Analyses of Immune Responses to Ontogeny Specific Antigens Using an Inbred Strain of Xenopus laevis J Strain

Yumi Izutsu and Mitsugu Maeno

Summary

In this chapter, the procedures for specific detection of ontogenic emerging antigens during animal development are described. Anuran metamorphosis has provided us with a good experimental model for investigation of the mechanisms of tissue remodeling. The establishment of a syngeneic strain of Xenopus laevis described in this chapter has enabled us to perform a unique experiment to develop antibodies that specifically react to ontogenic antigens by immunizing syngeneic animals. This strategy was successful because the antibody repertoires produced in the adult frog serum were well subtracted by a number of common antigens expressed in syngeneic larvae. Here we show, using results of immuno-histochemical and T-cell proliferation analyses that adult frogs exhibit humoral and cellmediated immune responses to larva- or metamorphosis-specific antigen molecules in epidermal cells.

Key Words: Larval antigens; epidermal cells; skin; metamorphosis; tissue remodeling; transformation; antiserum; antibodies; transplantation; skin graft; immunohistochemistry; T-cell proliferation; immunological recognition; Xenopus laevis; strain; immune system; larva; adult; ontogeny; subtraction.

1. Introduction

Metamorphosis of anuran amphibians involves morphological and functional tissue remodeling from larval to adult in various organs for adaptation from aquatic to terrestrial life. For instance, epidermal cells in the larval tail are completely resorbed at metamorphosis, and epidermal cells in the larval head are replaced by adult cells. Metamorphic changes also take place in the immune system. The larval immune system easily becomes tolerant to antigens. The immune system in the adult frog generates strong immune responses to various types of antigens comparable to those of the mammalian immune system. Therefore, we assumed that the adult-type immune system recognizes ontogenic antigens and takes part in the elimination of larval cells (1). Experiments using a major histocompatibility complex-homozygous inbred strain of Xenopus laevis were conducted to verify this possibility. This unique strain, named J

From: Methods in Molecular Medicine, Vol. 105: Developmental Hematopoiesis: Methods and Protocols. Edited by: M. H. Baron © Humana Press Inc., Totowa, NJ

strain ("J" for Japan), was established over a period of 30 yr by Drs. Tochinai and Katagiri at Hokkaido University (2). Adult J strain animals are completely histocom-patible and do not reject skin grafts from adult animals of the same strain, whereas we have demonstrated that young adults rejected skin grafts from syngeneic larvae (1). The fact that an accelerated secondary response was induced after repeated skin grafts indicates that the rejection of the skin grafts occurred as a result of immunological responses. It has also been shown that these animals produced specific antibodies against larval skin antigens (3). Therefore, the J strain provides us with a unique experimental system to identify development- or metamorphosis-specific tissue antigens. Here we describe the protocols for the transplantation technique and preparation of antiserum to examine immune responses to larva-specific antigens. We also describe the methods used for an adult T-cell proliferation assay to detect larval antigens.

2. Materials

2.1. Animals

The MHC-homozygous J strain of Xenopus laevis (2,4) was used. The J strain animals used are completely histocompatible, and no rejection of skin grafts occurs among the same strain of adult animals (1). Larvae and adults were reared at 23 ± 1°C. Tadpoles were staged according to the Normal Table of Nieuwkoop and Faber (5).

2.2. Reagents and Equipment

1. Gonadotropic hormones (Teikoku Zoki Co. Ltd., Tokyo, Japan).

2. Mashed green peas (Kagome Co. Ltd., Tokyo, Japan).

3. Fish meal (Oriental Yeast Co. Ltd., Tokyo, Japan).

4. MS222 (3-aminobenzoic acid ethyl ester, cat. no. A-5040, Sigma, St Louis, MO): 1% stock solution is stable for several months if stored in the dark at 4°C.

