Immunology Letters, 28 (1991) 5 7 - 6 4 Elsevier IMLET 01555
Protective immunity to Schistosoma mansoni in mice is dependent on antibody and complement but not on radiosensitive leukocytes A l f r e d o M. G o e s a n d E J. R a m a l h o - P i n t o Departamento de Bioquimica-lmunologia, 1CB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil (Received 9 August 1990; revision received 8 November 1990; accepted 11 December 1990)
1. Summary
2. Introduction
The role of complement in the control of the Schistosoma mansoni infection in mice was investigated in vivo. The number of schistosomula recovered from the lung 5 days post-infection was used as a parasitological criterion of immunity. A significant difference in worm burden was observed between normal and immune mice. In contrast, when cobra venom factor (CVF) was injected into normal or immune mice 3 h before challenge, a significant increase in worm burden was noticed compared to untreated mice. We also investigated the protective mechanisms in mice that had been exposed to 650 fads of 6°Co g a m m a radiation before challenge infection. Our results show that -z-irradiated immune mice, depleted of more than 90°70 of their circulating or tissue leukocytes, are still able to destroy most of the parasites of a challenge infection with cercariae, suggesting that the radiosensitive leukocytes are not essential in the effector mechanisms of this protective immunity to S. mansoni. These results provide evidence of a role for the complement system, in association with radioresistant effector cells, in protective immunity occurring in the first hours after infection with S. mansoni.
Schistosomiasis is a helminth infection which alters host immunity and produces a granulomatous tissue reaction. Experimental murine schistosomiasis has been widely used to investigate parasite biology, host-parasite interactions, including immunology and morbidity, and strategies for treating or preventing this c o m m o n tropical disease of man [1]. It is generally agreed that an understanding of the basic mechanisms of the immune response in humans and in experimental animal models is essential for the development of anti-schistosome vaccines [2, 3]. Several studies have demonstrated that a variety of experimental hosts develop an acquired immunity to schistosome infection after either a natural primary infection [1, 4, 5] or immunization with irradiated larvae [ 6 - 8] or as shown recently, with isolated antigens [2, 9-13]. Protective immunity to Schistosoma mansoni in mice can be mediated by antibody as shown by the passive transfer of resistance with polyclonal and monoclonal antibodies [9, 14]. About half the resistance to reinfection can be passively transferred to normal recipients with whole immune serum or with the IgG fraction of immune serum [14]. In addition to antibodies, there are suggestions that a non-sensitized cell, either the neutrophil, the eosinophil or the macrophage, is involved in the rejection of a challenge infection in different hosts [2, 16]. The suggestion that eosinophils may have an important role in the destruction of invading helminths by the immune host is derived from studies on the interaction of normal human or rat eosinophils and schistosomula in vitro [17, 18]. These in vitro studies have shown that the
Key words." Schistosoma mansoni; Mouse; Complement; Cobra venom factor; Gamma-irradiation; Immunity
Correspondence to: Dr. Alfredo Miranda de Goes, Departamento de Bioquimica-lmunologia, ICB, UFMG, Cx. Postal 2486, 30161 Belo Horizonte, MG, Brazil. Abbreviations." CVF~ cobra venom factor; PBS, phosphatebuffered saline; NMS, normal mouse serum; IMS, immune mouse serum; Ig, immunoglobulin. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
57
antibody-dependent cell cytotoxic (ADCC) effect is secondary to the adherence of the eosinophil to the schistosomulum and the release o f the specific granule contents from the granulocytes on the surface of the worm [18, 19]. A series of reports has implicated peroxidase [18, 20] and the major basic protein (MBP) [21] from the eosinophil granule as mediators of this cytotoxic activity. These findings from in vitro studies, together with a report that a monospecific anti-mouse eosinophil serum (AES) decreases immunity to S. mansoni [22] in immune mice, have strongly suggested the participation of eosinophils in protective immunity to schistosomes. However, several reports have suggested a role for the complement system in anti-schistosome immunity [23-26]. In this communication, evidence is presented that an intact complement system is essential not only for specific concomitant immunity but also for the expression of innate resistance to S. mansoni in mice. Furthermore, our results indicate that 7-irradiated immune mice depleted of more than 90°70 of their circulating or tissue granulocytes are still able to destroy most of the parasites of a challenge infection with cercariae, suggesting that the radio-sensitive leukocytes are not essential in the effector mechanisms of this protective immunity to S. mansoni. 3. Materials and Methods
