May 3, 1982 - Chambers of Guinea Pigs Immunized with a Ribosomal. Preparation from ..... grown in vivo to ingestion and digestion by phagocytes of.
Vol. 37, No. 2
INFECTION AND IMMUNITY, Aug. 1982, p. 469-473 0019-9567/82/080469-05$02.00/0
Characterization of the Immune Response in Subcutaneous Chambers of Guinea Pigs Immunized with a Ribosomal Preparation from Neisseria gonorrhoeae MICHAEL J. WANNEMUEHLER,lt RICHARD D. MILLER,'* AND MORRIS D. COOPER2 Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, Kentucky 40292,' and Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois 627082
Received 18 January 1982/Accepted 3 May 1982
Immunization of guinea pigs with ribosomal preparations has been previously shown to be protective against chamber infection with Neisseria gonorrhoeae, and this protection could be correlated with the presence of serum bactericidal antibody. Analysis of the chamber fluids from ribosome-immunized and nonimmunized guinea pigs has demonstrated that the chamber fluid may accurately reflect serum antibody levels and proteins. At least one major population of antiribosomal antibodies is present in both immune serum and chamber fluid as revealed by lines of identity between these components. Antibody- and complement-mediated bactericidal assays revealed that matched chamber fluids and sera from immune animals had comparable bactericidal titers. These results suggest that the antibody-complement-mediated bactericidal activity plays a major role in the protection against N. gonorrhoeae infection. Protection could not be explained on the basis of the cellular components of the inflammatory response since both immune and nonimmune chambers had comparable increases in polymorphonuclear neutrophils, monocytes, and lymphocytes after challenge. Previous work with the guinea pig subcutaneous (s.c.) chamber model has demonstrated the ability of gonococcal ribosomal preparations to elicit a protective immune response to a subsequent challenge with Neisseria gonorrhoeae (8, 9). In agreement with others (1, 5, 6, 15, 27), the protective response seen in the s.c. chamber model correlated with the presence of hemagglutinating and bactericidal antibodies in the serum of immunized animals. However, the nature of the protection in the chamber fluid has not been
fully investigated. Studies by several investigators have shown that gonococci adapted to grow in guinea pig s.c. chambers become more resistant to the complement-mediated bactericidal action of human serum (17, 18). The role of complement in the protection of these chambers against infection by N. gonorrhoeae is also supported by the work of Arko et al. (3), who showed the avirulent type 3 gonococci could infect the chambers only after the chambers had become tissue encapsulated with diminished complement levels. Polymorphonuclear neutrophils (PMNs) may also play a major role in protection against gonococcal infections. Novotny et al. (14) ret Present address: Department of Microbiology, The University of Alabama in Birmingham, Birmingham, AL 35294.
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ported that PMNs are very efficient in destroying ingested gonococci, although macrophages often release viable gonococci. The inflammatory response that develops in guinea pig s.c. chambers after challenge with N. gonorrhoeae has been previously described (24). However, differences in the cellular response between immunized and control animals have not been reported. The present investigation was undertaken to investigate the role of the humoral and cellular responses in the s.c. chambers of immunized guinea pigs which are responsible for the protection elicited against challenge with N. gonorrhoeae. MATERIALS AND METHODS Organism. Colony type 1 and colony type 4 of N. gonorrhoeae 120-94 were used during the investigation. The cultures were maintained on G C medium base (Difco Laboratories, Detroit, Mich.) supplemented with 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.). N. gonorrhoeae type 1 cells for ribosomal preparation and challenge of animals were grown in a liquid medium comprised of 0.1% soluble starch, 0.1% KH2PO4, 0.5% NaCl, and 1.5% proteose peptone no. 3 (Difco Laboratories). This was supplemented after autoclaving to give final concentrations of 0.5% glucose, 0.042% NaHCO3, and 1% IsoVitaleX.
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WANNEMUEHLER, MILLER, AND COOPER
These organisms (type 1) grew as opaque colonies according to the classification scheme described by Swanson (21). Animals. Female Hartley guinea pigs weighing approximately 600 g were used throughout the study. Each guinea pig was implanted with two dorsal s.c. tube chambers of modified polyallomer as previously described (8). Preparation of ribosomes. The ribosomal preparations were prepared by a modification of the method of Youmans and Youmans (28) as previously described
(8, 9).
