INFECTION AND IMMUNITY, Aug. 1978, p. 655-658
Vol. 21, No.2
0019-9567/78/0021-0655$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
NOTES Disruption of the Permeability Barrier (Zonula Occludens) Between Intestinal Epithelial Cells by Lethal Doses of Endotoxin RICHARD I. WALKER* AND MARTIN J. PORVAZNIK Experimental Hematology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014 Received for publication 17 January 1978
A freeze-fracture technique was used to examine the most apical intercellular junctional complex between intestinal epithelial cells (the tight or occluding junction) in mice after challenge with endotoxin. Some of these junctions were disrupted after challenge and may be a site for leakage of microbial agents from the lumen.
Persistence of low titers of endotoxin in circulation after challenge may be a serious event during endotoxemia. This concept is supported by the report of decreased hepatic uptake of the toxin when lethal doses are administered, and also by the fact that animals made tolerant to endotoxin will clear large challenges of the toxin from the bloodstream faster than will normal animals (2). Furthermore, when endotoxin is injected into animals intramuscularly, relatively little reaches the liver, but toxin persists in the blood over extended periods of time and is eventually lethal (12). Fine and his co-workers have suggested that a lethal dose of endotoxin stresses an animal to an extent that the barrier function of the gut is lost and endotoxin from the intestinal flora enters the systemic circulation (3, 4, 7, 8). Thus, animals die with more endotoxin in their livers than was injected, but this mortality can be obviated by treatment with oral antibiotics to eliminate gut flora capable of producing endotoxin. We hypothesized that endotoxin-induced inflammation may disrupt the zonula occludens or tight junctions between intestinal epithelial cells and thereby provide a channel or route for microbial agents in the lumen to enter host tissues. This event could be particularly serious because it would permit endotoxin entry via the systemic circulation (6, 9), a route by which animals are more sensitive to the toxin than when it is administered via the portal circulation (11, 13). Male B6CBF1 mice were inoculated intraperitoneally with 0.8 mg (>80% lethal dose) of Sal-
monella typhosa lipopolysaccharide (Difco Laboratories, Detroit, Mich.). At appropriate times the mice were sacrificed by cervical dislocation, and 1-inch (ca. 2.54-cm) segments of the ileum just anterior to the cecum were removed. Tissues were fixed by immersion in cold 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.2). After fixation, the ilea were washed in 0.1 M cacodylate buffer (pH 7.2) and cryoprotected with 30% glycerol, also in 0.1 M cacodylate buffer (pH 7.2). Samples of ilea from at least four experimentally treated animals and four control animals were placed in standard 3-mm gold holders, which were rapidly frozen in Freon 22 followed by liquid nitrogen, fractured at -105oC, and replicated in a Balzers 360 M freeze-etch instrument (Balzers High Vacuum Corp., Liechtenstein) equipped with an electron gun. The intestinal tissue was removed from the replicas by solubilization in methanol for 15 min followed by digestion overnight in chlorine bleach (50% strength). Subsequently, replicas were washed three times in distilled water, placed on 200-mesh copper grids, and examined in a Philips EM 400 electron microscope (Eindhoven, The Netherlands). Cross-fractured tight junctions were not considered since they did not reveal the internal structure of the junctional membrane. Membrane faces were labeled as either protoplasmic fracture faces or exoplasmic fracture faces (1). All micrographs were approxmately oriented with the direction of platinum shadowing from the bottom unless otherwise indicated by an encircled arrow. 655
W7~~ ~ ~ ~ ~
~ ~ ~
__
~
~
~
~
~
~
~
*-P.
-
~~.pi
FIG. 1. Tight junctional complex between epithelial cells of the ileum in control preparations (unstressed mice). Denoted are the lumenal space (L), apical microvillus (M), protoplasmic membrane fracture face (P), exoplasmic membrane fracture face (E), tight junction fibril (), and groove (g). Encircled arrowhead denotes the direction of platinum shadowing. x47,300; bar = 0.5 Imn. FIG. 2. Tight junctional complex between epithelial cells of the ileum from experimentally treated mice (18 h postchallenge with endotoxin). Notice the unraveled appearance of the tight junctional fibrils (f) and their particulate form (arrow). Denoted are the lumenal surface of the epithelial cell (LJ) and cross-fractured apical microvillus (M). x48,860; bar = 0.5 pim. 656
e
I.,CA
A ,' ,,i ,
'-, 4'
Jo.,
4,4.
