Immune Suppression and Induction of Gamma Interferon by Pertussis ...

Vol. 49, No. 1

INFECTION AND IMMUNITY, JUlY 1985, p. 90-97 0019-9567/85/070090-08$02.00/0 Copyright © 1985, American Society for Microbiology

Immune Suppression and Induction of Gamma Interferon by Pertussis Toxin FREDERICK R. VOGEL,lt THOMAS W.

KLEIN,'* WILLIAM E. STEWART II,' TAKESHI IGARASHI,2 AND HERMAN FRIEDMAN1

Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida 336121 and Showa University Research Institute for Biomedicine in Florida, St. Petersburg, Florida 337022 Received 13 September 1984/Accepted 27 March 1985 It has been suggested that pertussis toxin is a virulence factor of Bordetella pertussis. Although extracts enriched in pertussis toxin activity have been reported to enhance immune responsiveness, other studies have demonstrated a suppressive ability, suggesting that the toxin may contribute to the virulence of B. pertussis through mechanisms involving immune suppression. We report that purified pertussis toxin suppressed the in vitro immunoglobulin M antibody response of mouse splenocytes to sheep erythrocytes. At submitogenic doses, the toxin also suppressed [3H]thymidine incorporation by splenocytes, suggesting that it interfered with antibody formation by inhibiting lymphocyte proliferation. Antiviral activity was detected in culture supernatants obtained from pertussis toxin-suppressed splenocyte cultures by using a cytopathic effect inhibition assay. This antiviral activity was virus nonspecific, sensitive to pH 2.0 treatment, stable to heating at 56°C, and neutralized by anti-gamma interferon antiserum. Finally, the fractionation of splenocytes by anti-immunoglobulin panning techniques suggested that Lyt2+ lymphocytes proliferated in response to pertussis toxin and produced interferon. Our results suggest that pertussis toxin may contribute to the virulence of B. pertussis through stimulation of Lyt2+ lymphocytes, resulting in the induction of gamma interferon and the subsequent inhibition of the primary antibody response.

Pertussis toxin (PT) is an oligomeric protein exotoxin that is produced by phase I Bordetella pertussis cells (40). This toxin, which is also referred to as pertussigen (1), accounts for many of the biological activities of B. pertussis, has been associated with the pathophysiology of clinical pertussis (31), and is believed to be a B. pertussis virulence factor (42). PT has also been demonstrated to be a major protective antigen in that antitoxin antibodies protect mice from either intracerebral infection (29) or aerosol infection (32) with virulent B. pertussis cells. B. pertussis vaccine has been recognized for decades as an adjuvant (10). In particular, this vaccine enhances immunoglobulin G (IgG) and IgE antibody responses (27, 39). Extracts of B. pertussis enriched in PT activity also augment IgG and IgE antibody responses (24, 28, 33); however, similar preparations have also been reported to suppress antibody formation. Asakawa (2) demonstrated that the primary antibody responses to either sheep erythrocytes (SRBC) or tetanus toxid were suppressed in mice pretreated with PT. Iwasaki and Nozima (14) demonstrated a similar effect of the toxin on antibody responsiveness to influenza virus. Suppression of in vitro antibody responses to SRBC was reported by Kumazawa and Mizunoe (21). These authors observed that PT suppressed both primary and secondary in vitro anti-SRBC responses of murine spleen cells. The suppression by the toxin was greatest during the inductive phase of the response and appeared to be related to a suppressor factor produced by T-lymphocytes. The results of these studies illustrate that B. pertussis subcellular components can suppress immune responsiveness rather than enhance it and suggest that these components may contri-

