Detection of Antibody-Producing Cells of Mice

Transcription

1 INFECTION AND IMMUNITY, Feb. 1970, p Copyright 1970 American Society for Microbiology Vol. 1, No. 2 Pritnted in U.S.A. Detection of Antibody-Producing Cells of Mice Injected with Antigens of Neisseria meningitidis1 DOLORES G. EVANS Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland Received for publication 4 September 1969 The technique of Jerne et al. was adapted to the study of the antibody response of mice to Neisseria meningitidis antigens. The mice were inoculated with heated group A bacteria or antigen concentrated from their culture fluid. Sheep erythrocytes were coated with bacterial lysate or culture fluid. These antigens were shown by immune hemolysis to be group-specific and appeared to be of identical specificity. The plaque-forming cell (PFC) response obtained with either antigen was dosedependent over a range of 10 to 300,ug per mouse. However, the maximum response, reached at 4 days, was quite low. The number of PFC per 106 nucleated cells, elicited by the heated bacteria and culture fluid, was 15 and 7, respectively. The number of PFC declined rapidly after the peak. A second injection of heated bacteria, given 18 days after the first injection, elicited a more rapid but otherwise similar reaction, apparently involving the production of only immunoglobulin M antibodies. No hemolytic antibodies were demonstrated in the sera of these mice. The passive hemolytic plaque assay here described provided the sensitivity necessary to study the details of the antibody response of mice to meningococcal antigen. The study of immunity to Neisseria meningitidis is hampered by the lack of a satisfactory experimental animal model. Nonhuman primates are susceptible to meningococci only when the bacteria are inoculated directly into the lumbar space (7). Although horses can be killed by an intravenous dose of meningococcus which rabbits tolerate, they do not develop meningitis (16). The small laboratory animals thus far tested are highly resistant to infection. Branham and Lillie (1, 2) reproduced the clinical and pathological picture of typical meningitis in rabbits and guinea pigs. However, the inoculum was given intracistemally and the minimal lethal doses were 2 X 108 to 3.5 X 108 viable bacteria for rabbits and 108 to 2 X 108 for guinea pigs. Mice appeared to be even more resistant; 3.5 X 108 meningococci, injected intraperitoneally, produced only slight illness (1). In 1933, Miller (14) reported that N. meningitidis suspended in gastric mucin produced bacteremia in mice, fatal within 24 hr. Under these experimental conditions, the minimal lethal dose for some strains of meningococci was decreased ' From the Bureau of Medicine and Surgery, Department of the Navy, research tasks MR B and M to 10 bacteria. This method has been widely used for the demonstration of passive protection of mice by hyperimmune serum from several sources (3, 13, 18). However, actively immunied mice survive only small challenge doses of viable bacteria suspended in mucin. Consequently, this method has been used in only a few instances and with moderate success to study active immunity of mice elicited by bacteria or antigens derived from N. meningitidis (9, 21; R. G. Considine, B. W. Hammond, and D. T. Kingsbury, Bacteriol. Proc., 1968, p. 90). Thus, there is a need for a more sensitive and reliable method for the evaluation of the antigenicity of meningococcal fractions. The modification of the technique of Jerne et al. (11) here described may satisfy this need. 151 MATERIALS AND METHODS Microorganisms. N. meningitidis group A (strain MK-204), group B (NOR-7), and group C (NOR-20) were employed in the present study. The NOR-7 strain, sulfadiaine-sensitive, was isolated in Norfolk, Va., in November 1964, by E. S. Dunbar, from the spinal fluid of a patient. The other two strains were described previously (21). The bacteria were grown in 45 ml of the medium of Watson and Scherp (20)

