ARTICLE |
Correspondence to: Hugo Vankelecom, Univ. of Leuven, Dept. of Molecular Cell Biology, Lab. of Cell Pharmacology, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium.
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Summary |
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In the context of immune-endocrine relationships, we have previously shown that interferon- (IFN-
) inhibits hormone secretion in anterior pituitary (AP) cell cultures. The non-hormone-secreting folliculostellate (FS) cells were found to mediate this inhibitory action. Because in the immune system IFN-
is a strong stimulator of nitric oxide (NO) release through the induction of NO synthase (NOS), we investigated whether the inducible form of NOS (iNOS) is present in (rat) AP cell cultures, and whether its expression is stimulated by IFN-
. Immunocytochemistry revealed that under basal in vitro conditions only a very few AP cells contained iNOS. Treatment with IFN-
caused a sixfold rise in the number of iNOS-positive cells and augmented the intensity of the staining. The increased number of iNOS-expressing cells was paralleled by elevated production of NO. Some of the iNOS-positive cells extended cytoplasmic processes between hormone-secreting cells, which is a characteristic of FS cells. Immunostaining of FS cell-poor and FS cell-enriched populations (obtained by gradient sedimentation) also suggested the presence of iNOS in a subpopulation of FS cells. By double immunofluorescence techniques we found that about 65% of iNOS-expressing cells were positive for S-100, a marker protein for FS cells. However, around 80% of the S-100-positive cells were not labeled for iNOS. On the other hand, the majority of the S-100-negative iNOS-containing cells could not be further identified by antisera against the classical AP hormones, suggesting the presence of iNOS in a still unidentified non-hormone-secreting cell type of the AP gland. This report is the first to demonstrate the expression of the inducible form of NOS in the AP gland. IFN-
upregulates this expression, showing that cytokines may use the same signaling mechanisms in both the immune and the endocrine system. In addition, a putative new function of a subpopulation of FS cells in the paracrine regulation of the AP gland is suggested. (J Histochem Cytochem 45:847-857, 1997)
Key Words: inducible nitric oxide synthase, nitric oxide, anterior pituitary gland, folliculostellate cells, S-100, immunocytochemistry, immunofluorescence, rat
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Introduction |
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Folliculostellate (FS) cells are a class of non-hormone-secreting cells in the anterior pituitary (AP) gland. They have a starlike morphology, protruding long cytoplasmic extensions between the hormone- secreting cells of the gland. FS cells can be identified by the presence of S-100, a cytoplasmic protein which, at least in rat and human AP, is found exclusively in FS cells ( (IFN-
) exerts an inhibitory effect on stimulated AP hormone secretion, an action most likely mediated by FS cells (
In the present study we addressed the question of whether IFN- uses the same signaling molecule(s) in the AP gland as it does in the immune system. One of the factors recently discovered and now well documented to play a prominent role in mediating the effects of IFN-
in the immune system is nitric oxide (NO) (for review see
produce large amounts of NO. IFN-
stimulates the expression of the inducible form of NO synthase (iNOS), one of the isozymes catalyzing the formation of NO from L-arginine (
In the present study we investigated whether IFN- is capable of inducing iNOS in AP cells. Studies were done in vitro because in vivo administration of IFN-
is known to result in the release of a plethora of other cytokines and factors that may complicate interpretation of the results. To identify iNOS-expressing cells, differentially enriched AP cell populations obtained by unit gravity sedimentation in a serum albumin gradient and double immunofluorescence techniques were used.
