Immunology 1979 38 467
The location of tick salivary antigens, complement and immunoglobulin in the skin of guinea-pigs infested with Dermacentor andersoni larvae
J. R. ALLEN, HANAA M. KHALIL & JOANNE E. GRAHAM Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Acceptedforpublication 24 May 1979
Summary. Using indirect immunofluorescence techniques, tick salivary gland antigens (SGA) were demonstrable in cement deposited on the skin by ticks and in all layers of the epidermis of infested guineapigs close to the sites where ticks attached. The antigen remained in these sites for several days after ticks had detached. In tick-resistant but not in normal guineapigs, SGA, complement and IgG were deposited at the dermo-epidermal junction even at some distance from the attachment site. Complement was also demonstrable in epidermal vesicles which developed beneath larvae attached to resistant guinea-pigs. It is suggested that antigen-antibody reaction and complement activation at these sites may play a role in the development of skin lesions and the attraction of basophils to these areas in challenged tick-resistant guinea-pigs. Tick antigens were also found to be associated with dendritic suprabasal cells in the epidermis of resistant guinea-pigs. It has previously been proposed that such SGA-trapping cells are Langerhans cells. These cells, in the presence of specific antibody and complement, could also initiate epidermal lesions in resistant
guinea-pigs. Correspondence: Professor J. R. Allen, Department of Veterinary Microbiology, University of Saskatchewan, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada S7N OWO.
0019-2805/79/1100-0467$02.00 C) 1979 Blackwell Scientific Publications
467
INTRODUCTION Guinea-pigs acquire resistance to the feeding activities of larvae of Dermacentor spp. (Trager, 1939; Allen, 1973). Resistance becomes evident after a primary infestation with tick larvae and is manifested by the guinea-pig allowing relatively very few larvae to engorge during a secondary infestation of a different skin site. Such acquired tick resistance is an immunological phenomenon, abrogated by immunosuppressive treatments of guinea-pigs during their primary infestation (Allen, 1973; Wikel & Allen, 1976b), and transferable between syngeneic guinea-pigs by lymphoid cells from resistant donors (Wikel & Allen, 1976a). A marked allergic response, similar to a cutaneous basophil hypersensitivity response occurs in the infested skin of tick-resistant guinea-pigs (Allen, 1973). Basophil leucocytes infiltrate the dermis and epidermis of challenged resistant hosts, and are demonstrable in very large numbers in epidermal vesicles which develop beneath attachment sites. Wikel & Allen (1977) provided evidence that complement plays some role in the tick resistance response. Cobra venom factor was administered to guinea-pigs and produced prolonged depletion (85-95%) of complement titres. Such treatment significantly reduced the expression of the resistance response in tick-resistant guinea-pigs. Wikel (1979) provided evidence that the alternative pathway of complement activation is
468
J. R. Allen, Hanaa M. Khalil & Joanne E. Graham
involved in the resistance response. The classical complement pathway apparently plays a relatively minor role. This report describes the use of indirect immunofluorescence techniques to demonstrate the location of tick salivary gland antigens (SGA), complement (C3) and immunoglobulins (IgG) in the skin of guinea-pigs undergoing primary (immunizing) and secondary (challenge) infestations with larvae of D. andersoni.
MATERIALS AND METHODS Guinea-pigs Randomly bred guinea-pigs of both sexes, weighing 400-500 g were obtained from the Animal Resources Centre, University of Saskatchewan. A total of sixty animals was used, in three separate experimental runs, in this project.
