Vol. 4, No. 4 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1976, p. 384-387 Copyright © 1976 American Society for Microbiology
Production of Antibody in Induced Granulomas K. L. WOLFF,* B. W. HUDSON, R. A. ORMSBEE,
AND
M. G. PEACOCK
Immunochemistry Branch, Vector-Borne Diseases Division, Center for Disease Control, Fort Collins, Colorado 80522,* and National Institute ofAllergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840
Received for publication 24 June 1976
A method of producing antibodies in artificially induced granulomas with various microbial antigens is examined.
The artificially induced granulomas developed by Garra and Baygorria have been used to produce antibodies to arboviruses (8), thyroid hormone (12), and coxsackievirus Al (10). This method has also been used for the in vivo cultivation of Neisseria gonorrheae (1). In addition, the granuloma fluid has been chemically analyzed (9). Our laboratory has used a similar technique as a method of antibody production. The purpose of this note is to present some of our findings pertaining to the efficacy of this method for the production of antibody to a variety of microbial antigens. Artificial granulomas were induced in 3month-old New Zealand white rabbits by subdermal surgical implantation of perforated plastic golf balls 4.25 cm in diameter (Target brand, Target Department Stores, Inc.). After the rabbits were anesthetized with pentobarbital sodium (Nembutal; Abbott Laboratories), a middorsal incision was made and the balls were inserted bilaterally on the flanks of the animals. Rabbits were held for 6 weeks after surgery to allow complete healing. Vascularization and granulation after surgical insertion of the balls formed a saclike granuloma in which 25 ml of fluid collected. The procedures used to induce antibody formation varied. In the first series of experiments, 5.0 mg of Yersinia pestis soluble protein fraction IA (FIA) (2) was injected into each of the two artificially induced granulomas, and the blood serum and granuloma fluids were collected periodically for serological analysis. A second challenge (0.5 mg of FIA) was administered 160 days after the first. Smears of the fluid were examined to determine the types of cells migrating into the granuloma during the initial phase (1 to 2 weeks) of the immune response. By packed cell volume and cell count techniques (3), the fluid was found to be composed of 2 to 4% cellular materials that consisted of 70% lymphocytes, 20 to 25% monocytes, and 5 to 10% epithelial cells and erythrocytes.
Antibodies in granuloma fluids were characterized by immunodiffusion (11) against the homologous antigen as well as against an extract of acetone-dried Y. pestis and extracts of other related bacteria (i.e., Y. pseudotuberculosis, Y. enterocolitica, and Pasteurella multocida). Single lines of identity formed between the fluid and FIA. Since the bacterial extracts contained complexes of soluble antigens, the formation of a single precipitin band with the plague extract was used as evidence for FIA specificity. Antibody titers to FIA were measured by the passive hemagglutination test (PHA) as recommended by the World Health Organization (13). The results are shown in Fig. 1. Normally, intravenous injection of 5.0 mg of Y. pestis FIA produced rabbit anbibody titers of 1:512; biweekly injections of 5.0 mg each for 6 weeks were required to produce titers of the magnitude shown for the granuloma fluids in Fig. 1. Injection of Y. pestis FIA into the granulomas induced a prolonged antibody response lasting 120 days. Antibody levels were greater in the fluid from inoculated granulomas than in the serum. Note that although the average PHA titer in the granuloma fluids of the nine test animals was 1:64,000 (Fig. 1), the maximum titer in any one animal was >1:100,000 (Table 1). In contrast to the serum antibody, the time of appearance of antibody in the granuloma fluid was delayed. After a second challenge with FIA, antibody levels showed a marked depression, with a return to prechallenge levels taking over 3 months. The initial depression may have been caused by the presence of antigen complexing with antibody as it was formed. Drainage of the granulomas at day 180 should have removed the major portion of any excess antigen present; however, the fluid formed by day 210 still showed a reduced antibody titer. Additionally, it should be pointed out that although the fluid collected at day 180 had a reduced antibody titer, it still contained sufficient free antibody for a PHA titer of .1:2,000.