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Materials and Methods |
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Antibodies
The polyclonal rabbit anti-iNOS antiserum was a gift from Dr. S. Moncada and Dr. V. Riveros-Moreno of Wellcome Research Laboratories (Beckenham, Kent, UK). The specificity of the antiserum, which was raised against a synthetic peptide fragment of the mouse iNOS molecule, has previously been shown (
Immunocytochemistry of Paraffin-embedded Sections from Rat Anterior Pituitary Cell Aggregates
Anterior pituitaries (AP) were obtained from 3-month-old random cycling female Wistar rats, and AP cells from pooled pituitaries were prepared as previously described ( (500 U/ml) (courtesy of Prof. P. Van Meideder ; ITRI, Rijswijk, Nederland) for 24 hr. Subsequently, they were fixed in Zamboni fluid (4% paraformaldehyde and 15% saturated picric acid solution in 0.1 M phosphate buffer, pH 7.4) for 4 hr, paraffin-embedded, and sectioned as previously described (
Immunostaining was performed as reported previously (
Quantitative data were obtained by counting the number of iNOS-positive cells per unit aggregate section. Of at least 15-20 sections per condition (control or IFN--treated), the surface area was measured using computer-assisted image analysis (Quantimet 500; Leica, Cambridge, UK) and the immunopositive cells in the section were counted. Data are expressed as number of iNOS-immunoreactive cells/mm2 section area (mean ± SE of the indicated number of independent experiments). Statistical analysis was performed using analysis of variance (ANOVA) methods as described (
Single and Double Immunofluorescence of Rat Anterior Pituitary Cells in Monolayer Culture
To avoid crossreactivity in double immunofluorescence, antibodies from different species were chosen in combination with distinct fluorophores. The mouse monoclonal anti-iNOS antibody (Transduction Laboratories), however, did not produce a signal in paraffin-embedded aggregate sections, probably due to destruction of the (single) epitope by the paraffin and organic solvent treatment. Different fixation protocols proved unsuccessful. Therefore, we switched to AP cells cultured as monolayers. Staining for hormones in monolayer cells has previously been shown (
To obtain AP cell monolayers, cells were prepared as described above, and were seeded on poly-L-lysine (Sigma)-coated coverslips in six-well tissue culture plates (Corning Glass Works; Corning, NY). Cells were kept in defined culture medium ( for 24 hr, washed, and fixed with 1% paraformaldehyde/1.5% glutaraldehyde in PBS for 10 min. To lower background staining, samples were treated with NaBH4 (0.5 mg/ml in PBS). Cells were permeabilized with Triton X-100 (0.1% in PBS) and aspecific binding blocked with BSA (3% in PBS). In the first experiments, staining with the monoclonal anti-iNOS antibody was compared to S-100 staining on separate cell samples, using a fluorescein (FITC)-labeled secondary antibody (rabbit anti-mouse Ig for iNOS and swine anti-rabbit Ig for S-100, both at a dilution of 1:40) (Dako). After the permeabilization and blocking reaction, cells were incubated with the primary antibody for 1 hr (at a dilution of 1:10 for iNOS and 1:500 for S-100 in 0.3% Triton X-100/3% BSA in PBS) and, after rinsing, with the secondary antibody for 30 min. After thorough washing, coverslips with the cell samples were mounted on a glass slide using glycerine (50% in PBS) with 25 mg/ml diazabicyclo[2.2.2]octane (DABCO; Sigma). Cells were examined on a Leica DMRB microscope (Leica; Wetzlar, Germany) using a Leica FITC filterset (I3), and images were recorded on a Leitz Orthomat E semiautomatic camera.
For double immunofluorescence, secondary antibodies labeled with different fluorophores were selected that showed only negligible overlap in absorption and emission spectra. iNOS was visualized with sheep anti-mouse IgG [F(ab')2 fragment] conjugated with 7-amino-4-methylcoumarin-3-acetic-N'-hydroxysuccinimide ester (AMCA) (1:10) (Boeh-ringer; Mannheim, Germany) giving a blue fluorescent signal (excitation optimum 345 nm; emission at 450 nm). For staining S-100 and the AP hormones, a goat anti-rabbit IgG [F(ab')2 fragment] conjugated with indocarbocyanine (Cy3) (1:800) (Jackson ImmunoResearch Laboratories; West Grove, PA) was used, which resulted in a red signal (excitation optimum 550 nm; emission at 570 nm). Images were viewed using Leica filters UVA for AMCA and N2.1 for Cy3. Cells stained in a single staining procedure for iNOS were blue but did not show red fluorescence using the Cy3 filter combination. When staining only for S-100 (or the AP hormones) was performed, a red signal was obtained, whereas no blue color was observed using the AMCA filter set. The procedure used for double immunofluorescence of iNOS and S-100 was similar to the one described for single staining. The procedure for double staining of iNOS and the AP hormones (PRL, GH, ACTH, TSH, LH-ß/FSH-ß) resulted in high background levels. Therefore, minor modifications were introduced, including extension of fixation time from 10 min to 1 hr and of incubation time with the primary antibodies from 1 hr to overnight, resulting in more satisfying hormone staining and lower backgrounds. Controls were performed in which one of the primary or the secondary antibodies was omitted. In addition, a non-sense mouse IgG2a (Dako) was used as a negative control for iNOS staining.
