0022-1554/93/$3.30 The Joumal of Histochemistry and Cytochemistry Copyright 0 1993 by The Histochcmicd Society, Inc.

Original Article

Vol. 41, NO. 2, pp. 151-156, 1993 Printed in USA.

Immunocytochemical Evidence That S-100-positive Cells of the Mouse Anterior Pituitary Contain Interleukin-6 Immunoreactivity’

HUGO VANKELECOM,’ PATRICK MATTHYS, JO VAN DAMME, HUBERTINE HEREMANS, ALFONS BILLIAU, and CARL DENEF Laboratory of Immunobiology (the Rega Institute) (HKPMJW,HH,AB), and Laboratoy of Cell Pharmacology (CO), Universaty of Leuven School of Medicine, B-3000 Leuven, Belgium.

Received for publication May 29, 1992 and in revised form August 25, 1992; accepted September 4, 1992 (2A2694).

We have previously shown that bioactive interleukin-6 (L6) is produced by rat and mouse (anterior) pituitary cells in vitro. Since the amount produced correlated with the pres- ence of S-100-containing folliculostellate (FS) cells, these cells were suggested to be a source of IL6 in the anterior pitu- itary (AP) lobe. In the present study we used immunocyto- chemical techniques to codirm this presumption. Freshly isolated mouse pituitary cells were subjected to immunocy- tochemical procedures whereby two &rent (neutralizing) monoclonal antibodies (MAb) against mouse IL6 (6M and 20F3) and a polyclonal antiserum raised against bovine S-100 were used as primary antibodies. Single immunostaining re- vealed a s m a l l portion of mouse pituitary cells (about 6.5%) to be positive for IL6 immunoreactivity with both antibod- ies. Importantly, the same proportion of cells was found to be IL6 positive if only the AP was used as the cell source. About 7.5% of the pituitary cells stained for the presence of S-100 immunoreactivity. Positive staining for IL6 was also found in pituitary cell samples from 2-day-old monolayer cultures and h m redispersed 9-day-old histotypic aggregates,

Introduction There is growing evidence that the immune system and the neu- roendocrine system form an integrated functional network (6). Both systems seem to be responsive to each other’s messengers. In this context, some of the immune messengers [cytokines such as interleukin-1 ( e l ) , interleukin-6 (L6), tumor necrosis factor (TNF), interferon-y (IFN-y)] have been found to affect the neuroendo- crine system and were therefore suggested to function as signals

Supported by grants from the Belgian National Fund for Scientific Research (NFWO), the National Loaery, the ASLKlCGER (General Sav- ings and Retirement Fund), and the Ministry of Science Policy (Concerted Research Actions) of Belgium. JVD is Research Associate and HV Research Assistant of the NFWO.

Correspondence to: Dr. Hugo Vankelecom, Rega Institute, Univ. of Leuven, Minderbroedersstraat 10, B-3000 Lcuven, Belgium.

which both secreted bioassayable IL6. In contrast, no IL6 staining was found in Aff -20 cells, an established ACTH- secreting tumor cell line of the mouse pituitary which did not secrete bioactive IL6. The specificity of the IL6 immu- nostaining was demonstrated by a total loss of staining when MAb 6B4 was omitted or replaced by imlepant rat IgG. Con- clusively, pre-adsorption of the anti-IL6 MAb (6B4) with recombinant mouse IL6 totally abolished staining of pitu- itary cells. Double immunostaining for IL6 and S-100 re- vealed that most if not all of the ILL-containing pituitary cells were positive for S-100. Few of the S-lOO-contahhg cells did not stain for IL6. These results confirm our previous hypothesis that FS cells, characterized by immunostaining of S-100 protein, contain bioactive and immunoreactive IL6 and therefore are very likely producers of IL6 in the AP. Fur- thermore, our results suggest that IL6 is implicated in the local regulatory role asaibed to FS cells in the pituitary gland. (J fiktochem Cytochem 41:151-156, 1993) KEY WORDS: Interleukin-6; Folliculostellate cells; S-100; Mouse pi- tuitary; Immunocytochemistry.

