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Gastroduodenal ulceration in rabbits producing antibodies to prostaglandins
PROSTAGLANDINS
GASTRODUODENAL ULCERATION IN RABBITS PRODUCING ANTIBODIES TO PROSTAGLANDINS G.A. Olson,1 C.W. Leffler', and A.M. Fletcher,1,2 1 Animal Resources Division 2 Department of Physiology and Biophysics University of Tennessee Center for the Health Sciences Memphis, Tennessee 38163 USA ABSTRACT The consistent occurrence of gastric and duodenal ulcers was observed in laboratory rabbits used for production of high-titer plasPerforations developed in 7 ma antibody to 6-keto PGFIu and PGE2. of 10 animals, usually just distal to the pyloroduodenal junction. The remaining rabbits showed gross and/or microscopic evidence of imperforate ulcers and erosions. These lesions appeared to be direct pathologic complications of an immune response directed against prostaglandins since animals immunized against met-enkephalin with similar methods had no ulcers. INTRODUCTION There is an increasing body of evidence that prostaglandins play a role in maintaining the gastroduodenal mucosa. In general, PGE2 and PGI2 are cytoprotective whereas inhibitors of prostaglandin synthesis are ulcerogenic (1,2,3) This report decribes the experimental production of severe gastric and duodenal ulcers secondary to induction of an immune response to PGE2 and 6-keto PGFIo coupled to thyroglobulin. MATERIALS AND METHODS The animals were young adult male New Zealand White rabbits purchased and maintained under Pasteurella-free conditions. PGE2 and 6-keto PGFIu, the stable hydrolysis product of prostacyclin (PGIz), (Upjohn, Kalamazoo, MI) were coupled to porcine thyroEach rabbit received globulin using the mixed anhydride method (4). 200 ug of conjugate, emulsified in Freund's complete adjuvant, injected at multiple intradermal sites. Similar injections were given 7 weeks later. Thereafter, rabbits were immunized subcutaneously 3 times with antigen in incomplete Freund's adjuvant at 10 week intervals. Seven days arter each booster injection of prostaglandinthyroglobulin conjugate, plasma was obtained by centrifugation of heparinized blood. Antibody titer was determined by combining plasma, at roughly doubling dilutions from 1:2000 to 1:120,000 in gelatin-tris buffered
MARCH
1985 VOL. 29 NO. 3
475
PROSTAGLANDINS saline (pH 7.4), with lo-‘” M (final concentration) of the appropriate tritiated prostaglandin (New England Nuclear, Boston, MA). Fo 1 lowing 16 hr incubation at 4” C, the labelled antigen bound to antibody was separatedfrom the unbound antigen by precipitation of the rabbit antibody with goat antirabbit gamma globulin and 60% saturated ammonium sulfate. The antibody titer was expressed as the denominator of the dilution at which 40% of the labelled antigen was bound to antibody (numerator 1). Rabbits examination
that died to determine
antibody titers under anesthesia
to the followed
spontaneously were given gross cause of death. Live rabbits prostaglandins by necropsy
were killed examination.
nized with met-enkephalin bound to thyroglobin, as for prostaglandin antibody production, also
and with
microscopic high plasma
by exsanguination Three rabbits immu-
using the same were examined.
methods
RESULTS Table 1 shows the plasma antibody titers of the rabbits immunized to prostaglandin-thyroglobulin conjugates. Nine of the 10 injected rabbits produced documented plasma antibodies that bound labelled proIn one rabbit (number 3), plasma was not drawn between staglandins. the third injection and death so that the antiprostaglandin E2 plasma activity is not known. Table with
Plasma prostaglandin 1. prostaglandin-thyroglobulin.