To confirm the specificity of the monoclonal anti-iNOS antibody, we performed pre-adsorption studies, using the mouse macrophage lysate that is provided by Transduction Laboratories as a positive control for iNOS immunoreactivity. Antigen (lysate at a dilution of 1:10) and antibody were incubated overnight at 4C. The mixture was further used for single immunofluorescence as described above. As a negative control antigen, human endothelial cell lysate was used (Transduction Laboratories). This preparation was shown by the supplying company to contain ecNOS but no iNOS. Staining for S-100 and pituitary hormones by polyclonal rabbit anti-rat antisera has in our previous work been shown to be specific (
Proportions of double (iNOS + S-100) stained cells and of single (iNOS or S-100) stained cells were obtained by counting as many cells per field as possible before fading occurred. In general, about 300-400 immunofluorescent cells were counted per independent experiment. Data are expressed as mean ± SE of four independent experiments. In the Results section, only a limited number of (representative) color microphotographs are shown; additional pictures are available on request.
Uptake of the Dipeptide ß-Ala-Lys-N-AMCA as a Marker for FS Cells
Recently, -AMCA. We used this dipeptide as an additional marker for FS cells in monolayer cultures. AP cells seeded on coverslips (see above) were incubated with 15 µM ß-Ala-Lys-N
-AMCA (kindly provided by Dr. C. Otto and Dr. K. Bauer; Max-Planck Institute, Hannover, Germany) for 3 hr. After thorough washing, cells were fixed and processed as described above.
Nitrite Concentration in AP Cell Monolayer Culture Supernatant as a Measurement of NO Production
AP cells were cultured for 2 days at a density of 200,000 cells/ml in serum-free defined medium. Medium was removed and cells were stimulated with IFN- (100 U/ml) for 24 hr. Nitrite (NO2-) levels in the supernatant were determined spectrophotometrically (absorbance at 540 nm) after mixing 100-µl samples with 100 µl Griess reagent [sulfanilamide 2% (w/v), N-(1-naphthyl)ethylenediamine 0.2% (w/v), phosphoric acid 4% (v/v); all products from Sigma]. NaNO2 (Sigma) was used as a standard. In preliminary experiments, nitrate (NO3-) levels were also measured by comparing NO2- levels in cell supernatants before and after treatment with nitrate reductase (Sigma) (
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Results |
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IFN- Induces iNOS Expression in AP Cells, Some of Which Resemble FS Cells
Cell re-aggregates from primary rat AP cells were treated with IFN- (500 U/ml) for 24 hr, and paraffin-embedded sections were stained for iNOS using a rabbit polyclonal anti-iNOS antiserum. As shown in Figure 1A, iNOS was detectable in AP cells under basal conditions. However, only a very small number of cells showing only a weak signal were detectable, i.e., 103 ± 11 cells/mm2 aggregate section area (mean ± SE; n = 4) (which corresponds to 5-10 cells per average section of one aggregate). Treatment with IFN-
(Figure 1B) clearly upregulated the expression of iNOS: the number of iNOS-positive cells increased sixfold (605 ± 13 cells/mm2 after IFN-
-treatment, n = 4; IFN-
-treated vs. control p<0.001 by ANOVA), and the staining became more intense.
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A closer look revealed that some of the iNOS-positive cells exhibited the morphology typical of FS cells (S-100-positive cells). They displayed thin cell processes protruding between the other AP cells (see arrows in Figure 2A). Other iNOS-containing cells displayed a round or polygonal morphology. In addition, some S-100-positive cells were observed as round cells without the typical stellate shape (for an example see Figure 2D, Figure 3A, and 4A).
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To obtain further evidence that iNOS is expressed in FS cells, immunostaining was performed on AP cell populations that were differentially enriched in FS cells as obtained by unit gravity sedimentation in a BSA gradient. iNOS-positive cells were observed in populations that contain a high number of FS cells [ Figure 1C-F: BSA gradient fractions (BSA fr) 2 and 3]. In BSA fr 3, 26 ± 3 cells/mm2 were positive for iNOS in control cultures, and 417 ± 24 cells/mm2 expressed iNOS after IFN- treatment (n = 3; IFN-
-treated vs control p<0.001 by ANOVA). For BSA fr 2, no quantitative data were collected because of relatively large necrotic areas in the center of the aggregates. Only very few iNOS-positive cells were found in populations that are poor in FS cells (Figure 1G-J: BSA fr 4 and 7-9) for proportions of FS cells in different BSA fr see Table 1 and (
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To test the functional activity of iNOS in IFN--treated AP cells, NO2- levels were determined in AP cell monolayer cultures as a measure of NO production (Table 1). NO production was stimulated by IFN-
, and the level of stimulation was highest in populations with the highest proportion of FS cells (BSA fr 2 and 3), confirming our immunocytochemical data.