It
:"
FIG. 3. Exposed tight junctional complex on the exoplasmic fracture face (E) of a goblet cell (18 h postchallenge ivith endotoxin). Notice the complete discontinuity of the tight junction grooves (arrows). Denoted are the apical microvillus (M) and cell cytoplasm of the terminal web (C). Encircled arrowhead denotes the direction ofplatinum shadowing. x42,300; bar = 0.5 Am. FIG. 4. Complete disarray of the apical tight junction complex is shown 18 h postchallenge with endotoxin. Large discontinuities are clearly demonstrated (arrow). x29,500; bar = 0.5 ,um.
657
658
NOTES
Freeze-fracture replicas of control preparations (i.e., nonstressed mice) revealed completely intact zonula occludens-type junctions between epithelial cells of the ileum (Fig. 1). Of 100 tight junctional structures observed, all were intact. This observation is consistent with previously reported descriptions of tight junctions in normal animals (14). However, preparations from endotoxin-challenged mice revealed 5% "leaky" or disrupted tight junctional complexes (5 out of 100 observed) at 5 h after inoculation and 5.4% (9 out of 169 observed) leaky tight junctional complexes 18 h after challenge with endotoxin. The leaky or discontinuous zonula occludens observed in endotoxin-challenged animals are represented in Fig. 2, 3, and 4. In Fig. 2 the concertina-like fibrils or grooves that were commonly observed in control fracture faces (Fig. 1) appeared primarily as loosely organized, linear strands of particles or segmented fibrils on protoplasmic fracture faces. The unraveled appearance and particulate nature of these structures suggested localized disruption of these intercellular junctions. In other cases (Fig. 3 and 4), complete discontinuities in the zonula occludens were observed. In these situations an indisputable pathway existed between the lumenal compartment and the submucosal compartment of the intestine, potentially allowing diffusion of matter into the circulatory system. Under normal circumstances, penetration of the intestinal epithelium by microbial agents in the lumen is thought to occur at epithelial discontinuities overlying the dome regions of gutassociated lymphoid tissue or at the tips of the villi and by pinocytosis (5). Our data indicate that during endotoxemia and possibly in other pathological circumstances, disruption of tight junctional barriers of the intestinal epithelium may be an important route for escape of lumenal contents. It is also noteworthy that injury to the zonula occludens induced by a single lethal injection of endotoxin persisted over an extended period of time and therefore may have contributed to death. Based on studies in the mudpuppy (Necturus maculosus), the normal regeneration time for tight junctions after cessation of stimuli for disruption may be less than 15 min (10). To our knowledge this report is the first evidence for the intercellular route of absorption of microbial factors from the lumen. Recently (manuscript in preparation) we found that the aseptic endotoxemia observed during the first 3
INFECT. IMMUN.
to 4 days after ionizing radiation (15) correlates exactly with the period of tight junction disruption seen after radiation. It is hoped that further studies will be made to establish the importance of this route of penetration of the epithelial barrier during stress. ACKNOWLEDGMENTS We thank M. W. Brightman and T. S. Reese from the Department of Neurocytology, National Institute of Neurological and Communicative Disorders and Stroke, for the use of their Balzers freeze-fracture instrument. LITERATURE CITED 1. Branton, D., S. Bullivant, N. B. Gilula, M. J. Karnovsky, H. Moor, K. Muhlethaler, D. H. Northcote, L. Packer, B. Satir, P. Satir, V. Speth, L. A. Staehelin, R. L. Steere, and R. S. Weinstein. 1975. Freezeetching nomenclature. Science 190:54-56. 2. Carey, F. J., A. I. Braude, and M. Zalesky. 1958. Studies with radioactive endotoxin. mI. The effect of tolerance on the distribution of radioactivity after intravenous injection of Escherichia coli endotoxin labelled with Cr51. J. Clin. Invest. 37:441-457. 3. Caridis, D. T., M. Ishiyama, P. W. H. Woodruff, and J. Fine. 1973. Role of the intestinal flora in clearance and detoxification of circulating endotoxin. RES, J. Reticuloendothel. Soc. 14:513-521. 4. Caridis, D. T., R. B. Reinhold, P. W. H. Woodruff, and J. Fine. 1972. Endotoxemia in man. Lancet i:1381-1386. 5. Cottier, H., T. Schaffner, A. D. Chanana, D. D. Joel, B. Sordat, B. J. Bryant, and M. W. Hess. 1975. Species differences in the effectiveness of intestinal barriers against penetration of inert particulates and bacteria, p. 80-85. In B. Urbaschek, R. Urbaschek, and E. Neter (ed.), Gram-negative bacterial infections and mode of endotoxin actions. Springer-Verlag, Vienna. 