Immunology 1979 38 547
Specific suppression of delayed hypersensitivity response to sheep erythrocytes by heterologous anti-lymphocyte serum
CAROLINE REUBEN & G. P. PHONDKE Bio-Medical Group, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Bombay, India
Acceptedjor publication 7 June 1979
Summary. The development of a heterologous antilymphocyte serum (ALS) capable of specifically suppressing the delayed hypersensitivity (DH) response is reported. This ALS, termed ALS(CMI), was prepared against lymph node cells from rats which had been immunized against sheep erythrocytes (SRBC) following treatment with cyclophosphamide which is known to enhance the DH response and suppress the humoral immune response. The effect of ALS(CMI) on the primary DH-response to SRBC using the footpad swelling test was studied. Its effect on the primary humoral immune response to SRBC was also studied using the Jerne plaque assay technique. ALS(CMI) suppressed the humoral antibody response to SRBC and the DH response to a third party antigen only when administered before the antigen, having no effect when administered post-antigenically. On the other hand, ALS(CMI) significantly suppressed the primary DH response to SRBC when administered either before or after the antigen. INTRODUCTION
Anti-lymphocyte serum (ALS) has been shown to be an Correspondence: Dr G. P. Phondke, Bio-Medical Group, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India. 0019-2805/79/1 100-0547$02.00 ©) 1979 Blackwell Scientific Publications
547
effective suppressor of cell mediated immune response (Turk & Willoughby, 1967; Turk, Willoughby & Stevens, 1968; Willoughby, Walters & Spector, 1965). This immunosuppression is effectively manifest provided the ALS is administered prior to the antigenic stimulus. Suppression of established cellular immune responses, on the other hand, is largely equivocal. Levey & Medawar (1966, 1967) and Nagaya & Sieker (1966) have reported that prolonged administration of ALS can result in significant supprtssion of cell mediated immunity. The first dose of ALS, however, has to be given along with the antigen for effecting such suppression. Furthermore, reversion to the sensitive state occurs immediately on discontinuation of ALS treatment. In earlier communications (Reuben, Sundaram & Phondke, 1978, 1979) development of a heterologous ALS capable of suppressing even established humoral immune response to sheep erythrocytes (SRBC) was reported. The immunosuppression manifest by this ALS, termed ALS(I), appeared to be mediated in a manner similar to that brought about by anti-idiotypic antisera. This indicated that even normal plasma cells carry surface antigens analogous to idiotypic determinants. The immunosuppressive ability of ALS(I) displayed another kind of specificity. ALS(I) failed to suppress delayed hypersensitivity (DH) responses even to the specific immunizing antigen if given after the antigen. This observation suggested that the effector cells responsible for the manifestation of DH re-
Caroline Reuben & G. P. Phondke
548
sponse might possess specific surface determinants. It was, therefore, of interest to investigate this possibility and also to attempt development of a heterologous ALS capable of specifically suppressing the DH response. MATERIALS AND METHODS
Animals Eight- to ten-week old Wistar rats were used. Antisera were raised in white Belgian rabbits of either sex. Preparation of antisera
ALS(N). Rabbit anti-rat lymphocyte serum was prepared essentially as described by Levey & Medawar (1966). Rabbits were injected i.p. with 2 x 108 lymph node cells from normal unimmunized Wistar rats. Three weekly injections were administered and the rabbits were bled 10 days after the last injection. ALS(I). This anti-lymphocyte serum was prepared using the same protocol as for raising ALS(N) except that lymph node cells from rats pre-immunized with SRBC were employed. (Reuben et al., 1978). Two subcutaneous injections of 0-2 ml of 10% SRBC were administered into each front footpad of the rats 3 weeks apart. Ten days later cell suspensions were prepared from the axillary lymph nodes. ARGG. Anti-rat gammaglobulin was prepared as reported earlier (Reuben et al., 1978) by the technique of Exum & Bowser (1970).