3.1. Mice and parasites Adult male C57B1/6, CBA/J and Swiss albino mice (25 - 30 g in weight) were used throughout the experiments. The cercariae of S. mansoni were obtained from Biomphalaria glabrata snails previously infected with miracidia of the L. E. strain, from Belo Horizonte, Brazil. Cercariae were shed from infected snails under bright artificial illumination. 3.2. Serum collection Immune serum was collected from chronically infected CBA/J mice 15 weeks after percutaneous exposure to 20 cercariae of S. mansoni (IMS). Samples were heat-inactivated at 56 °C for 30 min, aliquotted and stored at - 2 0 °C until required. Normal CBA/J mice serum (NMS) was prepared in the same way. The IMS obtained was tested for antibody specifici58
ty by immunofluorescence. Briefly, samples of approximately 1000 schistosomula prepared in vitro [27] in 50 tzl PBS 0.15 M, pH 7.4, were incubated with 50/zl of IMS or NMS for 30 min at 4 °C. After incubation, the schistosomula were washed three times with PBS-I% BSA (Sigma, St. Louis, MO, U.S.A.) and incubated with 50 tA FITC-conjugated rabbit anti-mouse Ig (Sigma, St. Louis, MO, U.S.A.) diluted 1/100 for 30 min at 4 °C. After incubation, the schistosomula were washed 3 times, as above, and fluorescence was detected by microscopy (Photomicroscope-III, Zeiss). 3.3. Irradiation of mice Five days before the challenge infection, groups of naive and immune mice (chronically infected) were treated with 3,-radiation from a 6°Co source. Conscious animals were loaded into perspex restrainers and exposed to 650 rads (dose rate 5 6 - 63 rads/min). We examined the effect of radiation on populations of leukocytes in the blood and the skin of both groups of mice daily for 12 days. Briefly, blood was sampled from the tail and diluted 20-fold with a solution of 1.5% (v/v) acetic acid/0.5% (v/v) Malachite green (Sigma, St. Louis, MO, U.S.A.) (saturated solution). Leukocyte numbers were determined using a hemocytometer. This radiation dose caused a sudden and drastic fall, by 9 0 - 9 5 % , in leukocyte populations, 24 h after irradiation and persisted for 7 - 8 days. Five days after challenge infection of naive and immune mice, the numbers of blood leukocytes were still reduced by 82 % in irradiated animals. This effect was operative against cells resident in the skin in both groups of mice as analyzed by histological methods [28]. 3.4. Complement depletion in mice Cobra venom factor (CVF) free of phospholipase A was prepared in our laboratory from Naja naja kaounthiavenom (Sigma, St. Louis, MO, U.S.A.) using a method of sequential chromatography on DEAE-cellulose (Pharmacia, Uppsala, Sweden), Sephacryl-S-200 (Pharmacia, Uppsala, Sweden) and hydroxylapatite developed by others [29]. Decomplementation was achieved by a single intravenous injection of 7 ~g of purified CVF in 0.1 ml of saline (50 units/100 g body wt.) 3 h before the
challenge infection. This treatment resulted in a marked depletion of complement (to less than 5% of the control levels) for at least 36 h, as measured by an electroimmunoassay using a monospecific sheep anti-mouse C 3 serum [30]. 3.5. Protective activity assay The ability of IMS to confer protection to S. mansoni infections in normal, irradiated or decomplemented mice was assayed as follows: groups of 8 mice not treated (normal controls) either submitted to irradiation or injected with CVF, as described before, were injected i.v. with 0.5 ml of NMS or IMS. Two hours later, all groups were percutaneously infected with 200, 300 or 600 S. mansoni cercariae as indicated in each experiment. Recovery of schistosomula from the lungs was performed 5 days after challenge infection [14]. The protective activity of each group assayed was evaluated by comparing the difference between the recoveries of schistosomula from passively immunized mice and from those that had received normal serum. The results, expressed as percentage immunity, were obtained from the calculation: mean schistosomula recoveredin experimental group °7oImmunity = 100
