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Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains
lnrernarional Journalfor Printed in Grror Brirarn
Parasrlology
Vol. 22, No. 7,pp.
1033-1035,
002%7519/92 $5.00 + 0.N Pergamon Press Lfd 10 1992 Au_srralian Socieryfor Parasirology
1992
RESEARCH NOTE
MUCOSAL MAST CELL RESPONSE TO HYMENOLEPIS DIMINUTA INFECTION IN DIFFERENT RAT STRAINS AKIRA ISHIH Department of Parasitology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 43 1-31,Japan (Received 29 May 1992; accepted 28 July 1992) Abstract-ISHIH A. 1992. Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains. International Journalfor Parasitology 22: 1033-1035. Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminufa from DA rats.
INDEX
KEY WORDS:
Hymenolepis diminuta; rat strain; mucosal mast cells; worm expulsion.
IN recent years the mechanisms by which hosts expel intestinal helminth parasites have been of considerable interest, yet many aspects of these processes remain poorly understood. It is known that the expulsion of enteric parasitic helminths from mammals is mediated immunologically and is accompanied by a variety of host cellular responses (Rothwell, 1989). One of the cellular responses associated with the expulsion of intestinal worms is the development of increased numbers of mucosal mast cells in the gut wall of infected hosts, the increase often being correlated with the time of rejection of the worms (Andreassen, Hindsbo & Ruitenberg, 1978; Befus & Bienenstock, 1979; Befus, Johnston & Bienenstock, 1979; Woodbury, Miller, Huntley, Palliser & Wakelin, 1984). In laboratory rats, Hymenolepis diminuta can grow and survive for the life of the host (Read, 1967) while in mice it does not survive to reach maturity, destrobilating on the 12th day (Hopkins, Subramanian & Stallard, 1972). Furthermore, rat strain-dependent variation in the establishment and survival of this cestode has been shown (Ishih, Nishimura & Sano, in press). Despite considerable interest in H. diminutainduced mastocytosis of the alimentary tract in mice, little is known ofthe mucosal mast cell response in rats infected with this cestode (Featherston & Copeman, 1990). The present experiment was designed to examine the intestinal mast cell responses in different rat strains to determine if these responses were related to worm establishment and survival. Two inbred rat strains, DA and F34l/N, were purchased from Japan SLC, Inc. and SPF male rats, 5
weeks old, were used. All rats were inoculated via stomach tube under light anesthesia with 10 H. diminuta cysticercoids in Hanks’ balanced salt solution. H. diminuta was maintained by cyclical passage through Wistar rats and the flour beetle, Tribolium confisum as described elsewhere (Ishih et aI., in press). For experiments, the cysticercoids, at least 4 weeks old when dissected from infected flour beetles, were pooled and administered immediately to the rats. Infected rats were arranged in groups of five, and fed ad fibitum on a commercial MM3 diet (Funabashi Farms, Japan) and water. Four rats in each strain were used as a non-infected group. At 3 and 6 weeks post-infection (p.i.), respectively, five rats from each infected group were killed for autopsy. Non-infected control rats were killed at week 6. The small intestine was removed from each rat, the worms flushed out with warm balanced salt solution, and the total number of scolices recovered from each rat recorded. A 2-3 cm long section was removed from the middle part of the small intestine, opened longitudinally, and fixed overnight in Carnoy’s fluid (Enerback, 1966). Specimens of tissue were dehydrated, embedded in paraffin, sections 6 pm thick cut along the long axis of the gut, and stained for 20 min in Astra Blue (Bloom & Kelly, 1960). Mast cells were counted in each of 20 villus-crypt units (Miller & Jarrett, 1971). The mean values were compared by the Student’s t-test. P values less than 0.05 were considered to be statistically significant. The results of the investigation are summarized in Table 1. At 3 weeks p.i., means of 96 and 98% of the
1033
1034 TABLE I
A.