sent by the immune system towards the neuroendocrine system (6,11,26,35). In addition, certain molecules previously considered to belong uniquely to one system have in fact been found to occur in both systems. Several neuroendocrine messengers (hormones, neuropeptides) have been found to be present in certain cells of the immune system (7). On the other hand, the presence of cer- tain lymphokines and monokines (IL1, IL6, TNF-a, IFN-y) has now been demonstrated in organs of the neuroendocrine system, such as in particular parts of the brain (hypothalamus) and in the pituitary gland (15,19,29,35,37). We have reported that bioactive IL6 is produced in reaggregate cell cultures of mouse pituitaries and rat anterior pituitaries (APs) (37). This fmding has subsequently been confirmed by several groups (27,40). Furthermore, we found that the presence of a special rat AP cell type, the folliculostellate (FS) cell, is essential for IL6 production to occur, and that the yield of IL6 increased with increasing proportions of FS cells in differen- tially enriched AP cell populations (37). FS cells are a category of

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cells in the AP lobe that do not produce any of the established hormones, and neither their origin nor their functional significance is well understood (2). Phagocytosis, trophic effects, and/or a role in ion transport are some of the characteristics that have been as- signed to these cells. Recently, arguments have been brought for- ward supporting kinship between FS cells and cells of the mono- nuclear phagocytic system (2,36). Thus, macrophage- and dendritic cell-specific markers were found to occur on FS cells (3; and Denef and Andries, unpublished observations). Our finding that FS cells are the plausible source of IL6 also supports the existence of such a relationship. Several studies suggest a local regulatory role for FS cells, mediated by paracrine factors (1,34). One of these factors may be IL6, which has indeed been demonstrated to affect pitu- itary secretory function (21,25,36).

IL6 is a cytokine which can be produced by immunocompe- tent cells (macrophages, lymphocytes) but also by a variety of other cell types (fibroblasts, endothelial cells) (for review see 11,38). In the present study we investigated the presence of IL6 in the mouse (anterior) pituitary gland by immunocytochemistry. Furthermore, we examined whether IL6 is co-localized with S-100, the cytoplas- mic protein which, in rat and human AP, is found exclusively in FS cells (8,16,31).

Materials and Methods

Cell Separation and Culture Pituitaries were taken from 8-week-old female NMRI mice, sacrificed by decapitation. Animals, bred under specific pathogen-free conditions (Ex- perimental Animal Breeding Facility, University of Leuven, Belgium), had been kept in an environment of constant temperature, humidity, and day-night cycle with water and food ad libitum. Dispersed pituitary cells were prepared as previously described (37). Cells were then plated on poly- L-lysine-coated (M, 180.000-540.000) (Sigma; St Louis, MO), &well tis- sue culture chamber slides (Lab-Tek) (Miles Laboratories; Naperville. IL) at a density of 40,000 in 20 p1 per well for immediate immunocytochemi- cal processing, or 100.000 in 20 pl per well when immunostaining was done on Day 2 of culture. For long-term cultures, pituitary cells were established in aggregate culture as described (37). Briefly, 2 x lo6 cells were brought into 2 ml of serum-free, chemically defined medium (culture medium) (with- out triiodothyronine or dexamethasone) in a petri dish and were allowed to reassociate on a gyratory shaker in a 1.5% CO2/air incubator. The three- dimensional aggregates obtained are known to display ultrastructural and functional integrity (9,33). After 2 days in culture, spent medium was partly replaced by fresh medium and the number of cells was reduced to 0.5 x 106/ml. Twice weekly the aggregates were re-fed with new medium. On Day 9, aggregates were redispersed into separate cells, as previously de- scribed (1). Then, redispersed cells were plated at a density of 40,000 in 20 p1 per well on coated Lab-Tek slides and were subsequently subjected to immunocytochemical processing.