Immunogen
Rabbit
antibody
No.
titers
following
Plasma
Titera
immunization
128 57 Days
271 Days
1 <2,000 50,000 (43) 2 <2,000 7,000 PGE2 3 <2,000 --(18) 4 <2,000 2,000 5 <2,000 3,000 ___-----~~~__------~~~~__------~~~_____-----~~__-------~~~__-__------
6-keto
PGFIo
6
<2,000
12,000
7 8
<2,000 33,000
60,000
(17)
9 10
<2,000 (2,000
6,000 30,000
(36)
50,000 _-_ ---
30,000 120,000
60,000
(15>b
1 week a Rabbits were immunized number in parentheses determination; following the last immunization at perforating ulcer.
20,000
prior to indicates which the
(28)
bleeding for titer the number of days rabbit died with a
b Evidence
of ulcer perforation in the first animal to die was Hepatic abscesses and peritonicircumstantial and retrospective. obsertis were seen on post mortem examination. However, direct vation of the gastrointestinal mucosa was not made and tissue was not saved.
476
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS against both PGE2 and 6-keto-PGFI, resulted in Immunization gastroduodenal ulcerat ion. Six of the ten rabbits immunized against four of five 6-keto PGFl,) died prostaglandins (two of five PGE2, spontaneously with perforating duodenal ulcers. Deaths occurred 2 to 6 weeks following booster immunizations (Table 1). The ulcers were located 1 cm distal to the pylorus in the right wall of the duodenum. They were round and 7 to 10 mm in diameter. Perforating duodenal ulcers usually were solitary but occasionally were accompanied by nonperforating ulcers (Fig. 1). Fibrinous adhesions, blood clots, and peritonitis were observed in the abdomen near the ulcer stoma. Careful examination of the intestinal mucosa revealed no gross lesions distal to the perforation sites.
1. Macroscopic appearance of a perforating Fig. from the mucosal side in a rabbit that died 6-keto-PGF1,-thyroglobulin immunization with 60,000). perforation.
A smaller
nonperforating
ulcer
is
duodenal ulcer viewed 17 days after the third conjugate (titer seen
immediately
below
the
The remaining 4 rabbits immunized against prostaglandins all had evidence of gastroduodenal ulceration upon postmortem examination. One (rabbit number 5) had a large irregular gastric ulcer adjacent to the esophagus with a well-developed adhesion to the liver. This lesion appeared to represent a perforating gastric ulcer that had sealed over and begun to heal. host gastric lesions were found in the mucosa of
MARCH
1985 VOL. 29 NO. 3
477
PROSTAGLANDINS
the lesser curvature. They were 2-4 mm in diameter and appeared as pink spots or streaks in the mucosa[ folds. The number of gastric lesions ranged from 1 to 5 per rabbit. Microscopic examination (Fig. 2) showed typical mucosal ulcers with loss of epithelium, disruption of the muscularis mucosae, and fibrosis of the ulcer base. Inflammatory ceils consisted of heterophils and focal accumulations of lymphocytes near the margins of the ulcers (Fig. 2). Developing ulcers and erosions were often associated with a perivascular lymphocytic cuff directly below or at the margin of the lesion.
Fig. 2. Light micrograph of a gastric ulcer in a rabbit immunized with PGE2-thyroglobulin conjugate. The rabbit was sacrificed two weeks after the fifth immunization over 271 days (titer 30,000). (25x). The same 6-8 month immunization protocol was used to produce met-enkephalin antiserum. No spontaneous deaths occurred and no gastrointestinal ulcers were found in 3 rabbits examined, although antibodies that bound met-enkephalin were present in the serum.
