ARTICLE |
Correspondence to: Eugenio Bertelli, Dept. of Biomedical Sciences, University of Siena, Via Aldo Moro, I-53100 Siena, Italy.
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Summary |
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The study of intermediate filament expression in the pancreatic epithelium has been previously focused almost exclusively on cytokeratins. Transient vimentin immunoreactivity has also been detected in duct cells of rat fetal pancreas. Here we report that, in rat pancreas, intense GFAP-like immunoreactivity is detectable in a subpopulation of endocrine cells located in the periphery of the islet of Langerhans. In addition, staining appeared to be preferentially localized to the apical pole of the cells. Two different polyclonal antibodies were employed in this study, with analogous results. Staining of consecutive sections with anti-GFAP, anti-glucagon, and anti-somatostatin antibodies demonstrates that GFAP-like immunoreactivity is present in glucagon-secreting cells. The relevance of this finding is discussed. (J Histochem Cytochem 48:259265, 2000)
Key Words: intermediate filaments, GFAP, pancreas, glucagon cells, immunocytochemistry, rat
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Introduction |
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The cytoskeleton of virtually all differentiated eukaryotic cells is made up of a network of three main filamentous structures. In addition to microtubules and actin microfilaments, the third major component is represented by intermediate filaments (IFs) (-internexin, and nestin; Type V nuclear lamins; Type VI phakinin and filensin (
GFAP has been reported to be specifically expressed in mature astrocytes (
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Materials and Methods |
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Antibodies
Two rabbit anti-GFAP polyclonal antibodies (PAbs) were used. One was purchased from Zymed Laboratories (San Francisco, CA). The other, from Sigma (St Louis, MO), was kindly provided by Dr. C. Nicoletti (Laboratory of Molecular Recognition, The Babraham Institute, Cambridge, UK). Rabbit anti-glucagon and anti-somatostatin PAbs were a generous gift from Dr. M. Bendayan (Department of Pathology and Cell Biology, University of Montreal, Montreal, PQ, Canada). Anti-vimentin MAb (clone V9) was kindly provided by Dr. C. Nicoletti. Alkaline phosphatase (AP)-conjugated anti-rabbit IgG was obtained from Roche Diagnostic (Monza, MI, Italy). AP-conjugated anti-mouse IgG was purchased from SigmaAldrich (Milan, Italy).
Animals and Tissue Processing
Four-month-old Wistar rats were used for this study. Rats were anesthesized with diethylether and decapitated. In some cases, the entire splenic portion of the pancreas was fixed in 50 ml of 10% formalin or Bouin's fixative overnight, for 2 days or for 5 days at room temperature (RT), and processed for routine embedding in paraffin. In other cases, the pancreas was trimmed in small blocks of 0.5 mm3, fixed for 3 hr at 4C in 5 ml of 4% paraformaldehyde dissolved in 0.1 M cacodylate buffer, and processed for ordinary embedding in Epoxy resin (Epon 812).
Immunocytochemistry
Deparaffinized sections (45 µm thick) were pretreated with 1% BSA in PBS for 30 min and then incubated with anti-GFAP PAb or anti-vimentin MAb (clone V9) for 4 hr at RT. After rinsing with 0.1% Triton X-100 in PBS, sections were treated with 1% BSA in PBS for 10 min and incubated with the appropriate AP-conjugated anti-rabbit or anti-mouse IgG. Slides were thoroughly rinsed with 0.1% Triton X-100 in PBS, and AP reaction was developed with NBT/BCIP stock solution (Roche Diagnostic) as chromogen diluted in 0.1 M Tris buffer, pH 9.5, 0.05 M MgCl2, and 0.1 M NaCl.
Consecutive semithin sections (1 µm thick) from Epon-embedded samples were also used. In this case, sections were mounted on well-degreased slides and Epon was removed according to the technique suggested by
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Results |
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GFAP-immunoreactive cells were detected in rat pancreas as Schwann cells of nerve fibers. In the islets of Langerhans, however, strong staining with anti-GFAP Abs was obtained in peripheral islet cells regardless of the type of antibody employed (Figure 1). Moreover, observation of these cells at higher magnification allowed recognition of an asymmetrical distribution of staining, which was stronger at the apical (perivascular) pole of the cells (Figure 2). The intensity of the staining was dependent on fixation time regardless of the fixative employed: the longer the fixation, the weaker the reaction. Fixation for as long as 2 days abolished immunoreactivity.
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To determine precisely the type of cell responsible for islet GFAP immunoreactivity, consecutive semithin sections were stained with anti-glucagon, anti-GFAP, and anti-somatostatin PAbs. By this procedure, it was evident that somatostatin-secreting cells did not stain with anti-GFAP Abs, whereas all glucagon-secreting cells expressed GFAP-like immunoreactivity as well (Figure 3). On the other hand, a limited number of cells expressing GFAP-like immunoreactivity scattered throughout the exocrine tissue were also identified as glucagon-secreting cells (Figure 4). All GFAP-immunoreactive cells detected in our series of experiments therefore appeared to be glucagon cells. Omission of primary antibodies or replacement with rabbit nonimmune serum abolished all the reactions. On the other hand, staining was not impaired on anti-GFAP Ab absorption with glucagon or with poly-L-lysine (
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None of the anti-GFAP Abs employed stained blood vessels (Figure 5) either in endothelial or in muscle cell components. We also performed a series of positive controls with sections of rat brain tissue incubated with anti-GFAP Abs that stained exclusively glial cells (Figure 6). Incubation of sections with anti-vimentin MAb resulted in a completely different pattern of staining, with no labeling of islet cells but with strong staining of endothelial cells lining intra-islet blood capillaries (Figure 7).
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Discussion |
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Our immunocytochemical findings provide evidence that pancreatic glucagon-secreting cells exhibit GFAP-like immunoreactive antigens. Eventual crossreactions of anti-GFAP antibodies employed in the present study with other known IFs appear to be unlikely on the basis of the following considerations. The pattern of staining obtained in the pancreas and in other tissues does not match that expected if antibodies recognized desmin (unstained muscle cells), neurofilaments (unstained neurons), or CKs (no known CK has been reported to be expressed exclusively in a single islet cell type; for a review on CK expression in pancreas see
Even though we can not completely exclude the detection of a peptide not strictly related to GFAP, we can affirm that GFAP-like immunoreactivity in these conditions should reveal actual GFAP (GFAP) or a protein derived from an alternative splicing of GFAP mRNA. In this regard, in addition to GFAPß located prevalently in the peripheral nervous system (
), lacking the entire exon 1, has been reported to be expressed in mouse bone marrow and spleen (
The presence of GFAP in the endocrine pancreas could prompt reconsideration in a new light of the theory of a neural crest origin of endocrine cells (
The exact role of GFAP in glucagon-secreting cells is not clear at the moment. GFAP expression has been linked to the stellate morphology of some cell types including astrocytes, periacinar stellate cells, pituitary folliculostellate cells, and perisinusoidal stellate cells (
A variety of evidence suggests that IFs confer on cells higher resistance to mechanical stress. This has been clearly demonstrated for CKs in epidermal cells and for desmin in muscle cells (
To date, therefore, there is no hypothesis on GFAP function that can explain its presence in all GFAP-immunoreactive cells. It is possible, however, that GFAP may serve several different functions according to the cell type. In this regard, the finding that GFAP immunoreactivity appears to be restricted to the secretory pole of A-cells raises the possibility that in this type of cell GFAP is involved in the secretory machinery.
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Acknowledgments |
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This work is dedicated to the memory of Prof Leonetto Comparini.
Received for publication June 1, 1999; accepted September 29, 1999.
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