-MEAN WORMRECOVERY ANDMEANNUMBERDP MUCOSALMASTCELLS (MMC) PER 20 YILLUS-CRYPT UNITS(VCU) INFECTED CONTROLS AND EXPERIMENTAL RATS INFECTED WITH 10 CYSTKERCOIIJS OF Hymenolepis diminutu
Rat strain
F344,‘N
DA
ISHIH
Week*
No. rats infected/ No. rats autopsied
6 3 6 6 3 6
O/4? 515 5/5 O/4? 515 4!5
Mean No. worms i SD. 9.6zlzO.5 10.OfO.O _ 9.8 rt 0.4 a 3.0f 1.9 a
PROM NON-
Mean No.MMC / 20 VCU iSO 104.5* 11.8 Y0.6f 16.6 114.2f21.9 120.3zt6.5 b 129.2f 16.6~ 179.6538.7 bc
* Figures refer to weeks post-infection.
t Non-infected controls. Figures with the same letters are statistically significantly different at the following (Student’s f-test). inoculated worms were recovered from F344jN and DA rat strains, respectively. At 6 weeks 100% of the inoculated worms were recovered from F344/N rats but only 30% were recovered from the DA rats. The mean counts of mucosal mast cells from F344/N rats ranged between 90.6 and 114.2 throughout the experiment and did not change significantly. In DA rats, mucosal mast cell numbers in the non-infected controls and rats 3 weeks pi. were similar. However, mean counts of mucosal mast cells significantly increased from 129.2 at 3 weeks p.i. to 179.6 at 6 weeks p.i. It has been suggested that an increase in the number of intestinal mucosal mast cells could be responsible for destrobilation and expulsion of H. diminutu from mice (Andreassen et nl., 1978). The increase in mucosal mast cells and worm expulsion found in DA strain rats in the present work is consistent with this suggestion. Hindsbo, Andreassen & Ruitenberg (1982) showed that counts of mucosal mast cells in Wistar rats infected with 100 cysticercoids of H. diminuta differed significantly from non-infected controls during a 20 day infection. Featherston & Copeman (1990) also reported H. diminuta-induced mastocytosis in Sprague-Dawley female rats infected with 40 cysticercoids from day 30 p.i. to day 47 p.i. with a reduction in worm recovery. The speed and intensity of mastocytosis may depend on the number of cysticercoids given to rats. In the present study, a strain-dependent variation in mast cell response to H. diminuta infection is suggested, and it seems probable that mucosal mast cells are linked to the expulsion of H. d~~~n~~~ from DA rats. From concurrent infections of Strongyloides ratti and ~i~~~strongy~~s brasiiiensis in nude mice, Nawa & Abe (1991) suggested that at least two different mechanisms, mucosal mast celldependent and -independent, seemed to be operating in host defence against intestinal nematodes. The
levels: a: P
b, c: P co.05
presence of other effector mechanisms in the expulsion of H. d~rn~nufucannot be excluded, and consequently, further investigations concerning non-hematogeneous cells as well as mucosal mast cells are at present being undertaken. REFERENCES ANDREASSEN
