Vol. 24, No. 2
INFECTION AND IMMUNITY, May 1979, p. 422426 0019-9567/79/05-0422/05 02.00/0
Suppression of Local Intestinal Immunoglobulin A Immune Response to Cholera Toxin by Subcutaneous Administration of Cholera Toxoids STANLEY R. HAMILTON,* JOHN H. YARDLEY, AND GERTRUDE D. BROWN Department of Pathology, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205 Received for publication 12 February 1979
Cholera toxin has been shown to modulate immune responses, generally producing enhancement when administered simultaneously with antigen and suppression when administered a day or more earlier. In a previous study using chronically isolated ileal loops in rabbits, we found that two subcutaneous (s.c.) "priming" and "boosting" doses of biologically active cholera toxin suppressed the local intestinal immunoglobulin A response to intraloop doses of cholera toxin. In the study reported here, two different biologically inactive but antigenically intact cholera toxoids, glutaraldehyde toxoid and choleragenoid, were administered s.c. by the same immunization schedule as for toxin in the earlier experiment. Suppression of local immune response to intraloop cholera toxin as compared with animals receiving no s.c. inoculations was again found. The results suggest that in this model suppression was immunological (mediated by an immunological mechanism) rather than toxigenic (mediated by biological activity of cholera toxin). In addition, the occurrence of suppression of local intestinal immune response after systemic immunization suggests that suboptimal protection against enteric infections could occur after s.c. vaccination.
The modulation by cholera toxin (CT) of primary and secondary humoral and cellular immune responses to a variety of alloantigens has been studied in vitro and in vivo (2, 3, 8, 10, 11, 13-15, 17, 24). Administration of CT simultaneously with, or up to 24 h before, administration of antigen has been found to enhance antibody response to the antigen (2, 3, 11, 14, 15, 17). However, suppression of antibody response occurred when CT was given earlier, i.e., 5 days to 12 h before the antigen (2, 3, 8, 10, 11, 14, 15). We have studied local intestinal and serum immune responses to CT using chronically isolated Thiry-Vella leal loops in rabbits (12, 25). We found that three or four weekly intraloop (i.l.) inoculations of CT resulted in the development of immunoglobulin A (IgA) antitoxin antibodies in loop secretions. The presence of these antibodies correlated with the CT neutralization titers of the secretions and was associated with protection of the loops against the diarrheagenic effect of CT in challenge experiments. However, when subcutaneous (s.c.) "priming" and "boosting" doses of CT were given 3 weeks apart in addition to i.L doses, lower levels of fluid IgA anti-CT and less protection against challenge occurred than with i.L inoculation alone. This suppression of local IgA immune response by s.c.
administration of CT occurred when the first s.c. dose of CT was given either 7 days before or 21 days before the first i. dose (25). Mechanisms which have been proposed for CT suppression of immune responses include: (i) direct effect on lymphoid cells via stimulation of adenyl cyclase (1-3, 13, 15); (ii) lymphoid tissue depletion due to direct cytotoxicity (14, 15, 24); (iii) lymphoid tissue depletion due to lympholytic effect of increased plasma adrenocorticoids stimulated by CT (14-16); (iv) dysfunction of helper T cells (11, 15); and (v) stimulation of suppressor T cells (25). To obtain further evidence regarding these hypotheses, we repeated our study using two different biologically inactive but antigenically intact cholera toxoids (glutaraldehyde toxoid and choleragenoid) instead of CT for s.c. priming and boosting inoculations. (This paper was presented at the meeting of the American Gastroenterological Association, Las Vegas, Nevada, 22 May 1978.) MATERIALS AND METHODS Preparation and care of loops. The procedures for preparation and care of loops were carried out as previously described in detail (12, 25). Stated briefly, an isolated 20-cm loop of distal ileum containing a
422
VOL. 24, 1979
TOXOID SUPPRESSION OF IgA ANTI-CHOLERA TOXIN
Peyer's patch was prepared in outbred New Zealand white rabbits of mixed sex weighing 3 to 4 kg. Silastic tubing (Dow Chemical Corp., Midland, Mich.) was ligated into the ends of the loop and brought out through the skin of the posterior neck. Accumulated loop secretions were collected, and the loop was flushed daily with saline through the tubing.