2 152 EVANS INFEC. IMMUN. in a 300-ml Erlenmeyer flask with a side arm. The inoculum was 0.35 ml of a 16-hr culture grown on Mueller-Hinton agar and resuspended in 4 ml of 0.01 M phosphate buffer, ph 7.0. The cultures were shaken at 120 cycles/min in a gyratory incubatorshaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 37 C for approximately 4 hr. Mice. Seven to 8-week-old Swiss white mice, inbred NIH-NMRI strain, weighing 22 to 25 g were employed. Preparation of crude antigens. Three types of antigens were employed. The supernatant fluid of the cultures was collected during the logarithmic phase of growth (optical density 0.8 at 640 nm) and sterilied by membrane filtration (0.2 Am pore sie). The antigen in the supernatant fluid was concentrated 60-fold by ultrafiltration (ultrafiltration cell 50 and PM-10 membrane, Aminco Co., Lexington, Mass.), divided into small samples, and kept at -70 C until injection of mice or sensitiation of erythrocytes. Bacterial cells of the same cultures were washed once with saline, adjusted to the desired concentration, and rendered nonviable by heating at 56 C for 1 hr. Appropriate amounts of this cell suspension were injected intravenously into mice on the day of preparation. Dry weight determinations were performed on all of the antigenic preparations after extensive dialysis against water. For the sensitiation of erythrocytes, the heatkilled bacteria were allowed to autolye at 4 C for 72 hr. The cellular debris was eliminated by centrifugation, and the supernatant fluid was distributed into small samples and kept at -70 C until needed. Sensitiation of sheep erythrocytes. Appropriate dilutions of culture fluid or of the bacterial autolysates were boiled for 1 hr immediately before use. A 0.5-ml amount of either antigen was mixed with 0.1 ml of packed sheep erythrocytes (Microbiological Associates, Bethesda, Md.) which had been washed three times with phosphate-buffered saline (PBS), ph 7.4. The mixture was incubated at 37 C for 30 min. The sensitied erythrocytes were then washed four times with 20 volumes of PBS and used the same day. Microhemolytic assay. The efficiency of the sensitiation procedure was tested by the microhemolytic assay method with equal volumes of rabbit hyperimmune serum preabsorbed on sheep erythrocytes (Communicable Disease Center, Atlanta, Ga.), 1% sensitied erythrocytes, and a 1:10 dilution of guinea pig complement (Microbiological Associates). The microassay plates were incubated at 37 C for 1 hr. The titer was expressed as the inverse of the greatest serum dilution eliciting 100% lysis. Inhibition of immune hemolysis was tested by incubating equal volumes of serial dilutions of antigen with 20 units of antiserum for 30 min at 37 C. One unit of antiserum is the minimum amount required to completely lyse a % suspension of sensitied erythrocytes. The excess antibody was then determined by addition of 1% erythrocytes sensitied with either antigen and a 1:10 dilution of complement. In all cases, the complement diluent employed was 0.15 M NaCl containing M CaCl2 and M MgCl2 (10). Hemolytic plaque technique. The hemolytic plaque assay described by Jerne et al. (11) was employed as follows. To Noble Agar (Difco), 1.4% in distilled water and maintained at 56 C, was added an equal volume of 2X Eagle minimal essential medium (MEM, Grand Island Biological Co., Grand Island, N.Y.), also kept at 56 C, and the ph was adjusted to 7.2 with 4% NaHCO3. This mixture was then distributed in 2.0-ml portions in mixing tubes, which were transferred to a water bath at 45 C 5 min before plating. To each of these tubes the following suspensions were added in this order: 0.1 ml of 1% diethylaminoethyl(deae)-dextran in 0.15 M NaCl, 0.2 ml of 40% sensitied sheep erythrocytes, and to 0.2-ml samples of a mouse spleen cell suspension. Control assays were performed as above except that unsensitied sheep erythrocytes were employed. The contents of the tubes were promptly mixed by rotation and poured over a solid basal layer maintained at 37 C. The basal layer consisted of 1 volume of 2.4% Noble Agar and 1 volume of 2X MEM (ph 7.2) and was used within 1 to 4 days of preparation. Plates were incubated for 1 hr at 37 C under 10% CO2 and 100% relative humidity and then were flooded with 3.0 ml of a 1:10 dilution of guinea pig complement. The plates were reincubated at 37 C for 1 hr, complement was poured off, and the numbers of plaques were enumerated at 7X magnification with the aid of a dissecting microscope. Results are expressed in terms of plaque-forming cells (PFC) per 101 viable nucleated spleen cells. Three or four mice were used for each determination. Each spleen cell suspension was plated in triplicate. RESULTS Sensitiation of sheep erythrocytes. The amounts of group A bacterial autolysate and of supernatant fluid required to sensitie sheep erythrocytes are shown in Table 1. Minimal amounts that produced optimal sensitiation were 20,ug of bacterial autolysate or 60 Ag of culture fluid per 0.1 ml of packed sheep erythro- TABLE 1. Concentration of group A N. meningitidis antigen required to sensitie sheep erythrocytes Amnt (jig) of antigen Bacterial autolysate Hemolytic titera Culture supernatant fluid 0 <2 < a Reciprocal of the highest dilution of group A hyperimmune rabbit serum that gave complete lysis of 1% sensitied erythrocytes.