J., HINDSBO 0. & RU~TENBERG E. J. 1978. Hymenolepis diminuta infections in congenitally athymic (nude) mice: worm kinetics and intestinal histopathology. Immunology 34: 105-l 13. BEFUS A. D. & BI~NENSTOCK J. 1979. Immunologically mediated intestinal mastocytosis in Nipposrrongyfus br~~iliensis infected rats. tmmunoiogy 3%: 95-101. BEFUS A. D., JOI~NSTON N. & BIENENSTOCK J. 1979. Nippostrongylus hrusiliensis: mast celis and histamine levels in tissue of infected and normal rats. E.xperimental Parasitology 48: l-8. BLOOM G. & KELLY J. W. 1960. The copper phthalocyanine dye ‘Astrablau’ and its staining properties, especially the staining of mast cells. Hi~fochem~e 2: 48-57. ENERBACK L. 1966. Mast cells in rat gastrointestinal mucosa. I. Effects of fixation. Acta Pathoiogiea et Microbiologicu Scandinavica 66: 289-302. FEATHERSTOND. W. & COPEMANC. N. 1990. Mucosal mast cells in Sprague-Dawley rats infected with Hymenolepis diminuta tapeworms. International Journa! for Pora~i~o~ogy20: 401-403. HINDSB~ O., ANDREASSEN J. 81 RLJITENEERG J. 1982. Immunological and histopathological reactions of the rat against the tapeworm Hymenolepis diminula and the effects of anti-thymocyte serum. Parasite Immunology 4: 59-76. HOPKINSC. A., SUBRAMANIAN G. & STALLARDH. E. 1972. The effect of immunosuppressants on the development of Hy~nolepis diminura in mice. P~rasif0~og.v65: 11 L-l 20. ISHIH A., NISHIMURA M. & SANO M. (in press) Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains. Journal of Helminthology. MILLER H. R. P. & JARRETTW. F. H. 1971. Immune reactions in mucous membranes. I. Intestinal mast cell response during he&ninth expulsion in the rat. Immunolqqvu): 277-288.
Research NAWA Y. & ABE T. 1991. Mucosal mastocytosis in the small intestine and its significance in the host defence mechanisms against infection. Journal of Clinical Electron Microscopy 24: 431432. READ C. P. 1967. Longevity of the tapeworm, Hymenolepis diminuta. Journal of Parasilology 53: 1055-1056. ROTHWELL T. L. W. 1989. Immune expulsion of parasitic
Note
1035
nematodes from the alimentary tract. International Journal for Parasitology 19: 139-168. W~~DBURY R. G., MILLER H. R. P., HUNTLEY J. F., PALLEER A. C. & WAKELIN D. 1984. Mucosal mast cells are functionally active during spontaneous expulsion of intestinal nematode infections in rats. Nature (London) 312: 45&452.
Parasrlology
Vol. 22, No. 7,pp.
1033-1035,
002%7519/92 $5.00 + 0.N Pergamon Press Lfd 10 1992 Au_srralian Socieryfor Parasirology
1992
RESEARCH NOTE
MUCOSAL MAST CELL RESPONSE TO HYMENOLEPIS DIMINUTA INFECTION IN DIFFERENT RAT STRAINS AKIRA ISHIH Department of Parasitology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 43 1-31,Japan (Received 29 May 1992; accepted 28 July 1992) Abstract-ISHIH A. 1992. Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains. International Journalfor Parasitology 22: 1033-1035. Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminufa from DA rats.
INDEX
KEY WORDS:
Hymenolepis diminuta; rat strain; mucosal mast cells; worm expulsion.