Figure 1. Photograph showing footpad swelling in rats used in the preparation of ALS(CMI) after treatment with cyclophosphamide. The footpad on the right was injected with 0 2 ml of 50% v/v SRBC and the footpad on the left with 0 2 ml of saline. The footpad swelling was observed to be 4 8 mm. No detectable haemagglutination titre was observed.
were prepared in physiological saline. Rabbits were injected i.p. with these cells following the same immunization schedule employed for raising ALS(I) and ALS(N). Three different lots of antisera were used in these experiments. As all three exhibited essentially similar behaviour the data were pooled. All antisera were absorbed with SRBC, inactivated at 56° for 30 min and stored in small aliquots at -20°.
Experimental procedure ALS(CMI). Eight- to ten-week old Wistar rats were injected i.p. with 100 mg of cyclophosphamide (Endoxan Asta, Asta Werke, AG, W. Germany) per kg body weight (Kerckhaert, Hofhuis & Willers, 1977). Eight hours later the rats were injected s.c. in the nuchal region with 2 x 109 SRBC. Four days later 0-2 ml of 50% v/v SRBC was injected (s.c.) into the left front footpad and 0 2 ml of saline into the right front footpad. The footpad swelling was found to be significantly greater than even the highest value recorded during normal primary or secondary response (see Fig. 1). At the same time the sera from these animals showed no detectable anti-SRBC antibody as assayed by the haemagglutination technique. The axillary and thoracic lymph nodes were removed from these animals. Single-cell suspensions
Haemolytic plaque assay. The Jerne plaque assay technique (Jerne, Nordin & Henry, 1963) as modified by Wortis, Taylor & Dresser (1968) was used. Details are described in our earlier communication (Reuben et al., 1978). Footpad swelling test. The technique for the assay of the DH response as described by Tamura & Egashira (1976) was used. Rats were sensitized i.p. with 2 x 109 SRBC and an eliciting dose of approximately 0-2 ml of 50% v/v SRBC in saline was injected 4 days later into the left front footpad subcutaneously with a 27 gauge needle. The control right footpad was injected simultaneously with an equal volume of saline. The footpad cross-sections were measured 24 h later with vernier
549
Suppression of DH response by ALS
callipers having a least count of 0-02 mm. The footpad swelling is the difference between the cross-sections of the test antigen footpad and the control saline footpad. In the series of experiments wherin the DH response to rabbit erythrocytes (RRBC) was measured the same experimental procedure was employed. Treatment of animals. Sheep erythrocytes (SRBC) preserved in Alsever's solution at 40 were used. Rats were immunized with 2 x 109 SRBC i.p. They were also injected (i.p.) with 04 ml of either ALS(I), ALS(N), ALS(CMI) or NRS I day before or I day after the antigen. The eliciting dose was administered on the fourth day of antigen administration. The footpad swelling was measured 24 h later. Twelve animals were included in each group with one lot of antiserum given to four animals. These experiments were repeated using RRBC as the antigen in place of SRBC. The effect of ALS on the primary humoral immune response to SRBC was also studied using the plaque assay technique.
ALS(I) and ALS(N) failed to have any immunosuppressive effect on the DH response to SRBC when administered one day after the antigen (Fig. 2). This is in accordance with our earlier finding (Reuben et al., 1979). ALS(CMI) on the other hand retained its ability to suppress the DH response even when administered postantigenically (Fig. 2). In order to check whether this differential immunosuppression was a result of varying potency of these antisera different amounts of ALS were administered postantigenically. It is seen from Fig. 3 that even higher amounts,
90
z
0
80
-
70
-
ALSI ALSN 0-O ALS(CMI) -
0 -
60
W 50
D 40
cn
RESULTS
*30
Effect of ALS on DH response All the three types of ALS used in these studies significantly suppressed the DH response to SRBC when administered before the antigen (Fig. 2). Of these
20 10 0 -10
I 04
30
L NRS
ALS(CMI)
M
ALS(I)
ALS(N)
E o20 0
z
U.
Figure 2. Effect of ALS on the primary DH response to SRBC. ALS was administered 1 day before the antigen (1) or I day after the antigen (2). Eliciting dose was administered 4 days after the antigen and footpad measurements taken 24 h later. Vertical bars indicate the standard errors of the mean of twelve observations.