3 VOL. 1, 1970 DETECTION OF ANTIBODY-PRODUCING CELLS TABLE 2. Group specificity of sensitied sheep erythrocytes Antigen used to sensitie' Serumb Bacterial Culture supernatant autolysate fluid A B C A B C Anti-A <2 <2 512 <2 <2 Anti-B Anti-C Fresh sheep erythrocytes were sensitied with culture supernatant fluids and bacterial autolysates of N. meningitidis. b Rabbit group-specific antisera were employed in the microhemolytic assay. U-) LU 2 Ld 0 IL LUI a cl LL D F Ix1o8 -j -j uj LLJ.LL J a_ ID 0 1i1 L) J I- (D 0 0 L-j 0~ 153 I MICROGRANMS OF ANTIGEN FIG. 2. Effect of dose of antigen on the plaqueforming-cell responise. Mice received various amounts of either heated bacteria or supernatant fluid and were * assayed 4 days later. Each symbol represents the mean value of triplicate plates. 2x.o8 NUMBER OF SPLEEN CELLS FIG. 1. Plaque-forming cells as a function of thle number of spleen cells used in the assay. Each symbol represents the mean obtained from triplicate plates from a single spleen. Four mice were injected intravenously with 120,ug of heated bacteria and tested 4 days later. cytes. The same degree of hemolysis was obtained when the cells were sensitied with concentrations of antigen 10 times greater. In the hemolytic plaque assay described below, red cells were treated with four times the smallest concentration required to sensitie sheep erythrocytes. Sensitiation of erythrocytes with group A, B, and C antigens was group-specific when tested in vitro with hemolytic antisera (Table 2). Hemolytic titers elicited by heterologous sera were low or negligible. The immune hemolysis of erythrocytes sensitied with supernatant fluid of N. meningitidis group A (6,ug of antigen per 1% erythrocytes per ml) was inhibited by concentrations of 80 jig of culture fluid per ml or 50,ug of bacterial autolysate per ml. Antigen derived from group B or C meningococci did not inhibit the reaction in concentrations as high as 240,g/ml Effect of spleen cell concentration on plaque formation. Mice were injected intravenously with 110,ug (dry weight) of heated N. meningitidis and tested 4 days later. Samples representing one-tenth, one-twentieth, and one-fiftieth of the total cell suspension of individual spleens were assayed for plaque formation. The results shown in Fig. 1 indicate that the number of antibody-producing cells was proportional to the number of spleen cells used in the assay. In subsequent experiments, one-tenth of the spleen cell suspension was used per plate. Effect of dose of antigen on plaque formation. Mice were injected with various concentrations of heated bacteria or culture fluid and tested 4 days later. It can be seen from results shown in Fig. 2 that the number of PFC increased with the concentration of antigen. The response to heated bacteria was almost twice that obtained with culture fluid. It is noteworthy that, even at the highest concentration of heated bacteria, 310 Mg, the number of PFC was relatively low, 16

4 154 EVANS INFEC. IMMUN. cf)7 J LLJ u 6 LI u 3 cr 2 0 LL LUJ D I a DAYS AFTER INJECTION FIG. 3. Kinetics of the plaque-forming-cell response of mice to a single injection oj culture supernatant fluid. Mice were iinjected intravenously withl 140 lig of antigen and their spleens were assayed at the indicated intervals. PFC per 106 nucleated cells. This represents a mean of about 2,400 PFC per spleen. Mice stimulated with 280 Mg of culture fluid exhibited a mean of about 8 PFC per 106 nucleated spleen cells. Kinetics of the antibody response. Mice received a single intravenous injection of 220 Mg of heated bacteria or 140 Mg of culture fluid and their spleens were assayed at various intervals of time for plaque formation with sensitied and unsensitied red cells. Erythrocytes were sensitied with bacterial autolysates when used for the assay of spleen cells of mice stimulated by heated bacteria or culture fluid when mice were stimulated by this antigen. Results of these experiments are depicted in Fig. 3 and 4A. (Note that an arithmetic scale was employed.) Spleens of uninjected control mice exhibited a mean value of 0.91 PFC per 106 cells with a range of 0.8 to 1 PFC per 106 cells. The same number of plaques was obtained with sensitied or unsensitied erythrocytes. The injection of either antigen elicited a significant increase in the number of plaques detected with sensitied erythrocytes 1 day after injection. Maximal responses, corresponding to 15 and 7 PFC per 106 cells, respectively, were elicited by heated bacteria and culture fluid, 4 days after 1 12 (I) I 16 A [ L i 2' 0 ċni w 0 6I) llj Z 18 cr 2,1 D I. 1- I / -I B UNSENSITIZED * ERYTHROCYTES i -- Ar- Q SECOND INJECTION/ DAYS AFTER INJECTION 22 FIG. 4. Kinetics of the plaque-forming-cell response of mice to heated bacteria. (A) Response to a first intravenous injection of 240 jig of this antigen. (B) Response to a second injection of 200 jag. -~~~~~~~~~~~~~~~~~~~~