bute to the virulence of B. pertussis through immune suppression mechanisms. Gamma interferon induced by T-lymphocyte mitogens, such as concanavalin A (Con A) and staphylococcal enterotoxin A (SEA), has been shown to suppress the in vitro antibody response to SRBC (15, 16). In light of the facts that PT is a T-cell mitogen (19, 20) and that B. pertussis cells (3) and components (18) induce interferon, we tested for involvement of interferon production in PT suppression of antibody formation. We report here that purified PT both suppresses in vitro antibody production to SRBC and induces gamma interferon production in the cultures. We present evidence which suggests that the cell type responding to PT is the Lyt2+ cell type. These results suggest that PT may contribute to the virulence of B. pertussis through stimulation of Lyt2+ lymphocytes, resulting in the induction of gamma interferon and subsequent inhibition of the primary antibody response. MATERIALS AND METHODS Mice. Female 18- to 20-g strain BDF1 (C57BL/6 x DBA/2) mice were used throughout this study; they were obtained from Laboratory Supply Co., Indianapolis, Ind. The mice were maintained on tap water and commercial animal food ad libitum. PT preparation. PT was isolated from phase I B. pertussis culture supernatants by hydroxylapatite column chromatography, as described by Yajima et al. (43). B. pertussis strain 3779B12S4 was supplied by John Munoz, Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, Hamilton, Mont. The bacteria were cultured under stationary conditions for 3 days in Stainer-Scholte medium (1) and were inactivated by adding 0.02% thimerosal (Eli Lilly & Co., Indianapolis, Ind.). Portions (10 liters) of the

* Corresponding author. t Present address: Institut Pasteur, Immunotherapie Experimentale, 75724 Paris, Cedex 15, France.

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supernatants of these cultures were collected by centrifugation, adjusted to pH 6.0, and applied to a hydroxylapatite column (1.5 by 14.0 cm; Bio-Rad Laboratories, Richmond, Calif.) that had been previously equilibrated with 0.01 M phosphate buffer (pH 6.0). Eluate fractions were assayed for leukocytosis-promoting and histamine-sensitizing activity in strain BDF1 mice. Biologically active material eluted from the column as a sharp peak after the application of 0.1 M phosphate buffer (pH 7) containing 0.5 M NaCl (43). The PT preparation used in this study was obtained by further purification of the active material eluted from the hydroxlapatite column. The active material was concentrated (Centriflo; Amicon Corp., Danvers, Mass.) and applied to a Sephadex G-200 column (2.6 by 50 cm) in 0.1 M phosphate buffer (pH 7.0) containing 0.5 M NaCl. Three peaks were eluted from the column. The first peak eluted in the void volume, the second peak eluted at approximately 92 kilodaltons, and the third peak eluted at 18.5 kilodaltons. Biological activity was confined to the second peak. Injecting 1 ,ug of protein of this material into mice resulted in blood leukocytosis of 100,000 cells per mm3 and sensitized 100% of the mice to the lethal effects of 1 mg of histamine base. Heating the material to 80°C for 30 min eliminated these effects. The toxin preparation was weakly positive in the Limulus lysate gelation assay, with a ratio of protein weight to endotoxin weight of 756, and formed a single band upon double radial immunodiffusion against B. pertussis polyvalent antiserum. When PT (10 jig of protein per lane) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (22), it displayed a major band at 30 kilodaltons and minor bands at 29, 26, and 24 kilodaltons. This pattern is similar to the pattern obtained for islets-activating protein analyzed in sodium dodecyl sulfate gels in the presence of 4 M urea (17, 40). Tissue culture media. The tissue culture media used were RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.), Hanks balanced salt solution (Flow Laboratories, Inc., McLean, Va.), and Dulbecco modified Eagle medium (DMEM) (Flow Laboratories). RPMI 1640 medium and DMEM were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Flow Laboratories or Hyclone Laboratories, Logan, Utah). The RPMI 1640 medium used for in vitro immunization contained 10% FBS and was supplemented with L-glutamine (GIBCO Laboratories), 2mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.) and 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (Sigma) (complete medium). All tissue culture media and balanced salt solutions were supplemented with 100 U of penicillin per ml and 100 jig of streptomycin per ml. In vitro immunization. In vitro sensitization and antibody formation to SRBC were accomplished in the following manner. Portions (2 ml) of RPMI 1640 medium supplemented with 10% FBS (complete medium) containing 107 BDF1 spleen cells were added to individual wells of 24-well culture plates (Linbro-Flow). PT was diluted in pyrogen-free saline and added in 0.1-ml volumes to experimental cultures. Positive and negative control cultures received 0.1 ml of saline. A 0.2-ml portion of a 0.1% suspension of SRBC (2 x 106 SRBC; Colorado Serum, Denver, Colo.) was added to each positive control and experimental culture. Each negative control culture received 0.2 ml of complete medium. The cultures were then incubated for up to 5 days at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Hemolytic plaque assay. Direct (IgM) plaque-forming cells (PFCs) were assessed by localized hemolysis in a gel, as

