Int. Archs Allergy appl. Immun. 51: 583-593 (1976)
Synergism of Immunogenic and Adjuvant-Active Components of Mycobacterial W ax D in the Induction of Adjuvant Arthritis T oshitaka K oga, A tushi T anaka and C arl M. P earson Department of Biochemistry, Kyushu University School of Dentistry, Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, Fukuoka, and Department of Medicine, UCLA School of Medicine, Los Angeles, Calif.
Abstract. Two derivatives of wax D, one possessing immunogenicity and the oth er adjuvant activity, were tested for the possible role in the induction o f adjuvant arthritis (AA) in rats. The former, a water-soluble arthritogenic and immunogenic component (WAC), in incomplete Freund’s adjuvant, was able to induce delayed hy persensitivity (DH) and mild AA, but failed to function as an adjuvant in rats. The latter, an acetylated wax D (AD) and its subfraction, AD6, did exert adjuvant activ ity, but were free from immunogenicity and arthritogenicity. The addition o f AD or AD6 to the WAC in incomplete Freund’s adjuvant, when injected into inguinal lymph nodes, resulted in the production of severe AA with high incidence. Other adjuvants such as pertussis vaccine and lipopolysaccharide could not replace AD6; they failed to enhance AA when combined with the WAC. Also, other mycobacteri al antigen, PPD, could not replace wax D-derived WAC; it did not induce AA when coupled with AD6, although it did induce DH to PPD.
Introduction A single injection of the Freund-type adjuvant containing whole tuber cle bacilli or its wax D fraction produced polyarthritis and ancillary le sions in the rat [12, 16]. In a previous report [6], we showed that a watersoluble arthritogenic component (WAC) derived from wax D of Myco bacterium tuberculosis strain H37Ra induced adjuvant arthritis (AA) and delayed hypersensitivity (DH) in the rat.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 5:25:11 AM
Received: December 5,1975.
584
K oga/T anaka/P earson
The present study was designed to evaluate a precise role of adjuvant activity and immunogenicity of wax D in production of AA and DH. For this purpose, an acetylated wax D (AD) or its subfraction, AD6, derived from wax D of M. tuberculosis human strain [14] was used as an adju vant. This adjuvant was considered to be advantageous for exploring the role of adjuvant activity and immunogenicity in AA production, since it had been demonstrated to lack arthritogenicity [2,16] and immunogenicity [4, 13]. Materials and Methods
1 Difference between F-IA and F-IB was found only in their molecular size. F-IA and F-IB gave the sedimentation constant of 2.4s and 1.4s, and the molecular weight o f both fractions estimated on the basis o f alanine content were 14,000 ~ 17,000 and 13,000, respectively. Other chemical compositions as well as immuno genic activities were found to be similar to each other [unpubl. data].
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 5:25:11 AM
Animals. Female Lewis inbred rats were obtained from Microbiological Asso ciates, Bethesda, Md. They were 9-12 weeks old at the start o f the experiment and were maintained in plastic cages in groups o f up to four and were fed ad libitum with standard laboratory chow and water. Immunogens and adjuvants. The wax D fraction was prepared by exhaustive ex traction of the crude wax D with boiling acetone, from M. tuberculosis strain H37Ra or H37R v which had been cultivated on Sauton medium for 4 weeks, according to the method o f A sselineau [1]. It contained no detectable tuberculin-sensitizing substance (as determined by guinea pig skin testing with PPD). Both WAC and AD or AD6 used in the present study were derived from this wax D preparation, except where otherwise specified. The water-soluble portion of wax D was obtained by a mild saponification of wax D and fractionated by gel filtration on Sephadex G-50 and G-100 columns [5]. Three fractions were obtained on the Sephadex G-50 col umn (F-I, F-II, and F-III). F-I was further subdivided on a Sephadex G-100 column into 2 fractions (F-IA and F-IB).1 F-I, F-IA and F-1B (higher molecular weight components) were found to be arthritogcnic in the preceding report [6] and desig nated as W AC in this study. On the other hand, F-II and F-III (lower molecular weight components) were nonarthritogenic [6]. A D and its subfractions, AD6 and hexane-insoluble AD, were prepared as described previously [14]. PPD from H 37R v strain cultures was obtained through the courtesy of ParkeDavis and Co., Detroit, Mich, and another PPD from strain C, DT, PN cultures through the courtesy o f Fisheries and Food Central Veterinary Laboratory, Weybridge, Surrey, England. Crystallized ovalbumin (OA) was purchased from Sigma Chemical Company. A ll of the above materials were lyophilized for varying periods of time and were reconstituted immediately before their use and kept at 4 °C. Pertussis vaccine, a suspension o f killed Bordetella pertussis organisms in saline, preserved with merthiolate 1:10,000, was purchased from Eli Lilly and Company,
Induction o f Adjuvant Arthritis
585