12 0-8 VOWME (ml)
Figure 3. The effect ofdifferent amounts of ALS administered postantigenically on the percentage suppression of the primary DH response to SRBC. Percentage suppression= f.p. sw. with NRS-f.p. sw. with ALS ^ 1(V)n *A 1V\ f.p. sw. with NRS
up to 1-2 ml, of ALS(I) or ALS(N) failed to bring about any significant decrease in the footpad swelling.
Specificity of ALS for the immunizing antigen The DH response to RRBC was likewise suppressed significantly by all the three types of ALS when administered prior to the antigen (Fig. 4). However, even the ALS(CMI) proved ineffective when given after immunization (Fig. 4).
550
Caroline Reuben & G. P. Phondke DISCUSSION _.20-
,L__ ..
It is clear from the data in Fig. 2 that ALS given before the antigen suppressed the primary DH response significantly. All the three different types of ALS used in the present investigations exhibited this immunosuppressive ability. Further, this ability did not appear to depend upon the nature of the immunizing antigen since the DH response to RRBC was also suppressed to a similar extent (Fig. 4). This is true even of ALS(I) which does possess antigenic specificity in its ability to suppress established humoral immune response (Reuben et al., 1978, 1979). The observed reduction in the footpad swelling therefore most likely stems from the interference caused by the ALS in the early recognition events. This conclusion is in agreement with the earlier reports of Lance, Medawar & Taub (1973) and Turk & Willoughby (1967). Administration of ALS subsequent to the immunizing dose of antigen brought out the differences in the behaviour of the various types of ALS. Whereas ALS(N) and ALS(I) proved ineffective under this schedule, ALS(CMI) retained its immunosuppressive ability (Fig. 2). The extent of suppression was of the same order as that obtained when the antiserum was given prior to the antigen. It is unlikely that this depression in the DH response is caused by anti-SRBC antibody as none of the antisera possessed any detectable anti-SRBC antibody. Furthermore, the antisera used in the present studies were absorbed with SRBC. Barth & Singla (1972) had also discounted the contribution of heterophile antibody in the immunosuppressive ability of the ALS.
_
E
zTT 10 0
a
Specific suppression of delayed hypersensitivity response to sheep erythrocytes by heterologous anti-lymphocyte serum
CAROLINE REUBEN & G. P. PHONDKE Bio-Medical Group, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Bombay, India
Acceptedjor publication 7 June 1979
Summary. The development of a heterologous antilymphocyte serum (ALS) capable of specifically suppressing the delayed hypersensitivity (DH) response is reported. This ALS, termed ALS(CMI), was prepared against lymph node cells from rats which had been immunized against sheep erythrocytes (SRBC) following treatment with cyclophosphamide which is known to enhance the DH response and suppress the humoral immune response. The effect of ALS(CMI) on the primary DH-response to SRBC using the footpad swelling test was studied. Its effect on the primary humoral immune response to SRBC was also studied using the Jerne plaque assay technique. ALS(CMI) suppressed the humoral antibody response to SRBC and the DH response to a third party antigen only when administered before the antigen, having no effect when administered post-antigenically. On the other hand, ALS(CMI) significantly suppressed the primary DH response to SRBC when administered either before or after the antigen. INTRODUCTION
Anti-lymphocyte serum (ALS) has been shown to be an Correspondence: Dr G. P. Phondke, Bio-Medical Group, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India. 0019-2805/79/1 100-0547$02.00 ©) 1979 Blackwell Scientific Publications
547
effective suppressor of cell mediated immune response (Turk & Willoughby, 1967; Turk, Willoughby & Stevens, 1968; Willoughby, Walters & Spector, 1965). This immunosuppression is effectively manifest provided the ALS is administered prior to the antigenic stimulus. Suppression of established cellular immune responses, on the other hand, is largely equivocal. Levey & Medawar (1966, 1967) and Nagaya & Sieker (1966) have reported that prolonged administration of ALS can result in significant supprtssion of cell mediated immunity. The first dose of ALS, however, has to be given along with the antigen for effecting such suppression. Furthermore, reversion to the sensitive state occurs immediately on discontinuation of ALS treatment. In earlier communications (Reuben, Sundaram & Phondke, 1978, 1979) development of a heterologous ALS capable of suppressing even established humoral immune response to sheep erythrocytes (SRBC) was reported. The immunosuppression manifest by this ALS, termed ALS(I), appeared to be mediated in a manner similar to that brought about by anti-idiotypic antisera. This indicated that even normal plasma cells carry surface antigens analogous to idiotypic determinants. The immunosuppressive ability of ALS(I) displayed another kind of specificity. ALS(I) failed to suppress delayed hypersensitivity (DH) responses even to the specific immunizing antigen if given after the antigen. This observation suggested that the effector cells responsible for the manifestation of DH re-
