parvum development
veterinary parasitology ELSEVIER
Veterinary Parasitology68 (1997) 305-308
The effects of IFN-'y activated mouse peritoneal and alveolar macrophages on Cryptosporidium parvum development F. Martinez, M.J. Rosales, J. Diaz, C. Mascaro * Institute of Biotechnology, Schoolof Science, Universityof Granada, E-18071 Granada, Spain
Received 6 March 1996; accepted2 August 1996
Abstract
Mouse peritoneal and alveolar macrophages were interacted in vitro with C. parvum oocysts and cultured in normal medium and in medium with IFN-~/. The results showed that in vitro activation of macrophages by IFN-~/ limits C. parvum development although the inhibitory effect is not as potent as in other intracellular parasitic protozoa. Keywords: Cryptosporidiumparvum; Macrophages;IFN-~/
1. Introduction Phenotypic and functional changes induced in macrophages in response to T-cells can be simulated in vitro by culturing macrophages with IFN-~/. In the case of Trypanosoma cruzi (Kuhn, 1994), Toxoplasma gondii (Beaman et al., 1994), Leishmania (Titus et al., 1994) and other protozoa and bacteria, in vitro macrophage activation by IFN-'y inhibits intracellular growth according to a dose time relationship. Both oxidative and nonoxidative mechanisms are involved in most cases. Human respiratory cryptosporidiosis is well known and intracellular Cryptosporidium oocysts have been detected in human alveolar macrophages (ViUanueva et al., 1984), suggesting a possible hematogenous spread of this agent. These data and the demonstrated development of C. parvum in mouse peritoneal macrophages (Martinez et al., 1992), lead us to study the action of IFN-~ on the development of Cryptosporidium in both peritoneal and alveolar macrophages. Previously, Ungar et al. (1991) suggested that IFN-~t is involved in the in vivo control of cryptosporidiosis, reporting that mice treated
* Corresponding author. Fax: + 34 58 243174; e-mail: [email protected]. 0304-4017/97/$17.00 Copyright © 1997 Elsevier Science B.V, All fights reserved. PII S0304-401 7(96)01087-4
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F. Martinez et a l . / Veterinary Parasitology 68 (1997) 305-308
with neutralizing anti-IFN-~ monoclonal antibody terminated the infection by enhanced oocysts shedding. 2. Materials and methods
Cryptosporidium parvum oocysts were obtained and purified according to previously described methodology (Rosales et al., 1994). Mouse peritoneal macrophages were obtained also as previously reported (Martinez et al., 1992). Mouse alveolar macrophages were obtained from Swiss albino mice, following Black et al. (1987). The cells were concentrated by centrifugation at 2500 × g for 5 min, and the sediment was resuspended in RPMI 1640 medium (Gibco, USA) with L-glutamine and 10% inactivated fetal calf serum (IFCS). The macrophages were then counted and distributed in flat-bottomed microtiter plates (Costal Cambridge, UK), with a pre-cut glass disk in the bottom of each well. Adhesion of the cells to the glass disk was confirmed by microscopy after 12 h, and C parvum oocysts were added to macrophages cultures in a 1:1 proportion. After 12 h the medium was replaced, and parasitized macrophages were cultured either in normal medium or in medium containing IFN-~/ (Sigma) at a concentration of 1000 Uml-i. Cultures were interrupted at different time intervals by fixation with methanol, and stained with alcian-blue and Giemsa for observation by light microscope (Rosales et al., 1993). For each culture the number of macrophages harboring C. parvum forms were counted per 100 macrophages (% MP). Since multiparasitism was seen in peritoneal macrophages, we calculated not only the percentage of parasitized macrophages in each condition, but also the Cryptosporidium/macrophage relationship (C/M). 3. Results and discussion
Table 1 shows the results obtained when peritoneal macrophages were allowed to interact with C. parvum oocysts and then cultured either in normal medium or in medium containing IFN-~/. Cultures were interrupted after 3, 24, 36 and 48 h. At three hours, activated macrophages had clearly increased their phagocytic capacity (36.8% of parasitized macrophages in control cultures vs. 50.5% in cultures containing IFN-~). The C / M relationship was also higher in control cultures than in cultures containing IFN-'y (0.5 vs. 0.7). Both relationship remained relatively constant throughout the experiments, suggesting that macrophages stimulated by IFN-~/ did not completely suppress parasite development. A detailed microscopic study (Table 1) revealed that at 24, 36 and 48 h, a certain proportion of Cryptosporidiura forms was still undifferentiated, without nuclear accumulations or differentiated merozoites, and that the undifferentiated forms were more frequent after 24 h in IFN-~/cultures (70.7, 50 and 32.4%) than in control cultures (22.7, 10.6 and 8.7%). This suggests that IFN-3, under the conditions in this study, did not stop the growth on C. parvum within the macrophages but increased the percentage of parasite forms that were destroyed when ingested. Free parasitic forms (Table 1) at first