Indianapolis, Ind. Lipopolysaccharide from Salmonella typhimurium was purchased from D ifco Laboratories, Detroit, Mich. Preparation of the water-in-oil (w/o) emulsion. Heavy mineral oil (Squibb) and Arlacel A (Hilltop Lab., Ohio) were used for the preparation of the emulsion. The emulsion was composed of 9.5 parts (by volume) mineral oil, 0.5 parts Arlacel A and 10 parts phosphate-buffered saline (PBS), pH 7.2 containing immunogen. AD or its subfractions (such as AD6) as adjuvants were dissolved in mineral oil with constant stirring in a mortar, followed by the addition o f Arlacel A. The PBS solu tion was then added slowly to give a crude emulsion. This emulsion was then passed repeatedly through a 30-gauge needle attached to a 1-ml syringe until it started to thicken. In the case of other adjuvants like lipopolysaccharide and pertussis vaccine, these were suspended in PBS along with the immunogen before they were added to the mixture o f mineral oil and Arlacel A. All wax D-derived immunogens were completely water-soluble and were dissolved in the PBS before emulsification. Immunization of rats. Intralymph node injection was used throughout this study. Injections were done as previously reported [7] and the volume usually approximat ed 0.005 ml per each of two inguinal lymph nodes. In this regard, the method of N ewbould [11] was followed. Evaluation of the arthritis. Observations and scoring of AA were done as de scribed earlier [6, 16]. Skin testing. All skin test antigens were dissolved in PBS. The method of skin testing and evaluation o f erythema and induration were reported earlier [7]. Macrophage migration inhibition test. This test was performed according to the methods o f D avid et al. [3] and T hor et al. [15]. Peritoneal exudate cells were ob tained from the guinea pigs injected with 1.0 mg of conventional wax D (C.DT.PN) and AD6 (H 37Ra) or with 0.02 mg PPD each incorporated in w /o emulsion by sub cutaneous injection into the hind footpad 40 days previously. These guinea pigs were injected with 20 ml i.p, of heavy mineral oil (Squibb) 3 days before harvesting of the cells. Sykes-Moore chambers containing capillaries packed with cells were filled with TC-199 medium containing 15% normal guinea pig serum. F-IA or PPD was added to the medium at the concentration o f 25 /
Synergism of Immunogenic and Adjuvant-Active Components of Mycobacterial W ax D in the Induction of Adjuvant Arthritis T oshitaka K oga, A tushi T anaka and C arl M. P earson Department of Biochemistry, Kyushu University School of Dentistry, Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, Fukuoka, and Department of Medicine, UCLA School of Medicine, Los Angeles, Calif.
Abstract. Two derivatives of wax D, one possessing immunogenicity and the oth er adjuvant activity, were tested for the possible role in the induction o f adjuvant arthritis (AA) in rats. The former, a water-soluble arthritogenic and immunogenic component (WAC), in incomplete Freund’s adjuvant, was able to induce delayed hy persensitivity (DH) and mild AA, but failed to function as an adjuvant in rats. The latter, an acetylated wax D (AD) and its subfraction, AD6, did exert adjuvant activ ity, but were free from immunogenicity and arthritogenicity. The addition o f AD or AD6 to the WAC in incomplete Freund’s adjuvant, when injected into inguinal lymph nodes, resulted in the production of severe AA with high incidence. Other adjuvants such as pertussis vaccine and lipopolysaccharide could not replace AD6; they failed to enhance AA when combined with the WAC. Also, other mycobacteri al antigen, PPD, could not replace wax D-derived WAC; it did not induce AA when coupled with AD6, although it did induce DH to PPD.
Introduction A single injection of the Freund-type adjuvant containing whole tuber cle bacilli or its wax D fraction produced polyarthritis and ancillary le sions in the rat [12, 16]. In a previous report [6], we showed that a watersoluble arthritogenic component (WAC) derived from wax D of Myco bacterium tuberculosis strain H37Ra induced adjuvant arthritis (AA) and delayed hypersensitivity (DH) in the rat.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 5:25:11 AM
Received: December 5,1975.
584
K oga/T anaka/P earson
The present study was designed to evaluate a precise role of adjuvant activity and immunogenicity of wax D in production of AA and DH. For this purpose, an acetylated wax D (AD) or its subfraction, AD6, derived from wax D of M. tuberculosis human strain [14] was used as an adju vant. This adjuvant was considered to be advantageous for exploring the role of adjuvant activity and immunogenicity in AA production, since it had been demonstrated to lack arthritogenicity [2,16] and immunogenicity [4, 13]. Materials and Methods
1 Difference between F-IA and F-IB was found only in their molecular size. F-IA and F-IB gave the sedimentation constant of 2.4s and 1.4s, and the molecular weight o f both fractions estimated on the basis o f alanine content were 14,000 ~ 17,000 and 13,000, respectively. Other chemical compositions as well as immuno genic activities were found to be similar to each other [unpubl. data].
