Immunology 1977 33 925
Suppression of cell-mediated
immune responses
by dextran sulphate
G. F. BABCOCK* & R. E. McCARTHY Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68105, U.S.A.
Received 21 April 1977; acceptedfor publication 5 May 1977
Summary. The effect of high molecular weight dextran sulphate (DS) on cell-mediated immune responses was studied. Two criteria were used to assess cell-mediated responses, skin graft rejection and intra-dermal (i.d.) skin tests. Mice receiving allografts and DS displayed a significant increase in graft survival time compared with untreated control animals. Similar results were obtained when the grafted animals differed at only the H-Y locus.
using adjuvants to stimulate the immune response is that of immunosuppression rather than stimulation. Several adjuvants, such as phytohaemagglutinin, Bacillus Calmette-Guerin (BCG), Bordetellapertussis, and Freund's complete adjuvant (FCA), have been shown to suppress as well as stimulate cell-mediated immune responses (Calapinto, 1975; Jolles & Paraf, 1973; Schwab, 1975). Recently our laboratory has shown that dextran sulphate (DS) enhanced the cellmediated immune response to sheep red blood cells and egg albumin in mice and guinea pigs (McCarthy, Arnold & Babcock, 1976). In this study we investigated the effect of DS on two classical cell-mediated immune reactions, the rejection of mouse skin allografts and the intradermal skin reaction of guinea-pigs to Mycobacterium tuberculosis. The results presented here show that DS is capable of suppressing cell-mediated immune responses in mice and guinea-pigs.
Guinea-pigs sensitized with Mycobacterium tuberculosis and treated with DS showed a greatly reduced delayed skin test response when challenged with purified protein derivative. DS was capable of suppressing this response even though, by the same route, dosage, and time of administration, it was previously shown to enhance a cell-mediated response in guinea-pigs sensitized with a different antigen. The results of this study indicate that the adjuvant DS is capable of suppressing cell-mediated immune responses in mice and guinea-pigs.
MATERIALS AND METHODS
INTRODUCTION
Animals Male and female mice from 6 to 10 weeks of age of the following strains were used: CFW (Carworth Farms, Portage, Michigan); C57BL/6J (B6, Jackson Memorial Laboratory, Bar Harbor, Maine); and MA (Eppley Cancer Institute, Omaha, Nebraska). Female Camm/Hartley guinea-pigs (Camm Research, Wayne, N.J.) weighing 450-550 g were used. All animals were allowed food and water ad libitum.
A problem often encountered by immunologists * Present address: Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A. Correspondence: Dr R. E. McCarthy, Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68105, U.S.A.
925
G. F. Babcock & R. E. McCarthy
926
Chemicals Dextran sulphate (DS, MW 500,000, Pharmacia, Uppsala, Sweden), chondroitin sulphate (Sigma Chemical Co., St Louis, Missouri), heparin sodium (Fisher Scientific Co., Fair Lawn, N.J.), and disodium ethylenediaminetetraacetate (EDTA, Fisher Scientific Co.) were prepared by dissolving in Hank's balanced salt solution (HBSS, Grand Island Biological Co., Grand Island, N.Y.) or in phosphatebuffered saline (PBS, pH 7-2, 0-01 M phosphate) at the appropriate concentration and used immediately. Freund's complete adjuvant H37Ra (FCA) containing M. tuberculosis was purchased from Difco, Detroit, Michigan and purified protein derivative (PPD) containing 5 tuberculin units/0-1 ml was obtained from Parke-Davis Co., Detroit, Michigan. Skin transplantation The free skin grafting technique of Billingham & Medawar (1951) was used in all experiments. Donor ear skin was grafted onto the back of the recipient animals and held in place with gauze pressure bandage. The bandages were removed after 7 days and the grafts were observed daily until rejection. Rejection was determined by observation in situ (Billingham & Silvers, 1970). In addition, representative grafts from each group of animals were excised, fixed in neutral formalin, embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically. The dosage and treatment schedule for the various chemicals used will be
described in the results section. Significance was determined using the Student's t-test at 95% confidence limits (Snedecor & Cochran, 1967). Delayed-type hypersensitivity skin reactions in guineapigs
Guinea-pigs were sensitized to M. tuberculosis by injecting 0 1 ml of FCA into each footpad. Animals given DS received 5 ,l/g body weight of a solution containing 20, 40 or 60 mg/ml DS so as to give a final dosage of 100, 200 or 300 mg/kg respectively. The DS was given in four equal aliquots subcutaneously (s.c.) in the inguinal and axillary regions immediately after injection with FCA. All animals were skin tested 17 days after immunization. Each animal was challenged at two sites with 0*1 ml of PPD injected intradermally into the clipped flanks with a 26 gauge needle. Skin reactions were observed and measured at 4, 24 and 48 h. The delayed hypersensitivity reactions (induration at 24 and 48 h) were graded as follows: -, no induration; ±, 0-5 mm; +, 5-10 mm; ++, 10-15 mm diameter.