6. Cuevas, P., and J. Fine. 1972. Route of absorption of endotoxin from the intestine in nonseptic shock. RES, J. Reticuloendothel. Soc. 11:535-538. 7. Cuevas, P., and J. Fine. 1973. Production of fatal endotoxic shock by vasoactive substances. Gastroenterology 64:285-291. 8. Fine, J. 1972. Endotoxaemia in man. Lancet ii:181. 9. Gans, H., and K. Matsumoto. 1974. The escape of endotoxin from the intestine. Surg. Gynecol. Obstet. 139:395-402. 10. Hudspeth, A. J. 1975. Establishment of tight junctions between epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 72:2711-2713. 11. Mori, K., K. Matsumoto, and H. Gans. 1973. On the in vivo clearance and detoxification of endotoxin by lung and liver. Ann. Surg. 177:159-163. 12. Noyes, H. E., C. R. McInturf, and G. J. Blahuta. 1959. Studies on distribution of Escherichia coli endotoxin in mice. Proc. Soc. Exp. Biol. Med. 100:65-68. 13. Rutenburg, S., R. Skarnes, C. Palmenio, and J. Fine. 1967. Detoxification of endotoxin by perfusion of liver and spleen. Proc. Soc. Exp. Biol. Med. 125:455-459. 14. Staehelin, L A. 1974. Structure and function of intercellular junctions. Int. Rev. Cytol. 39:191-283. 15. Walker, R. I., G. D. Ledney, and C. B. Galley. 1975. Aseptic endotoxemia in radiation injury and graft-vshost disease. Radiat. Res. 62:242-249.
Vol. 21, No.2
0019-9567/78/0021-0655$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
NOTES Disruption of the Permeability Barrier (Zonula Occludens) Between Intestinal Epithelial Cells by Lethal Doses of Endotoxin RICHARD I. WALKER* AND MARTIN J. PORVAZNIK Experimental Hematology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014 Received for publication 17 January 1978
A freeze-fracture technique was used to examine the most apical intercellular junctional complex between intestinal epithelial cells (the tight or occluding junction) in mice after challenge with endotoxin. Some of these junctions were disrupted after challenge and may be a site for leakage of microbial agents from the lumen.
Persistence of low titers of endotoxin in circulation after challenge may be a serious event during endotoxemia. This concept is supported by the report of decreased hepatic uptake of the toxin when lethal doses are administered, and also by the fact that animals made tolerant to endotoxin will clear large challenges of the toxin from the bloodstream faster than will normal animals (2). Furthermore, when endotoxin is injected into animals intramuscularly, relatively little reaches the liver, but toxin persists in the blood over extended periods of time and is eventually lethal (12). Fine and his co-workers have suggested that a lethal dose of endotoxin stresses an animal to an extent that the barrier function of the gut is lost and endotoxin from the intestinal flora enters the systemic circulation (3, 4, 7, 8). Thus, animals die with more endotoxin in their livers than was injected, but this mortality can be obviated by treatment with oral antibiotics to eliminate gut flora capable of producing endotoxin. We hypothesized that endotoxin-induced inflammation may disrupt the zonula occludens or tight junctions between intestinal epithelial cells and thereby provide a channel or route for microbial agents in the lumen to enter host tissues. This event could be particularly serious because it would permit endotoxin entry via the systemic circulation (6, 9), a route by which animals are more sensitive to the toxin than when it is administered via the portal circulation (11, 13). Male B6CBF1 mice were inoculated intraperitoneally with 0.8 mg (>80% lethal dose) of Sal-
monella typhosa lipopolysaccharide (Difco Laboratories, Detroit, Mich.). At appropriate times the mice were sacrificed by cervical dislocation, and 1-inch (ca. 2.54-cm) segments of the ileum just anterior to the cecum were removed. Tissues were fixed by immersion in cold 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.2). After fixation, the ilea were washed in 0.1 M cacodylate buffer (pH 7.2) and cryoprotected with 30% glycerol, also in 0.1 M cacodylate buffer (pH 7.2). Samples of ilea from at least four experimentally treated animals and four control animals were placed in standard 3-mm gold holders, which were rapidly frozen in Freon 22 followed by liquid nitrogen, fractured at -105oC, and replicated in a Balzers 360 M freeze-etch instrument (Balzers High Vacuum Corp., Liechtenstein) equipped with an electron gun. The intestinal tissue was