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The use of cholera toxin for obtaining rabbit secretory immunoglobulins
Journal of Immunological Methods 4 (1974) 207--212. © North-Holland Publishing Company
THE USE OF CHOLERA TOXIN FOR OBTAINING RABBIT SECRETORY IMMUNOGLOBULINS* Scott WOOD** and L. William CLEM*** Department of Immunology and Medieal Microbiology, College of Medicine, University of Florida, Gainesville, Florida 32610, U.S.A.
Received 18 June 1973
Accepted 29 September 1973
The work described here involved the use of cholera toxin to stimulate segments of the rabbit small intestine in vivo to obtain fluids for immunological study. These fluids were found to contain considerable 11 S IgA and by utilizing the entire small intestine it should be possible to obtain about 300 mg of this protein from a single rabbit.
1. INTRODUCTION Since the discovery of secretory lgA (Chodirker and Tomasi, Jr., 1963) and the demonstration that it is synthesized locally on secretory surfaces (Tomasi et al., 1965), much work has been directed toward the study of this protein. Secretory IgA is the predominant immunoglobulin in the secretions of mucous membranes, but constitutes a relatively small portion of the serum immunoglobulins. Heretofore, colostrum has served to be the most abundant source of secretory IgA. Several different methods for the purification of colostral IgA have been described, and in general the yields are 3 . 0 4 . 5 mg per ml clarified colostrum or 60 90 mg per rabbit (Hanson and Johannson, 1962; Axelsson et al., 1966; Cebra and Robbins, 1966; Sell, 1967). In the intestinal mucosa of the rabbit, it has been shown that c~-chain containing cells comprise 80 90% of the immunoglobulin-containing cells (Crandall et al., 1967). The rabbit intestinal mucosa, therefore, may provide a source for obtaining large quantities of secretory immunoglobulin. The study reported here describes the use of cholera toxin into the rabbit ileal loop as a non-
* This investigation was supported by NSF Grant GB 29095 and NIH Grant 5 TI AI 0128. ** This work represented as portion of a thesis submitted by Scott Wood to the Graduate School of the University of Florida in partial fulfillment of the requirements for the Master of Science Degree. *** To whom correspondence should be addressed. 207
208
S. WOODand L.W.CLEM
specific method of obtaining rabbit intestinal immunoglobulins in relatively large amounts.
2. MATERIALS AND METHODS 2.1. Cholera toxin A commercial freeze-dried (Merck) preparation of a crude culture •trate of the 569B strain of Vibrio cholerae was provided by the National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Md. 2.2. A nimals Eleven New Zealand white male rabbits were used for the studies. The animals were young adults, and weighed 1.2-2.0 kg. Each animal was starved for 48 hr prior to the operation, but water was supplied ad libitum throughout the study. 2.3. Ileal loops The operative technique was similar to that used by other investigators (Burrows and Musteikis, 1966; Carpenter et al., 1968) under 'Metofane' anesthesia, and the small bowel was brought out of the abdominal cavity and ligated approximately 15 cm above the ileo-caecal junction. Three segments each about 20 cm long and approximating distal, medial, and proximal segments of the ileum were injected with 0.1-0.3 g lyophilized crude toxin in 1 ml distilled water. Formation of the loop was made by ligation distal to the site of inoculation. The canine model of experimental cholera (Carpenter et al., 1968) has shown that maximum secretion of intestinal fluid into the loops is reached and maintained 9 hr after injection of the toxin. Based on this model the rabbits were :exanguinatedl and intestinal fluids were collected by syringe 9 - 1 2 hr after the operations. Ileal loops of similar lengths in two control rabbits were washed with 15 ml of 0.86% sodium chloride and specimens were immediately collected. 2.4. Specimens Ileal fluids were tested for occult blood using guaiac tablets (Cambridge Chemical Products, Detroit, Michigan). Since a weak 1+ reading was found to correspond roughly to a 1:6000 dilution of whole peripheral blood, specimens having a higher reading were not used. Fluid volumes were measured after clarification by centrifugation at 2500 rev/min for 10 rain in a Model V International Centrifuge. Sodium azide and soybean trypsin inhibitor were each added to a final concentration of 0.01%. The samples were then frozen at 20°C.