IN recent years the mechanisms by which hosts expel intestinal helminth parasites have been of considerable interest, yet many aspects of these processes remain poorly understood. It is known that the expulsion of enteric parasitic helminths from mammals is mediated immunologically and is accompanied by a variety of host cellular responses (Rothwell, 1989). One of the cellular responses associated with the expulsion of intestinal worms is the development of increased numbers of mucosal mast cells in the gut wall of infected hosts, the increase often being correlated with the time of rejection of the worms (Andreassen, Hindsbo & Ruitenberg, 1978; Befus & Bienenstock, 1979; Befus, Johnston & Bienenstock, 1979; Woodbury, Miller, Huntley, Palliser & Wakelin, 1984). In laboratory rats, Hymenolepis diminuta can grow and survive for the life of the host (Read, 1967) while in mice it does not survive to reach maturity, destrobilating on the 12th day (Hopkins, Subramanian & Stallard, 1972). Furthermore, rat strain-dependent variation in the establishment and survival of this cestode has been shown (Ishih, Nishimura & Sano, in press). Despite considerable interest in H. diminutainduced mastocytosis of the alimentary tract in mice, little is known ofthe mucosal mast cell response in rats infected with this cestode (Featherston & Copeman, 1990). The present experiment was designed to examine the intestinal mast cell responses in different rat strains to determine if these responses were related to worm establishment and survival. Two inbred rat strains, DA and F34l/N, were purchased from Japan SLC, Inc. and SPF male rats, 5
weeks old, were used. All rats were inoculated via stomach tube under light anesthesia with 10 H. diminuta cysticercoids in Hanks’ balanced salt solution. H. diminuta was maintained by cyclical passage through Wistar rats and the flour beetle, Tribolium confisum as described elsewhere (Ishih et aI., in press). For experiments, the cysticercoids, at least 4 weeks old when dissected from infected flour beetles, were pooled and administered immediately to the rats. Infected rats were arranged in groups of five, and fed ad fibitum on a commercial MM3 diet (Funabashi Farms, Japan) and water. Four rats in each strain were used as a non-infected group. At 3 and 6 weeks post-infection (p.i.), respectively, five rats from each infected group were killed for autopsy. Non-infected control rats were killed at week 6. The small intestine was removed from each rat, the worms flushed out with warm balanced salt solution, and the total number of scolices recovered from each rat recorded. A 2-3 cm long section was removed from the middle part of the small intestine, opened longitudinally, and fixed overnight in Carnoy’s fluid (Enerback, 1966). Specimens of tissue were dehydrated, embedded in paraffin, sections 6 pm thick cut along the long axis of the gut, and stained for 20 min in Astra Blue (Bloom & Kelly, 1960). Mast cells were counted in each of 20 villus-crypt units (Miller & Jarrett, 1971). The mean values were compared by the Student’s t-test. P values less than 0.05 were considered to be statistically significant. The results of the investigation are summarized in Table 1. At 3 weeks p.i., means of 96 and 98% of the
1033
1034 TABLE I
A.
-MEAN WORMRECOVERY ANDMEANNUMBERDP MUCOSALMASTCELLS (MMC) PER 20 YILLUS-CRYPT UNITS(VCU) INFECTED CONTROLS AND EXPERIMENTAL RATS INFECTED WITH 10 CYSTKERCOIIJS OF Hymenolepis diminutu
Rat strain
F344,‘N
DA
ISHIH
Week*
No. rats infected/ No. rats autopsied
6 3 6 6 3 6
O/4? 515 5/5 O/4? 515 4!5
Mean No. worms i SD. 9.6zlzO.5 10.OfO.O _ 9.8 rt 0.4 a 3.0f 1.9 a
PROM NON-
Mean No.MMC / 20 VCU iSO 104.5* 11.8 Y0.6f 16.6 114.2f21.9 120.3zt6.5 b 129.2f 16.6~ 179.6538.7 bc
* Figures refer to weeks post-infection.