removed from the replicas by solubilization in methanol for 15 min followed by digestion overnight in chlorine bleach (50% strength). Subsequently, replicas were washed three times in distilled water, placed on 200-mesh copper grids, and examined in a Philips EM 400 electron microscope (Eindhoven, The Netherlands). Cross-fractured tight junctions were not considered since they did not reveal the internal structure of the junctional membrane. Membrane faces were labeled as either protoplasmic fracture faces or exoplasmic fracture faces (1). All micrographs were approxmately oriented with the direction of platinum shadowing from the bottom unless otherwise indicated by an encircled arrow. 655
W7~~ ~ ~ ~ ~
~ ~ ~
__
~
~
~
~
~
~
~
*-P.
-
~~.pi
FIG. 1. Tight junctional complex between epithelial cells of the ileum in control preparations (unstressed mice). Denoted are the lumenal space (L), apical microvillus (M), protoplasmic membrane fracture face (P), exoplasmic membrane fracture face (E), tight junction fibril (), and groove (g). Encircled arrowhead denotes the direction of platinum shadowing. x47,300; bar = 0.5 Imn. FIG. 2. Tight junctional complex between epithelial cells of the ileum from experimentally treated mice (18 h postchallenge with endotoxin). Notice the unraveled appearance of the tight junctional fibrils (f) and their particulate form (arrow). Denoted are the lumenal surface of the epithelial cell (LJ) and cross-fractured apical microvillus (M). x48,860; bar = 0.5 pim. 656
e
I.,CA
A ,' ,,i ,
'-, 4'
Jo.,
4,4.
It
:"
FIG. 3. Exposed tight junctional complex on the exoplasmic fracture face (E) of a goblet cell (18 h postchallenge ivith endotoxin). Notice the complete discontinuity of the tight junction grooves (arrows). Denoted are the apical microvillus (M) and cell cytoplasm of the terminal web (C). Encircled arrowhead denotes the direction ofplatinum shadowing. x42,300; bar = 0.5 Am. FIG. 4. Complete disarray of the apical tight junction complex is shown 18 h postchallenge with endotoxin. Large discontinuities are clearly demonstrated (arrow). x29,500; bar = 0.5 ,um.
657
658
NOTES
Freeze-fracture replicas of control preparations (i.e., nonstressed mice) revealed completely intact zonula occludens-type junctions between epithelial cells of the ileum (Fig. 1). Of 100 tight junctional structures observed, all were intact. This observation is consistent with previously reported descriptions of tight junctions in normal animals (14). However, preparations from endotoxin-challenged mice revealed 5% "leaky" or disrupted tight junctional complexes (5 out of 100 observed) at 5 h after inoculation and 5.4% (9 out of 169 observed) leaky tight junctional complexes 18 h after challenge with endotoxin. The leaky or discontinuous zonula occludens observed in endotoxin-challenged animals are represented in Fig. 2, 3, and 4. In Fig. 2 the concertina-like fibrils or grooves that were commonly observed in control fracture faces (Fig. 1) appeared primarily as loosely organized, linear strands of particles or segmented fibrils on protoplasmic fracture faces. The unraveled appearance and particulate nature of these structures suggested localized disruption of these intercellular junctions. In other cases (Fig. 3 and 4), complete discontinuities in the zonula occludens were observed. In these situations an indisputable pathway existed between the lumenal compartment and the submucosal compartment of the intestine, potentially allowing diffusion of matter into the circulatory system. Under normal circumstances, penetration of the intestinal epithelium by microbial agents in the lumen is thought to occur at epithelial discontinuities overlying the dome regions of gutassociated lymphoid tissue or at the tips of the villi and by pinocytosis (5). Our data indicate that during endotoxemia and possibly in other pathological circumstances, disruption of tight junctional barriers of the intestinal epithelium may be an important route for escape of lumenal contents. It is also noteworthy that injury to the zonula occludens induced by a single lethal injection of endotoxin persisted over an extended period of time and therefore may have contributed to death. Based on studies in the mudpuppy (Necturus maculosus), the normal regeneration time for tight junctions after cessation of stimuli for disruption may be less than 15 min (10). To our knowledge this report is the first evidence for the intercellular route of absorption of microbial factors from the lumen. Recently (manuscript in preparation) we found that the aseptic endotoxemia observed during the first 3
INFECT. IMMUN.
