Antibody-Mediated and Delayed-Type Hypersensitivity

Transcription

1 INFECriON AND IMMUNITY, Feb. 1975, p Copyright American Society for Microbiology Vol. 11, No. 2 Printed in U.S.A. Antibody-Mediated and Delayed-Type Hypersensitivity Reactions to Brucella Skin Test Antigens in Guinea Pigs LOIS M. JONES AND DAVID T. BERMAN* Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin Received for publication 8 July 1974 Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions to these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component. Although the ability of proteins to induce and elicit specific delayed-hypersensitivity reactions is generally recognized, there is controversy surrounding the role of polysaccharides (PS; 3) and lipopolysaccharides (LPS; 8) that are unassociated with proteins. Most brucella allergens contain varying amounts of LPS in addition to proteins and nucleoproteins (2). Others consist almost entirely of PS (15). Since animals infected with brucella usually have serum antibodies to PS determinants of the cell wall LPS, it is possible that dermal reactions to some allergens involve overlapping antibodymediated and delayed-type reactions. Preparations of brucella LPS contain lipid A (13) as well as varying amounts of polypeptide, some of which is tightly bound (M. S. Redfearn, Ph.D. thesis, Univ. of Wisconsin, Madison, 1960). Previous studies on the dermal response of sensitized guinea pigs to brucella LPS (10) suggested that the inflammation was a combination of reactions comprising those due to the toxicity of lipid A and specific delayed hypersensitivity to the polypeptide associated with the antigen. In the present study, a brucella PS antigen that had minimal toxicity for normal guinea pigs was examined concurrently with LPS antigens. These antigens, containing varying amounts of protein, were compared with a protein allergen that lacked LPS components. The experiments were designed to determine 36C whether brucella LPS and PS elicit delayed hypersensitivity reactions, or whether the dermal reactions elicited by these antigens are antibody mediated in whole or in part. MATERIALS AND METHODS Bacterial strains. All brucella cultures have been used in our previous studies (4, 5). Their significant characteristics are as follows. Smooth strains Brucella melitensis 16M, B. abortus 544, B. abortus 2308, B. abortus , and B. abortus 19 all contain smooth LPS and stimulate production of antibody specific for this surface component (4). B. abortus 544 produces generalized brucellosis in guinea pigs inoculated with 102 to 103 organisms (11). B. melitensis rough strain B115, B. abortus rough strain llr, and B. abortus rough strain 45/20 lack the smooth LPS (5). Antigens. Protein (P) antigen was prepared from B. melitensis rough strain B115 by extraction in cold saline (2) and characterized as a heat-stable protein allergen containing no detectable LPS (12). Protein content, as determined by the Lowry method (19), was 56%. Two different preparations of LPS antigen, extracted from B. melitensis smooth strain 16M by the modified hot phenol-water method (14), were used. LPS-1 contained 25% protein. LPS-2 had been sedimented at 60,000 x g for 12 h and contained 5% protein. PS antigen was prepared from B. abortus smooth strain by a method (15) modified by E. Scheibner (personal communication), which consisted of trichloroacetic acid extraction of the washed

2 VOL. 11, 1975 BRUCELLA SKIN TEST ANTIGENS 361 whole cells followed by acetic acid hydrolysis to remove lipids and proteins. The final product contained 1.4% protein. LPS antigens extracted by the hot phenol-water method from Yersinia enterocolitica types 8 and 9 were obtained from R. Diaz. They were included because LPS from smooth brucella strains and LPS from Y. enterocolitica type 9 (but not type 8) show cross-reactivity (6, 9) but the protein antigens from these genera do not (6). Antisera. Antisera were obtained from rabbits hyperimmunized with acetone-killed rough B. abortus strain 11R in Freund incomplete adjuvant (FIA), with killed smooth B. abortus strain 2308 in FIA, and by infection with living smooth B. abortus strain 19 (5). Sera were also obtained from sensitized guinea pigs before they were tested intradermally. Sensitization of guinea pigs. Guinea pigs of the Hartley strain