RESULTS Effect of DS and other selected polyanions on skin graft rejection The effect of DS and other selected polyanions and anticoagulants on the survival time of primary skin
Table 1. Effect of DS and other selected polyanions and anticoagulants on the survival time of primary female MA ear skin allografts on female CFW recipients
Day until rejection
Treatment
Injection schedule (h Group
Compound
1 2 3 4 5 6 7 8
DS DS DS DS Chondroitin sulphate
Dosage*
post
grafting)
10
6
50/treatment
6, 24, 48, 72, 96
200
6 6, 24 6 6 6 -
200/treatment 300
Heparin
150t
EDTA None
150
No. of hosts 14 14 25 36 15 15 15 48
Mean
Range
P
Suppression of cell-mediated
immune responses
by dextran sulphate
G. F. BABCOCK* & R. E. McCARTHY Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68105, U.S.A.
Received 21 April 1977; acceptedfor publication 5 May 1977
Summary. The effect of high molecular weight dextran sulphate (DS) on cell-mediated immune responses was studied. Two criteria were used to assess cell-mediated responses, skin graft rejection and intra-dermal (i.d.) skin tests. Mice receiving allografts and DS displayed a significant increase in graft survival time compared with untreated control animals. Similar results were obtained when the grafted animals differed at only the H-Y locus.
using adjuvants to stimulate the immune response is that of immunosuppression rather than stimulation. Several adjuvants, such as phytohaemagglutinin, Bacillus Calmette-Guerin (BCG), Bordetellapertussis, and Freund's complete adjuvant (FCA), have been shown to suppress as well as stimulate cell-mediated immune responses (Calapinto, 1975; Jolles & Paraf, 1973; Schwab, 1975). Recently our laboratory has shown that dextran sulphate (DS) enhanced the cellmediated immune response to sheep red blood cells and egg albumin in mice and guinea pigs (McCarthy, Arnold & Babcock, 1976). In this study we investigated the effect of DS on two classical cell-mediated immune reactions, the rejection of mouse skin allografts and the intradermal skin reaction of guinea-pigs to Mycobacterium tuberculosis. The results presented here show that DS is capable of suppressing cell-mediated immune responses in mice and guinea-pigs.
Guinea-pigs sensitized with Mycobacterium tuberculosis and treated with DS showed a greatly reduced delayed skin test response when challenged with purified protein derivative. DS was capable of suppressing this response even though, by the same route, dosage, and time of administration, it was previously shown to enhance a cell-mediated response in guinea-pigs sensitized with a different antigen. The results of this study indicate that the adjuvant DS is capable of suppressing cell-mediated immune responses in mice and guinea-pigs.
MATERIALS AND METHODS
INTRODUCTION
Animals Male and female mice from 6 to 10 weeks of age of the following strains were used: CFW (Carworth Farms, Portage, Michigan); C57BL/6J (B6, Jackson Memorial Laboratory, Bar Harbor, Maine); and MA (Eppley Cancer Institute, Omaha, Nebraska). Female Camm/Hartley guinea-pigs (Camm Research, Wayne, N.J.) weighing 450-550 g were used. All animals were allowed food and water ad libitum.
A problem often encountered by immunologists * Present address: Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A. Correspondence: Dr R. E. McCarthy, Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68105, U.S.A.
925
G. F. Babcock & R. E. McCarthy
926
Chemicals Dextran sulphate (DS, MW 500,000, Pharmacia, Uppsala, Sweden), chondroitin sulphate (Sigma Chemical Co., St Louis, Missouri), heparin sodium (Fisher Scientific Co., Fair Lawn, N.J.), and disodium ethylenediaminetetraacetate (EDTA, Fisher Scientific Co.) were prepared by dissolving in Hank's balanced salt solution (HBSS, Grand Island Biological Co., Grand Island, N.Y.) or in phosphatebuffered saline (PBS, pH 7-2, 0-01 M phosphate) at the appropriate concentration and used immediately. Freund's complete adjuvant H37Ra (FCA) containing M. tuberculosis was purchased from Difco, Detroit, Michigan and purified protein derivative (PPD) containing 5 tuberculin units/0-1 ml was obtained from Parke-Davis Co., Detroit, Michigan. Skin transplantation The free skin grafting technique of Billingham & Medawar (1951) was used in all experiments. Donor ear skin was grafted onto the back of the recipient animals and held in place with gauze pressure bandage. The bandages were removed after 7 days and the grafts were observed daily until rejection. Rejection was determined by observation in situ (Billingham & Silvers, 1970). In addition, representative grafts from each group of animals were excised, fixed in neutral formalin, embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically. The dosage and treatment schedule for the various chemicals used will be
described in the results section. Significance was determined using the Student's t-test at 95% confidence limits (Snedecor & Cochran, 1967). Delayed-type hypersensitivity skin reactions in guineapigs
Guinea-pigs were sensitized to M. tuberculosis by injecting 0 1 ml of FCA into each footpad. Animals given DS received 5 ,l/g body weight of a solution containing 20, 40 or 60 mg/ml DS so as to give a final dosage of 100, 200 or 300 mg/kg respectively. The DS was given in four equal aliquots subcutaneously (s.c.) in the inguinal and axillary regions immediately after injection with FCA. All animals were skin tested 17 days after immunization. Each animal was challenged at two sites with 0*1 ml of PPD injected intradermally into the clipped flanks with a 26 gauge needle. Skin reactions were observed and measured at 4, 24 and 48 h. The delayed hypersensitivity reactions (induration at 24 and 48 h) were graded as follows: -, no induration; ±, 0-5 mm; +, 5-10 mm; ++, 10-15 mm diameter.
RESULTS Effect of DS and other selected polyanions on skin graft rejection The effect of DS and other selected polyanions and anticoagulants on the survival time of primary skin
Table 1. Effect of DS and other selected polyanions and anticoagulants on the survival time of primary female MA ear skin allografts on female CFW recipients
Day until rejection
Treatment
Injection schedule (h Group
Compound
1 2 3 4 5 6 7 8
DS DS DS DS Chondroitin sulphate
Dosage*
post
grafting)
10
6
50/treatment
6, 24, 48, 72, 96
200
6 6, 24 6 6 6 -
200/treatment 300
Heparin
150t
EDTA None
150
No. of hosts 14 14 25 36 15 15 15 48
Mean
Range
P