478
MARCH 1985 VOL. 29 NO. 3
PROSTAGLANDINS DISCUSSION in all rabbits Observation of gastroduodenal ulcers immunized with prostaglandin-thyroglobulin conjugates suggests a direct correlation between the immune response and ulcerogenesis. Rabbits immunized with met-enkephalin-thyroglobulin did not develop ulcers. Therefore, it would appear that it is the prostaglandin moiety of the immunizing complex that causes subsequent ulcer development. Since the use of cyclooxygenase inhibitors can result in gastric it is not unexpected that an immune reerosion and/or ulcerat ion (5)) sponse directed at prostaglandins might have a similar effect. Several endogenous prostaglandins have been shown to have regulatory roles in gastric acid secretion (6) and mucosal cytoprotection (2). They have been associated with increased mucus production (71, increased bicarbonate transport from gastric mucosa (81, and reduced epithelial shedding after irritant exposure (9). All these effects would contribute to lower instances of gastrcduodenal ulceration. It is reasonable, therefore, to suggest that immune-mediated interference with prostaglandin synthesis or function can result in gastrointestinal disturbances that lead to ulceration. These experiments do not allow determination of the specific proThe cross reactivities of the antibodies with staglandinc a.1 involved. PG12 were not determined. The cross reactivities with each other and with other known, naturally occurring prostaglandins consistently was concentrations, large less than 1%. However) at these high antibody quantities of antigen with relatively low affinity for the antibody can be removed. Since it is reported that both PGI2 and PGE2 are important in protecting the gastroduodenal tract from ulcers (3), simultaneous immunization with both prostaplandins in each rabbit should increase the incidence and rapidity of ulcer development greatly. It may be possible to prevent or delay ulcer development in animals used for prostaglandin antibody production through the therapeutic use of anticholinergics, antacids, and/or histamine H2 blockers. This would prevent unnecessary pain or death and might allow the collection of greater amounts of serum with higher antibody titers. An alternative solution would be to exsanguinate the animals once adequate working titers are produced. ACKNOWLEDGEMENTS We acknowledge the excellent technical assistance of S. Adams and postmortem examination M. Jackson. We thank Dr. L. Share for allowing of rabbits immunized with met-enkephalin. Dr. Leffler is an Established Investigator of the American Heart Association with funds contributed in part by the Tennessee AffiliThe research was supported in part by Grants-in-aid from the ate. American Heart Association and from the Tennessee Affiliate.
MARCH
1985 VOL. 29 NO. 3
479
PROSTAGLANDINS
REFERENCES 1.
Gastrointestinal Miller, T.A., and E.D. Jacobson, tion by prostaglandins. Gut -20: 75, 1979.
2.
Robert, A. Cytoprotection by prostaglandins. -77: 761. 1979.
3.
Whittle, B.J.R., N.K. Broughton-Smith, S. Moncada, and J.R. Actions of its product Vane. prostacyclin (PGI2) and 6-oxo-PGF1, on the rat gastric mucosa in vivo and in vitro. Prostaglandins -15: 955, 1978.
4.
Prostaglandins E, A and F. In: Jaffe, B.M. and H.R. Behrman. Academic Press, New York, Methods of hormone radioimmunoassay. 1974.
5.
Bauer, Brodie, D.A., P.G. Cook, B.J. and G.E. Dagle. Tox. Appl. Indomethacin-induced intestinal lesions in the rat. Pharm. -17: 615, 1970.
6.
Robert, A. Inhibition of gastric Amer. J. Dig. Dis. -12: 359, 1978.
7.
Stimulation of mucus Bolton, J.P., D. Palmer, and M.M. Cohen. and nonparietal cell secretion by the E2 prostaglandins. Am. J. Dig. Dis. 23: 359, 1978.
8.
Stimulation Johansson, C., A. Aly, E. Nilsson, and G. Flemstrom. of gastric bicarbonate secretion by E2 prostaglandins in man. Adv. Prostaglandin Thromboxane Leukotriene Res -12: 395, 1983.
9.
CytoRobert, A., J.E. Negamis, C. Lancaster, and A.J. Huchar. Prevention of gastric protection by prostaglandins in rats. necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology -77: 433. 1978.
Editor: P. Piper
480
secretion
Received: 11-20-84
MARCH
cytoprotec-
Gastroenterology:
by
prostaglandins.
