Immunology 1977 32 963

Dextran sulphate:

an

adjuvant for cell-mediated immune responses

R. E. McCARTHY, L. W. ARNOLD & G. F. BABCOCK* Department of Medical Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A.

Received

11

October 1976; acceptedfor publication 5 November 1976

Summary. The effect of high mol. wt dextran sulphate (DS) on cell-mediated immune responses was studied. Three criteria were used to assess cellmediated delayed-type hypersensitivity: footpad swelling, i.d. skin tests and macrophage migration inhibitory factor (MIF) production. Guinea-pigs sensitized with egg albumin (EA) and treated with DS showed strong positive delayed skin tests. Control animals given only EA showed negative skin tests. Lymphocytes from mice sensitized s.c. with EA and treated with DS showed an increase in MIF production. Delayed footpad swelling responses in mice sensitized s.c. with sheep red blood cells (SRBC) and treated with DS were increased when the mice were challenged 8 days after sensitization. Doses of DS which were effective in increasing delayed footpad swelling ranged from 50-200 mg DS/kg body weight. DS was only capable of increasing delayed footpad swelling responses when both SRBC and DS were injected s.c. at the same site. Intraperitoneal injection of both SRBC and DS or injection of both s.c. but at different sites did not result in increased delayed footpad swelling. DS was capable of augmenting footpad swelling responses when given s.c. as much as 6 days before SRBC. The optimal time for administration of DS * 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.

963

was 2 days before SRBC. Injection of DS 2 days or more after SRBC resulted in no increase in delayed footpad swelling responses. The results of this study indicate that dextran sulphate is a potent adjuvant for cell-mediated delayed-type hypersensitivity immune responses in both mice and guinea-pigs.

INTRODUCTION

The search for materials which will enhance the immune response to any given antigen has been an objective of immunologists for many years. While many compounds are known which will effectively enhance antibody production, such as aluminum salts (April & Wardlaw, 1966), Mycobacteria (White, Coons & Connolly, 1955) and polyanions (Diamantstein, Wagner, Boyse, Odenwald, & Schulz, 1971a; Diamantstein, Wagner, Boyse, Odenwald & Schulz, 1971b), adjuvants which effectively enhance cell-mediated immune responses have been much harder to find. The most effective and widely used adjuvant for increasing cellmediated immune responses is Freund's complete adjuvant. One complicating factor in the use of Freund's adjuvant is the complex chemical nature of the adjuvant (Raffel, 1948). A chemically more well-defined adjuvant for cell-mediated immune responses would definitely be advantageous to the study of adjuvant action in this type of immune response.

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R. E. McCarthy, L. W. Arnold & G. F. Babcock

In the present study we have investigated the use of dextran sulphate as an adjuvant for cell-mediated delayed-type hypersensitivity immune responses (DTH). Three criteria were used to assess the influence of DS on cell-mediated delayed-type hypersensitivity: footpad swelling, i.d. skin tests and macrophage migration inhibitory factor (MIF) production. The results presented here show that DS is a potent adjuvant for cell-mediated delayed-type hypersensitivity immune responses.

MATERIALS AND METHODS

Animals Male and female random-bred white mice from 6-10 weeks of age of the following strains were used: ECI (Eppley Cancer Institute, Omaha, Nebraska); CFW and CF-1 (Carworth Farms, Portage, Michigan); SCH :ARS (ICR)f (ARS/ Sprague Dawley, Madison, Wisconsin); and Camm S/W (Camm Swiss/Webster, Camm Research, Wayne, New Jersey). Female Camm/Hartley guineapigs (Camm Research, Wayne, New Jersey) weighing 450-550 g were used. Antigens Sheep red blood cells (SRBC) in modified Alsever's solution (Colorado Serum Co., Denver, Colorado) were washed three times in phosphate-buffered saline (PBS, pH 7 2, 0 01 M phosphate) and resuspended to the required concentration in either PBS or Hanks's balanced salt solution (HBSS, Grand Island Biological Co., Grand Island, New York). Egg albumin (EA, Nutritional Biochemical Corporation, Cleveland, Ohio, lot 9705) at the appropriate concentration was dissolved in PBS or HBSS and used immediately.