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described by Yamada and Yamada (44). Briefly, 0.6 ml of 0.7% type II agarose (Sigma) in DMEM containing 10% FBS (52°C) was added to 0.3 ml of a 7% SRBC suspension and 0.1 ml of a spleen cell suspension (37°C). The resulting cell suspension was mixed with a Pasteur pipette (Corning Glass Works, Corning, N.Y.) and inoculated dropwise from a height of 60 cm onto plastic petri dishes (100 by 15 mm). Three plates were used for each sample, with each plate receiving 10 to 12 drops. The plates were incubated at 37°C for 2 to 3 h in a humidified atmosphere containing 95% air and 5% CO2. After incubation, IgM plaques were developed by adding guinea pig complement diluted 1:10 in Hanks balanced salt solution. The plates were then incubated for an additional 1 h. The number of plaques per sample was counted, and the data were expressed as the number of PFCs per 106 viable spleen cells. The spleen cell viability of each sample was determined by trypan blue dye exclusion. Lymphocyte blastogenesis. Spleen cells were cultured in flat-bottomed micro tissue culture plates (A/S Nunc, Roskilde, Denmark). Quadruplicate wells each contained 2 x 105 spleen cells and PT. Complete medium was used in these experiments. The plates were incubated for 72 h at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Each culture was pulsed with 0.5 ,uCi of [3H]thymidine (2 Ci/mmol; New England Nuclear Corp., Boston, Mass.) for 18 h before harvest. The cells were then collected on glass fiber filters (Whatman) by repeated washing with deionized water, using a model M12 cell harvester (Brandel, Inc., Rockville, Md.). The filter disks were dried at 100°C for 30 min, and the filters were placed in vials containing 5 ml of Instagel scintillation cocktail (Packard Instrument Co., Inc., Downers Grove, Ill.). Samples were counted with a Packard Tri-Carb liquid scintillation spectrometer. Antigen-specific proliferation was measured in a manner similar to that described above. In these studies SRBC at a concentration of 2 x 106 cells per ml were used in place of mitogens. Interferon assay. The antiviral activities of spleen cell culture supernatants were assessed by a semimicro cytopathic effect (CPE) inhibition assay, as described by Stewart (37). A 50-,ul portion of DMEM was added to each well of a flat-bottomed microtiter plate (Flow Laboratories). The supernatants to be examined for antiviral activity were diluted by half-log serial dilutions in 96-well flat-bottomed culture plates. As positive controls, some of the wells contained half-log1o dilutions of interferon reference standard G002-902-026 produced by the Research Resources Branch, National Institute of Allergy and Infectious Diseases. After the dilutions were made, the plates were exposed to sterilizing UV lamp for 10 min. L-929 cells, which were kindly supplied by P. T. Allen (Frederick Cancer Research Institute, Frederick, Md.), were suspended at a concentration of 2 x 105 cells per ml in DMEM containing 10% FBS. A 0.1-ml portion of this suspension was added to each microtiter well. The plates were then incubated for 24 h at 37°C in a humidified atmosphere containing 95% air and 5% CO2. After incubation cell control wells received 50 pl1 of DMEM. All other wells received 50 ,il of vesicular stomatitis virus (VSV) in DMEM diluted 1:300 from a VSV stock suspension. In some experiments, a murine encephalomyocarditis virus was used in interferon assays. The murine encephalomyocarditis virus was diluted for use to 10' of stock concentrations. After the virus was added, the microtiter plates were incubated for an additional 24 h. Interferon titers were determined for each sample by comparing the CPE observed in test wells with the tnaximal CPE observed in virus control wells and the minimal CPE ob-

