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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0695fje. |
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Dipartimento di Scienze Biochimiche, CNR Centro di Biologia Molecolare, and Istituto di Anatomia Umana, Università ‘La Sapienza’, 00185 Roma, Italy
2Correspondence: Dipartimento di Scienze Biochimiche, Università ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma, Italy.
SPECIFIC AIM
In the present study, we investigated the synthesis of antimicrobial peptides in skin glands of the frog Rana esculenta under different conditions. We show by HPLC analysis, detection of antimicrobial activity, and gland morphology that frogs kept in a sterile environment do not synthesize these peptides; their synthesis is induced by the presence of microorganisms.
PRINCIPAL FINDINGS
1. Induction of peptide synthesis by microorganisms
To study the synthesis of antimicrobial peptides in vivo,
specimens of R. esculenta were repeatedly stimulated to
deplete skin glands. Frogs were then exposed to different environmental
conditions. A first group was kept in water in the presence of bacteria
of the natural flora at 106 cfu/ml, and a second
group was maintained for the same amount of time in water containing
tetracycline and erythromycin at a final concentration of 80 and 150
µg/ml, respectively. After 1 and 2 wk, the antimicrobial activity,
expressed as cecropin A units, as well as the reversed-phase HPLC
profiles of the secretions were determined. In frogs kept under sterile
conditions, the amount of antimicrobial peptides decreases to very low
levels. When these frogs were subsequently re-exposed to bacteria of
the natural flora, the synthesis of antimicrobial peptides in the skin
was restored, as shown by HPLC analysis and determination of the
antimicrobial activity of the secretion. This effect is not caused by
the presence of antibiotics in the water. This was shown by adding
cells of Candida guiller-mondii, a yeast not affected by
antibiotics, to frogs kept under sterile conditions for 2 wk. In the
presence of yeast cells, the synthesis of antimicrobial peptides was
restored and the HPLC profile of the skin secretions returned to normal
levels. However, re-exposure to C. guiller-mondii must be
performed gradually to avoid killing of the animals.
2. Morphology of the glands
The morphology of the glands was analyzed in frogs kept under
different conditions, as described above. As expected, the glands of
frogs kept in the presence of bacteria have a normal structure, with
nuclei located at the periphery of the cells and cytoplasmic granules
slowly filling the lumen (Fig. 1A
, C
). Conversely, in the presence of antibiotics, the
regeneration of the glands is impaired. Gland cells are not enlarged,
nuclei stay in the center, and cytoplasmic granules (where the
antimicrobial peptides are stored) are absent (Fig. 1B
, D
).
Addition of C. guiller-mondii to frogs kept in
the presence of antibiotics also induces the formation of glands with a
normal morphology. It is noteworthy that in skin sections taken from
different parts of the animal, the morphology of the regenerated glands
was very similar. This indicates that the effects exerted by the
microorganisms are simultaneously transmitted to all glands.
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3. Morphology of glands from skin treated with glucocorticoids
We have shown previously that treatment of R. esculenta
skin with a systemically acting glucocorticoid (Clobesol, Glaxo,
containing 0.05 g clobetasol propionate/100 g cream) inhibits the
transcription of all genes encoding antibacterial peptides by inducing
the synthesis of I
B
. We also checked the morphology of glands
after this treatment. As in the case of frogs kept under sterile
conditions, the regeneration of glands is inhibited by treatment with
glucocorticoids. However, the morphology of the glands is different in
these two cases. When peptide synthesis is blocked by glucocorticoids,
the nuclei are thicker and the cells seem to be organized in columns
with a decreased amount of cytoplasm (Fig. 2B
). In frogs kept under sterile conditions, nuclei are
reduced in size and the cytoplasm appears to be empty (Fig. 1B
, D
). Furthermore, the total number of glands is appreciably lower.
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It could also be shown by immunohistology that, after treatment with
glucocorticoids, the glands strongly react with an antiserum against
IB (Fig. 2D
) whereas no signal could be observed in
normal glands (Fig. 2C
). The accumulation of IB in the
cytoplasm of the glands explains the observed blockade of peptide
synthesis.
4. Electrophoretic mobility shift assay
To further verify the involvement of a NF-B-mediated mechanism
in the induction of antimicrobial peptide synthesis, we tested for the
presence of NF-B-like proteins in the secretion. It is known that
upon electrical stimulation, the entire content of the gland, including
the cytoplasm of gland cells, is released. Aliquots of skin secretions,
corresponding to 
10 µg of proteins, were incubated with a
double-stranded deoxyoligonucleotide labeled with
[
-32P]ATP using T4 polynucleotide kinase and
resolved by electrophoresis on 5% nondenaturing polyacrylamide gels. A
B binding activity was present in the secretion of frogs kept in
bacteria-containing water, but absent in animals kept under sterile
conditions. When the frogs were re-exposed to either C.
guiller-mondii or natural bacterial flora, a strong signal
reappeared.
CONCLUSIONS
The present study demonstrates that the synthesis of antimicrobial
peptides in skin glands of R. esculenta is dependent on the
presence of microorganisms (see Fig. 3
). However, in contrast to what has
been reported in Drosophila, the nature and amount of
peptides synthesized in skin glands appear to be independent of the
microbial species to which the frogs are exposed. These findings
represent the first in vivo demonstration of the induction of defense
peptides in a vertebrate.
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These experiments open the way to study the mechanism by which
microorganisms interact with the frog skin. A direct effect of bacteria
on cells seems unlikely since the regeneration of glands appears to be
synchronized. We assume that the NF-B cascade is activated via an
unknown signaling event acting on the cells of all glands, which in
turn stimulates transcription of the genes coding for antimicrobial
peptides. Frogs may therefore represent interesting model organisms to
study the regulation of the innate immune defense in
vertebrates.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0695fje ; to cite this
article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0695fje 
This article has been cited by other articles:
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