were sensitized by infection with 10l organisms of smooth, virulent B. abortus strain 544 or by immunization with killed rough B. abortus strain 45/20 in FIA as previously described (11). They were tested by intradermal tests or for active cutaneous anaphylaxis (ACA) from 7 to 11 weeks after sensitization. Intradermal tests in guinea pigs. A constant volume of 0.1 ml of antigen was injected per site and the dose was expressed as micrograms (dry weight) in 0.1 ml of pyrogen-free saline. The mean diameter of erythema standard error was the parameter of skin response (11). ACA. Infected and adjuvant-immunized guinea pigs were injected intradermally with 0.1 ml of several doses of three or more antigens immediately after the intracardial injection of 0.5 ml of 1% Evan's blue in 0.85% saline (16). A blue ring appearing within a few minutes at the intradermal site of one of the antigens indicated that the intracardial injection had been successful. An intradermal injection of saline was included on each animal as a control to rule out the possibility of nonspecific bluing reactions. The animals were observed for 30 min. Normal guinea pigs given intracardial injections of Evan's blue followed by intradermal injections with the same antigens did not develop bluing reactions. Passive cutaneous anaphylaxis (PCA). Individual or pooled sera from normal, infected, and immunized guinea pigs, taken before intradermal or ACA tests, were diluted 1:25, 1:100, and 1:400, and 0.1 ml was injected intradermally into normal guinea pigs (16). Serum dilutions heated for 30 min at 56 C were also injected with unheated serum dilutions on some animals. Three to 4 h later the animals were injected intracardially with 1 ml containing 100 to 500 jug of one of the antigens and 0.5 ml of 1% Evan's blue solution. Only those guinea pigs showing a bluing reaction at at least one of the intradermal sites within 30 min were included in the results. RESULTS Antigenic analysis. Analysis of the brucella LPS preparation by immunoelectrophoresis with hyperimmune anti-smooth brucella serum showed slowly diffusing lines with the characteristic cathodic mobility that have been identified as LPS antigens (4), as well as several fast-moving lines associated with the P antigens (4). The PS antigen developed only a single line with cathodic mobility similar to that observed with LPS. The P antigen preparation did not develop the LPS line with the anti-smooth brucella serum from infected animals or those hyperimmunized with either smooth or rough organisms. P antigen developed a number of lines, as illustrated previously (12), when tested with either hyperimmune anti-smooth or antirough sera. Immunodiffusion tests were set up to determine the cross-reactivity of the LPS and PS lines, as well as the identity of the P components, in the various antigenic preparations. Table 1 summarizes the numbers of lines observed with all of the combinations of sera and antigens. It shows that sera from rabbits hyperimmunized with killed rough B. abortus did not precipitate with PS or yersinia antigens and that sera from rabbits infected with smooth B. abortus did not precipitate with brucella P antigen. Sera from rabbits hyperimmunized with killed smooth B. abortus precipitated with all of the antigens except Y. enterocolitica type 8 LPS, which was not precipitated with any of the sera. Figure 1 is from a representative experiment with sera from rabbits infected with smooth B. abortus. It illustrates the reaction of identity of one of the components of each of LPS-1 and yersinia type 9 LPS with the single component in brucella LPS-2 and PS antigens. Sera from rabbits hyperimmunized with killed rough brucella developed reactions of identity with at least one of the lines in brucella P, LPS-1, and LPS-2 antigens. The reactions with guinea pig sera were the same as those with the rabbit sera. That is, sera from infected guinea pigs had precipitins to the TABLE 1. Number of lines in immunodiffusion tests of antigens with rabbit antisera Test antigena Immunizing antigen Killed Killed Living rough smooth smooth B.abortus B.abortus B.abortus + FIA ± FIA Brucella P... >3 > 3 0 Brucella LPS-1... >3 >3 1-2 Brucella LPS Brucella PS Yersinia type 9-LPS Yersinia type 8-LPS a Antigens were prepared as described in Materials and Methods.