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served in cell control wells. The interferon titer of each sample was the reciprocal of the highest dilution which produced 50% CPE. The number of international units of interferon per milliliter for each sample was estimated by comparing sample titers with the known number of units of interferon activity of the reference standard interferon at a 50% endpoint. Separation of T- and B-lymphocytes by antiimmunoglobulin panning. Cells bearing surface immunoglobulin (sIg+) were separated from whole spleen cells by the anti-immunoglobulin panning technique of Mage et al. (26). Affinity-purified goat anti-mouse immunoglobulins (Cappel Laboratories, Malvern, Pa.) containing 50 ,ug of gentamicin (Schering Corp., Kenilworth, N.J.) per ml were diluted to concentrations of 1 mg/ml in sterile phosphatebuffered saline. Portions (2 ml) of the anti-mouse immunoglobulin solution were added to 25-cm2 tissue culture flasks (Corning), and the flasks were incubated broad side down for 18 h at 40C. After incubation, the excess, unbound anti-mouse immunoglobulin was transferred to identical flasks for further use. The antibody-coated flasks were washed three times with 5 ml of sterile phosphate-buffered saline. Spleen cells were diluted to a concentration of 1.5 x 107 cells per ml in RPMI 1640 medium containing 10% FBS and 2mM HEPES but lacking sodium bicarbonate (HEPES medium). A 3-ml portion of the spleen cell suspension was added to each anti-mouse immunoglobulin-coated flask. The flasks were incubated for 1 h at 4°C with gentle agitation of the cell suspension after 30 min. The nonadherent cells (sIg-) were removed with a Pasteur pipette, washed twice, and suspended in HEPES medium. Adherent (sIg+) cells were subsequently washed five times with sterile phosphate-buffered saline and removed from the flasks by incubation with lidocaine (Astra Pharmaceutical Products, Inc., Worcester, Mass.). The adherent (sIg+) cells were incubated at room temperature for 15 min with sterile phosphate-buffered saline containing 4 mg of lidocaine per ml. After incubation, the cells were removed from the surface of the flask by forceful pipetting with a Pasteur pipette. The sIg+ cells were then washed twice and suspended in complete medium. Separation of T-cell Lyt subsets by panning. T-lymphocytes bearing various Lyt surface antigens were selectively depleted from the sIg- cell populations by incubation with monoclonal anti-Lytl.1, anti-Lytl.2, anti-Lyt2.1, and antiLyt2.2 antibodies (Accurate Chemical and Scientific Corp., Westbury, N.Y.), with subsequent removal by anti-immunoglobulin panning as described above. Surface Ig- cells were incubated at 0°C for 45 min in HEPES medium containing an anti-Lyt antibody diluted to a concentration that was 1 log higher than the 50% cytotoxic concentration for thymocytes. The cells were then washed twice and suspended in 3 ml of HEPES medium. The cell suspension was then added to an anti-immunoglobulin-coated flask and incubated as described above. After incubation the nonadherent Lytdepleted sIg- cells were removed from the flask with a Pasteur pipette. These cells were washed twice and suspended in complete medium. Functional assays of the various separated populations were carried out in complete medium. The percentage of B-lymphocytes in each population was determined by EAC (erythrocyte-antibody complement) rosette formation (11). Statistics. Statistical significance was determined by Student's t test. P values less than 0.05 were considered significant. All experiments were conducted a minimum of three times.

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PT (ugnmO FIG. 1. Effect of PT (0) and PT heated to 80°C for 30 min (0) on the primary in vitro antibody response to SRBC. The asterisks indicate P values of