F. Martinez et al. / Veterinary Parasitology 68 (1997) 305-308
307
Table 1 Percentage of parasitized mouse peritonealmaerophages and of different Cryptosporidium developmentstages in cultures inoculatedwith oocysts PT=3h % MP C/M F I Nd
PT = 24h
PT= 36h
PT= 48 h
C
T
C
T
C
T
C
T
36.8 0.5 23.4 52.9 23.4
50.5 0.7 11.6 59.7 28.5
30.4 0.5 19.5 27.4 22.7
79.7 1.3 2.3 26.7 70.7
16.8 0.3 41.3 47.9 10.6
48.2 0.5 2.8 47 50
54.1 0.6 64.9 26.1 8.7
79.4 1.5 5.1 61.7 32.4
PT = post-inoculationtime; C = control; T = cultures treated with IFN-~ (1000 U/ml); % MP = percentage of macrophages harboring Cryptosporidium forms; C/M = relationship Cryptosporidium/macrophage; F = free forms; I = intracellularforms; Nd = undifferentiatedforms. were probably oocysts, but afterwards were primarily forms released into the medium after the death of the host cell. The number of free forms was always higher in control cultures than in IFN-~/ cultures. This was probably due to an increased phagocytic capacity of IFN-~ treated macrophages. Alveolar macrophages were allowed to interact with oocysts for 12 and 24 h either in normal medium or in medium containing IFN-~/. It was not possible to maintain alveolar macrophage cultures in good condition for more than 24 h; after this time, they began to die and to show structural alterations. The general aspects of alveolar macrophages cultured with C. parvum were quite different from those of peritoneal macrophages cultured with C. parvum. In contrast to Cryptosporidium cultured with peritoneal macrophages, (Martinez et al., 1992), a high percentage of forms adhered to the cytoplasmatic membrane in a typical Cryptosporidium position, i.e. intracellular but extracytoplasmatic, similar to the location this protozoan in the intestinal, bronchial or allantoic epithelia, This indicates the expression of specific receptors in murine alveolar macrophages which fix the invasive stages of C. parvum to the cell membrane and which are absent in peritoneal macrophages. Only a few Cryptosporidium forms were found intracytoplasmatic in alveolar macrophages, in both control and IFN-~/ cultures. Extracellular parasites were frequent in alveolar macrophage cultures, being mostly unicellular forms, elongated or rounded, isolated or grouped; they are interpreted as Table 2 Percentage of parasitized mouse alveolar macrophages and of different Cryptosporidium developmentstages in cultures inoculatedwith oocysts PT= 12h % MP S F 1
PT = 24h
C
T
C
T
40.5 I 1.2 85.9 2.9
13.5 43.4 54.3 2.2 "
35.2 59.2 22.4 18.3
16.8 76.7 17.6 3.8
PT = post-inoculationtime; C = control; T = cultures treated with ll~l-T (1000 U/ml); % MP = percentage of macrophages harboring Cryptosporidium forms; S = extracellular forms; F = free forms; I = intracellular forms.
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F. Martinez et aL / Veterinary Parasitology 68 (1997) 305-308
infectious merozoites, released from parasitized macrophages. The percentage of alveolar macrophages harboring Cryptosporidium from 12 h cultures was higher in control (40.5%) than in IFN-~/ cultures (13.5%) (Table 2), and this was noted again at 24 h (35.2% vs. 16.8%). The differences in the percentages of intracellular developmental forms in alveolar macrophages were considered irrelevant due their low numbers. It is clear that although IFN-~/did not completely inhibit the growth of C. parvum on alveolar macrophages, it considerably hampered it. Thus, the in vitro activation of macrophage by addition of IFN--y, limits the intracellular development of C. parvum. This inhibitory effect is however, not as distinct as in other intracellular parasitic protozoa. The observed differences between the two macrophage populations are apparently due to the higher sensitivity of alveolar macrophage to activation by IFN-~. More work is needed to ascertain whether this action is due directly to IFN-~/ action or to the induction of a-TNF, as has been demonstrated in other protozoan infections (Langermans et al., 1992).
Acknowledgements This work was supported by de CICYT (PM 91-0116).
