Acta Physiol Scand 1991, 141, 443444
ADONIS
000167729100067X
Cecropin PI -like immunoreactivity in epithelial cells of duodenum and glucagon cells of the pancreas J . M . L U N D B E R G , A.HEMSEN, M.ANDERSSON, T . H O K F E L T , V. M U T T and H. G. BOMAN Departments of Pharmacology, Histology & Neurobiology and Biochemistry, Karolinska Institute and Department of Microbiology, University of Stockholm, Sweden Cecropins are potent basic antibacterial peptides that play a key role in insect immunity (Boman & Hultmark 1987). Recently an antibacterial peptide with 33% homology to insect cecropins was isolated from extracts of the pig upper small intestine (Lee et al. 1989). Furthermore, it was postulated that production of this cecropin (named P1 for porcine) and other antibacterial peptides could contribute to reguIation of the concentration of e.g. Eschenchga coEi in the upper part of the intestine. T o further elucidate this hypothesis we have raised antibodies to cecropin P1 and localized cecropin P1-like immunoreactivity (LI) by immunohistochemistry. Antiserum against cecropin P1 was produced in rabbits immunized with synthesis peptide (Anderson 1990). Small pieces of porcine duodenum, pancreas, distal colon and lung ( n = 4) were immersion-fixed for 2 h at + 4 "C in buffered formalin. Male Sprague Dawley rats were perfusion fixed with this solution and the corresponding tissues dissected out. After rinsing cryostat sections (14 pm) were processed for indirect immunohistochemistry. Antibodies to cecropin P1 raised in rabbits (antisera cec 2-2310 or cec 41 127) and to glucagon and insulin raised in guineapigs (UCB Bioproducts, Brussels, Belgium) or monoclonal antibodies to somatostatin (Buchan et al. 1985) were used in dilutions 1:200. After incubation for 24 h at + 4 "C with the primary antibody, the sections were rinsed and incubated for 30 min at 37 "C with F I T C - or Rhodamine-labelled goat anti-rabbit antibodies (Boehringer Mannheim Scandinavia), swine anti guinea-pig antibodies labelled with Rhodamine (Nordic, Tilburg, The Netherlands) or FITC-labelled sheep antimouse antibodies (Amersham, UK). They were then mounted and examined in a fluorescence
microscope. Control sections were incubated with antibodies (diluted 1:200) preabsorbed with synthetic cecropin P1 or glucagon (40 pg ml-l). In the porcine small intestine epithelial cells with an endocrine-like appearance contained cecropin PI-LI (Fig. la, c). In addition some epithelial cells localized at the apical end of the villi were cecropin P1 positive (Fig. 1a, c). Some variation in the number of detected cells was present between individual animals ranging from few to many (Fig. la). In the pig colon and lung or in rat duodenum, colon and lung no cells were found to contain cecropin PI-LI (not shown). A distinct population of cells in the Langerhans islets of the pancreas was consistently cecropin P1immunoreactive in both pig and rat (Fig. 1 b). Doublelabelling revealed in both species that these pancreatic cells containing cecropin PI-LI also were glucagonimmunoreactive (IR) (Fig. 1b, d), but not insulin(Fig. I f ) or somatostatin-IR (not shown). Preabsorption of the cecropin P1 antisera with synthetic cecropin P1, but not glucagon, abolished the immunostaining in both the pancreas and intestine (Fig. le). The present data show that cecropin PI-LI is consistently found in pancreatic endocrine cells also containing glucagon-LI in both pig and rat. Although the IR material remains to be biochemically characterized this opens up the possibility that a peptide antibiotic is secreted as a hormone in relation to e.g. blood glucose levels. Furthermore, the presence of cecropin P1-LI in epithelial cells in the intestine suggests that cecropin PI may be secreted onto the intestinal mucosal surface to exert local antibacterial activity. The mechanisms underlying the variation in the presence of intestinal epithelial cells with cecropin P1-LI remain to be further elucidated.
Received 26 September 1990, accepted 29 September 1990. Correspondence : Jan M. Lundberg, Department of Pharmacology, Karolinska Institute, Box 60400, 104 01 Stockholm. Sweden.