Accepted: 12-18-84
1985VOL.29NO.3
GASTRODUODENAL ULCERATION IN RABBITS PRODUCING ANTIBODIES TO PROSTAGLANDINS G.A. Olson,1 C.W. Leffler', and A.M. Fletcher,1,2 1 Animal Resources Division 2 Department of Physiology and Biophysics University of Tennessee Center for the Health Sciences Memphis, Tennessee 38163 USA ABSTRACT The consistent occurrence of gastric and duodenal ulcers was observed in laboratory rabbits used for production of high-titer plasPerforations developed in 7 ma antibody to 6-keto PGFIu and PGE2. of 10 animals, usually just distal to the pyloroduodenal junction. The remaining rabbits showed gross and/or microscopic evidence of imperforate ulcers and erosions. These lesions appeared to be direct pathologic complications of an immune response directed against prostaglandins since animals immunized against met-enkephalin with similar methods had no ulcers. INTRODUCTION There is an increasing body of evidence that prostaglandins play a role in maintaining the gastroduodenal mucosa. In general, PGE2 and PGI2 are cytoprotective whereas inhibitors of prostaglandin synthesis are ulcerogenic (1,2,3) This report decribes the experimental production of severe gastric and duodenal ulcers secondary to induction of an immune response to PGE2 and 6-keto PGFIo coupled to thyroglobulin. MATERIALS AND METHODS The animals were young adult male New Zealand White rabbits purchased and maintained under Pasteurella-free conditions. PGE2 and 6-keto PGFIu, the stable hydrolysis product of prostacyclin (PGIz), (Upjohn, Kalamazoo, MI) were coupled to porcine thyroEach rabbit received globulin using the mixed anhydride method (4). 200 ug of conjugate, emulsified in Freund's complete adjuvant, injected at multiple intradermal sites. Similar injections were given 7 weeks later. Thereafter, rabbits were immunized subcutaneously 3 times with antigen in incomplete Freund's adjuvant at 10 week intervals. Seven days arter each booster injection of prostaglandinthyroglobulin conjugate, plasma was obtained by centrifugation of heparinized blood. Antibody titer was determined by combining plasma, at roughly doubling dilutions from 1:2000 to 1:120,000 in gelatin-tris buffered
MARCH
1985 VOL. 29 NO. 3
475
PROSTAGLANDINS saline (pH 7.4), with lo-‘” M (final concentration) of the appropriate tritiated prostaglandin (New England Nuclear, Boston, MA). Fo 1 lowing 16 hr incubation at 4” C, the labelled antigen bound to antibody was separatedfrom the unbound antigen by precipitation of the rabbit antibody with goat antirabbit gamma globulin and 60% saturated ammonium sulfate. The antibody titer was expressed as the denominator of the dilution at which 40% of the labelled antigen was bound to antibody (numerator 1). Rabbits examination
that died to determine
antibody titers under anesthesia
to the followed
spontaneously were given gross cause of death. Live rabbits prostaglandins by necropsy
were killed examination.
nized with met-enkephalin bound to thyroglobin, as for prostaglandin antibody production, also
and with
microscopic high plasma
by exsanguination Three rabbits immu-
using the same were examined.
methods
RESULTS Table 1 shows the plasma antibody titers of the rabbits immunized to prostaglandin-thyroglobulin conjugates. Nine of the 10 injected rabbits produced documented plasma antibodies that bound labelled proIn one rabbit (number 3), plasma was not drawn between staglandins. the third injection and death so that the antiprostaglandin E2 plasma activity is not known. Table with
Plasma prostaglandin 1. prostaglandin-thyroglobulin.