In some experiments the neurointermediate lobe of the mouse pitu- itary gland was removed by careful dissection under a stereomicroscope. Thus, only the cells of the AP were then used for immunochemical exami- nation.

The ACTH-producing AtT-20 cell line, derived from a mouse pituitary tumor, was found incapable of producing IL6 (unpublished observations) and therefore was used as a negative control for IL6 staining. The cell line (obtained from American Type Culture Collection, Rockville, MD) was propagated in DMEM, pH 7.4 (containing 4.5 glliter glucose), to which 10% fetal calf serum (FCS) (Gibcq Grand Island, NY) was added. Dis-

persed cells were plated on coated Lab-Tek chamber slides at a density of 40,000 in 20 pl per well and then subjected to immunostaining.

Immanocytochemistry Single Immunostaining of IL-6 and S-100. Cells were plated on the Lab-

Tek chamber slides and allowed to settle for at least 30 min in the 1.5% CO2 lair incubator. The cells were immunostained either immediately or after 2 days in culture. A slowly rocking platform was employed in all steps of this procedure. All incubations were done at room temperature. After washing with PBS, pH 7.4, cells were fixed by incubation for 3 hr 30 min in Zamboni fluid [4% (wlv) paraformaldehyde and 15% (v/v) saturated picric acid solution in 0.1 M phosphate buffer, pH 7.41. Fixative was re- moved by three washes with PBS. Pre-immune sheep serum (Dako; Car- pinteria, CA) (1:6 in PBS) in the IL6-staining procedure and pre-immune swine serum (Dako; Glostrup, Denmark) (1:5 in PBS) for S-100-staining were applied for 30 min. The primary antibodies against IL6 or S-100 were incubated overnight at a final dilution of, respectively, 1:1W and 1500 in PBS.

For immunostaining of IL6-containing cells, two monoclonal rat anti- mouse-IL6 antibodies (6B4 and 2OF3) were available. The (rat x mouse) hybridoma cell line D6906B4.M (abbreviated 6B4) was kindly provided by Dr. J Van Snick (Ludwig Institute, UCL, Woluwe, Belgium) (39). The anti- bodies were produced by propagation of the bB4 cells in athymic nude mice (nulnr). The ascites fluid collected was purified by affinity chromatogra- phy on a mouse anti-rat K-chain MAb-Sepharosc column (antibody MARK- 1) (14). The neutralizing potential of the 6B4 batch used was lo4,' against 10 U/ml natural mouse IL-6, as measured in the hybridoma growth factor (HGF) assay (32). The antibody was characterized as an IgGl (protein con- tent 270 pg/ml after Bradford). The (rat x mouse) cell line (MP5-2OF3.11) producing the 20F3 antibodies (28) was provided by American Type Cul- ture Collection with permission of DNAX Research Corporation (Palo Alto, CA). Purified antibodies, which were also of the IgGl type, were obtained in a similar manner as the 6% antibodies. The batch of 20F3 used for im- munocytochemistry exhibited a neutralizing potential of against 10 U/ml natural mouse IL6, and a protein content of 410 pg/ml. The poly- clonal rabbit antiserum (Ig fraction) against bovine S-100 protein was pur- chased from Dakopatts (Glostrup, Denmark).

After overnight incubation with the primary antibody, cells were washed with Tris-buffered saline [0.01 M Tris-buffer, 0.9% NaCI, 0.1% Triton X-100 (BDH Chemicals; Poole, UK), pH 7.61 and then subjected to the staining procedure

For immunocytochemical demonstration of IL6, we chose the sensitive avidin-biotin compla (ABC) method, with 3-amino-9-ethykarbaole @ins- sen Chimica; Beerse, Belgium) as a red color reagent. Cells were treated for 20 min with the secondary antiserum, i.e., biotinylated sheep anti-rat Ig antiserum (Ametsham; Poole, UK), at a dilution of 1:150 in Xis-saline. Subsequently, cells were washed, incubated with avidin-biotinylated horse- radish peroxidase complex (ABC-HRP; Dakopatts) for 25 min, and washed again. Finally, the bound antibody complex was demonstrated with a color reagent composed of 0.027% 3-amino-9-ethylcarbazole, 6.6% dimethyl- formamide (Aldrich-Chemie; Steinheim, Germany), and 0.04% H202 in acetate buffer (0.1 M, pH 5.2) .