5 VOL. I1, 1970 DETECTION OF ANTIBODY-PRODUCING CELLS 155 injection. From the 5th day on, the decline in PFC was relatively rapid. In mice stimulated with heated bacteria (Fig. 4A), the number of plaques was two or three times the background level at 8 and 12 days. Mice receiving the culture fluid appeared to respond in a similar manner (Fig. 3). Stimulation with either meningococcal antigen produced a small but reproducible increase, at 2, 3, and 4 days, in the number of plaques detected with unsensitied erythrocytes. The response of mice injected with 200 jig of heated bacteria 18 days after the first injection of 240,ug is shown in Fig. 4B. The PFC response to the second injection reached its peak on the 2nd day and declined rapidly. The maximal number of PFC detected was not greater than after the first injection. Synthesis of immunoglobulin (Ig)G as a response to meningococcal antigens was explored by the use of rabbit or goat antimouse IgG (Microbiological Associates). Portions of the spleen suspensions of mice that received either one or two injections (Fig. 4A, 4B) were plated and incubated for an additional 1 hr at 37 C with a 1:100 dilution of rabbit or goat antimouse IgG before the addition of complement. Neither antimouse IgG serum increased the number of detectable PFC after the first or second injection, but rather decreased the number of plaques detected with complement alone. These results suggest that meningococcal antigens did not induce an IgG response (6, 19). Hemolytic antibodies to sensitied or unsensitied red cells were not detected in the sera of mice used in experiments depicted in Fig. 4A and 4B. Inhibition of plaque formation by free antigen. Mice were injected with 110 Mg of N. meningitidis TABLE 3. Inhibition of hemolytic plaque formation by free antigen Plaque forming cells/2 X 107 spleen cells detected in the presence of Mousea Homologous Heterologousb No addition Autolysate Supernatant Supernatant (240 lg) fluid (80 pg) fluid, (100 jg) I llg of N. meniingi- a Mice were injected with 110 tidis group A culture fluid and assayed 4 days later. b The heterologous antigen was culture supernatant fluid of N. meningitidis group B. culture fluid. Four days later, spleen cells were assayed for plaque formation in the usual manner. In a parallel series of plates, 80 Mug of free bacterial autolysate (unadsorbed to erythrocytes) or 240 Mg of free culture fluid was also added. Plaque formation was inhibited by the presence of either free homologous antigen, but not by heterologous antigen (Table 3). DISCUSSION Kabat et al. (12), Scherp and Rake (18), and Watson and Scherp (20) have indicated that N. meningitidis contains group-specific carbohydrate antigens which can be detected in the bacterial cell and in the supematant fluids of the cultures. Recently, successful purification of group-specific meningococcal carbohydrate antigen has been achieved (8, 21). In the present work, it was possible to sensitie fresh sheep erythrocytes with autolysates and culture fluid without the use of conjugating agents. Sensitied erythrocytes were employed in the passive hemolytic plaque assay. This method has been successfully employed to study the antibody response of soluble carbohydrate antigens of gram-negative bacteria (5, 15, 17). The fact that conjugating agents were not required to sensitie fresh erythrocytes probably indicates that the meningococci antigens here studied were carbohydrate in nature. These antigens also proved to be groupspecific. The similarity in the cytokinetics of the primary antibody response of mice to heated bacteria and to culture fluids is interpreted as an indication that these antigen preparations contain either the same or very closely related antigens. This idea is further supported by the fact that the detection of hemolytic plaques with erythrocytes sensitied with culture fluid was inhibited by the addition of homologous bacterial autolysate. It is true that the response produced by heated bacteria was relatively greater than that produced by the culture fluid. This may be attributed to the adjuvant effect of endotoxin, which, presumably, was in higher concentration in the heated bacteria. It is noteworthy that the PFC response obtained against both heated bacteria and culture fluids was rather low. The highest concentration of antigen tested, 310 Mg of heated bacteria, produced only a mean of about 16 PFC per 106 cells (Fig. 1). Even so, the fact that the PFC response is dose-dependent suggests that the range of antigen concentrations used probably did not induce a tolerant state. It should be noted that a low PFC response was detected when viable bacteria were used as the target system (D. G.