Tick infestations Dermacentor andersoni larvae were derived from the pathogen-free colony at the Rocky Mountain Laboratory, NIH, NIAID, Hamilton, Montana, U.S.A., and were maintained as previously described (Wikel & Allen, 1976b). Each guinea-pig was subjected to a primary infestation of 100 larvae on the right ear. The larvae were confined to the ear within a plastic capsule, approximately 15 mm in diameter, held in place by adhesive tape. Guinea-pigs were prevented from grooming the infested ears. After the 5 day primary infestation, larvae were removed from the ear, weighed and categorized as engorged or unengorged by examination under a stereoscopic microscope. Guinea-pigs were maintained tick-free for the next 7 days and then subjected to secondary infestations with 100 larvae applied to each left ear. Following the 5 day secondary infestation the weights of larvae, and the numbers engorged, were recorded. The acquisition of tick resistance was confirmed, as before (Wikel & Allen, 1976a) by comparing results from guinea-pigs undergoing secondary infestations with those from control guinea-pigs subjected to concurrent primary infestations with larvae from the same batch. Sites of tick attachment to the skin were biopsied at intervals throughout this infestation schedule. Biopsies from right ears (which received the primary infestations) were taken on days 1 through 19, and on days 21, 23 and 25. Biopsies from left ears (which received the secondary infestations) were taken on days 12
through 23 and on day 25. Biopsies were also obtained from normal uninfested guinea-pigs. Processing ofskin biopsies Biopsies including the sites of attachment of D. andersoni larvae were trimmed, frozen in vials above liquid nitrogen and stored at - 700 until sections were cut. Cryostat sections (3-5 pm) were allowed to dry on coverglasses, fixed in acetone and then subjected to indirect immunofluorescence procedures. The optimal working dilutions of reagents for each procedure were established by chess-board titrations. The unconjugated specific antiserum, diluted in phosphate-buffered saline, 0 01 M in 0 15 M NaCl, pH 7 4 (PBS), was layered on sections for 30 min at room temperature. Sections were then washed in PBS for 90 min and treated with the corresponding fluorescein conjugate, diluted in PBS, for 30 min. Following washing in PBS and mounting in glycerol: PBS (9: 1), slides were examined with a Leitz Orthoplan fluorescence microscope, using BG 38 and K515 filters. Kodak Tri-X or Ektachrome 200 film was used for photomicrographs. Antibody controls were included in each procedure. Normal goat or rabbit sera were used in place of the unlabelled specific antisera. Staining controls were also included, in which sections were treated with the specific antiserum, with the corresponding unlabelled anti-IgG and finally with the corresponding FITC anti-IgG. For detection of SGA in sections, anti-SGA sera were raised in rabbits. SGA was prepared as described by Wikel, Graham & Allen (1978). Six injections, each of 400-500 pg SGA protein were administered to rabbits over a period of 3 months. Freund's complete adjuvant was used with the first inoculation and Freund's incomplete adjuvant with subsequent injections. Anti-SGA serum produced in this way reacted strongly with SGA, producing multiple bands in double diffusion tests. The anti-SGA serum, diluted 1:4 in PBS was used in conjunction with fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit IgG (Cappel Laboratories Inc., Cochranville, Pa, U.S.A.) diluted 1:3 in PBS to demonstrate the location of SGA in sections. For the demonstration of IgG deposits in skin sections, goat anti-guinea-pig (heavy and light chains) from Cappel Laboratories Inc. was diluted 1:4 in PBS and used in conjunction with FITC rabbit anti-goat IgG, from the same source, diluted 1:4 in PBS. Complement deposits in sections were located using goat anti-guinea-pig C3 (1:3 dilution in PBS) with
Tick salivary antigens, C and Ig in guinea-pigs FITC rabbit anti-goat IgG (1:3 dilution in PBS). Both antisera were obtained from Cappel Laboratories Inc. RESU LTS
Salivary gland antigens were located in the cement deposited by larvae at their attachment sites and within the epidermis close to these sites in both primary and secondary infestations. These antigens were found also in nearby hair follicles, including the outer root sheath of the follicle. This is illustrated in Fig. 1, a section taken on the fourth day of a primary infestation. Fluorescence specific for SGA occurred in the epidermis for at least a week after ticks had detached. Figure 2 illustrates epidermal fluorescence in a site from which a tick had detached 7 days previously. Deposits of SGA were detected at the dermo-epidermal junction close to and at distances up to 1 2 mm from the attachment site. This occurred in guinea-pigs which had been infested for at least 5 days. Similar deposits of IgG were found at these sites at similar times. The occurrence of these deposits in biopsies taken at various times through the infestation schedule is shown in Fig. 3. In Fig. 4, a section taken from a left ear on day 20,
;'n
469
SGA is shown in deposits at the dermo-epidermal
junction and also associated with a dendritic cell in the suprabasal layer of the epidermis. Such 'antigen trapping' cells were found in areas adjacent to the attachment site in secondary infestations, but not in primary infestations of guinea-pigs. Fluorescence specific for guinea-pig IgG is shown at the dermo-epidermaljunction in Fig. 5, a section taken on day 5 of a primary infestation. Complement deposition at the dermo-epidermal junction near to attachment sites was consistently demonstrable from day 5 onwards (see Fig. 3). Complement was also detected within the epidermal vesicles which developed beneath attached larvae during secondary infestations. Figure 6 shows fluorescence specific for C3 within a large epidermal vesicle and at the dermo-epidermal junction nearby. This section came from a left ear on day 13. Both antibody and staining controls for the procedures used to locate SGA, IgG and complement were consistently negative, and skin sections from control uninfested guinea-pigs also produced negative results. Guinea-pigs that completed their secondary infestations were shown to have acquired tick resistance. The percentage of larvae engorging, and the larval weights were significantly lower (P < 0 01, Student's t
~~~~~~'
Figure 1. Section of tick attachment site on the skin of a guinea-pig. The biopsy was taken on the fourth day of a primary infestation. Fluorescence specific for SGA is shown in the cement deposited by the tick on the skin surface (C) and in the section of a hair follicle (arrow). Magnification x 500.