For staining of S-100, a different method was required to avoid interfer- ence in the double-staining procedure. Therefore, we used the alkaline phos- phatase method with Fast Blue BB Salt (Serva; Heidelberg, Germany) as a blue dye. After overnight incubation with the anti-S-100 antiserum, cell samples were washed with Xis-saline (without Triton X-100). incubated for 45 min with an alkaline phosphatase-conjugated swine anti-rabbit Ig antiserum (Dakopatts) as the secondary antibody, washed again, and fi- nally stained as described (1).

To investigate the specificity of the IL6 staining, experiments were car- ried out in which the primary 6B4 antibody was either omitted, or replaced

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INTERLEUKIN-6 IN FOLLICULOSTELLATE CELLS 153

by an irrelevant rat IgG sample (Sigma) at about the same protein concen- tration. Ultimate proof was provided by a pre-adsorption experiment. Be- fore application to the cells, a 1:lOO dilution of the 6B4 antibody was in- cubated for 3 hr at 37'C in the presence of about 2500 U ( z 2 . 5 ng) recombinant mouse IL6. The specificity of the staining with anti-S-100 antiserum has been demonstrated in previous work (1). According to the specifications giwn by the manufacturer (Dakopatts), the antiserum against bovine S-100 is also able to recognize the S-100 protein of other animals (human, rat, mouse, chicken).

Double Immunostaining. Double immunostaining for IL6 and S-100 was facilitated by the fact that the respective antibodies were of different animal species (rat and rabbit, respectively). The cell preparations were in- cubated overnight with both primary antibodies simultaneously. After stain- ing for IL6, using the ABC method, a second staining sequence for s-100 protein, using the alkaline phosphatase method, was applied. In parallel, double stainjngs were performed in which one primary antibody was omitted.

Each staining, i.e., performed on an independent (anterior) pituitary cell preparation, was done in duplicate, and at least 400-600 cells/well were counted.

Results

Immunostaining of IL6 Dispersed mouse pituitary cells were stained for IL6 immunoreac- tivity by the ABC method, with use of a specific (neutralizing) MAb against mouse IL6 (6B4). IL6 immunoreactivity was found to be present in the cytoplasm of a small proportion of the cells (Figures 1A and ID). Freshly isolated pituitary cells as well as cells from 2-day-old monolayer cultures and from redispersed 9-day-old ag- gregates were stainable. In monolayer and aggregate cultures, production of bioactive IL6 coincided with positive immunostain- ing. The proportion of IL6-immunoreactive cells amounted to 6.55 i 0.46% (mean i SE; n = 9), and 10.40 2 0.33% (mean 2 S E n = 4) in, respectively, freshly isolated and 9-day-old redispersed pituitary cells. In 2-day-old monolayer cultures, correct counting was precluded by cell clumping. In addition, the mouse pituitary cell line AtT-20, which does not produce IL6, failed to stain with 6B4 (Figure IC), whereas it did stain with anti-ACTH and anti- gastrin-releasing peptide antibodies (results not shown), a finding that has previously been reported by Houben and Denef (17).

