Original Paper Int Arch Allergy Immunol 1992;98:262-265
Departments of Parasitology and Experimental Medicine, Postgraduate Insitute of Medical Education and Research, Chandigarh, India
Key Words Brugia malayi Filarial antigens Mast cells Mastomys natelensis
Response of Mast Cells against Filarial Antigens from Experimentally Infected Mastomys natelensis with Brugia malayi
Abstract The role of antigens of Brugia malayi adult worms in induction of histamine re lease from mast cells was studied. Both peritoneal and lung mast cells were pas sively sensitized using immune serum collected from Mastomys natelensis on different days after infection with B. malayi. A significant release of histamine both with crude worm and 60-kD antigens was shown. However, the role of the 43-kD antigen in histamine release was comparable to that of control. When the sera were heat inactivated, the histamine release was minimal, thus in dicating the heat-labile nature of the antibodies. Furthermore, the responses of peritoneal and lung mast cells to filarial antigens were similar.
Introduction Lymphatic filariasis is a disease of tropical and subtrop ical countries, affecting more than 100 million people [1]. The clinical manifestations range from filarial fever with lymphangitis and lymphadenitis during the acute phase to elephantiasis, hydrocele, and chyluria during the chronic phase of infection. Yet another manifestation is tropical pulmonary eosinophilia [2], These clinical symptoms are closely related to the diversity of the host immune re sponse to the parasite [3], Mast cell infiltration of various tissues and elevation of serum IgE levels are associated with many helminthic infections [4], The mast cells medi ate both immediate and delayed T cell dependent immu nological reactions [5]. Lymphatic filariasis might also be linked with the formation of parasite-specific IgE and re lease of histamine from sensitized mast cells [6], Because the role of parasite antigens in allergic reac
tions of filariasis is poorly understood, in the present study we attempted to investigate the response of mast cells to wards defined filarial antigens of adult Brugia malayi un der in vitro conditions. Both lung and peritoneal mast cells were taken and passively sensitized with immune scrum collected at various time intervals after infection. The study also attempts to highlight the role of antigen frac tions of B. malayi adult worms in the release of histamine at different stages of infection.
Materials and Methods Animals and Parasites 3- to 4-week-old male Mastomys natelensis rats (GRA strain) bred by conventional methods in the departmental animal house were used in this study. The animals were infected subcutaneously with 100 3rd-stage larvae of B. malayi obtained from Aedes aegypti mosqui toes. Microfilarial count was done using 20 mm3 of blood collected
Correspondence to: Dr. R.C. Mahajan Department of Parasitology Postgraduate Institute of Medical Education and Research 160012 Chandigarh (India)
© 1992 S. Karger AG, Basel 1018-2438/92/0983-0262 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:18:26 PM
P. Muralidhara* N. Mallei* N.K. Gangulyh R.C. Mahajana
Preparation o f Crude Worm Extract Antigen H. maiayi adult worms obtained from M. natelensis were washed extensively with phosphate-buffered saline (pH 7.2), lyophilized, and suspended in extraction buffer (50 mM Hcpcs, 100 mM glycine, pH 7.2) which contained phenylmethyl sulfonyl fluoride (2 mAi), leupcptin (0.2 m/W), and EDTA (1 mM) as protease inhibitors. Parasites were homogenized in a tissue homogenizer. The homogenate was in cubated overnight at 4°C with constant agitation and centrifuged at 100,0(X) g for 1 h. The supernatant was dialyzed against phosphatebuffered saline (pH 7.4) and the protein concentration deter mined [8], Characterization and Purification o f Antigen The crude worm antigen was separated by sodium dodccyl sul fate-polyacrylamide gel electrophoresis (SDS-PAGE); in 4% stack ing gcl/10% running gel in the presence of p-mercaptoethanol. To obtain the required antigens (43 and 60 kD), gel segments of corresponding lanes were cut out and macerated [9]. This was later suspended in phosphate-buffered saline (pH 7.2) and dialyzed against the buffer overnight to remove SDS. Later on, dialyzed mate rial was centrifuged at 10,000 g for 30 min. The gel-free supernatant was colllccted and used as fractionated antigen for histamine release assay. Isolation o f Mast Cells An enzyme-digested tissue cell supension was prepared accord ing to the