Immunogen
Rabbit
antibody
No.
titers
following
Plasma
Titera
immunization
128 57 Days
271 Days
1 <2,000 50,000 (43) 2 <2,000 7,000 PGE2 3 <2,000 --(18) 4 <2,000 2,000 5 <2,000 3,000 ___-----~~~__------~~~~__------~~~_____-----~~__-------~~~__-__------
6-keto
PGFIo
6
<2,000
12,000
7 8
<2,000 33,000
60,000
(17)
9 10
<2,000 (2,000
6,000 30,000
(36)
50,000 _-_ ---
30,000 120,000
60,000
(15>b
1 week a Rabbits were immunized number in parentheses determination; following the last immunization at perforating ulcer.
20,000
prior to indicates which the
(28)
bleeding for titer the number of days rabbit died with a
b Evidence
of ulcer perforation in the first animal to die was Hepatic abscesses and peritonicircumstantial and retrospective. obsertis were seen on post mortem examination. However, direct vation of the gastrointestinal mucosa was not made and tissue was not saved.
476
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS against both PGE2 and 6-keto-PGFI, resulted in Immunization gastroduodenal ulcerat ion. Six of the ten rabbits immunized against four of five 6-keto PGFl,) died prostaglandins (two of five PGE2, spontaneously with perforating duodenal ulcers. Deaths occurred 2 to 6 weeks following booster immunizations (Table 1). The ulcers were located 1 cm distal to the pylorus in the right wall of the duodenum. They were round and 7 to 10 mm in diameter. Perforating duodenal ulcers usually were solitary but occasionally were accompanied by nonperforating ulcers (Fig. 1). Fibrinous adhesions, blood clots, and peritonitis were observed in the abdomen near the ulcer stoma. Careful examination of the intestinal mucosa revealed no gross lesions distal to the perforation sites.
1. Macroscopic appearance of a perforating Fig. from the mucosal side in a rabbit that died 6-keto-PGF1,-thyroglobulin immunization with 60,000). perforation.
A smaller
nonperforating
ulcer
is
duodenal ulcer viewed 17 days after the third conjugate (titer seen
immediately
below
the
The remaining 4 rabbits immunized against prostaglandins all had evidence of gastroduodenal ulceration upon postmortem examination. One (rabbit number 5) had a large irregular gastric ulcer adjacent to the esophagus with a well-developed adhesion to the liver. This lesion appeared to represent a perforating gastric ulcer that had sealed over and begun to heal. host gastric lesions were found in the mucosa of
MARCH
1985 VOL. 29 NO. 3
477
PROSTAGLANDINS
the lesser curvature. They were 2-4 mm in diameter and appeared as pink spots or streaks in the mucosa[ folds. The number of gastric lesions ranged from 1 to 5 per rabbit. Microscopic examination (Fig. 2) showed typical mucosal ulcers with loss of epithelium, disruption of the muscularis mucosae, and fibrosis of the ulcer base. Inflammatory ceils consisted of heterophils and focal accumulations of lymphocytes near the margins of the ulcers (Fig. 2). Developing ulcers and erosions were often associated with a perivascular lymphocytic cuff directly below or at the margin of the lesion.
Fig. 2. Light micrograph of a gastric ulcer in a rabbit immunized with PGE2-thyroglobulin conjugate. The rabbit was sacrificed two weeks after the fifth immunization over 271 days (titer 30,000). (25x). The same 6-8 month immunization protocol was used to produce met-enkephalin antiserum. No spontaneous deaths occurred and no gastrointestinal ulcers were found in 3 rabbits examined, although antibodies that bound met-enkephalin were present in the serum.