3 362 JONES AND BERMAN INFECT. IMMUN. FIG. 1. Immunodiffusion tests of brucella LPS-1, LPS-2, PS, and Y. enterocolitica type 9-LPS (Y9) antigens with serum from a rabbit infected with living smooth B. abortus. LPS component but not to the P components, and sera from guinea pigs immunized with rough brucella in adjuvant had precipitins to P components but not to LPS. Intradermal tests. The characteristics of the dermal responses of guinea pigs to P and LPS antigens have been described previously (10). To investigate the nature of reactions of PS antigen in the same way, doses of 1, 10, and 100 ug were injected singly into non-sensitized controls and guinea pigs sensitized either by infection with living smooth brucella or by immunization with rough brucella in FIA. The 100-,ug dose produced pale erythematous reactions at 3 to 6 h in all animals. Only those infected guinea pigs with circulating anti-smooth antibody, as detected by agglutination tests or by immuno- -diffusion with LPS, developed larger and more marked erythematous reactions that persisted TABLE 2. for 24 h. Doses of 10 and 1,ug produced minimal reactions that disappeared by 24 h. To establish the qualitative characteristics of the reactions to the three types of antigens, groups of guinea pigs were injected simultaneously with two doses of each of the P, LPS-1, and PS preparations (Table 2). Although the numbers of animals were small, certain trends were apparent. Within 5 min of intradermal injection, a red bleb was observed at the site of LPS and PS injections in infected guinea pigs and at the site of P and LPS injections in adjuvant-immunized guinea pigs. These immediate reactions had almost completely disappeared by 3 h after injection, but erythema was again apparent at these sites at 5 h. Infected guinea pigs showed maximum reactions at 24 h with P and LPS antigens but at 5 h with PS antigen. In the adjuvant-immunized animals Dermal reactionsa to protein, lipopolysaccharide, and polysaccharide antigens of brucella in sensitized and normal guinea pigs Test Dose Infected (4)b Adjuvant immunized (3) Normal (4) antigen C(g) 5 min 5h 24h 5 min 5h 24h 5min 5h 24h P ± LPS ± i ± ± ± ± _ PS _ a Dermal reactions are designated as + (red bleb at 5 min) and - (no reaction). Figures are mean diameter of erythema (in millimeters) ± standard error of the mean. 'Number of animals tested.

4 VOL. 11, 1975 BRUCELLA SKIN TEST ANTIGENS there were not significant differences in the magnitude of the reactions between 5 and 24 h with either the P or LPS antigens, and there were no reactions with PS antigens. Normal guinea pigs responded to LPS antigen only. ACA. Infected guinea pigs manifested positive ACA reactions to all antigens containing the PS components in LPS, PS, or the crossreactive yersinia type 9 LPS, but negative reactions to P or yersinia type 8 LPS (Table 3). In contrast, the guinea pigs immunized with rough brucella in adjuvant had positive ACA reactions only with the P and LPS antigens containing the brucella proteins. PCA. Sera from infected guinea pigs produced positive PCA reactions in all animals injected intracardially with PS antigen, in some animals injected with LPS antigen, but in none of the animals given P antigen (Table 4). Sera from adjuvant-immunized guinea pigs produced positive PCA reactions in animals injected intracardially with P and LPS antigens. Sera from adjuvant-immunized guinea pigs were absorbed with P antigen. These absorbed sera did not give PCA reactions with either P or LPS antigens, although the unabsorbed sera tested on the same animals were positive with these antigens. This indicated that the P component in LPS was antigenically the same as that in P antigen. Serum dilutions that had been heated gave the same results as nonheated serum dilutions tested simultaneously on the same guinea pigs, i.e., positive at a 1:100 dilution. This minimizes the probability that immunoglobulin E was involved. TABLE 4. TABLE 3. Active cutaneous anaphylaxis reactions in sensitized guinea pigs Method of sensitization Test antigen Dose (gg) Adjuvant Infection immunization Brucella P /9a 10/10 1 0/5 7/8 Brucella LPS /8 7/7 1 4/8 7/10 Brucella LPS /8 3/4 1 1/6 3/5 Brucella PS /9 0/10 1 8/9 0/7 Yersinia LPS /5 0/2 Yersinia LPS /5 0/2 Saline 0/9 0/10 a Numerator, Number of guinea pigs giving positive reactions at site of intradermal injection of test