Izutsu Y et al. (2002), Larval antigen molecules recognized by adult im...

XB-ART-6005

Dev Growth Differ 2002 Dec 01;446:477-88. doi: 10.1046/j.1440-169x.2002.00660.x.

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Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis.

Izutsu Y , Tochinai S , Maéno M , Iwabuchi K , Onoé K .

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It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti-LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti-LS antiserum were localized only in the larval epidermal cells. Of 53 and 59-60 kDa acidic proteins that were reactive with anti-LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval-specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS-immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell-enriched splenocytes from normal frogs or in natural killer (NK)-cell-enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti-LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval-specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.

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Species referenced: Xenopus laevis
Genes referenced: actl6a ighx myh6 ouro1 tnfrsf10b
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	Fig. 1. Immunohistochemical staining using adult frog antiserum against larval skin (LS). Red signals of sections through epidermis of tail (A) and back skin of trunk (B) at stage 64, showing the larval antigens expressed on all of the larval tail epidermal cells but not in the adult-type epidermis of the back region. Sections were counterstained with quinacrine (green). Thickness of the epidermis (E) and dermis (D) are shown by the white lines on the right side. G, granular glands that are characteristic of adult-type skin. Bar, 100 μm.
	Fig. 2. Western blot analysis using adult frog antiserum against larval skin (LS) of larval and adult skin lysates. (a) Larvae at stage (St.) 55/56 (Larva) and whole skin of 2–3-month-old froglets (Adult), showing the larval-specific proteins (25 μg/lane) were separated by sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) at approximately 53 and 56–60 kDa (arrows). (b) Developmental expression of the larvalspecific bands during metamorphosis, showing their persistent expression in the tail but their decrease or loss in the trunk by metamorphic climax stage 65.
	Fig. 3. 2-D gel electrophoresis and western blot analysis using frog antiserum against larval skin (LS) of the lysates from tail of tadpoles at stage 55/56 (A,C) and from whole skins of 2–3-monthold froglets (B,D). Gels were stained with silver (C,D) or reacted with ECL Western Blotting Detection Reagents with 5 s exposure (A,B). Open white squares indicate actin. Circles represent the larval-specific spots on which amino acid sequences were analyzed (around the area at pH 4.5 and approximately 59 kDa).
	Fig. 4. (a) Alignment of amino acid sequences based on analysis of a larval-specific spot shown in Fig. 3A. Undistinguishable regions of leucine (L) and isoleucine (I) in the sequence are indicated in the squares. (b,c) Proliferative responses to synthetic peptides by adult splenic T cells primed with larval skin (LS) grafts. The index represents the relative ratio of proliferating splenocytes to that cultured without stimulators (W/O). Responders were adult splenocytes, including antigenpresenting cells (APC). (b) Dose-dependent proliferative responses to the peptide 1 shown in (a). Adult splenocytes were obtained from 1–2-year-old frogs that had been previously immunized by skin transplantation of larval tail () or normal frogs (). Values represent mean ± SD obtained from four different experiments for 10 μM peptide and those of two independent experiments for other concentrations of the peptide. (c) Stimulator-dependent proliferative response by adult splenocytes obtained from frogs preimmunized with ( ) or without () LS grafts. Splenocytes were co-cultured with syngeneic larval tail tissues at stage 55/56 (La) including MHC class II-positive apical cells or with peptides 1–4 shown in (a) at the concentration of 10 μM. Values represent mean ± SD obtained from four different experiments. There were statistically significant differences of preimmunized splenocyte responses to the peptides: between peptides 1 and 3 (P < 0.001), 1 and 4 (P < 0.001), 2 and 3 (P < 0.002), and 2 and 4 (P < 0.002).
	Fig. 5. Detection of larval-specific peptide by rat antiserum by western blot (a) and immunohistochemistry (b–e). (a) Western blots after sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE; 25 μg/lane) of the lysates from larvae at stage 55/56 (La) and the whole skin of 2–3-month-old froglets (Ad), showing that the antilarval peptide (LP) rat serum recognizes a 59 kDa band in the larval skin (LS) but not in the adult (antipeptide, La). The reactivity disappeared completely when the antiserum was pretreated with the peptide 2 used for immunogen (antipeptide + P2), but not with an unrelated control peptide (antipeptide + BMP). No signal was observed in the reaction with preimmune rat serum (normal rat serum). (b–e) Immunostained (red signals) and quinacrine counterstained (green) sections through epidermis of tail (b,d) and back skin of trunk (c,e) of larvae at stage 64, showing a prominent expression of the peptide in all for the tail epidermal cells (b) but not in the adult-type epidermis of the trunk except for the most upper layer of the epidermis (c). The signal in the most upper layer was not eliminated when the antisera were preabsorbed with 100 μg/μL peptide 2 (d,e), indicating that the fluorescence in the upper layer represents non-specific binding. Areas of epidermis are shown by the white lines on the left side. Bar, 100 μm.
	Fig. 6. Effect of antilarval skin (LS) antiserum on adult splenocyte- induced degeneration of larval tail tissues. The areas of larval tail pieces were measured before and after 2 days of coculture with adult splenocytes from which antigen-presenting cells (APC) such as macrophages, dendritic cells and B cells had been removed by nylon wool columns before culture. Frog anti-LS antiserum (5%) was added to the culture media including 5% normal adult Xenopus serum (+ Ab, ). Others included 10% normal adult Xenopus serum in the media (). (a) Co-cultured with (Sp) or without (W/O) normal adult splenocytes. (b) Cocultured with (Sp) or without (W/O) early thymectomized (E-txd) adult splenocytes. Values represent mean ± SD obtained from four different experiments. Asterisks at the top of columns indicate a statistically significant difference between –*(P < 0.005). (c) Typical features of larval fin pieces after 2 days of co-culture with (Sp) or without (W/O) E-txd splenocytes in the presence (+ Ab) or absence of anti-LS antiserum. Bar, 2 mm.