Caroline Reuben & G. P. Phondke
548
sponse might possess specific surface determinants. It was, therefore, of interest to investigate this possibility and also to attempt development of a heterologous ALS capable of specifically suppressing the DH response. MATERIALS AND METHODS
Animals Eight- to ten-week old Wistar rats were used. Antisera were raised in white Belgian rabbits of either sex. Preparation of antisera
ALS(N). Rabbit anti-rat lymphocyte serum was prepared essentially as described by Levey & Medawar (1966). Rabbits were injected i.p. with 2 x 108 lymph node cells from normal unimmunized Wistar rats. Three weekly injections were administered and the rabbits were bled 10 days after the last injection. ALS(I). This anti-lymphocyte serum was prepared using the same protocol as for raising ALS(N) except that lymph node cells from rats pre-immunized with SRBC were employed. (Reuben et al., 1978). Two subcutaneous injections of 0-2 ml of 10% SRBC were administered into each front footpad of the rats 3 weeks apart. Ten days later cell suspensions were prepared from the axillary lymph nodes. ARGG. Anti-rat gammaglobulin was prepared as reported earlier (Reuben et al., 1978) by the technique of Exum & Bowser (1970).
Figure 1. Photograph showing footpad swelling in rats used in the preparation of ALS(CMI) after treatment with cyclophosphamide. The footpad on the right was injected with 0 2 ml of 50% v/v SRBC and the footpad on the left with 0 2 ml of saline. The footpad swelling was observed to be 4 8 mm. No detectable haemagglutination titre was observed.
were prepared in physiological saline. Rabbits were injected i.p. with these cells following the same immunization schedule employed for raising ALS(I) and ALS(N). Three different lots of antisera were used in these experiments. As all three exhibited essentially similar behaviour the data were pooled. All antisera were absorbed with SRBC, inactivated at 56° for 30 min and stored in small aliquots at -20°.
Experimental procedure ALS(CMI). Eight- to ten-week old Wistar rats were injected i.p. with 100 mg of cyclophosphamide (Endoxan Asta, Asta Werke, AG, W. Germany) per kg body weight (Kerckhaert, Hofhuis & Willers, 1977). Eight hours later the rats were injected s.c. in the nuchal region with 2 x 109 SRBC. Four days later 0-2 ml of 50% v/v SRBC was injected (s.c.) into the left front footpad and 0 2 ml of saline into the right front footpad. The footpad swelling was found to be significantly greater than even the highest value recorded during normal primary or secondary response (see Fig. 1). At the same time the sera from these animals showed no detectable anti-SRBC antibody as assayed by the haemagglutination technique. The axillary and thoracic lymph nodes were removed from these animals. Single-cell suspensions
Haemolytic plaque assay. The Jerne plaque assay technique (Jerne, Nordin & Henry, 1963) as modified by Wortis, Taylor & Dresser (1968) was used. Details are described in our earlier communication (Reuben et al., 1978). Footpad swelling test. The technique for the assay of the DH response as described by Tamura & Egashira (1976) was used. Rats were sensitized i.p. with 2 x 109 SRBC and an eliciting dose of approximately 0-2 ml of 50% v/v SRBC in saline was injected 4 days later into the left front footpad subcutaneously with a 27 gauge needle. The control right footpad was injected simultaneously with an equal volume of saline. The footpad cross-sections were measured 24 h later with vernier
549
Suppression of DH response by ALS
callipers having a least count of 0-02 mm. The footpad swelling is the difference between the cross-sections of the test antigen footpad and the control saline footpad. In the series of experiments wherin the DH response to rabbit erythrocytes (RRBC) was measured the same experimental procedure was employed. Treatment of animals. Sheep erythrocytes (SRBC) preserved in Alsever's solution at 40 were used. Rats were immunized with 2 x 109 SRBC i.p. They were also injected (i.p.) with 04 ml of either ALS(I), ALS(N), ALS(CMI) or NRS I day before or I day after the antigen. The eliciting dose was administered on the fourth day of antigen administration. The footpad swelling was measured 24 h later. Twelve animals were included in each group with one lot of antiserum given to four animals. These experiments were repeated using RRBC as the antigen in place of SRBC. The effect of ALS on the primary humoral immune response to SRBC was also studied using the plaque assay technique.