to 4 days after ionizing radiation (15) correlates exactly with the period of tight junction disruption seen after radiation. It is hoped that further studies will be made to establish the importance of this route of penetration of the epithelial barrier during stress. ACKNOWLEDGMENTS We thank M. W. Brightman and T. S. Reese from the Department of Neurocytology, National Institute of Neurological and Communicative Disorders and Stroke, for the use of their Balzers freeze-fracture instrument. LITERATURE CITED 1. Branton, D., S. Bullivant, N. B. Gilula, M. J. Karnovsky, H. Moor, K. Muhlethaler, D. H. Northcote, L. Packer, B. Satir, P. Satir, V. Speth, L. A. Staehelin, R. L. Steere, and R. S. Weinstein. 1975. Freezeetching nomenclature. Science 190:54-56. 2. Carey, F. J., A. I. Braude, and M. Zalesky. 1958. Studies with radioactive endotoxin. mI. The effect of tolerance on the distribution of radioactivity after intravenous injection of Escherichia coli endotoxin labelled with Cr51. J. Clin. Invest. 37:441-457. 3. Caridis, D. T., M. Ishiyama, P. W. H. Woodruff, and J. Fine. 1973. Role of the intestinal flora in clearance and detoxification of circulating endotoxin. RES, J. Reticuloendothel. Soc. 14:513-521. 4. Caridis, D. T., R. B. Reinhold, P. W. H. Woodruff, and J. Fine. 1972. Endotoxemia in man. Lancet i:1381-1386. 5. Cottier, H., T. Schaffner, A. D. Chanana, D. D. Joel, B. Sordat, B. J. Bryant, and M. W. Hess. 1975. Species differences in the effectiveness of intestinal barriers against penetration of inert particulates and bacteria, p. 80-85. In B. Urbaschek, R. Urbaschek, and E. Neter (ed.), Gram-negative bacterial infections and mode of endotoxin actions. Springer-Verlag, Vienna. 6. Cuevas, P., and J. Fine. 1972. Route of absorption of endotoxin from the intestine in nonseptic shock. RES, J. Reticuloendothel. Soc. 11:535-538. 7. Cuevas, P., and J. Fine. 1973. Production of fatal endotoxic shock by vasoactive substances. Gastroenterology 64:285-291. 8. Fine, J. 1972. Endotoxaemia in man. Lancet ii:181. 9. Gans, H., and K. Matsumoto. 1974. The escape of endotoxin from the intestine. Surg. Gynecol. Obstet. 139:395-402. 10. Hudspeth, A. J. 1975. Establishment of tight junctions between epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 72:2711-2713. 11. Mori, K., K. Matsumoto, and H. Gans. 1973. On the in vivo clearance and detoxification of endotoxin by lung and liver. Ann. Surg. 177:159-163. 12. Noyes, H. E., C. R. McInturf, and G. J. Blahuta. 1959. Studies on distribution of Escherichia coli endotoxin in mice. Proc. Soc. Exp. Biol. Med. 100:65-68. 13. Rutenburg, S., R. Skarnes, C. Palmenio, and J. Fine. 1967. Detoxification of endotoxin by perfusion of liver and spleen. Proc. Soc. Exp. Biol. Med. 125:455-459. 14. Staehelin, L A. 1974. Structure and function of intercellular junctions. Int. Rev. Cytol. 39:191-283. 15. Walker, R. I., G. D. Ledney, and C. B. Galley. 1975. Aseptic endotoxemia in radiation injury and graft-vshost disease. Radiat. Res. 62:242-249.