478
MARCH 1985 VOL. 29 NO. 3
PROSTAGLANDINS DISCUSSION in all rabbits Observation of gastroduodenal ulcers immunized with prostaglandin-thyroglobulin conjugates suggests a direct correlation between the immune response and ulcerogenesis. Rabbits immunized with met-enkephalin-thyroglobulin did not develop ulcers. Therefore, it would appear that it is the prostaglandin moiety of the immunizing complex that causes subsequent ulcer development. Since the use of cyclooxygenase inhibitors can result in gastric it is not unexpected that an immune reerosion and/or ulcerat ion (5)) sponse directed at prostaglandins might have a similar effect. Several endogenous prostaglandins have been shown to have regulatory roles in gastric acid secretion (6) and mucosal cytoprotection (2). They have been associated with increased mucus production (71, increased bicarbonate transport from gastric mucosa (81, and reduced epithelial shedding after irritant exposure (9). All these effects would contribute to lower instances of gastrcduodenal ulceration. It is reasonable, therefore, to suggest that immune-mediated interference with prostaglandin synthesis or function can result in gastrointestinal disturbances that lead to ulceration. These experiments do not allow determination of the specific proThe cross reactivities of the antibodies with staglandinc a.1 involved. PG12 were not determined. The cross reactivities with each other and with other known, naturally occurring prostaglandins consistently was concentrations, large less than 1%. However) at these high antibody quantities of antigen with relatively low affinity for the antibody can be removed. Since it is reported that both PGI2 and PGE2 are important in protecting the gastroduodenal tract from ulcers (3), simultaneous immunization with both prostaplandins in each rabbit should increase the incidence and rapidity of ulcer development greatly. It may be possible to prevent or delay ulcer development in animals used for prostaglandin antibody production through the therapeutic use of anticholinergics, antacids, and/or histamine H2 blockers. This would prevent unnecessary pain or death and might allow the collection of greater amounts of serum with higher antibody titers. An alternative solution would be to exsanguinate the animals once adequate working titers are produced. ACKNOWLEDGEMENTS We acknowledge the excellent technical assistance of S. Adams and postmortem examination M. Jackson. We thank Dr. L. Share for allowing of rabbits immunized with met-enkephalin. Dr. Leffler is an Established Investigator of the American Heart Association with funds contributed in part by the Tennessee AffiliThe research was supported in part by Grants-in-aid from the ate. American Heart Association and from the Tennessee Affiliate.
MARCH
1985 VOL. 29 NO. 3
479
PROSTAGLANDINS
REFERENCES 1.
Gastrointestinal Miller, T.A., and E.D. Jacobson, tion by prostaglandins. Gut -20: 75, 1979.
2.
Robert, A. Cytoprotection by prostaglandins. -77: 761. 1979.
3.
Whittle, B.J.R., N.K. Broughton-Smith, S. Moncada, and J.R. Actions of its product Vane. prostacyclin (PGI2) and 6-oxo-PGF1, on the rat gastric mucosa in vivo and in vitro. Prostaglandins -15: 955, 1978.
4.
Prostaglandins E, A and F. In: Jaffe, B.M. and H.R. Behrman. Academic Press, New York, Methods of hormone radioimmunoassay. 1974.
5.
Bauer, Brodie, D.A., P.G. Cook, B.J. and G.E. Dagle. Tox. Appl. Indomethacin-induced intestinal lesions in the rat. Pharm. -17: 615, 1970.
6.
Robert, A. Inhibition of gastric Amer. J. Dig. Dis. -12: 359, 1978.
7.
Stimulation of mucus Bolton, J.P., D. Palmer, and M.M. Cohen. and nonparietal cell secretion by the E2 prostaglandins. Am. J. Dig. Dis. 23: 359, 1978.
8.
Stimulation Johansson, C., A. Aly, E. Nilsson, and G. Flemstrom. of gastric bicarbonate secretion by E2 prostaglandins in man. Adv. Prostaglandin Thromboxane Leukotriene Res -12: 395, 1983.
9.
CytoRobert, A., J.E. Negamis, C. Lancaster, and A.J. Huchar. Prevention of gastric protection by prostaglandins in rats. necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology -77: 433. 1978.
Editor: P. Piper
480
secretion
Received: 11-20-84
MARCH
cytoprotec-
Gastroenterology:
by
prostaglandins.
Accepted: 12-18-84
1985VOL.29NO.3