423
enzyme conjugates were highly specific for their respective isotypes and detected 30 ng or less of the appropriate immunoglobulin per ml. Specimens from animals in the ILCT, SCGT, and SCCD groups were tested in each run. Control samples of immune fluid and sera were included in each run, and their absorbance values were adjusted to an arbitrary absorbance of 2.00 by a correction factor. This factor was then applied to the specimen values to compensate for day-to-day variations in the test system. As described previously (25), in preliminary studies relative absorbances of specimens at any given dilution generally showed the same relationships as the titers of the specimens, i.e., the specimens with the highest titers usually had the highest absorbances at any given dilution. On the basis of this relationship, the lowest specimen dilution in which the enzymelinked immunosorbent assay system was generally not exhausted of substrate for each type of specimen and each isotype was chosen as the test dilution. Thus, IgA anti-CT in fluids and sera and IgG anti-CT in fluids were assayed at 1:40 whereas IgG anti-CT in sera was assayed at 1:1,600. The absorbance values of the IgA test system could not be compared directly with those for IgG. However, the time course of the IgA anti-CT and IgG anti-CT response could be followed in both sera and fluids. Within-day coefficients of variation for the test systems, as calculated from standard deviations of the differences of control specimens, were about 8%.
Immunization of animals and specimen preparation. Immunization schedules and antigens are summarized in Table 1. Three weekly i.l. inoculations of 100 ,ug of purified CT (Schwarz-Mann Division of Becton, Dickinson & Co., Orangeburg, N.Y.) in 4 ml of 0.01 M phosphate-buffered saline, pH 7.2, were given to all animals on days 0, 7, and 14. This was the same dose as in our earlier experiment (25). Animals in the ILCT group received only i.l. inoculations of CT. Animals in the SCGT group received 400 1Lg of glutaraldehyde toxoid (22) in protamine sulfate and aluminum chloride adjuvant (lot 20201, Wyeth Laboratories, provided by Division of Biological Standards, National Institutes of Health, Bethesda, Md.) s.c. 1 week before (day -7) and 2 weeks after (day 14) the first i.L inoculation of CT (day 0). Animals in the SCCD group received 30 ,ug of choleragenoid (5, 6) (lot GB76 provided by Richard Finklestein, Southwestern Medical School, Dallas, Tex.) in 1 ml of phosphate-buffered saline s.c. on the same schedule. Both toxoid preparations were free of demonstrable CT activity (5, 6, 22). Subcutaneous dosages were selected after preliminary studies showed that the amount of glutaraldehyde toxioid or choleragenoid given produced serum RESULTS IgG anti-CT levels similar to those resulting from 30 ug of CT given s.c. in our earlier studies (25). IgA anti-CT in fluids from ILCT animals rose Loop fluids were collected on the day of first i.L after the second i.l. dose of CT and reached high 3 to 4 thereafter and inoculation (day 0) every days until the experiments were ended on day 28. Speci- mean enzyme-linked immunosorbent assay valmens were stored at -20'C. After thawing, mucus and ues after the third dose (Fig. 1). The high levels debris were removed by centrifugation, yielding clear were sustained until the end of the experiment. supernatants which were used for antibody assay. On the other hand, IgA anti-CT in fluids from Blood for serum was collected before first s.c. inocula- SCGT animals was significantly lower than in tion (day -7) and at weekly intervals thereafter. ILCT animals in the latter of the experiEnzyme-linked immunosorbent assay for an- ment (P < 0.05 on day 17 andpart onward). Further, tibodies to CT. As described in detail previously (25), IgA and IgG antibodies to cholera toxin were assayed the levels were not well maintained and fell in fluids and sera by incubation of diluted specimens during the last week of the experiment. In conwith CT-coated polystyrene tubes followed by alkaline trast to the other two groups, IgA anti-CT in phosphatase-conjugated goat anti-rabbit alpha chain fluids from SCCD animals was slightly elevated or gamma chain and then nitrophenyl phosphate sub- prior to the first ii. dose of CT. However, similar strate. Relative quantities of IgA or IgG antitoxin were to the SCGT group, fluid IgA anti-CT in SCCD expressed as absorbance at 400 nm of the chromagen animals rose slowly and was poorly sustained, resulting from the enzyme activity on the colorless and the values were significantly less than in substrate. The anti-alpha chain and anti-gamma chain ILCT animals on days 17, 24, and 28 (P < 0.05). Serum IgA anti-CT was low in all three groups TABLE 1. Immunization schedules and antigens at all times (Fig. 1); the greatest rise occurred in i.l. inoculations s.c. inoculations the ILCT group. Expt group (Days 0, 7, 14) (Days -7 and 14) In contrast to fluid IgA anti-CT, fluid IgG anti-CT remained low in all three groups Cholera toxin, 100 None ILCT (6) throughout the experiment (Fig. 2). The animals 'Ag SCGT (6) Cholera toxin, 100 Glutaraldehyde in the SCGT group showed the greatest rise, toxoid, 400 ,ug 1Ag which occurred during the last 2 weeks of the SCCD (5) Cholera toxin, 100 Choleragenoid, 30 experiment. Serum IgG anti-CT in ILCT aniAg lAg mals rose after day 14 (Fig. 2), but the values for the s.c. immunized groups rose earlier and beaNumber of animals in parentheses.