Specificity of the IL6 immunostaining was ascertained by the following experiments. Staining intensity diminished when the con- centration of the monoclonal anti-IL6 antibody added to the sam- ples was reduced, all other components of the reaction remaining equal. Moreover, no stained cells were observed when the primary antibody (6B4) and/or the secondary (bridging) antibody (bi- otinylated sheep anti-rat Ig) were omitted, or when irrelevant rat IgG was used as primary antibody instead of 6B4. The specificity of the IL6 staining was also ascertained by immunoadsorption studies. Before application of the anti-mouse IL6 antibody (6B4) to the cells, it was pre-adsorbed to recombinant mouse IL6. This procedure completely abolished staining of the mouse pituitary cells (Figure 1B). Additional proof for specific staining of the IL6 protein was the observation that the use ofa different rat anti-mouse IL6 antibody (20F3) resulted in the same number of positively stained cells (6.72 * 0.42%) (mean 2 SE; n = 4). With this anti-

body, however, background staining was more intense than with the 6B4 antibody.

In further experiments, we investigated whether the IL6- containing cell is situated in the anterior or in the posterior lobe of the pituitary gland. Therefore, mouse pituitaries were dissected under a stereomicroscope and the neurointermediate lobe was care- fully discarded. In the cell samples prepared from the anterior lobe, IL6-positive staining was found in, respectively, 6.4 and 7.2% (n = 2) of the cells, a number that does not differ from that found in cell preparations from the total pituitary gland.

Identification of the Pituitary Cell Type Containing IL6 Immunoreactivity Double immunostaining studies, performed on freshly isolated AP cells and cells from redispersed 9-day-old aggregates, revealed that IL6 and S-100 are co-localized. It was found that almost all of the stained cells were double stained. In freshly isolated cells (Figure LF), their proportion amounted to 6.09 i 0.43% (mean i SE; n = 6). About 2% (range 1.2-3.146) ofthe cells were S-1OO-positive (blue) cells that failed to stain for IL6, whereas almost all IL6 positive (red) cells stained for S-100 (range of only IL6-containing cells 0.1- 0.6%). When stainingwas done only for S-100 (Figure lE), 7.39 i 0.40% (mean i S E n = 4) ofthe pituitary cellswere found positive. Double staining revealed itself as an intense mixed color of bor- deaux red, rather than separate blue and red. This color did not reflect nonspecific background reactivity. Indeed, after the double- staining procedure, some cells were only single stained and many others were not stained at all. In addition, cells undergoing the double-staining method, in which one of the primary antibodies (6B4, respectively anti-S-100 antiserum) was omitted, displayed a pure blue, respectively red, but not a bordeaux red, color (not shown).

Discussion 11-6 immunoreactivity was found to be present in 6.5% of mouse pituitary cells, as demonstrated by staining of freshly dispersed cells with a specific MAb against mouse IL6 (6B4). Specificity of the staining was ascertained by several experiments: omission of pri- mary and secondary antibody, use of irrelevant antibodies as pri- mary antibody and, conclusively, immunoadsorption, in which staining was abolished by previous adsorption of the antibody to its appropriate antigen (recombinant mouse IL6). In addition, AtT-20 cells, which were found not to produce IL6 (unpublished observations), failed to stain for IL6. Furthermore, a different anti- mouse IL6 antibody (20F3) revealed IL6 positivity in the same number of cells. Identification of the IL6-containing cells was per- formed by double immunostaining procedures. They showed that IL6 immunoreactivity is localized in the cells that contain S-100 protein. In human and rat, S-100-containing cells of the AP have been thoroughly characterized as FS cells (8,13,16,18,20,22,31). Al- though FS cells in the mouse pituitary have been electron microscop- ically characterized (10,12,41), expression of S-100 by these cells has thus far not been verified. As a consequence, we can only speak in terms of a co-localization of IL6 with S-100. Nevertheless, the number of S-100-positive cells in the pituitary of female adult mice

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(about 7.5%) correlates with the proportion of FS cells in the AP lobe of the female adult rat (7-8%).