Adjuvants and chemicals Dextran sulphate (DS, mol. wt 500,000, Pharmacia, Uppsala, Sweden), DEAE-dextran (mol. wt 500,000, Pharmacia) and dextran (mol. wt. 500,000, Pharmacia were prepared by dissolving in HBSS and used immediately. Freund's complete adjuvant (FCA) was purchased from Difco, Detroit, Michigan. Heparin-sodium (Fisher Scientific Co., Fair Lawn, New Jersey) was prepared in PBS. Polyadenylic acid and polyuridylic acid were purchased from Miles Laboratories, Incorporated, Elkart, Indiana. Poly A :U was prepared by mixing poly A and

poly U in equal molar concentration. Complex formation was noted by the increased viscosity of the solution (Johnson & Johnson, 1971). Immediate and delayed-type hypersensitivity reactions determined by mouse footpad swelling Mice were sensitized on day 0 either s.c. or i.p. as indicated in the results with 0 02 ml/g body weight of 5% (v/v) SRBC in HBSS. Adjuvant in HBSS was injected either separately or mixed with SRBC as indicated in the results. Control mice received only SRBC. On day 7 blood was collected by bleeding from the retro-orbital venous plexus. The blood from mice of the same group was pooled and the serum obtained and used to determine haemagglutinin titres (see below). On day 8 the mice were challenged in the right hind footpad with 0 05 ml of a 20% (vfv) suspension of SRBC in PBS and in the left hind footpad with 0 05 ml PBS, using a 27 gauge needle. The thickness of the footpads was measured at 4, 24, and 48 h after challenge with Vernier calipers. The difference in thickness between the left and right footpads of each mouse was expressed as a percentage. The statistical validity of considering both hind feet to be identical in thickness before the challenge injections has been demonstrated (Nelson & Mildenhall, 1967). The average per cent footpad swelling (APFS) for each group of mice was determined by dividing the sum of the individual swellings of the mice by the number of mice in the group. The average percentage increase (API) was determined by subtracting the APFS of the control group from the APFS of the treated group. Unless otherwise stated, all data is presented as the average percentage increase over controls. Significance was determined using the Student's t-test at 95% confidence limits (Snedecor & Cochran, 1967). Positive footpad swelling reactions occurring at 4 h are immediate-type hypersensitivity reactions (antibody-mediated). Positive reactions occurring at 24 and 48 h are delayed-type hypersensitivity reactions (cell-mediated) (Nelson & Mildenhall, 1967; Crowle, 1975).

Haemagglutinin titre Haemagglutinating antibodies were determined using the microtitre method. All tests were performed in V-bottom microtitre plates (Cooke Engineering Co., Alexandria, Virginia). Two-fold dilutions of sera (obtained as described above) were made in bovine serum albumin saline (100 mg BSA in 100

965

Dextran sulphate ml PBS, Nutritional Biochemicals Corporation). The titre was considered to be the reciprocal of the highest dilution showing positive agglutination. MIF assay MIF production was assayed by the method of Houck & Chang (1973). Macrophages were collected by the method of Argyris (1967) with slight modification. Mouse peritoneal cells were stimulated by injecting non-immunized female SCH:ARS (ICR)f mice i.p. with 3 ml of sterile 3 Y. fluid thioglycollate medium (TG, Difco). Six days after the administration of the TG, mice were killed by cervical dislocation. Peritoneal cells were collected by washing the peritoneal cavity twice with 3 ml cold HBSS containing 100 u/ml penicillin and 100 ug/ml streptomycin (Grand Island Biological Co.) and placed in cold polycarbonate centrifuge tubes. The peritoneal cells were washed once in HBSS and resuspended to a final concentration of 1 5-2 0 x 106 cells/ml in TC199 culture medium (Grand Island Biological Co.) which contained 20% foetal calf serum (FCS, Grand Island Biological Co.) 100 u/ml penicillin, 100 ,ug/ml streptomycin and 25 mm HEPES buffer (Grand Island Biological Co.). The cells were placed in 16 x 83 mm Leighton tubes (2 0 ml/tube) (Bellco Biological Glassware, Vineland, New Jersey) and incubated at 370 for 1 h. The culture tubes were then rinsed three times with HBSS and once with TC199 (20 ml/rinse). The remaining adherent cells were considered macrophages. Two ml of TC199 was then added to each tube and incubated for 18 h at 370 before further use. Lymphocytes were collected from ECI female mice which had been previously sensitized with EA or EA plus adjuvant s.c. in the scruff of the neck. The mice were killed by cervical dislocation and the inguinal and axillary lymph nodes were removed aseptically and placed in a small volume of RPMI 1640 (Grand Island Biological Co.) which contained 10% FCS, 100 u/ml penicillin and 100 ug/ml streptomycin. The lymph nodes were gently teased apart with sterile dissecting needles and forced through an 80-mesh stainless steel wire screen (Cistron Corp., Elmsford, New York) with the plunger from a disposable 12-ml syringe. The screens were washed with RPMI 1640 and the cell suspension was placed in a sterile centrifuge tube. The cells were washed once and resuspended in RPMI 1640. The concentration of mononuclear cells was adjusted to 1-2 x 107 mononuclear cells/ml.