References Beaman, M.H., Subauste, C.S., Wong, S.Y. and Remington, J.S., 1994. Toxoplasma-macrophage interactions. In: B.S. Zwilling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 475-493. Black, C.M., Catteral, J.R. and Remington, J.S., 1987. In vivo and in vitro cultivation of alveolar macrophages by recombinmlt interferon-gamin& J. Immunol., 138: 491-495. Kulm, R.E., 1994. Macrophages in experimental Chagas' Disease. In: B.S. Zwiiling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 495-502. Langermans, J.A.M., Van der Hulst, M.E.B., Nibbering, P. H., Hiemstra, P.S., Fransen, L. and Furth, R.V., 1992. IFN--/-induced l-arginine-dependent uaxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor-necrosis-factor-alfa. J. lmmunol., 148: 568-574. Martinez, F., Mascar6, C., Rosales, MJ., Diaz, J., Cifuentes, J. and Osuna, A., 1992. In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages. Vet. Parasitoi., 42: 27-31. Rosales, M.J., Cifuentes, J. and Mascar6, C., 1993. Crypto~poridium parvum: culture in MDCK cells. Exp. Parasitol., 76: 209-212. Rosales, M.J., Mascar6, C., Amedo, T. and Castilla, J.J., 1994. Isolation and identification of Cryptosporidium parvum oocysts with continuous percoll gradients and combined alcian-blue Giemsa staining. Acta Trop., 56: 371-373. Titus, R.G., Theodos, C.M., Shankar, A.H. and Hall, L.R., 1994. Interactions between Leishmania major and macrophages. In: B.S. Zwilling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 437-459. Ungar, B.L.P., Kao, T.C., Burris, J.A. and Finkelman, F.D., 1991. Cryptosporidium infection in an adult-mouse model, independent roles for IFN-gamma and CIM + lymphocytes in protective immunity. J. lmmunol., 147: 1014-- 1022. Villanueva, T.G., Kaufraan, D. and Giilooley, J.F., 1984. Respiratory cryptosporidiosis in the acquired immune deficiency syndrome. Use of modified cold Kinyonn and bemacolor stains for rapid diagnosis. J. Am. Med. Assoc., 252" 1298-301.
Veterinary Parasitology68 (1997) 305-308
The effects of IFN-'y activated mouse peritoneal and alveolar macrophages on Cryptosporidium parvum development F. Martinez, M.J. Rosales, J. Diaz, C. Mascaro * Institute of Biotechnology, Schoolof Science, Universityof Granada, E-18071 Granada, Spain
Received 6 March 1996; accepted2 August 1996
Abstract
Mouse peritoneal and alveolar macrophages were interacted in vitro with C. parvum oocysts and cultured in normal medium and in medium with IFN-~/. The results showed that in vitro activation of macrophages by IFN-~/ limits C. parvum development although the inhibitory effect is not as potent as in other intracellular parasitic protozoa. Keywords: Cryptosporidiumparvum; Macrophages;IFN-~/
1. Introduction Phenotypic and functional changes induced in macrophages in response to T-cells can be simulated in vitro by culturing macrophages with IFN-~/. In the case of Trypanosoma cruzi (Kuhn, 1994), Toxoplasma gondii (Beaman et al., 1994), Leishmania (Titus et al., 1994) and other protozoa and bacteria, in vitro macrophage activation by IFN-'y inhibits intracellular growth according to a dose time relationship. Both oxidative and nonoxidative mechanisms are involved in most cases. Human respiratory cryptosporidiosis is well known and intracellular Cryptosporidium oocysts have been detected in human alveolar macrophages (ViUanueva et al., 1984), suggesting a possible hematogenous spread of this agent. These data and the demonstrated development of C. parvum in mouse peritoneal macrophages (Martinez et al., 1992), lead us to study the action of IFN-~ on the development of Cryptosporidium in both peritoneal and alveolar macrophages. Previously, Ungar et al. (1991) suggested that IFN-~t is involved in the in vivo control of cryptosporidiosis, reporting that mice treated
* Corresponding author. Fax: + 34 58 243174; e-mail: [email protected]. 0304-4017/97/$17.00 Copyright © 1997 Elsevier Science B.V, All fights reserved. PII S0304-401 7(96)01087-4
306
F. Martinez et a l . / Veterinary Parasitology 68 (1997) 305-308
with neutralizing anti-IFN-~ monoclonal antibody terminated the infection by enhanced oocysts shedding. 2. Materials and methods