t Non-infected controls. Figures with the same letters are statistically significantly different at the following (Student’s f-test). inoculated worms were recovered from F344jN and DA rat strains, respectively. At 6 weeks 100% of the inoculated worms were recovered from F344/N rats but only 30% were recovered from the DA rats. The mean counts of mucosal mast cells from F344/N rats ranged between 90.6 and 114.2 throughout the experiment and did not change significantly. In DA rats, mucosal mast cell numbers in the non-infected controls and rats 3 weeks pi. were similar. However, mean counts of mucosal mast cells significantly increased from 129.2 at 3 weeks p.i. to 179.6 at 6 weeks p.i. It has been suggested that an increase in the number of intestinal mucosal mast cells could be responsible for destrobilation and expulsion of H. diminutu from mice (Andreassen et nl., 1978). The increase in mucosal mast cells and worm expulsion found in DA strain rats in the present work is consistent with this suggestion. Hindsbo, Andreassen & Ruitenberg (1982) showed that counts of mucosal mast cells in Wistar rats infected with 100 cysticercoids of H. diminuta differed significantly from non-infected controls during a 20 day infection. Featherston & Copeman (1990) also reported H. diminuta-induced mastocytosis in Sprague-Dawley female rats infected with 40 cysticercoids from day 30 p.i. to day 47 p.i. with a reduction in worm recovery. The speed and intensity of mastocytosis may depend on the number of cysticercoids given to rats. In the present study, a strain-dependent variation in mast cell response to H. diminuta infection is suggested, and it seems probable that mucosal mast cells are linked to the expulsion of H. d~~~n~~~ from DA rats. From concurrent infections of Strongyloides ratti and ~i~~~strongy~~s brasiiiensis in nude mice, Nawa & Abe (1991) suggested that at least two different mechanisms, mucosal mast celldependent and -independent, seemed to be operating in host defence against intestinal nematodes. The
levels: a: P
b, c: P co.05
presence of other effector mechanisms in the expulsion of H. d~rn~nufucannot be excluded, and consequently, further investigations concerning non-hematogeneous cells as well as mucosal mast cells are at present being undertaken. REFERENCES ANDREASSEN
J., HINDSBO 0. & RU~TENBERG E. J. 1978. Hymenolepis diminuta infections in congenitally athymic (nude) mice: worm kinetics and intestinal histopathology. Immunology 34: 105-l 13. BEFUS A. D. & BI~NENSTOCK J. 1979. Immunologically mediated intestinal mastocytosis in Nipposrrongyfus br~~iliensis infected rats. tmmunoiogy 3%: 95-101. BEFUS A. D., JOI~NSTON N. & BIENENSTOCK J. 1979. Nippostrongylus hrusiliensis: mast celis and histamine levels in tissue of infected and normal rats. E.xperimental Parasitology 48: l-8. BLOOM G. & KELLY J. W. 1960. The copper phthalocyanine dye ‘Astrablau’ and its staining properties, especially the staining of mast cells. Hi~fochem~e 2: 48-57. ENERBACK L. 1966. Mast cells in rat gastrointestinal mucosa. I. Effects of fixation. Acta Pathoiogiea et Microbiologicu Scandinavica 66: 289-302. FEATHERSTOND. W. & COPEMANC. N. 1990. Mucosal mast cells in Sprague-Dawley rats infected with Hymenolepis diminuta tapeworms. International Journa! for Pora~i~o~ogy20: 401-403. HINDSB~ O., ANDREASSEN J. 81 RLJITENEERG J. 1982. Immunological and histopathological reactions of the rat against the tapeworm Hymenolepis diminula and the effects of anti-thymocyte serum. Parasite Immunology 4: 59-76. HOPKINSC. A., SUBRAMANIAN G. & STALLARDH. E. 1972. The effect of immunosuppressants on the development of Hy~nolepis diminura in mice. P~rasif0~og.v65: 11 L-l 20. ISHIH A., NISHIMURA M. & SANO M. (in press) Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains. Journal of Helminthology. MILLER H. R. P. & JARRETTW. F. H. 1971. Immune reactions in mucous membranes. I. Intestinal mast cell response during he&ninth expulsion in the rat. Immunolqqvu): 277-288.
Research NAWA Y. & ABE T. 1991. Mucosal mastocytosis in the small intestine and its significance in the host defence mechanisms against infection. Journal of Clinical Electron Microscopy 24: 431432. READ C. P. 1967. Longevity of the tapeworm, Hymenolepis diminuta. Journal of Parasilology 53: 1055-1056. ROTHWELL T. L. W. 1989. Immune expulsion of parasitic
Note
1035
nematodes from the alimentary tract. International Journal for Parasitology 19: 139-168. W~~DBURY R. G., MILLER H. R. P., HUNTLEY J. F., PALLEER A. C. & WAKELIN D. 1984. Mucosal mast cells are functionally active during spontaneous expulsion of intestinal nematode infections in rats. Nature (London) 312: 45&452.