× 100 mean schistosomula recoveredin control group
3.6. Statistical method The data were subjected to Student's t-test to determine the significant differences between groups. P value of < 0.05 was considered significant. 4. Results
4.1. The effect of decomplementation on acquired immunity in mice C57B1/6 mice which had been infected with S. mansoni cercariae for 24 weeks presented a concomitant immunity that would eliminate 60°-/0 of the parasites of a challenge infection, as assessed by the lung assay. This marked degree of active immunity in C57B1/6 mice was completely abolished by the intravenous injection of CVF, 3 h before the cercarial
challenge (Fig. 1A). The results in Fig. 1A also show that the injection of CVF interfered not only with the acquired immunity of infected mice, but also caused a marked block in the mechanisms of innate immunity, as measured by 5-day lung recovery, to schistosomes in normal and immune mice. The numbers of schistosomula recovered from the lungs of immune or normal mice treated with CVF was significantly higher than in mice with normal levels of complement. 4.2. The effect of CVF on passively transferred immunity in mice The transfer of IMS to normal mice was used to establish whether CVF would interfere with passively transferred immunity. CBA/J which received CVF 2 h before the transfer of IMS showed no detectable immunity to a challenge infection with 500 cercariae, whereas normal mice which received IMS alone presented a significant level of immunity (Fig. 1B). As previously observed, the lungs of all CVF-treated mice yielded significantly more schistosomula than the lungs of untreated animals, either immune or normals. The experiments demonstrate the importance of an intact complement system in this mechanism of immunity to S. mansoni. 4.3. The effect of radiation on mechanisms of protective immunity to S. mansoni As a previous report showed that the treatment with anti-eosinophil sera abolished concomitant immunity in mice [22] and complement can kill schistosomula in vitro, the participation of granulocytes in the mechanisms of protective immunity was investigated. To determine if, in addition to antibody and complement, a cell-dependent mechanism was operative in mice, a series of cell depletion experiments using whole body -y-irradiated animals was performed. Two different strains of mice and 2 doses of cercariae were used to assess this effect. Mice were given 650 rad 5 days previous to the transfer of serum and the challenge with cercariae. This dose of irradiation depleted mice of 90-95°7o of their circulating white blood cells for the period of the experiment. From 48 h after irradiation until the day before lung recovery, no eosinophils were found in the peripheral blood of mice which received 650 rad, 59
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Protective immunity to Schistosoma mansoni in mice is dependent on antibody and complement but not on radiosensitive leukocytes A l f r e d o M. G o e s a n d E J. R a m a l h o - P i n t o Departamento de Bioquimica-lmunologia, 1CB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil (Received 9 August 1990; revision received 8 November 1990; accepted 11 December 1990)
1. Summary
2. Introduction
The role of complement in the control of the Schistosoma mansoni infection in mice was investigated in vivo. The number of schistosomula recovered from the lung 5 days post-infection was used as a parasitological criterion of immunity. A significant difference in worm burden was observed between normal and immune mice. In contrast, when cobra venom factor (CVF) was injected into normal or immune mice 3 h before challenge, a significant increase in worm burden was noticed compared to untreated mice. We also investigated the protective mechanisms in mice that had been exposed to 650 fads of 6°Co g a m m a radiation before challenge infection. Our results show that -z-irradiated immune mice, depleted of more than 90°70 of their circulating or tissue leukocytes, are still able to destroy most of the parasites of a challenge infection with cercariae, suggesting that the radiosensitive leukocytes are not essential in the effector mechanisms of this protective immunity to S. mansoni. These results provide evidence of a role for the complement system, in association with radioresistant effector cells, in protective immunity occurring in the first hours after infection with S. mansoni.