method described by Fox et al. [10], with slight modifica tions. Lung tissue was washed twice with phosphate-buffered saline (pH 7.2) and cut into 2- to 5-mm pieces. These pieces were washed twice with 50 ml of calcium and magnesium free Hank’s balanced salt solution (CMF-HBSS) containing 25 mM Hcpcs and 1.3xl()'J M EDTA (pH 12-1 A) to remove epithelial cells and then digested once with pronase (2 mg/ml) for 1 h and twice with collagenase (1 mg/ml) to obtain free cells. The residual fragments were mechanically dis rupted by repeated syringing. Supernatants from enzymatic digestion were pooled with the cells obtained from mechanical disruption. The resulting suspension was rapidly filtered through a 10-ml syringe con taining loosely packed nylon wool. The cells were washed with CMFHBSS. The mast cells thus obtained were further purified by flotation through discontinuous Pcrcoll gradient and centrifuged at 400 g for 12 min. The mast cells found between the 50 and 70% interphases (up to 95% purity) were washed twice with CMF-HBSS. Mast cells were also obtained from peritoneal washings in CMFHBSS, washed tw ice, and purified by Percoll gradient. The viability of isolated mast cells was checked with 0.1% trypan blue and the purity assessed by staining with 0.5% toluidine blue in 0.5 M HCI. The purity was more than 90%. Histamine Release Studies Both lung and peritoneal mast cells in HBSS (1-2 x 105 cells/ml) were preincubatcd at 37°C for 10 min. They were sensitized with test sera collected at different stages of infection and incubated at 37°C for 30 min [11]. A parallel set of experiments was carried out using the above scrum, but heat inactivated. Sera from normal uninfected, agematched animals were taken as controls and used with each experi
mental group. The different antigen fraction crude worm extracts, 60 and 43 kD were added to activate the mast cells and incubated for 30 min. These were centrifuged at 400g for 10 min and the supernatant was assayed for histamine release. To assess the total histamine, the cells were lysed with 0.3 N perchloric acid. The released histamine was measured fluorometrically after con densation with 0.1% o-phthaldchydc according to the method de scribed by Shore ct al. [12]. The released histamine was expressed as percent histamine in supernatant ------------------------- - ---------------- x 100. histamine in supernatant + lysate The results shown arc for lung mast cells and representative of six experiments for each experimental setup. Student’s ‘t’ test (un paired) has been used for analysis of data.
Results Parasitic Load Microfilariae started appearing in peripheral blood 4 months after infection, with a gradual increase until 5 months after infection, after which time there was a de cline in microfilarial count. However, a low level of micro filaremia was maintained thereafter (fig. 2). Histamine Release Assay Sera collected from M. natelensis 2, 4, 5, and 6 months after infection were used for passive immunization of both lung and peritoneal mast cells. These sensitized mast cells, when treated with crude worm extract antigen, showed ac tivation, as evidenced by the release of histamine in super natant. When crude worm extract was used to activate the mast cells, there was a significant amount of histamine release by cells incubated with sera collected 5 months after in fection (fig. 3). Heat inactivation of immune sera resulted in loss of activity and subsequent histamine release. The antibodies appeared only 5 months after infection. When the 60- and 43-kD antigen fractions were compared, the 60-kD antigen fraction triggered a more significant hista mine release than the 43-kD antigen (fig. 4,5).
Discussion The kinetics of mast cell response in human filarial in fection is not fully understood. Attempts to characterize and define pathogenic mechanisms associated with these phenomena were hampered by the many difficulties en countered in dealing with human populations and sequen-
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periodically from the tail veins from 90 days after infection onwards [7]. Blood samples were collected at various periods after infection from the retro-orbital plexus; serum was separated and stored at -70 °C.
160-1
Fig. 1. SDS-PAGE pattern of B. malayi adult worm antigen stained with Coomassie blue. Lane A: molecular weight markers; Lane B: crude saline extract of adult worm.
Fig. 2. Course of microfilaremia in M. natelensis infected with L3 stages of B. malayi. Points and bars indicate mean values ± SD; n = 6.