Cholera induced secretory immunoglobulins
209
2.5. Immunodiffusion Double diffusion in agar was performed according to Ouchterlony (1962). IgA and IgG immunoglobulin levels were quantified by single radial immunodiffusion (SRID) (Fahey and McKelvey, 1965). The IgA standard was 11 S IgA prepared by passage of clarified rabbit colostrum through a Sephadex G-200 column (Cebra and Robbins, 1966). The unabsorbed peak of rabbit whole serum off a DEAE-cellulose column was used as the source of IgG standard. Sheep anti-rabbit IgA, IgG, and IgM were absorbed with 0.3 mg rabbit F(ab')2/ml of antiserum. The resulting antisera were specific for s-chain, 7-chain, and ~t-chain as determined by double diffusion in agar*. 2.6. Gel filtration Twenty ml of intestinal fluid was mixed with an equal volume of saturated (NH4)2SO 4 and incubated overnight in the cold. The resulting precipitate was pelleted by centrifugation and washed twice with cold 50% saturated (NH4)2SO 4. The precipitate was then dissolved in 5 ml of buffer (0.15 M NaC1, 0.015 M Tris HC1, pH 7.4) and gel filtered on a column of Sephadex G-200 (Pharmacia, Uppsala, Sweden) equilibrated with the same buffer. Protein elution was monitored by reading absorbance of 5 ml fractions at 280 nm. Appropriate fractions were pooled and concentrated to 3 ml by pressure dialysis in the cold. The fractions were then analyzed for immunoglobulins by double diffusion in agar and SRID. 2.7. A nalytical ultracentrifugation Sedimentation velocity studies were performed in the 'Spinco' model E analytical ultracentrifuge, employing the Schlieren optical system at 20°C. The buffer was 0.14 M NaC1, 0.015 M Tris-HC1, pH 7.4.
3. RESULTS 3.1. Immunoglobulin con tent o f intestinal secretions The average concentrations of IgA and IgG in ileal loop fluids are represented in table 1. Statistical analysis showed no significant difference in immunoglobulin concentrations among the fluids obtained from proximal, medial or distal loops. * Sheep anti-rabbit lgA was a gift from M. Schulkind and was prepared as described by Cebra and Robbins (1966). Sheep anti-rabbit lgM was a gift from P.A. Small and was the same antiserum used previously (Small and Lamm, 1966). Anti-rabbit IgG was obtained from sheep two weeks after the last of two-weekly injections of 5 mg alum-precipitated rabbit IgG.
S. WOOD and L.W. CLEM
210
Table 1 Average concentrations of IgA and IgG in ileal loop fluids. Concentrations of immunoglobulins (mg/ml + SD) Ig
Proximal loop
Medial loop
Distal loop
Saline wash
IgA
0.76 (-+ 0.26)
0.68 (-+ 0.13)
0.56 (-+ 0.14)
0.40 (+- 0.15)
IgG
0.39 (-+ 0.02)
0.40 (-+ 0.04)
0.43 (-+ 0.04)
0.34 (± 0.02)
IgA/IgG
1.9
1.7
1.3
1.2
Av. vol.