384
NOTES
VOL. 4, 1976 o-o
Antibody Titer in Combined Gronuloma Fluids
cr w
I.I.-
9
!iZ n
TIME (DAYS)
--
FIG. 1. Average prolonged immune response in granuloma fluid and blood serum in nine rabbits inoculated with Y. pestis FIA.
In standard PHA tests, purified FIA is used as a specific inhibition control; the concentration required to inhibit serums with PHA titers of 1:1,000 to 1:2,000 is 50 ,ug of FIA per 25 ,lI of serum. Assuming that the granuloma yields 25 ml of fluid, an estimated 50+ mg of FIA would be required to account for the observed depression in titer by antigen-antibody complexes. This amount is at least 100 times greater than the 0.5 mg of FIA actually administered. It should also be noted that there is no evidence of the anamnestic response characteristic of standard intravenous challenge. In a second series of experiments, various Y. pseudoturberculosis and Y. enterocolitica antigens were used. The preparation of whole bacteria, bacterial lipopolysaccharide (LPS), and somatic 0 antigens as well as the determination of hemagglutination (HA) antibody titers to the type-specific LPS were done with minor modifications of techniques described by Currie et al. (6). Intravenous inoculation of rabbits with any of the three antigen types usually resulted in type-specific LPS HA titers of 2 1:2,048. When whole bacteria were inoculated into the granulomas only, minimal immune responses (HA s 1:128) were obtained in the granuloma fluid and no immune response was noted in the blood serum. In a further effort to obtain a significant immune response, sequential inoculations of LPS and 0 antigens were given in the granulomas. Although severe inflammatory reactions that
385
required treatment with hydrocortisone appeared at the inoculation sites in 40% of the rabbits, the same minimal serological responses were noted at the end of 90 days. However, the simultaneous inoculation of any one of the antigens by intravenous, intramuscular, and granuloma routes resulted in high antibody levels in the blood serum; but, again, only minimal levels of antibody were found in the granuloma fluid. These latter results are summarized in Table 1. In a third series of experiments, single 20-,ug doses of particulate rickettsial antigens (R. Ormsbee, M. Peacock, R. Philip, E. Casper, J. Plorde, T. Gabre-Kidan, and L. Wright, submitted for publication) in 0.15 M NaCl were inoculated into only the right granulomas of one group of rabbits; the left granulomas remained uninoculated. Blood serum and fluids from inoculated and uninoculated granulomas were collected and tested separately. Agglutinin titers were determined by the microagglutination (MA) technique of Fiset et al. (7). Rickettsial antigens from members of the spotted fever and typhus groups induced only minimal antibody titers in inoculated granuloma fluids; in contrast, Coxiella burnetii antigens, both phase I and phase II, were effective in causing the production of large amounts of antibody. The preceding results are also summarized in Table 1. Given subcutaneously or intramuscularly, 50-,ug amounts of typhus and spotted fever group antigens and phase II C. burnetii required the presence of incomplete Freund adjuvant (IFA) for the production of moderate antibody levels (transient MA titers of 1:256 to 1:4,096). In contrast, phase I C. burnetii (whole organism) antigen appeared to contain its own adjuvant. MA titers of 1:2,048 were reached and maintained for 20 to 30 days whether or not IFA was used. The two rabbits that received single doses (20 ,ug) of phase I C. burnetii via the granuloma route responded with extremely high phase II titers and moderate phase I titers (Table 2). This is similar to the typical response pattern in guinea pigs, except for the magnitude of the phase II titers which are truly impressive. It was also interesting to note the lack of antibody response in both the blood serum and the uninoculated granuloma fluid, especially when compared with the extremely high titers in the inoculated granuloma fluid. A second group of rabbits was inoculated intramuscularly (not into the granulomas) with 400 ug of the rickettsial antigens. These animals responded with predictable, but not remarkable, serum antibody titers (l1:2,048),