5. Steinberg's solution: (3.4 g/L NaCl, 0.05 g/L KCl, 0.08 g/L Ca(NO3)24H2O, 0.1025 g/L MgSO4, 0.56 g/L Tris-HCl, pH 7.5, 10 mg/L phenol red), 10X stock solution is stable for a few months at room temperature. The solution should be stored at 4°C after being supplemented with antibiotics.

6. Penicillin G and streptomycin sulfate mixture (100X solution, cat. no. Gibco 15240-062, Invitrogen Co., Carlsbad, CA).

7. 10-cm Petri dishes (Falcon35-3803, Becton Dickinson and Co., Franklin Lakes, NJ).

8. Forceps (0508-L5-PO, Natsume Seisakusho Co., Tokyo, Japan).

9. Scissors (Napox B-12H, Natsume Seisakusho Co).

11. Needled suture (cat. no. 5-O Nylon-blue color, Azwell, Osaka, Japan).

12. Cotton stick (Johnson and Johnson Co.).

15. 1.5-mL Centrifuge tube (Asahi Techno Glass Co., Tokyo, Japan).

16. 0.2-^m Pore filter (Millipore Japan, Tokyo, Japan).

17. OCT compound (cat. no. 4583, Sakura Seiki Co., Tokyo, Japan).

18. Tissue Tek (Plastic-Tissue-Tek cryomold, Cat. No. 4565, Miles Inc. Elkhart, IN).

19. Liquid nitrogen.

20. 50-mL Plastic tube (Falcon 35-2070, Becton Dickinson and Co.).

21. Precleaned slide glasses (cat. no. S-2445, Matsunami Glass Ind., Osaka, Japan).

22. Cryostat (Leica CM1850, Leica Instruments, Nussloch, Germany).

24. Fetal calf serum (FCS; Gibco, Invitrogen Co.).

25. Phosphate-buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4, pH 7.2.

26. Bovine serum albumin (BSA; Fraction V, cat. no. A-3311, Sigma).

28. Monoclonal antibody 11D5 (a mouse anti-Xenopus IgY [IgG analog], (6)).

29. Cy3-conjugated secondary antibodies against mouse Ig (cat. no. AP130C, Chemicon International, Temecula, CA).

30. Quinacrine dihydrochloride (cat. no. Q-3251, Sigma).

31. MacIlivain solution: 3.81 g/L citric acid, 23.24 g/L Na2HPO4, pH 7.

32. Sucrose (cat. no. 196-00015, Wako Pure Chem. Ind.).

33. Fluorescent microscope (BX51, Olympus Co., Japan).

34. Laminar flow hood.

35. L-15 Medium (Leibovitz's L-15 medium, cat. no. Gibco 11415-064, Invitrogen Co.). The medium mixed with supplements is freshly prepared and store at 4°C.

36. 7.5% NaHCO3 (7.5% sodium bicarbonate solution, cat. no. Gibco 25080-094, Invitro-gen Co.).

37. 1 M HEPES (cat. no. Gibco 15630-080, Invitrogen Co.).

38. Razor blade (Ophthalmic blade 21BZ0082, Feather Safety Razor Co., Japan).

39. Glass Petri dish (Iwaki glass dish, Asahi Techno Glass Co., Tokyo, Japan).

40. 96-Well U plate culture dish (Falcon cat. no. 35-3077, Becton Dickinson and Co.).

41. 35-mm Plastic petri dishes (Falcon35-3801, Becton Dickinson and Co.).

42. Rubber policeman (cat. no. 125-50-69-21, Tokyo Garasu Kiki Co., Tokyo, Japan).

43. Cell strainer (Falcon 35-2350, Becton Dickinson and Co.).

44. Plastic pipets (5 mL; Falcon 35-7543, Becton Dickinson and Co.).

45. 15-mL Plastic tubes (Falcon 35-2096, Becton Dickinson and Co.).

46. Hemocytometer.

47. 28°C CO2 Incubator (Benchtop low-temperature CO2 incubator, 9100, Wakenyaku Co., Kyoto, Japan).