424
INFECT. IMMUN.
HAMILTON, YARDLEY, AND BROWN
SERA( :40)
ELISA MEAN
ABSORBANCE 400nm 1.0
-7
0
14
7
DAYS
DAYS
A IL INOC. SC INOC. A
A
A
A .
A A
A
A
FIG. 1. IgA anti-CT in loop fluids and sera. ELISA, Enzyme-linked immunosorbent assay.
LOOP FLUIDS(1:40) m-- IL TOXIN 0- -o IL TOXIN +SC CHOLERAGENOID -@ IL TOXIN 4 SC
GLUTAR. TOXOID
ELISA MEAN ABSORBANCE 400nm
A IL INOC. SC INOC. A
DAYS
DAYS A A
A
A
A
A
AL
FIG. 2. IgG anti-CT in loop fluids and sera. ELISA, Enzyme-linked immunosorbent assay. came higher; serum IgG anti-CT in SCGT and SCCD animals was significantly greater than in ILCT animals on day 7 (P < 0.05) and day 14 (P 0.01), respectively, and onward to the end of the experiment. In addition, serum IgG antiCT in SCGT animals was greater than in SCCD animals on day 14 (P < 0.01). DISCUSSION Suppression by CT of immune responses to alloantigens appears to be the result of the biological activity of CT, i.e., is toxigenic, because heat inactivation (3, 17), administration of antitoxin (13), and administration of choleragenoid
INFECTION AND IMMUNITY, May 1979, p. 422426 0019-9567/79/05-0422/05 02.00/0
Suppression of Local Intestinal Immunoglobulin A Immune Response to Cholera Toxin by Subcutaneous Administration of Cholera Toxoids STANLEY R. HAMILTON,* JOHN H. YARDLEY, AND GERTRUDE D. BROWN Department of Pathology, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205 Received for publication 12 February 1979
Cholera toxin has been shown to modulate immune responses, generally producing enhancement when administered simultaneously with antigen and suppression when administered a day or more earlier. In a previous study using chronically isolated ileal loops in rabbits, we found that two subcutaneous (s.c.) "priming" and "boosting" doses of biologically active cholera toxin suppressed the local intestinal immunoglobulin A response to intraloop doses of cholera toxin. In the study reported here, two different biologically inactive but antigenically intact cholera toxoids, glutaraldehyde toxoid and choleragenoid, were administered s.c. by the same immunization schedule as for toxin in the earlier experiment. Suppression of local immune response to intraloop cholera toxin as compared with animals receiving no s.c. inoculations was again found. The results suggest that in this model suppression was immunological (mediated by an immunological mechanism) rather than toxigenic (mediated by biological activity of cholera toxin). In addition, the occurrence of suppression of local intestinal immune response after systemic immunization suggests that suboptimal protection against enteric infections could occur after s.c. vaccination.
The modulation by cholera toxin (CT) of primary and secondary humoral and cellular immune responses to a variety of alloantigens has been studied in vitro and in vivo (2, 3, 8, 10, 11, 13-15, 17, 24). Administration of CT simultaneously with, or up to 24 h before, administration of antigen has been found to enhance antibody response to the antigen (2, 3, 11, 14, 15, 17). However, suppression of antibody response occurred when CT was given earlier, i.e., 5 days to 12 h before the antigen (2, 3, 8, 10, 11, 14, 15). We have studied local intestinal and serum immune responses to CT using chronically isolated Thiry-Vella leal loops in rabbits (12, 25). We found that three or four weekly intraloop (i.l.) inoculations of CT resulted in the development of immunoglobulin A (IgA) antitoxin antibodies in loop secretions. The presence of these antibodies correlated with the CT neutralization titers of the secretions and was associated with protection of the loops against the diarrheagenic effect of CT in challenge experiments. However, when subcutaneous (s.c.) "priming" and "boosting" doses of CT were given 3 weeks apart in addition to i.L doses, lower levels of fluid IgA anti-CT and less protection against challenge occurred than with i.L inoculation alone. This suppression of local IgA immune response by s.c.