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 5:25:11 AM
Animals. Female Lewis inbred rats were obtained from Microbiological Asso ciates, Bethesda, Md. They were 9-12 weeks old at the start o f the experiment and were maintained in plastic cages in groups o f up to four and were fed ad libitum with standard laboratory chow and water. Immunogens and adjuvants. The wax D fraction was prepared by exhaustive ex traction of the crude wax D with boiling acetone, from M. tuberculosis strain H37Ra or H37R v which had been cultivated on Sauton medium for 4 weeks, according to the method o f A sselineau [1]. It contained no detectable tuberculin-sensitizing substance (as determined by guinea pig skin testing with PPD). Both WAC and AD or AD6 used in the present study were derived from this wax D preparation, except where otherwise specified. The water-soluble portion of wax D was obtained by a mild saponification of wax D and fractionated by gel filtration on Sephadex G-50 and G-100 columns [5]. Three fractions were obtained on the Sephadex G-50 col umn (F-I, F-II, and F-III). F-I was further subdivided on a Sephadex G-100 column into 2 fractions (F-IA and F-IB).1 F-I, F-IA and F-1B (higher molecular weight components) were found to be arthritogcnic in the preceding report [6] and desig nated as W AC in this study. On the other hand, F-II and F-III (lower molecular weight components) were nonarthritogenic [6]. A D and its subfractions, AD6 and hexane-insoluble AD, were prepared as described previously [14]. PPD from H 37R v strain cultures was obtained through the courtesy of ParkeDavis and Co., Detroit, Mich, and another PPD from strain C, DT, PN cultures through the courtesy o f Fisheries and Food Central Veterinary Laboratory, Weybridge, Surrey, England. Crystallized ovalbumin (OA) was purchased from Sigma Chemical Company. A ll of the above materials were lyophilized for varying periods of time and were reconstituted immediately before their use and kept at 4 °C. Pertussis vaccine, a suspension o f killed Bordetella pertussis organisms in saline, preserved with merthiolate 1:10,000, was purchased from Eli Lilly and Company,
Induction o f Adjuvant Arthritis
585
Indianapolis, Ind. Lipopolysaccharide from Salmonella typhimurium was purchased from D ifco Laboratories, Detroit, Mich. Preparation of the water-in-oil (w/o) emulsion. Heavy mineral oil (Squibb) and Arlacel A (Hilltop Lab., Ohio) were used for the preparation of the emulsion. The emulsion was composed of 9.5 parts (by volume) mineral oil, 0.5 parts Arlacel A and 10 parts phosphate-buffered saline (PBS), pH 7.2 containing immunogen. AD or its subfractions (such as AD6) as adjuvants were dissolved in mineral oil with constant stirring in a mortar, followed by the addition o f Arlacel A. The PBS solu tion was then added slowly to give a crude emulsion. This emulsion was then passed repeatedly through a 30-gauge needle attached to a 1-ml syringe until it started to thicken. In the case of other adjuvants like lipopolysaccharide and pertussis vaccine, these were suspended in PBS along with the immunogen before they were added to the mixture o f mineral oil and Arlacel A. All wax D-derived immunogens were completely water-soluble and were dissolved in the PBS before emulsification. Immunization of rats. Intralymph node injection was used throughout this study. Injections were done as previously reported [7] and the volume usually approximat ed 0.005 ml per each of two inguinal lymph nodes. In this regard, the method of N ewbould [11] was followed. Evaluation of the arthritis. Observations and scoring of AA were done as de scribed earlier [6, 16]. Skin testing. All skin test antigens were dissolved in PBS. The method of skin testing and evaluation o f erythema and induration were reported earlier [7]. Macrophage migration inhibition test. This test was performed according to the methods o f D avid et al. [3] and T hor et al. [15]. Peritoneal exudate cells were ob tained from the guinea pigs injected with 1.0 mg of conventional wax D (C.DT.PN) and AD6 (H 37Ra) or with 0.02 mg PPD each incorporated in w /o emulsion by sub cutaneous injection into the hind footpad 40 days previously. These guinea pigs were injected with 20 ml i.p, of heavy mineral oil (Squibb) 3 days before harvesting of the cells. Sykes-Moore chambers containing capillaries packed with cells were filled with TC-199 medium containing 15% normal guinea pig serum. F-IA or PPD was added to the medium at the concentration o f 25 /