40.6 (+- 10.8)
42.8 (+-12.0)
37.8 (_+6.7)
14.5 (-+ 0.2)
The range of values was great, having a total loop average c o n c e n t r a t i o n for IgA of 0.67 (-+ 0.09) mg/ml. The average ratio of IgA to IgG was therefore approximately 1.7. This ratio is comparable to values reported in the literature for secretory i m m u n o g l o b u l i n s (Tomasi and Bienenstock, 1968). IgM levels in the u n d i l u t e d fluids were not detected at concentrations representing greater than a 1:32 dilution of normal rabbit serum, i.e., less than 0.06 mg/ml assuming normal rabbit serum to have an upper limit of 2 mg/ml IgM. 3.2. The nature of lgA in ileal loop fluids Gel filtration chromatography on Sephadex G-200 of 20 ml of ileal fluid (contain-
0.50-
anti_.~
anti.'~
TF
I
7I
0
0
0
lifo
T]
0.40-
0 antt_~
TwO
X 0
0 anti_i
°
A2BOnm 050-
0.20
-
O.IO-
000o
,b
2b
3b
FRACTION
4%
s'o
6'0
7b
8'0
NUMBER (5 ml)
Fig. 1. Sephadex G-200 gel filtration of 20 ml ileal loop fluid precipitated at 50% saturated (NH4)2SO4. Insert is double diffusion in agar of pooled fractions I, II, III, and IV,
Cholera induced secretory immunoglobulins
211
ing approx. 5.8 mg lgA) precipitated at 50% saturation of (NH4)2SO 4 is shown in fig. 1. Double diffusion in agar of the concentrated fractions showed that IgA is present only in the excluded peak (fraction I). IgG determinants appeared to be found in all fractions except I|, and that present in fraction I, presumably representing aggregated material, was estimated by gel diffusion to account for less than 15% of the protein, By SRID, the amount of IgA in fraction I was found to be 2.3 mg, a 41% recovery. Analytical ultracentrifugation of fraction 1 (at a concentration of approximately 2 mg/ml) revealed a single boundary having a s20,w = 11.2 S.
4. DISCUSSION The results of this study demonstrate the usefulness of cholera toxin for obtaining large amounts of IgA from the rabbit intestinal mucosa. After precipitation of the ileal loop fluid at 50% saturated (NH4)2SO4, the IgA obtained was shown to be excluded from Sephadex G-200 and to have an S value of ~11 S. The yield was approximately 41% and only slight contamination with IgG was observed. Cholera toxin stimulation of ileal loops may be extended to include the entire small intestine of the rabbit. Allowing as many as ten loops per rabbit to be employed for stimulation, (individual loops limit possible blood contamination to individual samples), one should readily be able to obtain quantities of secretory IgA approaching 0.34 g from a single rabbit. Hyperimmunization of the rabbit intestine followed by the method of immunoglobulin collection described here, could provide large quantities of secretory IgA antibody. Unfortunately, a preliminary study (to be reported elsewhere) has indicated that oral immunization of rabbits with dinitrophenylated streptococcal cells followed by ileal loop studies resulted in anti-dinitrophenyl antibodies almost exclusively of the IgG class. Further studies are obviously required to test the validity of the hypothesis that the method described here would be useful in collecting large amounts of IgA antibodies to a given antigenic determinant. In conclusion it should be mentioned that the studies reported here, when compared to other studies on the immunoglobulin levels in intestinal secretions from cholera patients (Waldman et al., t971), tend to substantiate the usefulness of the ileal loop as a valid model for the biological activity of cholera toxin.
REFERENCES Axelsson, H., B.G. Joharmson and L. Rymo, 1966, Acta Chem. Scand. 20, 2339. Burrows, W. and G.M. Musteikis, 1966, J. Infect. Dis. 116,183. Carpenter, C.C.J., R.B. Sack, J.C. Feeley and R.W. Steenberg, 1968, J. Clin. Invest. 47, 1210. Cebra, J.J. and J.B. Robbins, 1966, J. Immunol. 97, 12. Chodirker, W.B. and T.B. Tomasi, Jr., 1963, Science, 142, 1080. CrandaU, R.B., J.J. Cebra and C.A. Crandall, 1967, Immunology, 12,149. Fahey, J.L. and E.M. McKelvey, 1965, J. Immunol. 94, 84.
212
S. WOOD and L.W. CLEM
Hanson, L.A. and B.G. Johannson, 1962, Intern. Arch. Allergy Appl. Immunol. 20, 65. Ouchterlony, O., 1962, Progr. Allergy 6, 30. Sell, S., 1967, Immunochemistry 4, 49. Small, P.A., Jr. and M.E. Lamm, 1966, Biochemistry 5,259. Tomasi, T.B., Jr. and J. Bienenstock, 1968, Advan. Immunol. 9, 1. Tomasi, T.B., Jr., E.M. Tan, A. Soloman and R.A. Predergast, 1965, J. Exptl. Med. 121,101. Waldman, R.H., Z. Bencic, R. Sakazaki, R. Sinha, R. Ganguly, B.C. Deb and S. Mukerjee, 1971 J. Infectious Dis. 123,579.