Because there were clearly more S-100-positive cells (FS cells) than iNOS-containing cells in the aggregates (for examples see Figure 2C and Figure 2D), we hypothesized that (part of) iNOS is localized in a subpopulation of FS cells. The finding that BSA fr 4 still contains a considerable amount of S-100-positive cells (3-4%) ( treatment (Figure 1J), some of them displaying cytoplasmic extensions (Figure 2E and Figure 2F).
As can be seen in Figure 1D, staining in BSA fr 2 was prominent along the peripheral borders of the aggregates, which may be partly due to the fact that there were some necrotic areas in the center of the aggregate. Necrotic zones have consistently been observed in aggregates highly enriched in FS cells (unpublished observations). However, in aggregates from BSA fr 3 cells (Figure 1F), as well as from unfractionated AP cell populations (Figure 1B), expression of iNOS was also most pronounced in the peripheral cell layers, and necrotic foci were not observed in these aggregates.
Morphological resemblance between iNOS-positive cells and S-100-containing (FS) cells could also be observed in AP monolayer cell cultures treated for 24 hr with IFN- and processed for immunofluorescence using the monoclonal antibody against iNOS (see Figure 3). Cultured in the presence of a small amount of FCS (1%), FS cells nicely displayed their starlike morphology (Figure 3A). Such morphology was also seen in some of the iNOS-positive cells (Figure 3B). Again, some non-stellate cells contained iNOS immunoreactivity. On the other hand, some non-stellate cells were also S-100-positive. Further evidence for the morphological resemblance was based on the recent observation that FS cells, but no other AP cells, bear a dipeptide carrier of which the presence can be made visible by uptake of the fluorescent dipeptide ß-Ala-Lys-N
-AMCA (
-AMCA (see Figure 3C) displayed the stellate morphology as observed for a portion of the iNOS-containing cells. However, some non-stellate cells also accumulated ß-Ala-Lys-N
-AMCA.
The specificity of the monoclonal anti-iNOS antibody was confirmed by pre-adsorption experiments: incubating the antibody with iNOS-containing macrophage lysate abolished the staining (Figure 3D), whereas human endothelial cell lysate known to contain ecNOS but no iNOS did not affect the staining (data not shown).
Localization of iNOS in a Subpopulation of FS Cells and in Still Unidentified Cells as Revealed by Double Immunofluorescence
To conclusively identify the iNOS-expressing cell type(s), double immunofluorescence was performed. The mouse monoclonal antibody against iNOS and the rabbit polyclonal antisera against AP hormones and S-100 were used. Immunostaining with the anti-iNOS monoclonal antibody was unsuccessful in paraffin-embedded aggregate sections. In contrast, AP cells cultured as monolayers and fixed by brief glutaraldehyde and paraformaldehyde treatment provided a suitable alternative (see Materials and Methods).
As shown in Figure 4A and Figure 4B, iNOS-positive (blue) cells were also S-100-positive (red). A large number of the S-100-positive cells (77.9 ± 4.3%, mean ± SE; n = 4) did not stain for iNOS. On the other hand, one third of the iNOS-positive cells (33.2 ± 1.6%; n = 4) failed to stain for S-100 (see arrowheads in Figure 4C and Figure 4D). Only very sporadically, iNOS immunoreactive material was seen in cells that stained for LH-ß/FSH-ß (Figure 4E and Figure 4F, arrow), or PRL (Figure 4G and Figure 4H; no double-stained cell shown). In contrast, iNOS was never found in cells staining for ACTH (Figure 4I and Figure 4J), GH, or TSH. Appropriate controls in which one of the primary antibodies or the secondary antibodies was omitted did not show any sign of aspecific staining or staining overlap. Substituting the mouse monoclonal anti-iNOS antibody with a mouse non-sense antibody of the same isotype (IgG2a) abolished blue immunofluorescent staining (data not shown).