Supported by grants from the Swedish Medical Research Council (14X-6554,04X-2887, 13X-01010). We thank Mrs Siv Nilsson for expert technical assistance and Mrs Evy Carlsson for secretarial help. We thank Prof J. C. Brown, MRC Regulatory Peptide
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et al.
Fig. 1. Immunofluorescence micrographs of the pig duodenum (a, c, e) and rat pancreas (b, d, f) after incubation with (a, b, c) antiserum to cecropin P1 (using antiserum CEC-22310), (e) cecropin P1 antiserum absorbed with synthetic cecropin P1 peptide, (d) glucagon antiserum (GLUC) or (f) insulin (INS) antiserum. Note the loss of cecropin PI-LI in epithelial intestinal cells after control (CON) absorption with synthetic peptide (e). In the double-labelling experiment on the same section cecropin P1- (b) and glucagon-LI (d) are present in the same cells in the periphery of Langerhans islets. Insulin-LI on the other hand is present in cells with a central location (f). Bars in (a) and (c) represent 50 pm, (a, e) and (b, c, d, f) have the same magnification, respectively. Group, UBC, Vancouver, B.C., Canada for a generous supply of somatostatin antibodies.
REFERENCES ANDERSON,M. 1990. Solid-phase synthesis of a 31residue mammalian cecropin and its C-terminally amidated analogue. MPSA 9, 359. BOMAN,H.G. & HULTMARK, D. 1987. Cell-free immunity in insects. Ann Rev Microbiol 41, 103-1 26.
BUCIiAN, A.M.J., SIKORA,L.K.J., LEVY, J.G., MCINTOSH, C.H.S., DYCK,I. & BROWN, J.C. 1985. An immunocytochemical investigation with monoclonal antibodies to somatostatin. Histochemistry 83, 175-180. LEE,J.Y., BOMAN,A., CHUANXIN, S., ANDERSON,M., JORNVALL, H., MUTT,11. & BOMAN,H.G. 1989. Antibacterial peptides from pig intestine : Isolation of a mammalian cecropin. Proc Natl Acad Sci, USA, 86, 9159-9162.
ADONIS
000167729100067X
Cecropin PI -like immunoreactivity in epithelial cells of duodenum and glucagon cells of the pancreas J . M . L U N D B E R G , A.HEMSEN, M.ANDERSSON, T . H O K F E L T , V. M U T T and H. G. BOMAN Departments of Pharmacology, Histology & Neurobiology and Biochemistry, Karolinska Institute and Department of Microbiology, University of Stockholm, Sweden Cecropins are potent basic antibacterial peptides that play a key role in insect immunity (Boman & Hultmark 1987). Recently an antibacterial peptide with 33% homology to insect cecropins was isolated from extracts of the pig upper small intestine (Lee et al. 1989). Furthermore, it was postulated that production of this cecropin (named P1 for porcine) and other antibacterial peptides could contribute to reguIation of the concentration of e.g. Eschenchga coEi in the upper part of the intestine. T o further elucidate this hypothesis we have raised antibodies to cecropin P1 and localized cecropin P1-like immunoreactivity (LI) by immunohistochemistry. Antiserum against cecropin P1 was produced in rabbits immunized with synthesis peptide (Anderson 1990). Small pieces of porcine duodenum, pancreas, distal colon and lung ( n = 4) were immersion-fixed for 2 h at + 4 "C in buffered formalin. Male Sprague Dawley rats were perfusion fixed with this solution and the corresponding tissues dissected out. After rinsing cryostat sections (14 pm) were processed for indirect immunohistochemistry. Antibodies to cecropin P1 raised in rabbits (antisera cec 2-2310 or cec 41 127) and to glucagon and insulin raised in guineapigs (UCB Bioproducts, Brussels, Belgium) or monoclonal antibodies to somatostatin (Buchan et al. 1985) were used in dilutions 1:200. After incubation for 24 h at + 4 "C with the primary antibody, the sections were rinsed and incubated for 30 min at 37 "C with F I T C - or Rhodamine-labelled goat anti-rabbit antibodies (Boehringer Mannheim Scandinavia), swine anti guinea-pig antibodies labelled with Rhodamine (Nordic, Tilburg, The Netherlands) or FITC-labelled sheep antimouse antibodies (Amersham, UK). They were then mounted and examined in a fluorescence