ALS(I) and ALS(N) failed to have any immunosuppressive effect on the DH response to SRBC when administered one day after the antigen (Fig. 2). This is in accordance with our earlier finding (Reuben et al., 1979). ALS(CMI) on the other hand retained its ability to suppress the DH response even when administered postantigenically (Fig. 2). In order to check whether this differential immunosuppression was a result of varying potency of these antisera different amounts of ALS were administered postantigenically. It is seen from Fig. 3 that even higher amounts,
90
z
0
80
-
70
-
ALSI ALSN 0-O ALS(CMI) -
0 -
60
W 50
D 40
cn
RESULTS
*30
Effect of ALS on DH response All the three types of ALS used in these studies significantly suppressed the DH response to SRBC when administered before the antigen (Fig. 2). Of these
20 10 0 -10
I 04
30
L NRS
ALS(CMI)
M
ALS(I)
ALS(N)
E o20 0
z
U.
Figure 2. Effect of ALS on the primary DH response to SRBC. ALS was administered 1 day before the antigen (1) or I day after the antigen (2). Eliciting dose was administered 4 days after the antigen and footpad measurements taken 24 h later. Vertical bars indicate the standard errors of the mean of twelve observations.
12 0-8 VOWME (ml)
Figure 3. The effect ofdifferent amounts of ALS administered postantigenically on the percentage suppression of the primary DH response to SRBC. Percentage suppression= f.p. sw. with NRS-f.p. sw. with ALS ^ 1(V)n *A 1V\ f.p. sw. with NRS
up to 1-2 ml, of ALS(I) or ALS(N) failed to bring about any significant decrease in the footpad swelling.
Specificity of ALS for the immunizing antigen The DH response to RRBC was likewise suppressed significantly by all the three types of ALS when administered prior to the antigen (Fig. 4). However, even the ALS(CMI) proved ineffective when given after immunization (Fig. 4).
550
Caroline Reuben & G. P. Phondke DISCUSSION _.20-
,L__ ..
It is clear from the data in Fig. 2 that ALS given before the antigen suppressed the primary DH response significantly. All the three different types of ALS used in the present investigations exhibited this immunosuppressive ability. Further, this ability did not appear to depend upon the nature of the immunizing antigen since the DH response to RRBC was also suppressed to a similar extent (Fig. 4). This is true even of ALS(I) which does possess antigenic specificity in its ability to suppress established humoral immune response (Reuben et al., 1978, 1979). The observed reduction in the footpad swelling therefore most likely stems from the interference caused by the ALS in the early recognition events. This conclusion is in agreement with the earlier reports of Lance, Medawar & Taub (1973) and Turk & Willoughby (1967). Administration of ALS subsequent to the immunizing dose of antigen brought out the differences in the behaviour of the various types of ALS. Whereas ALS(N) and ALS(I) proved ineffective under this schedule, ALS(CMI) retained its immunosuppressive ability (Fig. 2). The extent of suppression was of the same order as that obtained when the antiserum was given prior to the antigen. It is unlikely that this depression in the DH response is caused by anti-SRBC antibody as none of the antisera possessed any detectable anti-SRBC antibody. Furthermore, the antisera used in the present studies were absorbed with SRBC. Barth & Singla (1972) had also discounted the contribution of heterophile antibody in the immunosuppressive ability of the ALS.
_
E
zTT 10 0
a