70-1
60< D 50 ■ CO CO 0.05); ** significant difference at the 0.001 level (p 0.05); ** significant differ ence at the 0.001 level (p
Departments of Parasitology and Experimental Medicine, Postgraduate Insitute of Medical Education and Research, Chandigarh, India
Key Words Brugia malayi Filarial antigens Mast cells Mastomys natelensis
Response of Mast Cells against Filarial Antigens from Experimentally Infected Mastomys natelensis with Brugia malayi
Abstract The role of antigens of Brugia malayi adult worms in induction of histamine re lease from mast cells was studied. Both peritoneal and lung mast cells were pas sively sensitized using immune serum collected from Mastomys natelensis on different days after infection with B. malayi. A significant release of histamine both with crude worm and 60-kD antigens was shown. However, the role of the 43-kD antigen in histamine release was comparable to that of control. When the sera were heat inactivated, the histamine release was minimal, thus in dicating the heat-labile nature of the antibodies. Furthermore, the responses of peritoneal and lung mast cells to filarial antigens were similar.
Introduction Lymphatic filariasis is a disease of tropical and subtrop ical countries, affecting more than 100 million people [1]. The clinical manifestations range from filarial fever with lymphangitis and lymphadenitis during the acute phase to elephantiasis, hydrocele, and chyluria during the chronic phase of infection. Yet another manifestation is tropical pulmonary eosinophilia [2], These clinical symptoms are closely related to the diversity of the host immune re sponse to the parasite [3], Mast cell infiltration of various tissues and elevation of serum IgE levels are associated with many helminthic infections [4], The mast cells medi ate both immediate and delayed T cell dependent immu nological reactions [5]. Lymphatic filariasis might also be linked with the formation of parasite-specific IgE and re lease of histamine from sensitized mast cells [6], Because the role of parasite antigens in allergic reac
tions of filariasis is poorly understood, in the present study we attempted to investigate the response of mast cells to wards defined filarial antigens of adult Brugia malayi un der in vitro conditions. Both lung and peritoneal mast cells were taken and passively sensitized with immune scrum collected at various time intervals after infection. The study also attempts to highlight the role of antigen frac tions of B. malayi adult worms in the release of histamine at different stages of infection.
Materials and Methods Animals and Parasites 3- to 4-week-old male Mastomys natelensis rats (GRA strain) bred by conventional methods in the departmental animal house were used in this study. The animals were infected subcutaneously with 100 3rd-stage larvae of B. malayi obtained from Aedes aegypti mosqui toes. Microfilarial count was done using 20 mm3 of blood collected
Correspondence to: Dr. R.C. Mahajan Department of Parasitology Postgraduate Institute of Medical Education and Research 160012 Chandigarh (India)
© 1992 S. Karger AG, Basel 1018-2438/92/0983-0262 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:18:26 PM
P. Muralidhara* N. Mallei* N.K. Gangulyh R.C. Mahajana
Preparation o f Crude Worm Extract Antigen H. maiayi adult worms obtained from M. natelensis were washed extensively with phosphate-buffered saline (pH 7.2), lyophilized, and suspended in extraction buffer (50 mM Hcpcs, 100 mM glycine, pH 7.2) which contained phenylmethyl sulfonyl fluoride (2 mAi), leupcptin (0.2 m/W), and EDTA (1 mM) as protease inhibitors. Parasites were homogenized in a tissue homogenizer. The homogenate was in cubated overnight at 4°C with constant agitation and centrifuged at 100,0(X) g for 1 h. The supernatant was dialyzed against phosphatebuffered saline (pH 7.4) and the protein concentration deter mined [8], Characterization and Purification o f Antigen The crude worm antigen was separated by sodium dodccyl sul fate-polyacrylamide gel electrophoresis (SDS-PAGE); in 4% stack ing gcl/10% running gel in the presence of p-mercaptoethanol. To obtain the required antigens (43 and 60 kD), gel segments of corresponding lanes were cut out and macerated [9]. This was later suspended in phosphate-buffered saline (pH 7.2) and dialyzed against the buffer overnight to remove SDS. Later on, dialyzed mate rial was centrifuged at 10,000 g for 30 min. The gel-free supernatant was colllccted and used as fractionated antigen for histamine release assay. Isolation o f Mast Cells An enzyme-digested tissue cell supension was prepared accord ing to the method described by Fox et al. [10], with