IL6 can be produced by various cell types like monocyteslmac- rophages, fibroblasts and endothelial cells (for review see 11,38). Since expression of S t O O protein also occurs in these cell types (4,5,23,30), we cannot exclude the possibility that one of them is responsible for production of IL6 by pituitary cell populations. However, expression of S-100 protein in endothelial cells has only occasionally been demonstrated, in bovine lymph nodes and spleen arteries (5). Furthermore, only fibroblast-like cells in certain tumors have been reported to contain S-100 immunoreactivity (4,23). Liter- ature data pertaining to S-100 expression by pituitary-derived fi- broblasts, endothelial cells, or macrophages (histiocytes) are not available. Importantly, staining for IL6 immunoreactivity was also observed in pituitary cells obtained after redispersion of 9-day-old aggregates. In this aggregate culture system, mesenchymal cells such as endothelial cells, fibroblasts, and histiocytes do not proliferate (33). Therefore, we consider it likely that the IL6-producing cell does not belong to these categories but is probably the FS cell.

Other possible sources for L 6 in the pituitary gland are the pitui- cytes of the posterior lobe, which also contain S-100 (31). Indeed, related cells such as astrocytes have been demonstrated to be capa- ble of producing IL6, e.g., after viral infection (15). The possibil- ity of pituicytes being the IL6 source could be excluded by experi- ments in which the neurointermediate lobe was discarded. The observation that equal proportions of cells were stained in cell prepa- rations from total pituitary and from only the AP makes it seem unlikely that IL6 staining in total pituitary cell populations was due to the presence of posterior lobe pituicytes.

We have not formally excluded the possibility that the presence of IL6 in the S-100-positive cells results from uptake from the en- vironment rather than endogenous production. However, staining of redispersed 9-day-old aggregates indicated that the IL6 im- munoreactivity did not originate from the blood circulation. In addition, our previous work demonstrated that the amount of bio- active IL6 in cultures of different AP cell populations (obtained by velocity and buoyant density sedimentation) did not correlate with the presence of any cell type other than the FS cell, thereby excluding the possibility of another AP cell type being the source of IL6 (37). Furthermore, this relationship remained evident in culture for more than 20 days (unpublished observations), clearly indicating that FS cells are the source.

The production of IL6 by AP cells has now been confirmed by several groups (27.40). In accordance with our finding that FS cells are the source, Spangelo et al. (24) reported release of IL6 in those rat AP cell populations, as obtained by gradient sedimen- tation, that did not overlap with the AP cell populations secreting classical hormones.

Several studies indicate that IL6 can play a local regulatory role within the pituitary gland. Treatment of rat AP cells with IL6 over 2 to 4 hr resulted in enhanced hormone secretion (growth hormone, prolactin, luteinizing hormone) (25). In contrast, when AP cell cul- tures were pre-incubated for 24 hr with L 6 , inhibition ofthe stimu- lated release of adrenocorticotropic hormone (ACTH) (36) (and also of prolactin and growth hormone; unpublished observations) was observed.

In conclusion, our study unambiguously demonstrates that IL6 in the AP has its origin in the FS cells and therefore provides fur- ther support for the hypothesis that IL6 is implicated in the local regulatory role assigned to FS cells in the AP.

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Figure 1. lmmunostaining of 11-6 in freshly isolated mouse pituitary cells and dispersed AtT-20 cells. (a-c) Specificity of the 11-6 immunostaining procedure. (a) Cells were immunostained with the monoclonal rat anti-mouse 11-6 antibody (684) following the ABC method. (b) Pre-adsorption of 684 to mouse 11-6 abolished specific staining. (c) AtT-20 cells, which do not produce 11-6 in culture, failed to stain with 684. (d-f) Co-localization of 11-6 and S-100 protein immunoreactivities by double immunostaining of freshly isolated mouse pituitary cells. (d) Cells were immunostained for 11-6 with the monoclonal rat anti-mouse 11-6 antibody (684) following the ABC method (red color). (e) Cells were immunostained for S-100 with the polyclonal rabbit anti-(bovine)-M00 antiserum following the alkaline phos- phatase method (blue color). (f) Cells were immunostained for both 11-6 (ABC method) and W O O (alkaline phosphatase method). Note that cells are positive for both proteins (bordeaux red color). Original magnifications: a-c x 350; d-f x 850. Bars = 10 pm.

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