administration of CT occurred when the first s.c. dose of CT was given either 7 days before or 21 days before the first i. dose (25). Mechanisms which have been proposed for CT suppression of immune responses include: (i) direct effect on lymphoid cells via stimulation of adenyl cyclase (1-3, 13, 15); (ii) lymphoid tissue depletion due to direct cytotoxicity (14, 15, 24); (iii) lymphoid tissue depletion due to lympholytic effect of increased plasma adrenocorticoids stimulated by CT (14-16); (iv) dysfunction of helper T cells (11, 15); and (v) stimulation of suppressor T cells (25). To obtain further evidence regarding these hypotheses, we repeated our study using two different biologically inactive but antigenically intact cholera toxoids (glutaraldehyde toxoid and choleragenoid) instead of CT for s.c. priming and boosting inoculations. (This paper was presented at the meeting of the American Gastroenterological Association, Las Vegas, Nevada, 22 May 1978.) MATERIALS AND METHODS Preparation and care of loops. The procedures for preparation and care of loops were carried out as previously described in detail (12, 25). Stated briefly, an isolated 20-cm loop of distal ileum containing a
422
VOL. 24, 1979
TOXOID SUPPRESSION OF IgA ANTI-CHOLERA TOXIN
Peyer's patch was prepared in outbred New Zealand white rabbits of mixed sex weighing 3 to 4 kg. Silastic tubing (Dow Chemical Corp., Midland, Mich.) was ligated into the ends of the loop and brought out through the skin of the posterior neck. Accumulated loop secretions were collected, and the loop was flushed daily with saline through the tubing.
423
enzyme conjugates were highly specific for their respective isotypes and detected 30 ng or less of the appropriate immunoglobulin per ml. Specimens from animals in the ILCT, SCGT, and SCCD groups were tested in each run. Control samples of immune fluid and sera were included in each run, and their absorbance values were adjusted to an arbitrary absorbance of 2.00 by a correction factor. This factor was then applied to the specimen values to compensate for day-to-day variations in the test system. As described previously (25), in preliminary studies relative absorbances of specimens at any given dilution generally showed the same relationships as the titers of the specimens, i.e., the specimens with the highest titers usually had the highest absorbances at any given dilution. On the basis of this relationship, the lowest specimen dilution in which the enzymelinked immunosorbent assay system was generally not exhausted of substrate for each type of specimen and each isotype was chosen as the test dilution. Thus, IgA anti-CT in fluids and sera and IgG anti-CT in fluids were assayed at 1:40 whereas IgG anti-CT in sera was assayed at 1:1,600. The absorbance values of the IgA test system could not be compared directly with those for IgG. However, the time course of the IgA anti-CT and IgG anti-CT response could be followed in both sera and fluids. Within-day coefficients of variation for the test systems, as calculated from standard deviations of the differences of control specimens, were about 8%.