antigens; denominator, number of sensitized guinea pigs successfully injected intracardially with Evan's blue. DISCUSSION The need for improvement of standardization of skin test antigens in general has been emphasized in recent World Health Organization reports (17, 18). Improvement of the techniques of standardization should include evaluation of the capacity of antigens used for assessing delayed hypersensitivity to induce immediate hypersensitivity reactions, as these may interfere with the interpretation of the delayed response. Our data show that guinea pigs infected with smooth B. abortus have circulating antibody to Passive cutaneous anaphylaxis tests Source and dilution of guinea pig serum injected intradermally Brucella test Intracardial dose. antigen ~ (,lgj (Mg Infected Adjuvant immunized Normal 1:25 1:100 1:400 1:25 1:100 1:400 1:25 1:100 P 100 0/2a 0/2 0/2 2/2 2/2 1/2 0/1 0/ /3 0/3 0/3 3/3 3/3 0/3 0/1 0/ /6 0/6 0/5 8/8 8/8 3/6 0/3 0/3 LPS /1 0/1 0/1 1/1 1/1 1/1 0/1 0/ /4 1/4 1/4 6/6 6/6 0/3 0/3 0/3 LPS /2 0/2 0/2 2/2 2/2 2/ /3 1/3 0/1 2/3 2/3 0/3 0/2 0/2 PS 100 1/1 1/1 0/1 0/1 0/1 0/1 0/1 0/ /5 3/5 1/5 0/5 0/5 0/5 0/5 0/ /1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 a Numerator, Number of guinea pigs giving positive reactions at site of intradermal injection of the specified serum; denominator, number of normal guinea pigs that were successfully injected intracardially with the test antigen and Evan's blue. 363

5 364 JONES AND BERMAN INFECT. IMMUN. the PS determinants of the cell wall LPS but not to the protein components. These animals have immediate reactions in ACA tests with LPS and PS but not with P antigens, and their sera give identical results in PCA tests. This establishes that, in infected animals, the reactions observed at 24 h in sites injected with P antigens are not antibody mediated. The reactions to LPS and PS antigens clearly have an antibody-mediated component, but the data do not permit the exclusion of an overlapping delayed-type reaction. Animals immunized with the rough organisms in adjuvant are not equivalent to infected animals in their responses to skin test antigens. They have circulating antibody to the P antigens but not to the PS determinants of the cell wall LPS. These animals have immediate reactions in ACA tests with P and LPS but not with PS antigens, and their sera give identical results in PCA tests. The abolition of PCA reactions to LPS by absorbing sera with P antigen shows that the antibody-mediated reactions in these animals are to the P component. These antibody-mediated reactions can interfere with the quantification and interpretation of the delayed-type hypersensitivity response to P antigen. It seems clear from these results that it is preferable to use infected, rather than adjuvant-immunized, guinea pigs for the standardization of antigens for delayed-type hypersensitivity testing in brucellosis. This and other studies have shown the superiority of LPS-free P antigen for tests of delayed hypersensitivity in brucellosis. There seems to be no useful purpose served by the inclusion of LPS in a test antigen, and there are clearly a number of disadvantages. Lipid A is toxic (7) and mitogenic for B cells (1), and the presence of the complete LPS or the PS can result in cutaneous anaphylaxis and Arthus reactions, or both, with resultant confusion of the interpretation. Also, the complete LPS in the antigen can stimulate antibody production that can interfere with subsequent serological diagnosis. ACKNOWLEDGMENTS This study was supported by grants from the World Health Organization and the Research Committee of the Graduate School with funds furnished by the Wisconsin Alumni Research Foundation. We are indebted to R. Diaz and G. Dubray for some of the antigens employed. LITERATURE CITED 1. Andersson, J., O., Sjoberg, and G. Moller Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharides. Eur. J. Immunol. 2: Bhongbhibhat, N., S. S. Elberg, and T. H. Chen Characterization of Brucella skin-test antigens. J. Infect. Dis. 122: Daniel, T. M., and C. F. Hinz, Jr Reactivity of purified proteins and polysaccharides from Mycobacterium tuberculosis in delayed skin test and cultured lymphocyte mitogenesis assays. Infect. Immun. 9: Diaz, R., L. M. Jones, D. Leong, and J. B. Wilson Surface antigens of smooth Brucellae. J. 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