THE USE OF CHOLERA TOXIN FOR OBTAINING RABBIT SECRETORY IMMUNOGLOBULINS* Scott WOOD** and L. William CLEM*** Department of Immunology and Medieal Microbiology, College of Medicine, University of Florida, Gainesville, Florida 32610, U.S.A.
Received 18 June 1973
Accepted 29 September 1973
The work described here involved the use of cholera toxin to stimulate segments of the rabbit small intestine in vivo to obtain fluids for immunological study. These fluids were found to contain considerable 11 S IgA and by utilizing the entire small intestine it should be possible to obtain about 300 mg of this protein from a single rabbit.
1. INTRODUCTION Since the discovery of secretory lgA (Chodirker and Tomasi, Jr., 1963) and the demonstration that it is synthesized locally on secretory surfaces (Tomasi et al., 1965), much work has been directed toward the study of this protein. Secretory IgA is the predominant immunoglobulin in the secretions of mucous membranes, but constitutes a relatively small portion of the serum immunoglobulins. Heretofore, colostrum has served to be the most abundant source of secretory IgA. Several different methods for the purification of colostral IgA have been described, and in general the yields are 3 . 0 4 . 5 mg per ml clarified colostrum or 60 90 mg per rabbit (Hanson and Johannson, 1962; Axelsson et al., 1966; Cebra and Robbins, 1966; Sell, 1967). In the intestinal mucosa of the rabbit, it has been shown that c~-chain containing cells comprise 80 90% of the immunoglobulin-containing cells (Crandall et al., 1967). The rabbit intestinal mucosa, therefore, may provide a source for obtaining large quantities of secretory immunoglobulin. The study reported here describes the use of cholera toxin into the rabbit ileal loop as a non-
* This investigation was supported by NSF Grant GB 29095 and NIH Grant 5 TI AI 0128. ** This work represented as portion of a thesis submitted by Scott Wood to the Graduate School of the University of Florida in partial fulfillment of the requirements for the Master of Science Degree. *** To whom correspondence should be addressed. 207
208
S. WOODand L.W.CLEM
specific method of obtaining rabbit intestinal immunoglobulins in relatively large amounts.
2. MATERIALS AND METHODS 2.1. Cholera toxin A commercial freeze-dried (Merck) preparation of a crude culture •trate of the 569B strain of Vibrio cholerae was provided by the National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Md. 2.2. A nimals Eleven New Zealand white male rabbits were used for the studies. The animals were young adults, and weighed 1.2-2.0 kg. Each animal was starved for 48 hr prior to the operation, but water was supplied ad libitum throughout the study. 2.3. Ileal loops The operative technique was similar to that used by other investigators (Burrows and Musteikis, 1966; Carpenter et al., 1968) under 'Metofane' anesthesia, and the small bowel was brought out of the abdominal cavity and ligated approximately 15 cm above the ileo-caecal junction. Three segments each about 20 cm long and approximating distal, medial, and proximal segments of the ileum were injected with 0.1-0.3 g lyophilized crude toxin in 1 ml distilled water. Formation of the loop was made by ligation distal to the site of inoculation. The canine model of experimental cholera (Carpenter et al., 1968) has shown that maximum secretion of intestinal fluid into the loops is reached and maintained 9 hr after injection of the toxin. Based on this model the rabbits were :exanguinatedl and intestinal fluids were collected by syringe 9 - 1 2 hr after the operations. Ileal loops of similar lengths in two control rabbits were washed with 15 ml of 0.86% sodium chloride and specimens were immediately collected. 2.4. Specimens Ileal fluids were tested for occult blood using guaiac tablets (Cambridge Chemical Products, Detroit, Michigan). Since a weak 1+ reading was found to correspond roughly to a 1:6000 dilution of whole peripheral blood, specimens having a higher reading were not used. Fluid volumes were measured after clarification by centrifugation at 2500 rev/min for 10 rain in a Model V International Centrifuge. Sodium azide and soybean trypsin inhibitor were each added to a final concentration of 0.01%. The samples were then frozen at 20°C.