Immunization and challenge of guinea pigs. Guinea pigs were immunized s.c. with 1 mg of ribosomal preparation as previously described (8, 9) with Freund incomplete adjuvant. For the immunodiffusion assay, sera were obtained from guinea pigs immunized intraperitoneally with a second injection of ribosomal material (200 jig of protein) without Freund incomplete adjuvant 20 days after the primary immunization. A third immunization (intraperitoneal, 200 ,ug of protein) was given 1 week later. Guinea pigs were challenged with 107 colony-forming units of opaque N. gonorrhoeae 120-94 type 1 on day 10 after immunization. Gonococci used in this procedure were prepared from agar-grown cultures. Colonies were washed from the agar plates with warm (37°C) saline and diluted to approximately 108 colonyforming units per ml. Saline was used for the suspending medium to minimize the amount of medium-induced inflammation in the chamber. Evaluation of the protection afforded to immunized guinea pigs was determined as previously described (8, 9). Examination of chamber fluid. At various times after immunization, 0.2 to 0.5 ml of fluid was withdrawn from the s.c. chambers for examination. Total leukocyte counts were performed using improved Neubauer hemacytometer. Differential cell counts were performed on smears stained with Wright stain. Lymphocytes, PMNs, and mononuclear cells were characterized on the basis of nuclear morphology, size, presence of granules, nucleus-to-cytoplasm ratio, and appearance of the cytoplasm. Results of leukocyte counts are expressed as the mean ± standard error of results from five animals (10 chambers). The significance of differences among means was determined by Student's t test. Samples of chamber fluid were centrifuged at 5,000 x g for 20 min to remove cells, and the supernatant fluid was collected. Supernatant fluid was stored at -70°C until used. Serum bactericidal assay. The serum bactericidal assay employed in this study was a modification of the procedure of Wong et al. (27) and was performed as follows. N. gonorrhoeae 120-94 type 4 was inoculated into agar plates and allowed to grow overnight at 37°C with 5% CO2. The cells were then washed from the plates with warm (37°C) buffered saline (0.45 g of NaCl-0.245 g of KH2PO4-0.809 g of Na2HPO4 per 100 ml of water; pH 7.2) containing 0.5% glucose, 0.01 M MgC92, and 0.01% bovine serum albumin. The cells were standardized to 300 to 325 Klett units (540 nm) and diluted in the same buffer to give approximately 10' colony-forming units per ml. A portion of the cell suspension (0.1 ml) was added to 0.1 ml of serum or chamber fluid (diluted in the same buffer). Normal guinea pig serum was used as a source of complement.
INFECT. IMMUN.
The reaction mixture was incubated at 37°C for 90 min while shaking. Samples (25 ,ul each) were plated in triplicate on agar plates. The plates were then incubated overnight at 37°C with 5% CO2. Killing was accorded to those dilutions of antisera which had at least a 90% reduction in colony-forming units when compared with controls. Immunodiffusion and immunoelectrophoresis. Immunodiffusion was carried out in 0.9% agarose (Sea-plaque, Marine Colloids, Rockland, Maine) with O.9o NaCl and 1% Triton-X-100 (Sigma Chemical Co., St. Louis, Mo.). Precipitin bands were stained with Coomassie blue R and destained in 7.5% acetic acid5% methanol. Immunoelectrophoresis was carried out in 1% agarose in Tricine IV buffer (Sigma Chemical Co.). Gels were run for 2 h at 17 V per slide. Rabbit anti-guinea pig serum was used to precipitate the chamber fluid and serum samples. The precipitin bands were stained with 0.50% bromphenol blue and destained with 2% acetic acid.
RESULTS Analysis of chamber fluids components. Chamber fluids from ribosome-immunized and nonimmunized guinea pigs were evaluated by immunoelectrophoresis. When chamber fluids or matched sera were electrophoresed and subsequently reacted with rabbit anti-guinea pig sera, their immunoelectrophoretic patterns were qualitatively similar (data not shown). Also, no differences were detected between ribosome-immunized and nonimmunized chamber fluids or sera. This indicated that the major antigenic components present in the sera were also represented in chamber fluids. In addition, when the antiribosomal activity in sera or chamber fluids was tested by immunodiffusion against homologous ribosomal preparations, both sera and fluid gave one major line of precipitation which formed lines of identity. Bactericidal activity of chamber fluids and sera. The bactericidal activity of chamber fluids and sera from ribosome-immunized guinea pigs is shown in Table 1. Both chamber fluids and sera from immunized animals had bactericidal titers ranging from 8 to 16. Chamber fluids from saline-immunized control animals had a low level of bactericidal activity (c4); however, sera from these same animals had undetectable activity (
