Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Growth Differ
2002 Dec 01;446:477-88. doi: 10.1046/j.1440-169x.2002.00660.x.
Show Gene links
Show Anatomy links
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
.
???displayArticle.abstract???
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 trunkepidermis 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 tailepidermis. 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 tailepidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.
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 2â3-month-old
froglets (Adult), showing the larval-specific proteins (25 μg/lane)
were separated by sodium dodecylsulfate (SDS)âpolyacrylamide
gel electrophoresis (PAGE) at approximately 53 and
56â60 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 2â3-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 1â2-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 1â4 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 *â**(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.
