Crystalloid Lysozyme Inclusions in Paneth Cells of Vitamin A-Deficient Rats HANS K. BIESALSKI,h MARTIN J. KOCH," ECKART STOFFT," HARALD WEISER,' HANS-P. DIENES," A N D BURKARD SCHULZ-DOBRICKd l'Oc~prrrtmi~nt of Anatomy hPiiy.sioIogicaI Chemistry ' Pothologicul Aniitomy and 'Minermlogy University o f Mainz 06500 Muinz, Giwnany "RcjsorrrchDeprirtment F . H(?ffintinn-L(rRoche Ltd. B r i s d , Sktitzerland
The influence of vitamin A deficiency on epithelial tissues has been studied extensively. I.? The mucous membranes of the respiratory tract are especially a f f e ~ t e d . ~ This ciiuscs typical functional changes in the tracheobronchial tract.4 In contrast to the tespiratory epithelium, less is known concerning changes in intestinal epithelium diiring vitamin A deficiency. In mild vitamin A deficiency, however, infectious diseases of the intestinal tract are a serious problem.' Because the intestinal mucoii\ membrane seems unaffected during vitamin A deficiency, a disturbance of 1oc;il immunity might be the reason for the increase in infectious diseases. Paneth cells provide a major barrier against bacterial infections. Paneth cells are found at the base of the crypts in the small intestine. They are one of the four cellular elements of the intestinal epithelium, the other three being enterocytes, goblet cells, and endocrine cells. It has been suggested that they are involved in protecting the intestine against infection' due to their secretory capacity for lysozyme and other substances. Lysozyme, the main secretory product of the Paneth cell,' is an enzyme capable of degrading the cell walls of most bacteria. In addition, Paneth cells secrete immunoglobulines' and other enzymes, such a s carboxylicester hydrolase,' pancreatic enzymes,"' and intestinal phospholipase A2." These enzymes also may be involved in the elimination of microorganisms in the small intestine.'."'
RESULTS AND DISCUSSION Roil-like particles were observed in granules of Paneth cells in six of the eight vitamin A-deficient animals (FIG.I ) . The particles were composed of electrondense parallel stripes. Mostly, the stripes were arranged diagonally along the length of the axis of the particles. In some cases, latice-like, dense network-like o r granulated, and ladder-rung-like particles were found. The periodicity was about 332
BIESALSKI et al.: PANETH CELLS
333
8-9 nm in the diagonally arranged particles (range 6.4 nm to 12.5 nm). The particles measured up to 2 p m in length and 0.5 pm in width. The particles occurred in vacuoles without traceable secretion products and in secretory granules, which also contained morphologically unaltered secretion
FIGURE 1. Figure shows different views of crystalloid particles in Paneth cells of vitamin A-depleted rats. Two groups of 4-week-old male Sprague-Dawley rats (8 animals each) were fed a vitamin A-free semisynthetic diet for about two months. One group received, in addition, 100 I U of vitamin A each day via pharyngeal tube (control group). At the time of sacrifice, the vitamin A-deficient animals were in an early stage of deficiency, and thus typical symptoms such as Bitot's spots or xerophthalmia were not yet manifest. Deficiency was verified by the determination of plasma retinol and liver retinyl-ester concentration with high performance liquid chromatography (data not shown). Specimens were taken from the jejunum 8 cm distal from the upper end of the radix mesenterii and postfixed by immersion in 2% osmium tetroxide in 0.2 M PBS (pH 7.4), then dehydrated in a graded series of ethanol and propylene oxide and subsequently embedded in Epon 812. Ultrathin sections were placed on 300-mesh copper grids and stained with uranyl acetate and lead citrate. Electron microscopy was performed with a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany).
