 1973. 16. Parish, C. R., J. Exp. Med. 134, 21, 1971. 17. Liew, F. Y., Immztnology 21, 1045, 1971. 18. Parish, C. R., and Liew, F. Y., J. Exp. Med. 135, 298, 1972. 19. Lagrange, P. H., MacKaness, G. B., and Miller, T. E., J. Exp. Med. 139, 528, 1974. 20. Asherson, G. L., and Stone, S. H., Immmzo1ogy 13, 469, 1967. 21. Dwyer, J. M., and Kantor, F. S., J. Exp. Med. 137, 32, 1972. 22. Radovich, J., and Talmage, D. W., Scirncc 158, 512, 1967. 23. Yoshida, T. O., and Anderson, B., Stand. J. Im~rz~~ol. 1, 401, 1972. N. A., and Tigelaar, R. E., Ia “Cellular Se24. Gorczynski, R., Kontiainen, S., Mitchison, lection and Regulation in the Immune Response.” (G. M. Edelman, Ed.), pp. 143-154. Raven Press, New York, 1974.