microscope. Control sections were incubated with antibodies (diluted 1:200) preabsorbed with synthetic cecropin P1 or glucagon (40 pg ml-l). In the porcine small intestine epithelial cells with an endocrine-like appearance contained cecropin PI-LI (Fig. la, c). In addition some epithelial cells localized at the apical end of the villi were cecropin P1 positive (Fig. 1a, c). Some variation in the number of detected cells was present between individual animals ranging from few to many (Fig. la). In the pig colon and lung or in rat duodenum, colon and lung no cells were found to contain cecropin PI-LI (not shown). A distinct population of cells in the Langerhans islets of the pancreas was consistently cecropin P1immunoreactive in both pig and rat (Fig. 1 b). Doublelabelling revealed in both species that these pancreatic cells containing cecropin PI-LI also were glucagonimmunoreactive (IR) (Fig. 1b, d), but not insulin(Fig. I f ) or somatostatin-IR (not shown). Preabsorption of the cecropin P1 antisera with synthetic cecropin P1, but not glucagon, abolished the immunostaining in both the pancreas and intestine (Fig. le). The present data show that cecropin PI-LI is consistently found in pancreatic endocrine cells also containing glucagon-LI in both pig and rat. Although the IR material remains to be biochemically characterized this opens up the possibility that a peptide antibiotic is secreted as a hormone in relation to e.g. blood glucose levels. Furthermore, the presence of cecropin P1-LI in epithelial cells in the intestine suggests that cecropin PI may be secreted onto the intestinal mucosal surface to exert local antibacterial activity. The mechanisms underlying the variation in the presence of intestinal epithelial cells with cecropin P1-LI remain to be further elucidated.
Received 26 September 1990, accepted 29 September 1990. Correspondence : Jan M. Lundberg, Department of Pharmacology, Karolinska Institute, Box 60400, 104 01 Stockholm. Sweden.
Supported by grants from the Swedish Medical Research Council (14X-6554,04X-2887, 13X-01010). We thank Mrs Siv Nilsson for expert technical assistance and Mrs Evy Carlsson for secretarial help. We thank Prof J. C. Brown, MRC Regulatory Peptide
+
443
444
3. M . Lundberg
et al.
Fig. 1. Immunofluorescence micrographs of the pig duodenum (a, c, e) and rat pancreas (b, d, f) after incubation with (a, b, c) antiserum to cecropin P1 (using antiserum CEC-22310), (e) cecropin P1 antiserum absorbed with synthetic cecropin P1 peptide, (d) glucagon antiserum (GLUC) or (f) insulin (INS) antiserum. Note the loss of cecropin PI-LI in epithelial intestinal cells after control (CON) absorption with synthetic peptide (e). In the double-labelling experiment on the same section cecropin P1- (b) and glucagon-LI (d) are present in the same cells in the periphery of Langerhans islets. Insulin-LI on the other hand is present in cells with a central location (f). Bars in (a) and (c) represent 50 pm, (a, e) and (b, c, d, f) have the same magnification, respectively. Group, UBC, Vancouver, B.C., Canada for a generous supply of somatostatin antibodies.
REFERENCES ANDERSON,M. 1990. Solid-phase synthesis of a 31residue mammalian cecropin and its C-terminally amidated analogue. MPSA 9, 359. BOMAN,H.G. & HULTMARK, D. 1987. Cell-free immunity in insects. Ann Rev Microbiol 41, 103-1 26.
BUCIiAN, A.M.J., SIKORA,L.K.J., LEVY, J.G., MCINTOSH, C.H.S., DYCK,I. & BROWN, J.C. 1985. An immunocytochemical investigation with monoclonal antibodies to somatostatin. Histochemistry 83, 175-180. LEE,J.Y., BOMAN,A., CHUANXIN, S., ANDERSON,M., JORNVALL, H., MUTT,11. & BOMAN,H.G. 1989. Antibacterial peptides from pig intestine : Isolation of a mammalian cecropin. Proc Natl Acad Sci, USA, 86, 9159-9162.