6 156 EVANS INFEC. IMMUN. Evans and E. Weiss, Bacteriol. Proc., 1969, p. 92). The requirement for a relatively high antigen concentration for induction of a PFC response is in agreement with the observations of Gotschlich et al. (8). They found that purified meningococcal carbohydrate antigen at concentrations from 0.05 to 1.5,tg did not protect mice against a challenge dose of viable bacteria or produce a rise in hemagglutinating titers. Recently, in this laboratory, Considine, Hammond, and Kingsbury (Bacteriol. Proc., 1968, p. 90) and Weiss and Long (21) succeeded in producing partial protection of mice which had received a minimum of 50 Mg of purified group-specific carbohydrate antigen. It is surprising that a second injection of meningococci produced only a transitory response of a low magnitude. The finding that the number of PFC was not enhanced by the addition of either rabbit or goat anti-mouse IgG, at any time after the first or second injection of antigen, suggests that the PFC response obtained was mainly due to the synthesis of IgM. From this work, it is apparent that meningococcal antigens induce a very weak antibody response in this strain of mice. Whether this is related to the rate of degradation of meningococcal antigens in mice is not known. These antigens enhanced antibody synthesis nonspecifically, as demonstrated by the increase of PFC detected with nonsensitied erythrocytes. This property may be due to the presence of endotoxin or to an endotoxin-like property of the antigens themselves. Endotoxins, besides being adjuvants, usually elicit low antibody responses (4). In the present investigation, the demonstration of the antibody response of mice to meningococcal antigens by a serological method was unsuccessful. Similar observations were obtained by Gotschlich et al. (8), who also used the mouse protection test. It is thus evident that the greater sensitivity of the hemolytic plaque assay permitted a successful approach to the problem of the antibody response of the mouse to meningococcal antigens. ACKNOWLEDGMENTS The author acknowledges the valuable advice given by Emilio Weiss and F. B. Gordon in the preparation of this manuscript. I especially thank Dr. Weiss for his many helpful discussions. Research was conducted while the author was pursuing an NRC-Navy Bureau of Medicine and Surgery Postdoctoral Research Associateship. LITERATURE CITED 1. Branham, S. E., and R. D. Lillie Observations on experimental meningitis in rabbits. U.S. Public Health Rep. 47: Branham, S. E., and R. D. Lillie Experimental meningitis in guinea pigs. U.S. Public Health Rep. 52: Branham, S. E., and M. Pitman Recommended procedure for the mouse protection test in evaluation of antimeningococcus serum. U.S. Public Health Rep. 55: Braun, W., and E. Cohen Regulation of antibody response. Charles C Thomas Publisher, Springfield, Ill. 5. Domingue, G., and E. Neter The plaque test for the demonstration of antibodies against enterobacterial common antigen produced by spleen and lymph node cells. Immunology 13: Dresser, D. W., and H. H. Wortis Use of an antiglobulin serum to detect cells producing antibody with low haemolytic efficiency. Nature (London) 208: Flexner, S Experimental cerebro-spinal meningitis in monkeys. J. Exp. Med. 9: Gotschlich, E. C., T. Y. Liu, and M. S. Artenstein Human immunity to the meningococcus. m. Preparation and immunochemical properties of the group A, group B, and group C meningococcal polysaccharides. J. Exp. Med. 129: Greenberg, L., and M. W. Cooper A somatic antigen vaccine for the prevention of meningococcal cerebro-spinal meningitis. World Health Organ. Rep. Ser. 33: HUbner, K. F., and N. 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