49. 5-Bromo-2'-deoxyuridine (BrdU; cat. no. B-5002, Sigma). The stock solution should be freshly prepared.

52. 2 N HCl (mixture of 100 mL of conc. HCl and 465 mL of distilled water).

53. Borate-buffered solution (pH 8.5): prepared by mixing approx 850 mL of 0.2 M boric acid and 700 mL of 0.05 M sodium borate to make a solution of pH 8.5. This solution is stable for at least one year if stored at room temperature).

54. Anti-BrdU mouse monoclonal antibody (cat. no. 8003; Sanbio, Netherlands).

All reagents should be sterilized prior to use and stored at 4°C. 3. Methods

The following methods outline 1) the operation of transplantation using the J strain of Xenopus laevis, 2) preparation of antiserum from immunized adult frogs, 3) immuno-histochemistry using antisera, and 4) the proliferation assay for adult T-cells to detect ontogenic antigens.

3.1. Transplantation of Larval Skin Tissues Into Adults

1. Obtain fertilized eggs after ovulation and mating induced by gonadotropic hormones.

2. Rear the larvae at 24 ± 1°C. Feed the tadpoles mashed green peas and commercial fish meal. For tadpoles after stage 58, when the forelimbs have begun to develop, feed fish meal only (same for froglets). Grind the fish meal into small pieces using a food mixer.

3. Keep the adult frogs unfed in clean dechlorinated tap water for 1-2 d before use (see Note 1).

4. Gently wash the tadpoles (stage 56/57) and 1- to 2-yr-old female frogs with deionized water 10 times.

5. Anesthetize the animals by immersing them in MS222 solution, 0.01% (for tadpoles) or 0.05% (for adults) in sterile Steinberg's solution containing 100 IU/mL penicillin G and 100 ^g/mL streptomycin sulfate. The duration of anesthesia should not exceed 10 min for larvae and 30 min for adults. All treatments should be done aseptically.

6. Cut the tails from the tadpoles using forceps and scissors that have been sterilized with 70% ethanol, and transfer the tails into a 10-cm Petri dish containing ice-cold Steinberg's solution supplemented with antibiotics.

7. Isolate skin tissues from five tails in the Petri dish under a dissecting microscope. Keep the Petri dish on ice.

8. Transfer a host frog into the 10-cm Petri dish, pinch a grafting point of the dorsal skin of the frog with forceps, and make a slit of approx 5 mm in width with scissors.

9. Immediately insert the pieces of tail skins through the slit into the subcutaneous space.

10. Sew up the slit at one or two points using needled suture (see Note 2).

11. Maintain the hosts in Steinberg's solution containing antibiotics for 2-3 d without feeding. The solution should be changed every day. Then rear the animals in clean dechlori-nated tap water as mentioned previously (see Notes 3 and 4).

12. Repeat grafting three times at one-month intervals into different sites of the dorsal skin of host animals.

3.2. Preparation of Antiserum

Antisera are obtained from 1- to-2-yr-old frogs (female) that have received repeated tadpole tissue grafts or injections of larval cells (see Note 2) three times.

1. One month after the final skin grafting, anesthetize the immunized animals in 0.05% MS222 in Steinberg's solution for 10-20 min.

2. Place each animal on its back in a 10-cm Petri dish with a sheet of clean Kim wipe. Keep the Petri dish on ice. Carefully dissect off a small area of skin around the heart and remove muscle layers to expose the heart. Use a cotton stick to pull up the heart without injury. Then tear off the thin integument membrane of the heart with fine forceps as shown in Fig. 1.

3. Pierce the point of the ventricle of the heart directly with a 24-gage needle and a 1-mL syringe and collect clean blood. Pull little by little in accordance with beats of the heart. About 1 to 1.5 mL of blood is usually collected from one adult J strain frog (see Note 5).

4. Transfer the blood into a 1.5-mL centrifuge tube and keep it over night at 4°C.

5. Centrifuge the tubes at 1000g for 15 min at 4°C and transfer the supernatant into a new tube. Spin the supernatant one more time to avoid contamination of the serum by the cell pellet.