Cryptosporidium parvum oocysts were obtained and purified according to previously described methodology (Rosales et al., 1994). Mouse peritoneal macrophages were obtained also as previously reported (Martinez et al., 1992). Mouse alveolar macrophages were obtained from Swiss albino mice, following Black et al. (1987). The cells were concentrated by centrifugation at 2500 × g for 5 min, and the sediment was resuspended in RPMI 1640 medium (Gibco, USA) with L-glutamine and 10% inactivated fetal calf serum (IFCS). The macrophages were then counted and distributed in flat-bottomed microtiter plates (Costal Cambridge, UK), with a pre-cut glass disk in the bottom of each well. Adhesion of the cells to the glass disk was confirmed by microscopy after 12 h, and C parvum oocysts were added to macrophages cultures in a 1:1 proportion. After 12 h the medium was replaced, and parasitized macrophages were cultured either in normal medium or in medium containing IFN-~/ (Sigma) at a concentration of 1000 Uml-i. Cultures were interrupted at different time intervals by fixation with methanol, and stained with alcian-blue and Giemsa for observation by light microscope (Rosales et al., 1993). For each culture the number of macrophages harboring C. parvum forms were counted per 100 macrophages (% MP). Since multiparasitism was seen in peritoneal macrophages, we calculated not only the percentage of parasitized macrophages in each condition, but also the Cryptosporidium/macrophage relationship (C/M). 3. Results and discussion
Table 1 shows the results obtained when peritoneal macrophages were allowed to interact with C. parvum oocysts and then cultured either in normal medium or in medium containing IFN-~/. Cultures were interrupted after 3, 24, 36 and 48 h. At three hours, activated macrophages had clearly increased their phagocytic capacity (36.8% of parasitized macrophages in control cultures vs. 50.5% in cultures containing IFN-~). The C / M relationship was also higher in control cultures than in cultures containing IFN-'y (0.5 vs. 0.7). Both relationship remained relatively constant throughout the experiments, suggesting that macrophages stimulated by IFN-~/ did not completely suppress parasite development. A detailed microscopic study (Table 1) revealed that at 24, 36 and 48 h, a certain proportion of Cryptosporidiura forms was still undifferentiated, without nuclear accumulations or differentiated merozoites, and that the undifferentiated forms were more frequent after 24 h in IFN-~/cultures (70.7, 50 and 32.4%) than in control cultures (22.7, 10.6 and 8.7%). This suggests that IFN-3, under the conditions in this study, did not stop the growth on C. parvum within the macrophages but increased the percentage of parasite forms that were destroyed when ingested. Free parasitic forms (Table 1) at first
F. Martinez et al. / Veterinary Parasitology 68 (1997) 305-308
307
Table 1 Percentage of parasitized mouse peritonealmaerophages and of different Cryptosporidium developmentstages in cultures inoculatedwith oocysts PT=3h % MP C/M F I Nd
PT = 24h
PT= 36h
PT= 48 h
C
T
C
T
C
T
C
T
36.8 0.5 23.4 52.9 23.4
50.5 0.7 11.6 59.7 28.5
30.4 0.5 19.5 27.4 22.7
79.7 1.3 2.3 26.7 70.7
16.8 0.3 41.3 47.9 10.6
48.2 0.5 2.8 47 50
54.1 0.6 64.9 26.1 8.7
79.4 1.5 5.1 61.7 32.4
PT = post-inoculationtime; C = control; T = cultures treated with IFN-~ (1000 U/ml); % MP = percentage of macrophages harboring Cryptosporidium forms; C/M = relationship Cryptosporidium/macrophage; F = free forms; I = intracellularforms; Nd = undifferentiatedforms. were probably oocysts, but afterwards were primarily forms released into the medium after the death of the host cell. The number of free forms was always higher in control cultures than in IFN-~/ cultures. This was probably due to an increased phagocytic capacity of IFN-~ treated macrophages. Alveolar macrophages were allowed to interact with oocysts for 12 and 24 h either in normal medium or in medium containing IFN-~/. It was not possible to maintain alveolar macrophage cultures in good condition for more than 24 h; after this time, they began to die and to show structural alterations. The general aspects of alveolar macrophages cultured with C. parvum were quite different from those of peritoneal macrophages cultured with C. parvum. In contrast to Cryptosporidium cultured with peritoneal macrophages, (Martinez et al., 1992), a high percentage of forms adhered to the cytoplasmatic membrane in a typical Cryptosporidium position, i.e. intracellular but extracytoplasmatic, similar to the location this protozoan in the intestinal, bronchial or allantoic epithelia, This indicates the expression of specific receptors in murine alveolar macrophages which fix the invasive stages of C. parvum to the cell membrane and which are absent in peritoneal macrophages. Only a few Cryptosporidium forms were found intracytoplasmatic in alveolar macrophages, in both control and IFN-~/ cultures. Extracellular parasites were frequent in alveolar macrophage cultures, being mostly unicellular forms, elongated or rounded, isolated or grouped; they are interpreted as Table 2 Percentage of parasitized mouse alveolar macrophages and of different Cryptosporidium developmentstages in cultures inoculatedwith oocysts PT= 12h % MP S F 1
PT = 24h
C
T
C
T
40.5 I 1.2 85.9 2.9
13.5 43.4 54.3 2.2 "
35.2 59.2 22.4 18.3
16.8 76.7 17.6 3.8
PT = post-inoculationtime; C = control; T = cultures treated with ll~l-T (1000 U/ml); % MP = percentage of macrophages harboring Cryptosporidium forms; S = extracellular forms; F = free forms; I = intracellular forms.