products. Secretory granules lacking particles showed no changes compared with controls. The particles occurred predominantly in granules of the Paneth cells. The electron-immunohistochemical evaluation showed a clear lysozyme immunoreactivity to the crystalloid particles. It was hypothesized by Otto and WeitzI2and Linss,I3 who detected similar structures in actinomycin-treated and zinc-deficient
334
ANNALS NEW YORK ACADEMY OF SCIENCES
rats, that these structures may reflect a crystalline form of lysozyme. Following this assumption, our immunoelectronrnicroscopic evaluation shows a clear lysozyme imniunoreactivity of the crystalloid particles (FIG.2 ) . Cr) stalloid particles in secretory granules of Paneth cells have been described in 20-day-old rats and have been found to display a periodicity of 8 nanometers.14
FIGURE 2. Rod-like crystalloid particles showing a strong lysozyme immunoreactivity. Stripes are covered by the immunoreaction product. For immunoelectron microscopy. three deficient rats (with the same diet as described above) were killed under ether anesthesia by decapitation. Specimens of jejunum were rapidly removed (as described above) and immersion-fixed in 2% periodate-lysine-paraformaldehyde,buffered in 0. I M PBS. For cryoprotection, samples were pretreated in a graded series of sucrose buffer (796, IS%’, 2092, 40%, and 60%, sucrose in 0. I M PBS) for 4 h in each concentration. The samples were then frozen in liquid nitrogen. The peroxidase-antiperoxidase method according to Sternberger.I8 using an antiserum against human lysozyme (cross-reaction with rat lysozyme) from rabbit, was perlormed. The dilution of the antiserum was I : 75 and I : 150; the second antiserum (anti-rabbit IgG antiserum, peroxide labeled, from goat) was diluted I : 50. Five pm-thick frozen scctions were incubated for 30 min at 20°C in a humidified chamber. Further procedures were carried out according Sternberger.Ix Conventional negative and positive controls were prepared. The frozen sections were dried and embedded for electronmicroscopy, and ultrathin sections were viewed unstained. All immune reagents were obtained from Dako (Heidelberg, Germany).
Paneth cells in rats following whole-body X-ray irradiation contain particles with a periodicity of 6 nanometers.” Tubular structures were seen with crystalloid particle\ in the Paneth cells of irradiated rats.” Furthermore, they were illustrated by Otto and Weitzl? in zinc-deficient rats, although a detailed description was
BIESALSKI er al.: PANETH CELLS
335
TABLE 1. Results of the Zinc Determination in Paneth cells Using X-ray Microanalysis"
Samples (ppm Zinc) Normal Deficient Normal Deficient
130 180
150 160
142 159
151 145
153 148
Mean
SD
P
146.2 162.8
6.5 18.7
0.016
148 195
147 140
149 146
147 180
145 175
Analyses were carried out in duplicate in normal nourished and vitamin A-deficient derived Paneth cells. "
missing. It seems likely that tubular structures are identical with crystalloid particles. It has been speculated'? that a crystallization of lysozyme might be inhibited by zinc, which is normally present in the Paneth ce11.I6 It was also observed that lowered plasma levels of vitamin A are correlated with zinc deficiency." A direct influence of vitamin A deficiency, however, on tissue zinc concentration has not yet been demonstrated. We measured the zinc concentrations in Paneth cells in vitamin A-deficient and vitamin A-supplemented rats using X-ray microanalysis; however, in the deficient group the zinc concentration was even slightly significantly higher than in the Paneth cells of the normal nourished group (TABLEI). It seems more reasonable to speculate that the occurrence of crystalloid particles in the Paneth cells during vitamin A deficiency is caused by a disturbance in the composition of the secretion products of Paneth cells due to this deficiency. Further studies are necessary of elucidate the mechanisms and the consequences of the occurrence of crystalloid particles on Paneth cell function. It could be speculated that crystalloid particles in Paneth cells during vitamin A deficiency may be associated with impaired immune function of the Paneth cell, inasmuch as following irradiation, zinc deficiency or actinomycin C treatment-showing similar morphological alteration-an impaired immune function of the Paneth cell has already been documented. If the function of lysozyme as a local defense mechanism against bacterial infections is impaired due to decreased secretion or impaired solubility, this will give an explanation for the increased risk of intestinal infections during vitamin A deficiency. REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9.