Schistosomiasis is a helminth infection which alters host immunity and produces a granulomatous tissue reaction. Experimental murine schistosomiasis has been widely used to investigate parasite biology, host-parasite interactions, including immunology and morbidity, and strategies for treating or preventing this c o m m o n tropical disease of man [1]. It is generally agreed that an understanding of the basic mechanisms of the immune response in humans and in experimental animal models is essential for the development of anti-schistosome vaccines [2, 3]. Several studies have demonstrated that a variety of experimental hosts develop an acquired immunity to schistosome infection after either a natural primary infection [1, 4, 5] or immunization with irradiated larvae [ 6 - 8] or as shown recently, with isolated antigens [2, 9-13]. Protective immunity to Schistosoma mansoni in mice can be mediated by antibody as shown by the passive transfer of resistance with polyclonal and monoclonal antibodies [9, 14]. About half the resistance to reinfection can be passively transferred to normal recipients with whole immune serum or with the IgG fraction of immune serum [14]. In addition to antibodies, there are suggestions that a non-sensitized cell, either the neutrophil, the eosinophil or the macrophage, is involved in the rejection of a challenge infection in different hosts [2, 16]. The suggestion that eosinophils may have an important role in the destruction of invading helminths by the immune host is derived from studies on the interaction of normal human or rat eosinophils and schistosomula in vitro [17, 18]. These in vitro studies have shown that the
Key words." Schistosoma mansoni; Mouse; Complement; Cobra venom factor; Gamma-irradiation; Immunity
Correspondence to: Dr. Alfredo Miranda de Goes, Departamento de Bioquimica-lmunologia, ICB, UFMG, Cx. Postal 2486, 30161 Belo Horizonte, MG, Brazil. Abbreviations." CVF~ cobra venom factor; PBS, phosphatebuffered saline; NMS, normal mouse serum; IMS, immune mouse serum; Ig, immunoglobulin. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
57
antibody-dependent cell cytotoxic (ADCC) effect is secondary to the adherence of the eosinophil to the schistosomulum and the release o f the specific granule contents from the granulocytes on the surface of the worm [18, 19]. A series of reports has implicated peroxidase [18, 20] and the major basic protein (MBP) [21] from the eosinophil granule as mediators of this cytotoxic activity. These findings from in vitro studies, together with a report that a monospecific anti-mouse eosinophil serum (AES) decreases immunity to S. mansoni [22] in immune mice, have strongly suggested the participation of eosinophils in protective immunity to schistosomes. However, several reports have suggested a role for the complement system in anti-schistosome immunity [23-26]. In this communication, evidence is presented that an intact complement system is essential not only for specific concomitant immunity but also for the expression of innate resistance to S. mansoni in mice. Furthermore, our results indicate that 7-irradiated immune mice depleted of more than 90°70 of their circulating or tissue granulocytes are still able to destroy most of the parasites of a challenge infection with cercariae, suggesting that the radio-sensitive leukocytes are not essential in the effector mechanisms of this protective immunity to S. mansoni. 3. Materials and Methods
3.1. Mice and parasites Adult male C57B1/6, CBA/J and Swiss albino mice (25 - 30 g in weight) were used throughout the experiments. The cercariae of S. mansoni were obtained from Biomphalaria glabrata snails previously infected with miracidia of the L. E. strain, from Belo Horizonte, Brazil. Cercariae were shed from infected snails under bright artificial illumination. 3.2. Serum collection Immune serum was collected from chronically infected CBA/J mice 15 weeks after percutaneous exposure to 20 cercariae of S. mansoni (IMS). Samples were heat-inactivated at 56 °C for 30 min, aliquotted and stored at - 2 0 °C until required. Normal CBA/J mice serum (NMS) was prepared in the same way. The IMS obtained was tested for antibody specifici58
ty by immunofluorescence. Briefly, samples of approximately 1000 schistosomula prepared in vitro [27] in 50 tzl PBS 0.15 M, pH 7.4, were incubated with 50/zl of IMS or NMS for 30 min at 4 °C. After incubation, the schistosomula were washed three times with PBS-I% BSA (Sigma, St. Louis, MO, U.S.A.) and incubated with 50 tA FITC-conjugated rabbit anti-mouse Ig (Sigma, St. Louis, MO, U.S.A.) diluted 1/100 for 30 min at 4 °C. After incubation, the schistosomula were washed 3 times, as above, and fluorescence was detected by microscopy (Photomicroscope-III, Zeiss). 