308
F. Martinez et aL / Veterinary Parasitology 68 (1997) 305-308
infectious merozoites, released from parasitized macrophages. The percentage of alveolar macrophages harboring Cryptosporidium from 12 h cultures was higher in control (40.5%) than in IFN-~/ cultures (13.5%) (Table 2), and this was noted again at 24 h (35.2% vs. 16.8%). The differences in the percentages of intracellular developmental forms in alveolar macrophages were considered irrelevant due their low numbers. It is clear that although IFN-~/did not completely inhibit the growth of C. parvum on alveolar macrophages, it considerably hampered it. Thus, the in vitro activation of macrophage by addition of IFN--y, limits the intracellular development of C. parvum. This inhibitory effect is however, not as distinct as in other intracellular parasitic protozoa. The observed differences between the two macrophage populations are apparently due to the higher sensitivity of alveolar macrophage to activation by IFN-~. More work is needed to ascertain whether this action is due directly to IFN-~/ action or to the induction of a-TNF, as has been demonstrated in other protozoan infections (Langermans et al., 1992).
Acknowledgements This work was supported by de CICYT (PM 91-0116).
References Beaman, M.H., Subauste, C.S., Wong, S.Y. and Remington, J.S., 1994. Toxoplasma-macrophage interactions. In: B.S. Zwilling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 475-493. Black, C.M., Catteral, J.R. and Remington, J.S., 1987. In vivo and in vitro cultivation of alveolar macrophages by recombinmlt interferon-gamin& J. Immunol., 138: 491-495. Kulm, R.E., 1994. Macrophages in experimental Chagas' Disease. In: B.S. Zwiiling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 495-502. Langermans, J.A.M., Van der Hulst, M.E.B., Nibbering, P. H., Hiemstra, P.S., Fransen, L. and Furth, R.V., 1992. IFN--/-induced l-arginine-dependent uaxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor-necrosis-factor-alfa. J. lmmunol., 148: 568-574. Martinez, F., Mascar6, C., Rosales, MJ., Diaz, J., Cifuentes, J. and Osuna, A., 1992. In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages. Vet. Parasitoi., 42: 27-31. Rosales, M.J., Cifuentes, J. and Mascar6, C., 1993. Crypto~poridium parvum: culture in MDCK cells. Exp. Parasitol., 76: 209-212. Rosales, M.J., Mascar6, C., Amedo, T. and Castilla, J.J., 1994. Isolation and identification of Cryptosporidium parvum oocysts with continuous percoll gradients and combined alcian-blue Giemsa staining. Acta Trop., 56: 371-373. Titus, R.G., Theodos, C.M., Shankar, A.H. and Hall, L.R., 1994. Interactions between Leishmania major and macrophages. In: B.S. Zwilling and T.K. Eisenstein (Editors). Macrophage-Pathogen Interactions. Marcel Dekker, New York, pp. 437-459. Ungar, B.L.P., Kao, T.C., Burris, J.A. and Finkelman, F.D., 1991. Cryptosporidium infection in an adult-mouse model, independent roles for IFN-gamma and CIM + lymphocytes in protective immunity. J. lmmunol., 147: 1014-- 1022. Villanueva, T.G., Kaufraan, D. and Giilooley, J.F., 1984. Respiratory cryptosporidiosis in the acquired immune deficiency syndrome. Use of modified cold Kinyonn and bemacolor stains for rapid diagnosis. J. Am. Med. Assoc., 252" 1298-301.