Cholera induced secretory immunoglobulins
209
2.5. Immunodiffusion Double diffusion in agar was performed according to Ouchterlony (1962). IgA and IgG immunoglobulin levels were quantified by single radial immunodiffusion (SRID) (Fahey and McKelvey, 1965). The IgA standard was 11 S IgA prepared by passage of clarified rabbit colostrum through a Sephadex G-200 column (Cebra and Robbins, 1966). The unabsorbed peak of rabbit whole serum off a DEAE-cellulose column was used as the source of IgG standard. Sheep anti-rabbit IgA, IgG, and IgM were absorbed with 0.3 mg rabbit F(ab')2/ml of antiserum. The resulting antisera were specific for s-chain, 7-chain, and ~t-chain as determined by double diffusion in agar*. 2.6. Gel filtration Twenty ml of intestinal fluid was mixed with an equal volume of saturated (NH4)2SO 4 and incubated overnight in the cold. The resulting precipitate was pelleted by centrifugation and washed twice with cold 50% saturated (NH4)2SO 4. The precipitate was then dissolved in 5 ml of buffer (0.15 M NaC1, 0.015 M Tris HC1, pH 7.4) and gel filtered on a column of Sephadex G-200 (Pharmacia, Uppsala, Sweden) equilibrated with the same buffer. Protein elution was monitored by reading absorbance of 5 ml fractions at 280 nm. Appropriate fractions were pooled and concentrated to 3 ml by pressure dialysis in the cold. The fractions were then analyzed for immunoglobulins by double diffusion in agar and SRID. 2.7. A nalytical ultracentrifugation Sedimentation velocity studies were performed in the 'Spinco' model E analytical ultracentrifuge, employing the Schlieren optical system at 20°C. The buffer was 0.14 M NaC1, 0.015 M Tris-HC1, pH 7.4.
3. RESULTS 3.1. Immunoglobulin con tent o f intestinal secretions The average concentrations of IgA and IgG in ileal loop fluids are represented in table 1. Statistical analysis showed no significant difference in immunoglobulin concentrations among the fluids obtained from proximal, medial or distal loops. * Sheep anti-rabbit lgA was a gift from M. Schulkind and was prepared as described by Cebra and Robbins (1966). Sheep anti-rabbit lgM was a gift from P.A. Small and was the same antiserum used previously (Small and Lamm, 1966). Anti-rabbit IgG was obtained from sheep two weeks after the last of two-weekly injections of 5 mg alum-precipitated rabbit IgG.
S. WOOD and L.W. CLEM
210
Table 1 Average concentrations of IgA and IgG in ileal loop fluids. Concentrations of immunoglobulins (mg/ml + SD) Ig
Proximal loop
Medial loop
Distal loop
Saline wash
IgA
0.76 (-+ 0.26)
0.68 (-+ 0.13)
0.56 (-+ 0.14)
0.40 (+- 0.15)
IgG
0.39 (-+ 0.02)
0.40 (-+ 0.04)
0.43 (-+ 0.04)
0.34 (± 0.02)
IgA/IgG
1.9
1.7
1.3
1.2
Av. vol.