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The location of tick salivary antigens, complement and immunoglobulin in the skin of guinea-pigs infested with Dermacentor andersoni larvae
J. R. ALLEN, HANAA M. KHALIL & JOANNE E. GRAHAM Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Acceptedforpublication 24 May 1979
Summary. Using indirect immunofluorescence techniques, tick salivary gland antigens (SGA) were demonstrable in cement deposited on the skin by ticks and in all layers of the epidermis of infested guineapigs close to the sites where ticks attached. The antigen remained in these sites for several days after ticks had detached. In tick-resistant but not in normal guineapigs, SGA, complement and IgG were deposited at the dermo-epidermal junction even at some distance from the attachment site. Complement was also demonstrable in epidermal vesicles which developed beneath larvae attached to resistant guinea-pigs. It is suggested that antigen-antibody reaction and complement activation at these sites may play a role in the development of skin lesions and the attraction of basophils to these areas in challenged tick-resistant guinea-pigs. Tick antigens were also found to be associated with dendritic suprabasal cells in the epidermis of resistant guinea-pigs. It has previously been proposed that such SGA-trapping cells are Langerhans cells. These cells, in the presence of specific antibody and complement, could also initiate epidermal lesions in resistant
guinea-pigs. Correspondence: Professor J. R. Allen, Department of Veterinary Microbiology, University of Saskatchewan, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada S7N OWO.
0019-2805/79/1100-0467$02.00 C) 1979 Blackwell Scientific Publications
467
INTRODUCTION Guinea-pigs acquire resistance to the feeding activities of larvae of Dermacentor spp. (Trager, 1939; Allen, 1973). Resistance becomes evident after a primary infestation with tick larvae and is manifested by the guinea-pig allowing relatively very few larvae to engorge during a secondary infestation of a different skin site. Such acquired tick resistance is an immunological phenomenon, abrogated by immunosuppressive treatments of guinea-pigs during their primary infestation (Allen, 1973; Wikel & Allen, 1976b), and transferable between syngeneic guinea-pigs by lymphoid cells from resistant donors (Wikel & Allen, 1976a). A marked allergic response, similar to a cutaneous basophil hypersensitivity response occurs in the infested skin of tick-resistant guinea-pigs (Allen, 1973). Basophil leucocytes infiltrate the dermis and epidermis of challenged resistant hosts, and are demonstrable in very large numbers in epidermal vesicles which develop beneath attachment sites. Wikel & Allen (1977) provided evidence that complement plays some role in the tick resistance response. Cobra venom factor was administered to guinea-pigs and produced prolonged depletion (85-95%) of complement titres. Such treatment significantly reduced the expression of the resistance response in tick-resistant guinea-pigs. Wikel (1979) provided evidence that the alternative pathway of complement activation is
468
J. R. Allen, Hanaa M. Khalil & Joanne E. Graham
involved in the resistance response. The classical complement pathway apparently plays a relatively minor role. This report describes the use of indirect immunofluorescence techniques to demonstrate the location of tick salivary gland antigens (SGA), complement (C3) and immunoglobulins (IgG) in the skin of guinea-pigs undergoing primary (immunizing) and secondary (challenge) infestations with larvae of D. andersoni.
MATERIALS AND METHODS Guinea-pigs Randomly bred guinea-pigs of both sexes, weighing 400-500 g were obtained from the Animal Resources Centre, University of Saskatchewan. A total of sixty animals was used, in three separate experimental runs, in this project.