slight modifica tions. Lung tissue was washed twice with phosphate-buffered saline (pH 7.2) and cut into 2- to 5-mm pieces. These pieces were washed twice with 50 ml of calcium and magnesium free Hank’s balanced salt solution (CMF-HBSS) containing 25 mM Hcpcs and 1.3xl()'J M EDTA (pH 12-1 A) to remove epithelial cells and then digested once with pronase (2 mg/ml) for 1 h and twice with collagenase (1 mg/ml) to obtain free cells. The residual fragments were mechanically dis rupted by repeated syringing. Supernatants from enzymatic digestion were pooled with the cells obtained from mechanical disruption. The resulting suspension was rapidly filtered through a 10-ml syringe con taining loosely packed nylon wool. The cells were washed with CMFHBSS. The mast cells thus obtained were further purified by flotation through discontinuous Pcrcoll gradient and centrifuged at 400 g for 12 min. The mast cells found between the 50 and 70% interphases (up to 95% purity) were washed twice with CMF-HBSS. Mast cells were also obtained from peritoneal washings in CMFHBSS, washed tw ice, and purified by Percoll gradient. The viability of isolated mast cells was checked with 0.1% trypan blue and the purity assessed by staining with 0.5% toluidine blue in 0.5 M HCI. The purity was more than 90%. Histamine Release Studies Both lung and peritoneal mast cells in HBSS (1-2 x 105 cells/ml) were preincubatcd at 37°C for 10 min. They were sensitized with test sera collected at different stages of infection and incubated at 37°C for 30 min [11]. A parallel set of experiments was carried out using the above scrum, but heat inactivated. Sera from normal uninfected, agematched animals were taken as controls and used with each experi
mental group. The different antigen fraction crude worm extracts, 60 and 43 kD were added to activate the mast cells and incubated for 30 min. These were centrifuged at 400g for 10 min and the supernatant was assayed for histamine release. To assess the total histamine, the cells were lysed with 0.3 N perchloric acid. The released histamine was measured fluorometrically after con densation with 0.1% o-phthaldchydc according to the method de scribed by Shore ct al. [12]. The released histamine was expressed as percent histamine in supernatant ------------------------- - ---------------- x 100. histamine in supernatant + lysate The results shown arc for lung mast cells and representative of six experiments for each experimental setup. Student’s ‘t’ test (un paired) has been used for analysis of data.
Results Parasitic Load Microfilariae started appearing in peripheral blood 4 months after infection, with a gradual increase until 5 months after infection, after which time there was a de cline in microfilarial count. However, a low level of micro filaremia was maintained thereafter (fig. 2). Histamine Release Assay Sera collected from M. natelensis 2, 4, 5, and 6 months after infection were used for passive immunization of both lung and peritoneal mast cells. These sensitized mast cells, when treated with crude worm extract antigen, showed ac tivation, as evidenced by the release of histamine in super natant. When crude worm extract was used to activate the mast cells, there was a significant amount of histamine release by cells incubated with sera collected 5 months after in fection (fig. 3). Heat inactivation of immune sera resulted in loss of activity and subsequent histamine release. The antibodies appeared only 5 months after infection. When the 60- and 43-kD antigen fractions were compared, the 60-kD antigen fraction triggered a more significant hista mine release than the 43-kD antigen (fig. 4,5).
Discussion The kinetics of mast cell response in human filarial in fection is not fully understood. Attempts to characterize and define pathogenic mechanisms associated with these phenomena were hampered by the many difficulties en countered in dealing with human populations and sequen-
263
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:18:26 PM
periodically from the tail veins from 90 days after infection onwards [7]. Blood samples were collected at various periods after infection from the retro-orbital plexus; serum was separated and stored at -70 °C.
160-1
Fig. 1. SDS-PAGE pattern of B. malayi adult worm antigen stained with Coomassie blue. Lane A: molecular weight markers; Lane B: crude saline extract of adult worm.
Fig. 2. Course of microfilaremia in M. natelensis infected with L3 stages of B. malayi. Points and bars indicate mean values ± SD; n = 6.
70-1
60< D 50 ■ CO CO 0.05); ** significant difference at the 0.001 level (p 0.05); ** significant differ ence at the 0.001 level (p