The percentage of viable mononuclear cells was determined by eosin Y exclusion (Wolstencroft & Dumonde, 1971) to assure approximately the same percent viability for each culture. Ten-millilitre aliquots of the cell suspension were placed in 100-ml glass bottles and 500 ug of EA was added to each bottle. The cultures were then incubated at 370 in a humid 5 % CO2 in air atmosphere for 48 h. The cultures were then centrifuged at 800 g for 15 min and the supernatant fluids collected. Leighton tubes containing macrophages prepared 18-24 h earlier were washed once in HBSS. Cells were removed from an area approximately 2 x 0 3 cm in the cell monolayer along the long axis of the tube using a bent sterile stainless steel spatula. The cultures were then washed twice in HBSS and once in TC199. Two-millitres of medium containing 1 6 ml of fresh TC199 and 0 4 ml of the appropriate lymphocyte culture supernatant were added to each tube. The number of cells in an area 0 7 x 0-7 mm was counted. The number of cells in this same area was then determined 24 h later. Each supernatant fluid to be tested was done in triplicate tubes and two areas of each tube were counted. The percentage migration was determined as follows: per cent migration = no. of cells at 24 h -no. of cells at 0 h 100 no. of cells at 24 h The percentage migration inhibition was calculated according to the formula of Glade, Broder, Grotsky, & Hirschhorn (1971) with slight modification: per cent migration inhibition = 1

-

average per cent

migration

experimentalX 100.

average per cent migration of control

Immediate and delayed-type hypersensitivity reactions determined by i.d. skin tests in guinea-pigs Guinea-pigs were sensitized to EA by injection in the footpad. When DS was used as the adjuvant the animal received 0 005 ml/g body wt of a solution of 40 mg/ml DS in HBSS so as to give a final dosage of 200 mg/kg. 01 ml of the total dosage was given in each footpad with the remaining amount distributed approximately equally s.c. in the inguinal and axillary areas. EA in PBS was injected immediately after the DS in the footpad, 0-1 ml per footpad, for a total dosage of 300 pg. When Freund's complete adjuvant (FCA), as a positive control, was used the EA was emulsified in the FCA using equal amounts

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R. E. McCarthy, L. W. Arnold & G. F. Babcock