ZILE,M., E. C. BUNGE& D. E. LUCA.1981. J . Nutr. 111: 777-788. CHYTIL,F. 1985. Acta Vitaminol. Enzymol. 7: 27-31. STOFFT,E., H. K. BIESALSKI, U. NIEDERAUER, A. ZSCHABITZ & H. WEISER.1992. Int. J . Vitam. Nutr. Res. 62: 134-142. BIESALSKI, H. K . & E. STOFFT.1992. Ann. N.Y. Acad. Sci. This volume. GORDON,J., T. H. INGALS.1988. J. Nutr. 114: 113-115. SANDOW, M. J. & R. WHITEHEAD. 1979. Gut 20: 420-431. SPEECE, A. J . 1964. J. Histochem. Cytochem. 12: 384-391. RODNING, C. B. & I. D. WILSON.1976. Lancet i: 984-987. L E C H ~ NDEE LA PORTE,P., H. LAFONT& D. LOMBARDO.1986. Histochemistry 86: 21 1-214.
336 10. 11.
12. 13. 14. 1s. 16. 17. 18.
ANNALS NEW YORK ACADEMY OF SCIENCES BOHEM., C. LINDSTROM & K. OHLSSON.1986. Scand. J . Gastroenterol. 21: 65-68. S ~ N ~ G A S - B A F., L AD. S . BALAS,R. VERGER, A. DE CARLO,C. FIGARELLA, F. FERRARO, P. LECHENE, C. BERTRAND & A. RIBET.1984. Histochemistry 81: 581-584. OTTO, H. F. & H. WEITZ.1972. Beitr. Pathol. 145: 336-349. L I N S SW. , 1976. 2. Mikrosk. Anat. Forsch. 85: 449-468. Bi H N K E , 0. & H. MOE. 1964. J. Cell Biol. 22: 633-652. LAMPRECHT. J . & Z. LEwIcKi. 1976. Ann. Med. Sect. Pol. Acad. Sci. 21: 69-70. DINSDALE, D. 1984. J . Histochem. Cytochem. 32: 139-145. S M I T H , J . C., E. D. BROWN, E. G. MCDANIEL & W. C H A N1976. . J . Nutr. 106: 569-574. SI ERNBERGER, L . A , , P. H. HARDY, J. J . CUCULIS & H. G . MEYER.1970. J. Histochem. Cytochem. 18: 3 15-333.
The influence of vitamin A deficiency on epithelial tissues has been studied extensively. I.? The mucous membranes of the respiratory tract are especially a f f e ~ t e d . ~ This ciiuscs typical functional changes in the tracheobronchial tract.4 In contrast to the tespiratory epithelium, less is known concerning changes in intestinal epithelium diiring vitamin A deficiency. In mild vitamin A deficiency, however, infectious diseases of the intestinal tract are a serious problem.' Because the intestinal mucoii\ membrane seems unaffected during vitamin A deficiency, a disturbance of 1oc;il immunity might be the reason for the increase in infectious diseases. Paneth cells provide a major barrier against bacterial infections. Paneth cells are found at the base of the crypts in the small intestine. They are one of the four cellular elements of the intestinal epithelium, the other three being enterocytes, goblet cells, and endocrine cells. It has been suggested that they are involved in protecting the intestine against infection' due to their secretory capacity for lysozyme and other substances. Lysozyme, the main secretory product of the Paneth cell,' is an enzyme capable of degrading the cell walls of most bacteria. In addition, Paneth cells secrete immunoglobulines' and other enzymes, such a s carboxylicester hydrolase,' pancreatic enzymes,"' and intestinal phospholipase A2." These enzymes also may be involved in the elimination of microorganisms in the small intestine.'."'