386
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Summary of maximum antibody responses in granuloma fluids and blood serum after one inoculation with various microbial antigens Antigen ~~~ Antigen type
Inoculation routea
Total amt inoculated
No. of rabbits inoculated
Serological
ployed
Maximum antibody responseb __________ Granuloma Blood serum
5.0 mg
9
PHA
>1:100,000
test em-
1:8,192
Y. pestis soluble protein fraction IA
IB
Y. pseudotuberculosis serotype IA Whole bacteria LPS Somatic 0
Simultaneous
IV, IM, IB IV, IM, IB IV, IM, IB
10.0 mg 5.0 mg 5.0 mg
1 1 1
HA HA HA
1:256 1:64 1:128
1:4,096 1:1,024 1:4,096
Y. enterocolitica serotype 8 Whole bacteria LPS Somatic 0
IV, IM, IB IV, IM, IB IV, IM, IB
10.0 mg 5.0 mg 5.0 mg
1 1 1
HA HA HA
1:128 1:128 1:128
1:1,024 1:2,048 1:8,192
Rickettsia akari R. conorii R. prowazekii R. rickettsii R. typhi C. burnetii, phase I C. burnetii, phase fl
IB IB IB IB IB IB IB
ug ug ug ug ,ug ug ,ug
2 2 2 2 2 2 2
MA MA MA MA MA MA MA
1:4 1:4 1:32 1:4 1:16 >1:1,000,000 1:500,000
Dengue virus 2 St. Louis encephalitis virus West Nile virus
IB (right only) IB (right only) IB (right only)
105 LD5o 105 LD50 105 LD50
1
HI HI HI
1:1,280
(right only) (right only) (right only) (right only) (right only) (right only) (right only)
20 20 20 20 20 20 20
1 1
1:32 1:8 1:64 1:16 1:64 1:64 1:16
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1976, p. 384-387 Copyright © 1976 American Society for Microbiology
Production of Antibody in Induced Granulomas K. L. WOLFF,* B. W. HUDSON, R. A. ORMSBEE,
AND
M. G. PEACOCK
Immunochemistry Branch, Vector-Borne Diseases Division, Center for Disease Control, Fort Collins, Colorado 80522,* and National Institute ofAllergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840
Received for publication 24 June 1976
A method of producing antibodies in artificially induced granulomas with various microbial antigens is examined.
The artificially induced granulomas developed by Garra and Baygorria have been used to produce antibodies to arboviruses (8), thyroid hormone (12), and coxsackievirus Al (10). This method has also been used for the in vivo cultivation of Neisseria gonorrheae (1). In addition, the granuloma fluid has been chemically analyzed (9). Our laboratory has used a similar technique as a method of antibody production. The purpose of this note is to present some of our findings pertaining to the efficacy of this method for the production of antibody to a variety of microbial antigens. Artificial granulomas were induced in 3month-old New Zealand white rabbits by subdermal surgical implantation of perforated plastic golf balls 4.25 cm in diameter (Target brand, Target Department Stores, Inc.). After the rabbits were anesthetized with pentobarbital sodium (Nembutal; Abbott Laboratories), a middorsal incision was made and the balls were inserted bilaterally on the flanks of the animals. Rabbits were held for 6 weeks after surgery to allow complete healing. Vascularization and granulation after surgical insertion of the balls formed a saclike granuloma in which 25 ml of fluid collected. The procedures used to induce antibody formation varied. In the first series of experiments, 5.0 mg of Yersinia pestis soluble protein fraction IA (FIA) (2) was injected into each of the two artificially induced granulomas, and the blood serum and granuloma fluids were collected periodically for serological analysis. A second challenge (0.5 mg of FIA) was administered 160 days after the first. Smears of the fluid were examined to determine the types of cells migrating into the granuloma during the initial phase (1 to 2 weeks) of the immune response. By packed cell volume and cell count techniques (3), the fluid was found to be composed of 2 to 4% cellular materials that consisted of 70% lymphocytes, 20 to 25% monocytes, and 5 to 10% epithelial cells and erythrocytes.