of EA and FCA. The animals received 01 ml of FCA-EA in each footpad so that the total dose of EA was 300 pg per animal. Additional control animals received only EA or DS alone in the identical manner described above for the DS-EA-treated animals. All animals were skin tested 17 days after immunization. Ten micrograms of EA in 01 ml PBS were injected i.d. into the clipped flanks of the guinea-pigs with a 26 gauge needle. Each animal was challenged at two sites. 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: ±, 0-5 mm; +, 5-10 mm; + +, 10-15 mm; + + +, 15-20 mm. RESULTS Toxicity of candidate adjuvants The toxicity of DS has been studied previously. DS of high mol. wt was found to be highly toxic when given i.v. (Walton, 1954). This toxic effect of DS was found to be primarily due to its anticoagulant activity and the precipitation of plasma proteins (Walton, 1954; Ricketts, 1952). In toxicity studies in our own laboratory DS was given either s.c. or i.p. When the dosage of DS given s.c. was much greater than 200 mg/kg body wt (300 mg/kg and higher) there was severe local and systemic damage ranging from extensive necrosis at the injection site to death of the animal. At 200 mg/kg body weight, the highest dosage we felt could be well tolerated, occasional animals did develop necrosis at the site of injection. The mice treated with 200 mg DS/kg body weight appeared in good health, however. Subcutaneous dosages of 100 mg/kg body weight or less produced no gross signs of toxic manifestations. When DS was injected i.p. the highest dose that could be well tolerated was 75 mg/kg body weight in ECI mice. DEAE-dextran induced occasional necrosis at the injection site in animals injected s.c. with 200 mg/kg body weight. Lower dosages produced no necrosis. Dextran, heparin and poly A:U produced no demonstrable gross toxic reactions at any of the dosages used in these studies. Effect of DS on footpad swelling The effects of DS on immediate and delayed-type hypersensitivity as determined by the footpad

swelling assay in mice are summarized in Table 1. The data in Table 1 are expressed as the average percentage increase over controls. ECI male and female mice treated s.c. with 200, 100, or 50 mg/kg body wt mixed with SRBC (groups 1, 2, 3, 5, 6, 7) showed a significant increase in delayed-type responses over controls both at 24 and 48 h after challenge. Camm S/W and CFW mice (male and female) treated with 200 and 100 mg DS/kg body weight and SRBC (groups 17-24) also showed significant increases in delayed-type footpad swelling responses over control mice. CF-1 male and female mice treated with 200, 100 and 50 mg DS/kg body weight and SRBC (groups 9, 10, 11, 13, 14, 15) showed a weaker and less consistent response. ECI and CF-1 males and females treated s.c. with 20 mg DS/kg body wt mixed with SRBC (groups 4, 8, 12, 16) did not show a significant increase in delayed-type responses over controls. Immediatetype footpad swelling responses measured at 4 h post-challenge, in general, were not increased over the control values. It should be noted however, that mice sensitized with SRBC and treated with DS often went into anaphylactic shock when challenged in the footpad and some mice died. These results have been reported elsewhere (McCarthy & Arnold, 1976). Mice sensitized with only DS and challenged with SRBC showed no increase in swelling over unsensitized mice. In most of the strains tested (ECI, Camm S/W and CFW) no great difference between male and female responses at 4 and 24 h after challenge was noted. Female mice generally gave greater 48-h swelling, however. ECI male and female mice were used in all subsequent studies with DS because of the consistently good results obtained and because of the availability of this strain. In another study, summarized in Table 2, the effect on footpad swelling of administration of antigen and adjuvant at different sites was determined. Only when DS and SRBC were injected s.c. at the same site was a significant adjuvant effect noted (groups 1-4 and 14). When DS and SRBC were given together i.p. (group 12) or at different sites (groups 5-10 and 13) no significant adjuvant effect was noted. A slight effect on footpad swelling was seen at 24 h post-challenge when DS (200 mg/kg) was given s.c. in the neck and SRBC was given i.p. (group 11). This response was very slight and was diminished at 48 h post-challenge as compared to groups 1-4. It should be noted that in

Dextran sulphate

967

Table 1. Effect of DS on footpad swelling in mice sensitized with SRBC

Footpad swelling* Group

Strain

Doset

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

ECI

200§ 100§ 50 20 200§ 100§ 50

ECI

CF-I

CF-I

CAMM S/W

20 200 100 50 20 200 100 50 20 200

Sex

9

9

100§ CAMM S/W CFW CFW

200 100§ 200 100 200 100

9

9

No. of mice+

24 h

P

Dextran sulphate: an adjuvant for cell-mediated immune responses.

Immunology 1977 32 963 Dextran sulphate: an adjuvant for cell-mediated immune responses R. E. McCARTHY, L. W. ARNOLD & G. F. BABCOCK* Department o...
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