RESULTS AND DISCUSSION Roil-like particles were observed in granules of Paneth cells in six of the eight vitamin A-deficient animals (FIG.I ) . The particles were composed of electrondense parallel stripes. Mostly, the stripes were arranged diagonally along the length of the axis of the particles. In some cases, latice-like, dense network-like o r granulated, and ladder-rung-like particles were found. The periodicity was about 332
BIESALSKI et al.: PANETH CELLS
333
8-9 nm in the diagonally arranged particles (range 6.4 nm to 12.5 nm). The particles measured up to 2 p m in length and 0.5 pm in width. The particles occurred in vacuoles without traceable secretion products and in secretory granules, which also contained morphologically unaltered secretion
FIGURE 1. Figure shows different views of crystalloid particles in Paneth cells of vitamin A-depleted rats. Two groups of 4-week-old male Sprague-Dawley rats (8 animals each) were fed a vitamin A-free semisynthetic diet for about two months. One group received, in addition, 100 I U of vitamin A each day via pharyngeal tube (control group). At the time of sacrifice, the vitamin A-deficient animals were in an early stage of deficiency, and thus typical symptoms such as Bitot's spots or xerophthalmia were not yet manifest. Deficiency was verified by the determination of plasma retinol and liver retinyl-ester concentration with high performance liquid chromatography (data not shown). Specimens were taken from the jejunum 8 cm distal from the upper end of the radix mesenterii and postfixed by immersion in 2% osmium tetroxide in 0.2 M PBS (pH 7.4), then dehydrated in a graded series of ethanol and propylene oxide and subsequently embedded in Epon 812. Ultrathin sections were placed on 300-mesh copper grids and stained with uranyl acetate and lead citrate. Electron microscopy was performed with a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany).
products. Secretory granules lacking particles showed no changes compared with controls. The particles occurred predominantly in granules of the Paneth cells. The electron-immunohistochemical evaluation showed a clear lysozyme immunoreactivity to the crystalloid particles. It was hypothesized by Otto and WeitzI2and Linss,I3 who detected similar structures in actinomycin-treated and zinc-deficient
334
ANNALS NEW YORK ACADEMY OF SCIENCES
rats, that these structures may reflect a crystalline form of lysozyme. Following this assumption, our immunoelectronrnicroscopic evaluation shows a clear lysozyme imniunoreactivity of the crystalloid particles (FIG.2 ) . Cr) stalloid particles in secretory granules of Paneth cells have been described in 20-day-old rats and have been found to display a periodicity of 8 nanometers.14
FIGURE 2. Rod-like crystalloid particles showing a strong lysozyme immunoreactivity. Stripes are covered by the immunoreaction product. For immunoelectron microscopy. three deficient rats (with the same diet as described above) were killed under ether anesthesia by decapitation. Specimens of jejunum were rapidly removed (as described above) and immersion-fixed in 2% periodate-lysine-paraformaldehyde,buffered in 0. I M PBS. For cryoprotection, samples were pretreated in a graded series of sucrose buffer (796, IS%’, 2092, 40%, and 60%, sucrose in 0. I M PBS) for 4 h in each concentration. The samples were then frozen in liquid nitrogen. The peroxidase-antiperoxidase method according to Sternberger.I8 using an antiserum against human lysozyme (cross-reaction with rat lysozyme) from rabbit, was perlormed. The dilution of the antiserum was I : 75 and I : 150; the second antiserum (anti-rabbit IgG antiserum, peroxide labeled, from goat) was diluted I : 50. Five pm-thick frozen scctions were incubated for 30 min at 20°C in a humidified chamber. Further procedures were carried out according Sternberger.Ix Conventional negative and positive controls were prepared. The frozen sections were dried and embedded for electronmicroscopy, and ultrathin sections were viewed unstained. All immune reagents were obtained from Dako (Heidelberg, Germany).