Tick infestations Dermacentor andersoni larvae were derived from the pathogen-free colony at the Rocky Mountain Laboratory, NIH, NIAID, Hamilton, Montana, U.S.A., and were maintained as previously described (Wikel & Allen, 1976b). Each guinea-pig was subjected to a primary infestation of 100 larvae on the right ear. The larvae were confined to the ear within a plastic capsule, approximately 15 mm in diameter, held in place by adhesive tape. Guinea-pigs were prevented from grooming the infested ears. After the 5 day primary infestation, larvae were removed from the ear, weighed and categorized as engorged or unengorged by examination under a stereoscopic microscope. Guinea-pigs were maintained tick-free for the next 7 days and then subjected to secondary infestations with 100 larvae applied to each left ear. Following the 5 day secondary infestation the weights of larvae, and the numbers engorged, were recorded. The acquisition of tick resistance was confirmed, as before (Wikel & Allen, 1976a) by comparing results from guinea-pigs undergoing secondary infestations with those from control guinea-pigs subjected to concurrent primary infestations with larvae from the same batch. Sites of tick attachment to the skin were biopsied at intervals throughout this infestation schedule. Biopsies from right ears (which received the primary infestations) were taken on days 1 through 19, and on days 21, 23 and 25. Biopsies from left ears (which received the secondary infestations) were taken on days 12
through 23 and on day 25. Biopsies were also obtained from normal uninfested guinea-pigs. Processing ofskin biopsies Biopsies including the sites of attachment of D. andersoni larvae were trimmed, frozen in vials above liquid nitrogen and stored at - 700 until sections were cut. Cryostat sections (3-5 pm) were allowed to dry on coverglasses, fixed in acetone and then subjected to indirect immunofluorescence procedures. The optimal working dilutions of reagents for each procedure were established by chess-board titrations. The unconjugated specific antiserum, diluted in phosphate-buffered saline, 0 01 M in 0 15 M NaCl, pH 7 4 (PBS), was layered on sections for 30 min at room temperature. Sections were then washed in PBS for 90 min and treated with the corresponding fluorescein conjugate, diluted in PBS, for 30 min. Following washing in PBS and mounting in glycerol: PBS (9: 1), slides were examined with a Leitz Orthoplan fluorescence microscope, using BG 38 and K515 filters. Kodak Tri-X or Ektachrome 200 film was used for photomicrographs. Antibody controls were included in each procedure. Normal goat or rabbit sera were used in place of the unlabelled specific antisera. Staining controls were also included, in which sections were treated with the specific antiserum, with the corresponding unlabelled anti-IgG and finally with the corresponding FITC anti-IgG. For detection of SGA in sections, anti-SGA sera were raised in rabbits. SGA was prepared as described by Wikel, Graham & Allen (1978). Six injections, each of 400-500 pg SGA protein were administered to rabbits over a period of 3 months. Freund's complete adjuvant was used with the first inoculation and Freund's incomplete adjuvant with subsequent injections. Anti-SGA serum produced in this way reacted strongly with SGA, producing multiple bands in double diffusion tests. The anti-SGA serum, diluted 1:4 in PBS was used in conjunction with fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit IgG (Cappel Laboratories Inc., Cochranville, Pa, U.S.A.) diluted 1:3 in PBS to demonstrate the location of SGA in sections. For the demonstration of IgG deposits in skin sections, goat anti-guinea-pig (heavy and light chains) from Cappel Laboratories Inc. was diluted 1:4 in PBS and used in conjunction with FITC rabbit anti-goat IgG, from the same source, diluted 1:4 in PBS. Complement deposits in sections were located using goat anti-guinea-pig C3 (1:3 dilution in PBS) with
Tick salivary antigens, C and Ig in guinea-pigs FITC rabbit anti-goat IgG (1:3 dilution in PBS). Both antisera were obtained from Cappel Laboratories Inc. RESU LTS
Salivary gland antigens were located in the cement deposited by larvae at their attachment sites and within the epidermis close to these sites in both primary and secondary infestations. These antigens were found also in nearby hair follicles, including the outer root sheath of the follicle. This is illustrated in Fig. 1, a section taken on the fourth day of a primary infestation. Fluorescence specific for SGA occurred in the epidermis for at least a week after ticks had detached. Figure 2 illustrates epidermal fluorescence in a site from which a tick had detached 7 days previously. Deposits of SGA were detected at the dermo-epidermal junction close to and at distances up to 1 2 mm from the attachment site. This occurred in guinea-pigs which had been infested for at least 5 days. Similar deposits of IgG were found at these sites at similar times. The occurrence of these deposits in biopsies taken at various times through the infestation schedule is shown in Fig. 3. In Fig. 4, a section taken from a left ear on day 20,
;'n
469
SGA is shown in deposits at the dermo-epidermal
junction and also associated with a dendritic cell in the suprabasal layer of the epidermis. Such 'antigen trapping' cells were found in areas adjacent to the attachment site in secondary infestations, but not in primary infestations of guinea-pigs. Fluorescence specific for guinea-pig IgG is shown at the dermo-epidermaljunction in Fig. 5, a section taken on day 5 of a primary infestation. Complement deposition at the dermo-epidermal junction near to attachment sites was consistently demonstrable from day 5 onwards (see Fig. 3). Complement was also detected within the epidermal vesicles which developed beneath attached larvae during secondary infestations. Figure 6 shows fluorescence specific for C3 within a large epidermal vesicle and at the dermo-epidermal junction nearby. This section came from a left ear on day 13. Both antibody and staining controls for the procedures used to locate SGA, IgG and complement were consistently negative, and skin sections from control uninfested guinea-pigs also produced negative results. Guinea-pigs that completed their secondary infestations were shown to have acquired tick resistance. The percentage of larvae engorging, and the larval weights were significantly lower (P < 0 01, Student's t
~~~~~~'
Figure 1. Section of tick attachment site on the skin of a guinea-pig. The biopsy was taken on the fourth day of a primary infestation. Fluorescence specific for SGA is shown in the cement deposited by the tick on the skin surface (C) and in the section of a hair follicle (arrow). Magnification x 500.
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