Antibodies in granuloma fluids were characterized by immunodiffusion (11) against the homologous antigen as well as against an extract of acetone-dried Y. pestis and extracts of other related bacteria (i.e., Y. pseudotuberculosis, Y. enterocolitica, and Pasteurella multocida). Single lines of identity formed between the fluid and FIA. Since the bacterial extracts contained complexes of soluble antigens, the formation of a single precipitin band with the plague extract was used as evidence for FIA specificity. Antibody titers to FIA were measured by the passive hemagglutination test (PHA) as recommended by the World Health Organization (13). The results are shown in Fig. 1. Normally, intravenous injection of 5.0 mg of Y. pestis FIA produced rabbit anbibody titers of 1:512; biweekly injections of 5.0 mg each for 6 weeks were required to produce titers of the magnitude shown for the granuloma fluids in Fig. 1. Injection of Y. pestis FIA into the granulomas induced a prolonged antibody response lasting 120 days. Antibody levels were greater in the fluid from inoculated granulomas than in the serum. Note that although the average PHA titer in the granuloma fluids of the nine test animals was 1:64,000 (Fig. 1), the maximum titer in any one animal was >1:100,000 (Table 1). In contrast to the serum antibody, the time of appearance of antibody in the granuloma fluid was delayed. After a second challenge with FIA, antibody levels showed a marked depression, with a return to prechallenge levels taking over 3 months. The initial depression may have been caused by the presence of antigen complexing with antibody as it was formed. Drainage of the granulomas at day 180 should have removed the major portion of any excess antigen present; however, the fluid formed by day 210 still showed a reduced antibody titer. Additionally, it should be pointed out that although the fluid collected at day 180 had a reduced antibody titer, it still contained sufficient free antibody for a PHA titer of .1:2,000.
384
NOTES
VOL. 4, 1976 o-o
Antibody Titer in Combined Gronuloma Fluids
cr w
I.I.-
9
!iZ n
TIME (DAYS)
--
FIG. 1. Average prolonged immune response in granuloma fluid and blood serum in nine rabbits inoculated with Y. pestis FIA.
In standard PHA tests, purified FIA is used as a specific inhibition control; the concentration required to inhibit serums with PHA titers of 1:1,000 to 1:2,000 is 50 ,ug of FIA per 25 ,lI of serum. Assuming that the granuloma yields 25 ml of fluid, an estimated 50+ mg of FIA would be required to account for the observed depression in titer by antigen-antibody complexes. This amount is at least 100 times greater than the 0.5 mg of FIA actually administered. It should also be noted that there is no evidence of the anamnestic response characteristic of standard intravenous challenge. In a second series of experiments, various Y. pseudoturberculosis and Y. enterocolitica antigens were used. The preparation of whole bacteria, bacterial lipopolysaccharide (LPS), and somatic 0 antigens as well as the determination of hemagglutination (HA) antibody titers to the type-specific LPS were done with minor modifications of techniques described by Currie et al. (6). Intravenous inoculation of rabbits with any of the three antigen types usually resulted in type-specific LPS HA titers of 2 1:2,048. When whole bacteria were inoculated into the granulomas only, minimal immune responses (HA s 1:128) were obtained in the granuloma fluid and no immune response was noted in the blood serum. In a further effort to obtain a significant immune response, sequential inoculations of LPS and 0 antigens were given in the granulomas. Although severe inflammatory reactions that
385