6. Incubate the serum at 56°C for 30 min for inactivation of complements. If it is necessary, sterilize the serum by filtration through a 0.2-^m pore filter and then dispense aliquots into 1.5-mL centrifuge tubes and store them at -80°C until use (see Notes 6 and 7).

Fig. 1. Flow chart diagrams of bleeding procedure. (A) Anesthetize the immunized animals in MS222 solution for 10 to 20 min. (B) Place each animal on its back in a 10-cm Petri dish and then dissect off a small area of skin around the heart. (C) Pull up the heart using a cotton stick. (D) Tear off the thin integument membrane of the heart with fine forceps by pulling on both sides. (E) Pierce the point of the ventricle of the heart directly with a needle and syringe.

Fig. 1. Flow chart diagrams of bleeding procedure. (A) Anesthetize the immunized animals in MS222 solution for 10 to 20 min. (B) Place each animal on its back in a 10-cm Petri dish and then dissect off a small area of skin around the heart. (C) Pull up the heart using a cotton stick. (D) Tear off the thin integument membrane of the heart with fine forceps by pulling on both sides. (E) Pierce the point of the ventricle of the heart directly with a needle and syringe.

3.3. Detection of Antibodies by Immunohistochemistry

To estimate the titer of antiserum, we usually employ an immunohistochemical assay (3,7).

1. Anesthetize animals in MS222 solution for 5 to 10 min.

2. Immediately dissect skin tissues, including both the tail and dorsal trunk junction regions, from tadpoles at stage 63, using forceps and scissors.

3. Transfer the tissues into OCT compound in plastic Tissue-Tek specimen molds after onetime wash with a sufficient amount of OCT compound.

4. After gently orientating the tissues with forceps, dip each specimen very slowly into liquid nitrogen. Store the mounted tissues in a 50-mL plastic tube at -80°C until use (see Note 8).

5. Prepare 4-^m thick frozen sections using a cryostat, transfer sections to precleaned glass slides (see Note 9), dry them by air blowing for 30 min at room temperature, and store dried, without fixation, in an airtight container at 4°C until use (within 1-2 wk).

6. Using a Dako Pen, trace a circle around the sectioned tissues and dry the slides by air blowing for 5 to 10 min at room temperature.

7. Apply 50 to 100 ^L of 10% FCS in PBS to the sections, and incubate the slides for 30 min in a moist chamber at room temperature for blocking.

8. Wipe off the blocking solution, and apply 50 to 100 ^L of 10-fold diluted frog serum in 0.1% BSA in PBS with 0.01% NaN3 onto the sections. Then incubate the slides for 12 h at 4°C in a moist chamber.

9. Wash the sections gently with PBS three times for 5 min each at room temperature.

10. Wipe off the remaining PBS and apply 50 to 100 ^L of 200-fold diluted monoclonal antibody 11D5 (a mouse anti-Xenopus IgY [IgG analog], [6]) in PBS with 0.1% BSA and 0.01% NaN3 onto each section. Then incubate the slides for 2 h at room temperature in a moist chamber.

11. Wash the sections gently with PBS three times for 5 min each at room temperature.

12. Wipe off the remaining PBS and apply 50 to 100 mL of Cy3-conjugated secondary antibodies against mouse Ig at a 300 to 600-fold dilution in PBS with 0.1% BSA and 0.01% NaN3 onto the sections. Then, incubate the slides for 30 min to 1 h at room temperature in a moist chamber in the dark.

13. Wash the sections gently with PBS for 5 min at room temperature three times.

14. Counterstain the tissues with 0.025% quinacrine dihydrochloride for 8 min and gently wash the slides with distilled water for 4 min by changing the water five to six times. Then, immerse the slides in Mcllivein solution for a few minutes.

15. Cover the sections with saturated sucrose solution (see Note 10).

16. Observe the sections under a fluorescent microscope (see Note 11).

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