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Discussion |
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In this study we demonstrated that in AP cell aggregate cultures a few cells express iNOS and that IFN- causes a drastic increase in the expression of this enzyme. The number of cells immunoreactive for iNOS increased by a factor of 6 and the intensity of the immunostaining significantly increased as well. Enhanced expression of the enzyme was paralleled by elevated NO production, indicating that immunoreactive material represents functional iNOS.
iNOS expression after IFN- treatment was found in (S-100-positive) FS cells, as indicated by the following observations. First, the morphology of some of the iNOS-positive cells resembled that of FS cells. Second, iNOS-containing cells co-sedimented with FS cells in a BSA gradient. Moreover, NO levels after IFN-
stimulation in differentially enriched AP cell populations were higher when more FS cells were present. Third, double immunofluorescence data clearly showed iNOS immunoreactivity in some of the cells positive for S-100, indicating expression of iNOS in a subpopulation of FS cells. Importantly, however, a portion of the iNOS-positive cells did not contain S-100. These particular iNOS-containing cells failed to stain for the AP hormones (ACTH, PRL, GH, LH/FSH, TSH) apart from some very rare cells exhibiting PRL or LH/FSH immunoreactivity. These findings suggest the presence of iNOS in an AP cell type that remains to be identified. An attractive hypothesis is that iNOS may be expressed in progenitor cells. It has recently been shown that nerve growth factor (NGF) induces NO production in PC12 cells and that NO is responsible for cell growth arrest (cytostasis), a prerequisite for entry into the terminal differentiation pathway (
has been shown to facilitate NGF-induced differentiation of PC12 cells (
. Furthermore, after NGF treatment PC12 cells produce an IFN-like product which, in turn, may induce NO production and trigger differentiation events in these cells (
In view of the reported heterogeneity of FS cells (-AMCA in non-stellate cells, we cannot rule out the possibility that iNOS is expressed in FS cells that are not identifiable by S-100. Along the same line, iNOS may be localized in hormone-secreting cells that have lost their capacity to synthesize hormones in culture. This problem, however, has been minimized by the use of short-term cultures (1-2 days), thereby reducing the probability of de-differentiation of specific AP cells.
Thus far, other investigators have not found iNOS immunoreactivity in the AP which is probably because no inducing stimulus such as IFN- was added to their test system (
The expression of iNOS by FS cells after IFN- stimulation is interesting in view of the putative macrophage/dendritic cell-like nature of at least part of the FS cells (
, which is the principal macrophage-activating factor in the immune system, also activates FS cells to exert inhibitory actions on hormone secretion, possibly mediated by (a) paracrine factor(s) (
The present study also showed that iNOS-positive cells clearly tend to be located at the periphery of the AP aggregates. This peculiar distribution is not the consequence of insufficient penetration of IFN- into the aggregate, because AP cell aggregates are fully permeable to macromolecules (
Several authors have reported that NO may be implicated in the regulation of AP hormone release ( on AP hormone secretion, as revealed by the use of specific iNOS inhibitors (Vankelecom et al., submitted for publication).
In summary, our findings demonstrate that the inducible form of NOS is expressed in the AP gland and is strongly upregulated by IFN-. It is detected in a subpopulation of FS cells and in other as yet unidentified cells. The presence of iNOS, in addition to the expression of cNOS as demonstrated by others, suggests an important regulatory role for NO in the AP gland. Moreover, the presence of both forms in one cell type, i.e., the FS cell, further emphasizes the significance of FS cells in the paracrine regulation of AP cell development, growth, and function, and in integrating hormonal responses to immune and other stress reactions.
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Acknowledgments |
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This work was supported by grants from the Fund for Scientific Research-Flanders, Belgium (FWO), and the Flemish Ministry of Science Policy (Concerted Research Actions). HV is a Postdoctoral Fellow of the FWO, and PM is a Postdoctoral Fellow of the University of Leuven.
We thank Dr C. Otto and Dr K. Bauer (Max-Planck Institute, Hannover, Germany) for providing the fluorescent dipeptide ß-Ala-Lys-N-AMCA, and for transferring fixation and fluorescent staining technology on AP cell monolayers. We also thank Dr J. Steel (ICRF; Histopathology, London, UK), Dr S. Moncada, and Dr V. Riveros-Moreno (Wellcome Research Laboratories, Beckenham, UK) for supplying the polyclonal anti-iNOS antibody. We further thank K. Rillaerts, L. Seghers, and T. Mitera for skilfull technical assistance.
Received for publication September 11, 1996; accepted December 18, 1996.
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