Paneth cells in rats following whole-body X-ray irradiation contain particles with a periodicity of 6 nanometers.” Tubular structures were seen with crystalloid particle\ in the Paneth cells of irradiated rats.” Furthermore, they were illustrated by Otto and Weitzl? in zinc-deficient rats, although a detailed description was
BIESALSKI er al.: PANETH CELLS
335
TABLE 1. Results of the Zinc Determination in Paneth cells Using X-ray Microanalysis"
Samples (ppm Zinc) Normal Deficient Normal Deficient
130 180
150 160
142 159
151 145
153 148
Mean
SD
P
146.2 162.8
6.5 18.7
0.016
148 195
147 140
149 146
147 180
145 175
Analyses were carried out in duplicate in normal nourished and vitamin A-deficient derived Paneth cells. "
missing. It seems likely that tubular structures are identical with crystalloid particles. It has been speculated'? that a crystallization of lysozyme might be inhibited by zinc, which is normally present in the Paneth ce11.I6 It was also observed that lowered plasma levels of vitamin A are correlated with zinc deficiency." A direct influence of vitamin A deficiency, however, on tissue zinc concentration has not yet been demonstrated. We measured the zinc concentrations in Paneth cells in vitamin A-deficient and vitamin A-supplemented rats using X-ray microanalysis; however, in the deficient group the zinc concentration was even slightly significantly higher than in the Paneth cells of the normal nourished group (TABLEI). It seems more reasonable to speculate that the occurrence of crystalloid particles in the Paneth cells during vitamin A deficiency is caused by a disturbance in the composition of the secretion products of Paneth cells due to this deficiency. Further studies are necessary of elucidate the mechanisms and the consequences of the occurrence of crystalloid particles on Paneth cell function. It could be speculated that crystalloid particles in Paneth cells during vitamin A deficiency may be associated with impaired immune function of the Paneth cell, inasmuch as following irradiation, zinc deficiency or actinomycin C treatment-showing similar morphological alteration-an impaired immune function of the Paneth cell has already been documented. If the function of lysozyme as a local defense mechanism against bacterial infections is impaired due to decreased secretion or impaired solubility, this will give an explanation for the increased risk of intestinal infections during vitamin A deficiency. REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9.
ZILE,M., E. C. BUNGE& D. E. LUCA.1981. J . Nutr. 111: 777-788. CHYTIL,F. 1985. Acta Vitaminol. Enzymol. 7: 27-31. STOFFT,E., H. K. BIESALSKI, U. NIEDERAUER, A. ZSCHABITZ & H. WEISER.1992. Int. J . Vitam. Nutr. Res. 62: 134-142. BIESALSKI, H. K . & E. STOFFT.1992. Ann. N.Y. Acad. Sci. This volume. GORDON,J., T. H. INGALS.1988. J. Nutr. 114: 113-115. SANDOW, M. J. & R. WHITEHEAD. 1979. Gut 20: 420-431. SPEECE, A. J . 1964. J. Histochem. Cytochem. 12: 384-391. RODNING, C. B. & I. D. WILSON.1976. Lancet i: 984-987. L E C H ~ NDEE LA PORTE,P., H. LAFONT& D. LOMBARDO.1986. Histochemistry 86: 21 1-214.
336 10. 11.
12. 13. 14. 1s. 16. 17. 18.
ANNALS NEW YORK ACADEMY OF SCIENCES BOHEM., C. LINDSTROM & K. OHLSSON.1986. Scand. J . Gastroenterol. 21: 65-68. S ~ N ~ G A S - B A F., L AD. S . BALAS,R. VERGER, A. DE CARLO,C. FIGARELLA, F. FERRARO, P. LECHENE, C. BERTRAND & A. RIBET.1984. Histochemistry 81: 581-584. OTTO, H. F. & H. WEITZ.1972. Beitr. Pathol. 145: 336-349. L I N S SW. , 1976. 2. Mikrosk. Anat. Forsch. 85: 449-468. Bi H N K E , 0. & H. MOE. 1964. J. Cell Biol. 22: 633-652. LAMPRECHT. J . & Z. LEwIcKi. 1976. Ann. Med. Sect. Pol. Acad. Sci. 21: 69-70. DINSDALE, D. 1984. J . Histochem. Cytochem. 32: 139-145. S M I T H , J . C., E. D. BROWN, E. G. MCDANIEL & W. C H A N1976. . J . Nutr. 106: 569-574. SI ERNBERGER, L . A , , P. H. HARDY, J. J . CUCULIS & H. G . MEYER.1970. J. Histochem. Cytochem. 18: 3 15-333.