3.3. Irradiation of mice Five days before the challenge infection, groups of naive and immune mice (chronically infected) were treated with 3,-radiation from a 6°Co source. Conscious animals were loaded into perspex restrainers and exposed to 650 rads (dose rate 5 6 - 63 rads/min). We examined the effect of radiation on populations of leukocytes in the blood and the skin of both groups of mice daily for 12 days. Briefly, blood was sampled from the tail and diluted 20-fold with a solution of 1.5% (v/v) acetic acid/0.5% (v/v) Malachite green (Sigma, St. Louis, MO, U.S.A.) (saturated solution). Leukocyte numbers were determined using a hemocytometer. This radiation dose caused a sudden and drastic fall, by 9 0 - 9 5 % , in leukocyte populations, 24 h after irradiation and persisted for 7 - 8 days. Five days after challenge infection of naive and immune mice, the numbers of blood leukocytes were still reduced by 82 % in irradiated animals. This effect was operative against cells resident in the skin in both groups of mice as analyzed by histological methods [28]. 3.4. Complement depletion in mice Cobra venom factor (CVF) free of phospholipase A was prepared in our laboratory from Naja naja kaounthiavenom (Sigma, St. Louis, MO, U.S.A.) using a method of sequential chromatography on DEAE-cellulose (Pharmacia, Uppsala, Sweden), Sephacryl-S-200 (Pharmacia, Uppsala, Sweden) and hydroxylapatite developed by others [29]. Decomplementation was achieved by a single intravenous injection of 7 ~g of purified CVF in 0.1 ml of saline (50 units/100 g body wt.) 3 h before the
challenge infection. This treatment resulted in a marked depletion of complement (to less than 5% of the control levels) for at least 36 h, as measured by an electroimmunoassay using a monospecific sheep anti-mouse C 3 serum [30]. 3.5. Protective activity assay The ability of IMS to confer protection to S. mansoni infections in normal, irradiated or decomplemented mice was assayed as follows: groups of 8 mice not treated (normal controls) either submitted to irradiation or injected with CVF, as described before, were injected i.v. with 0.5 ml of NMS or IMS. Two hours later, all groups were percutaneously infected with 200, 300 or 600 S. mansoni cercariae as indicated in each experiment. Recovery of schistosomula from the lungs was performed 5 days after challenge infection [14]. The protective activity of each group assayed was evaluated by comparing the difference between the recoveries of schistosomula from passively immunized mice and from those that had received normal serum. The results, expressed as percentage immunity, were obtained from the calculation: mean schistosomula recoveredin experimental group °7oImmunity = 100
× 100 mean schistosomula recoveredin control group
3.6. Statistical method The data were subjected to Student's t-test to determine the significant differences between groups. P value of < 0.05 was considered significant. 4. Results
4.1. The effect of decomplementation on acquired immunity in mice C57B1/6 mice which had been infected with S. mansoni cercariae for 24 weeks presented a concomitant immunity that would eliminate 60°-/0 of the parasites of a challenge infection, as assessed by the lung assay. This marked degree of active immunity in C57B1/6 mice was completely abolished by the intravenous injection of CVF, 3 h before the cercarial
challenge (Fig. 1A). The results in Fig. 1A also show that the injection of CVF interfered not only with the acquired immunity of infected mice, but also caused a marked block in the mechanisms of innate immunity, as measured by 5-day lung recovery, to schistosomes in normal and immune mice. The numbers of schistosomula recovered from the lungs of immune or normal mice treated with CVF was significantly higher than in mice with normal levels of complement. 4.2. The effect of CVF on passively transferred immunity in mice The transfer of IMS to normal mice was used to establish whether CVF would interfere with passively transferred immunity. CBA/J which received CVF 2 h before the transfer of IMS showed no detectable immunity to a challenge infection with 500 cercariae, whereas normal mice which received IMS alone presented a significant level of immunity (Fig. 1B). As previously observed, the lungs of all CVF-treated mice yielded significantly more schistosomula than the lungs of untreated animals, either immune or normals. The experiments demonstrate the importance of an intact complement system in this mechanism of immunity to S. mansoni. 4.3. The effect of radiation on mechanisms of protective immunity to S. mansoni As a previous report showed that the treatment with anti-eosinophil sera abolished concomitant immunity in mice [22] and complement can kill schistosomula in vitro, the participation of granulocytes in the mechanisms of protective immunity was investigated. To determine if, in addition to antibody and complement, a cell-dependent mechanism was operative in mice, a series of cell depletion experiments using whole body -y-irradiated animals was performed. Two different strains of mice and 2 doses of cercariae were used to assess this effect. Mice were given 650 rad 5 days previous to the transfer of serum and the challenge with cercariae. This dose of irradiation depleted mice of 90-95°7o of their circulating white blood cells for the period of the experiment. From 48 h after irradiation until the day before lung recovery, no eosinophils were found in the peripheral blood of mice which received 650 rad, 59
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