Immunization of animals and specimen preparation. Immunization schedules and antigens are summarized in Table 1. Three weekly i.l. inoculations of 100 ,ug of purified CT (Schwarz-Mann Division of Becton, Dickinson & Co., Orangeburg, N.Y.) in 4 ml of 0.01 M phosphate-buffered saline, pH 7.2, were given to all animals on days 0, 7, and 14. This was the same dose as in our earlier experiment (25). Animals in the ILCT group received only i.l. inoculations of CT. Animals in the SCGT group received 400 1Lg of glutaraldehyde toxoid (22) in protamine sulfate and aluminum chloride adjuvant (lot 20201, Wyeth Laboratories, provided by Division of Biological Standards, National Institutes of Health, Bethesda, Md.) s.c. 1 week before (day -7) and 2 weeks after (day 14) the first i.L inoculation of CT (day 0). Animals in the SCCD group received 30 ,ug of choleragenoid (5, 6) (lot GB76 provided by Richard Finklestein, Southwestern Medical School, Dallas, Tex.) in 1 ml of phosphate-buffered saline s.c. on the same schedule. Both toxoid preparations were free of demonstrable CT activity (5, 6, 22). Subcutaneous dosages were selected after preliminary studies showed that the amount of glutaraldehyde toxioid or choleragenoid given produced serum RESULTS IgG anti-CT levels similar to those resulting from 30 ug of CT given s.c. in our earlier studies (25). IgA anti-CT in fluids from ILCT animals rose Loop fluids were collected on the day of first i.L after the second i.l. dose of CT and reached high 3 to 4 thereafter and inoculation (day 0) every days until the experiments were ended on day 28. Speci- mean enzyme-linked immunosorbent assay valmens were stored at -20'C. After thawing, mucus and ues after the third dose (Fig. 1). The high levels debris were removed by centrifugation, yielding clear were sustained until the end of the experiment. supernatants which were used for antibody assay. On the other hand, IgA anti-CT in fluids from Blood for serum was collected before first s.c. inocula- SCGT animals was significantly lower than in tion (day -7) and at weekly intervals thereafter. ILCT animals in the latter of the experiEnzyme-linked immunosorbent assay for an- ment (P < 0.05 on day 17 andpart onward). Further, tibodies to CT. As described in detail previously (25), IgA and IgG antibodies to cholera toxin were assayed the levels were not well maintained and fell in fluids and sera by incubation of diluted specimens during the last week of the experiment. In conwith CT-coated polystyrene tubes followed by alkaline trast to the other two groups, IgA anti-CT in phosphatase-conjugated goat anti-rabbit alpha chain fluids from SCCD animals was slightly elevated or gamma chain and then nitrophenyl phosphate sub- prior to the first ii. dose of CT. However, similar strate. Relative quantities of IgA or IgG antitoxin were to the SCGT group, fluid IgA anti-CT in SCCD expressed as absorbance at 400 nm of the chromagen animals rose slowly and was poorly sustained, resulting from the enzyme activity on the colorless and the values were significantly less than in substrate. The anti-alpha chain and anti-gamma chain ILCT animals on days 17, 24, and 28 (P < 0.05). Serum IgA anti-CT was low in all three groups TABLE 1. Immunization schedules and antigens at all times (Fig. 1); the greatest rise occurred in i.l. inoculations s.c. inoculations the ILCT group. Expt group (Days 0, 7, 14) (Days -7 and 14) In contrast to fluid IgA anti-CT, fluid IgG anti-CT remained low in all three groups Cholera toxin, 100 None ILCT (6) throughout the experiment (Fig. 2). The animals 'Ag SCGT (6) Cholera toxin, 100 Glutaraldehyde in the SCGT group showed the greatest rise, toxoid, 400 ,ug 1Ag which occurred during the last 2 weeks of the SCCD (5) Cholera toxin, 100 Choleragenoid, 30 experiment. Serum IgG anti-CT in ILCT aniAg lAg mals rose after day 14 (Fig. 2), but the values for the s.c. immunized groups rose earlier and beaNumber of animals in parentheses.
424
INFECT. IMMUN.
HAMILTON, YARDLEY, AND BROWN
SERA( :40)
ELISA MEAN
ABSORBANCE 400nm 1.0
-7
0
14
7
DAYS
DAYS
A IL INOC. SC INOC. A
A
A
A .
A A
A
A
FIG. 1. IgA anti-CT in loop fluids and sera. ELISA, Enzyme-linked immunosorbent assay.
LOOP FLUIDS(1:40) m-- IL TOXIN 0- -o IL TOXIN +SC CHOLERAGENOID -@ IL TOXIN 4 SC
GLUTAR. TOXOID
ELISA MEAN ABSORBANCE 400nm
A IL INOC. SC INOC. A
DAYS
DAYS A A
A
A
A
A
AL
FIG. 2. IgG anti-CT in loop fluids and sera. ELISA, Enzyme-linked immunosorbent assay. came higher; serum IgG anti-CT in SCGT and SCCD animals was significantly greater than in ILCT animals on day 7 (P < 0.05) and day 14 (P 0.01), respectively, and onward to the end of the experiment. In addition, serum IgG antiCT in SCGT animals was greater than in SCCD animals on day 14 (P < 0.01). DISCUSSION Suppression by CT of immune responses to alloantigens appears to be the result of the biological activity of CT, i.e., is toxigenic, because heat inactivation (3, 17), administration of antitoxin (13), and administration of choleragenoid