40.6 (+- 10.8)
42.8 (+-12.0)
37.8 (_+6.7)
14.5 (-+ 0.2)
The range of values was great, having a total loop average c o n c e n t r a t i o n for IgA of 0.67 (-+ 0.09) mg/ml. The average ratio of IgA to IgG was therefore approximately 1.7. This ratio is comparable to values reported in the literature for secretory i m m u n o g l o b u l i n s (Tomasi and Bienenstock, 1968). IgM levels in the u n d i l u t e d fluids were not detected at concentrations representing greater than a 1:32 dilution of normal rabbit serum, i.e., less than 0.06 mg/ml assuming normal rabbit serum to have an upper limit of 2 mg/ml IgM. 3.2. The nature of lgA in ileal loop fluids Gel filtration chromatography on Sephadex G-200 of 20 ml of ileal fluid (contain-
0.50-
anti_.~
anti.'~
TF
I
7I
0
0
0
lifo
T]
0.40-
0 antt_~
TwO
X 0
0 anti_i
°
A2BOnm 050-
0.20
-
O.IO-
000o
,b
2b
3b
FRACTION
4%
s'o
6'0
7b
8'0
NUMBER (5 ml)
Fig. 1. Sephadex G-200 gel filtration of 20 ml ileal loop fluid precipitated at 50% saturated (NH4)2SO4. Insert is double diffusion in agar of pooled fractions I, II, III, and IV,
Cholera induced secretory immunoglobulins
211
ing approx. 5.8 mg lgA) precipitated at 50% saturation of (NH4)2SO 4 is shown in fig. 1. Double diffusion in agar of the concentrated fractions showed that IgA is present only in the excluded peak (fraction I). IgG determinants appeared to be found in all fractions except I|, and that present in fraction I, presumably representing aggregated material, was estimated by gel diffusion to account for less than 15% of the protein, By SRID, the amount of IgA in fraction I was found to be 2.3 mg, a 41% recovery. Analytical ultracentrifugation of fraction 1 (at a concentration of approximately 2 mg/ml) revealed a single boundary having a s20,w = 11.2 S.
4. DISCUSSION The results of this study demonstrate the usefulness of cholera toxin for obtaining large amounts of IgA from the rabbit intestinal mucosa. After precipitation of the ileal loop fluid at 50% saturated (NH4)2SO4, the IgA obtained was shown to be excluded from Sephadex G-200 and to have an S value of ~11 S. The yield was approximately 41% and only slight contamination with IgG was observed. Cholera toxin stimulation of ileal loops may be extended to include the entire small intestine of the rabbit. Allowing as many as ten loops per rabbit to be employed for stimulation, (individual loops limit possible blood contamination to individual samples), one should readily be able to obtain quantities of secretory IgA approaching 0.34 g from a single rabbit. Hyperimmunization of the rabbit intestine followed by the method of immunoglobulin collection described here, could provide large quantities of secretory IgA antibody. Unfortunately, a preliminary study (to be reported elsewhere) has indicated that oral immunization of rabbits with dinitrophenylated streptococcal cells followed by ileal loop studies resulted in anti-dinitrophenyl antibodies almost exclusively of the IgG class. Further studies are obviously required to test the validity of the hypothesis that the method described here would be useful in collecting large amounts of IgA antibodies to a given antigenic determinant. In conclusion it should be mentioned that the studies reported here, when compared to other studies on the immunoglobulin levels in intestinal secretions from cholera patients (Waldman et al., t971), tend to substantiate the usefulness of the ileal loop as a valid model for the biological activity of cholera toxin.
REFERENCES Axelsson, H., B.G. Joharmson and L. Rymo, 1966, Acta Chem. Scand. 20, 2339. Burrows, W. and G.M. Musteikis, 1966, J. Infect. Dis. 116,183. Carpenter, C.C.J., R.B. Sack, J.C. Feeley and R.W. Steenberg, 1968, J. Clin. Invest. 47, 1210. Cebra, J.J. and J.B. Robbins, 1966, J. Immunol. 97, 12. Chodirker, W.B. and T.B. Tomasi, Jr., 1963, Science, 142, 1080. CrandaU, R.B., J.J. Cebra and C.A. Crandall, 1967, Immunology, 12,149. Fahey, J.L. and E.M. McKelvey, 1965, J. Immunol. 94, 84.
212
S. WOOD and L.W. CLEM
Hanson, L.A. and B.G. Johannson, 1962, Intern. Arch. Allergy Appl. Immunol. 20, 65. Ouchterlony, O., 1962, Progr. Allergy 6, 30. Sell, S., 1967, Immunochemistry 4, 49. Small, P.A., Jr. and M.E. Lamm, 1966, Biochemistry 5,259. Tomasi, T.B., Jr. and J. Bienenstock, 1968, Advan. Immunol. 9, 1. Tomasi, T.B., Jr., E.M. Tan, A. Soloman and R.A. Predergast, 1965, J. Exptl. Med. 121,101. Waldman, R.H., Z. Bencic, R. Sakazaki, R. Sinha, R. Ganguly, B.C. Deb and S. Mukerjee, 1971 J. Infectious Dis. 123,579.