required treatment with hydrocortisone appeared at the inoculation sites in 40% of the rabbits, the same minimal serological responses were noted at the end of 90 days. However, the simultaneous inoculation of any one of the antigens by intravenous, intramuscular, and granuloma routes resulted in high antibody levels in the blood serum; but, again, only minimal levels of antibody were found in the granuloma fluid. These latter results are summarized in Table 1. In a third series of experiments, single 20-,ug doses of particulate rickettsial antigens (R. Ormsbee, M. Peacock, R. Philip, E. Casper, J. Plorde, T. Gabre-Kidan, and L. Wright, submitted for publication) in 0.15 M NaCl were inoculated into only the right granulomas of one group of rabbits; the left granulomas remained uninoculated. Blood serum and fluids from inoculated and uninoculated granulomas were collected and tested separately. Agglutinin titers were determined by the microagglutination (MA) technique of Fiset et al. (7). Rickettsial antigens from members of the spotted fever and typhus groups induced only minimal antibody titers in inoculated granuloma fluids; in contrast, Coxiella burnetii antigens, both phase I and phase II, were effective in causing the production of large amounts of antibody. The preceding results are also summarized in Table 1. Given subcutaneously or intramuscularly, 50-,ug amounts of typhus and spotted fever group antigens and phase II C. burnetii required the presence of incomplete Freund adjuvant (IFA) for the production of moderate antibody levels (transient MA titers of 1:256 to 1:4,096). In contrast, phase I C. burnetii (whole organism) antigen appeared to contain its own adjuvant. MA titers of 1:2,048 were reached and maintained for 20 to 30 days whether or not IFA was used. The two rabbits that received single doses (20 ,ug) of phase I C. burnetii via the granuloma route responded with extremely high phase II titers and moderate phase I titers (Table 2). This is similar to the typical response pattern in guinea pigs, except for the magnitude of the phase II titers which are truly impressive. It was also interesting to note the lack of antibody response in both the blood serum and the uninoculated granuloma fluid, especially when compared with the extremely high titers in the inoculated granuloma fluid. A second group of rabbits was inoculated intramuscularly (not into the granulomas) with 400 ug of the rickettsial antigens. These animals responded with predictable, but not remarkable, serum antibody titers (l1:2,048),
386
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Summary of maximum antibody responses in granuloma fluids and blood serum after one inoculation with various microbial antigens Antigen ~~~ Antigen type
Inoculation routea
Total amt inoculated
No. of rabbits inoculated
Serological
ployed
Maximum antibody responseb __________ Granuloma Blood serum
5.0 mg
9
PHA
>1:100,000
test em-
1:8,192
Y. pestis soluble protein fraction IA
IB
Y. pseudotuberculosis serotype IA Whole bacteria LPS Somatic 0
Simultaneous
IV, IM, IB IV, IM, IB IV, IM, IB
10.0 mg 5.0 mg 5.0 mg
1 1 1
HA HA HA
1:256 1:64 1:128
1:4,096 1:1,024 1:4,096
Y. enterocolitica serotype 8 Whole bacteria LPS Somatic 0
IV, IM, IB IV, IM, IB IV, IM, IB
10.0 mg 5.0 mg 5.0 mg
1 1 1
HA HA HA
1:128 1:128 1:128
1:1,024 1:2,048 1:8,192
Rickettsia akari R. conorii R. prowazekii R. rickettsii R. typhi C. burnetii, phase I C. burnetii, phase fl
IB IB IB IB IB IB IB
ug ug ug ug ,ug ug ,ug
2 2 2 2 2 2 2
MA MA MA MA MA MA MA
1:4 1:4 1:32 1:4 1:16 >1:1,000,000 1:500,000
Dengue virus 2 St. Louis encephalitis virus West Nile virus
IB (right only) IB (right only) IB (right only)
105 LD5o 105 LD50 105 LD50
1
HI HI HI
1:1,280
(right only) (right only) (right only) (right only) (right only) (right only) (right only)
20 20 20 20 20 20 20
1 1
1:32 1:8 1:64 1:16 1:64 1:64 1:16
