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Endocrinology, doi:10.1210/en.2004-0946
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Endocrinology Vol. 146, No. 1 35-43
Copyright © 2005 by The Endocrine Society

Macrophage Migration Inhibitory Factor Is Released from Pituitary Folliculo-Stellate-Like Cells by Endotoxin and Dexamethasone and Attenuates the Steroid-Induced Inhibition of Interleukin 6 Release

Tanya Tierney, Reshma Patel, Caroline A. S. Stead, Lin Leng, Richard Bucala and Julia C. Buckingham

Department of Cellular and Molecular Neuroscience (T.T., R.P., C.A.S.S., J.C.B.), Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom; and Department of Internal Medicine and Pathology (L.L., R.B.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Prof. Julia Buckingham, Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: j.buckingham{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by peripheral immune cells and also by endocrine cells in the anterior pituitary gland. MIF exerts its proinflammatory actions in the host-defense system by blocking the inhibitory effects of glucocorticoids on the release of other proinflammatory cytokines (e.g. IL-1, IL-6, TNF{alpha}). Reports that pituitary folliculo-stellate (FS) cells share many characteristics with immune cells led us to propose that these cells may serve as an additional source of MIF in the pituitary and that pituitary-derived MIF may act in an autocrine or paracrine manner to modulate endotoxin-induced cytokine release from FS cells. In the present study we addressed this hypothesis by using 1) immunohistochemistry to localize MIF in primary pituitary tissue and 2) well-characterized FS (TtT/GF), corticotroph (AtT20), and macrophage/monocyte (RAW 264.7) cell lines to explore the effects of CRH, endotoxin, and dexamethasone on MIF release and to examine the effects of MIF on IL-6 release. Our immunohistochemical study showed that MIF is expressed in abundance in S100-positive FS cells and also in other pituitary cell types. All three cell lines expressed MIF protein and responded to endotoxin (10–1000 ng/ml, 24 h) and dexamethasone (100 pM to 10 nM, 24 h) with concentration-dependent increases in MIF release. CRH (10–100 nM) also stimulated MIF release from AtT20 cells but, unlike endotoxin and dexamethasone, it had no effect on MIF release from TtT/GF or RAW cells. Recombinant MIF did not affect the basal release of IL-6 from TtT/GF cells; however, it effectively reversed the inhibitory effects of dexamethasone (1 nM) on the endotoxin-induced release of IL-6 from these cells. The results suggest that the FS cells are both a source of and a target for MIF and raise the possibility that MIF serves as a paracrine/autocrine factor in the pituitary gland that contributes to the protective neuroendocrine response to endotoxin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECIPROCAL COMMUNICATION BETWEEN the neuroendocrine and immune systems is of fundamental importance to homeostasis. Not surprisingly, in the light of the powerful antiinflammatory and immunosuppressive properties of glucocorticoids, particular attention has focused on the interface between the hypothalamo-pituitary-adrenocortical (HPA) axis and the host-defense system. It is now well established that proinflammatory cytokines released in response to an immune challenge cause marked activation of the HPA axis (reviewed in Refs. 1 and 2) and that the resulting rise in glucocorticoid secretion plays a critical role in tempering the immune response (2). This is amply illustrated by reports that adrenalectomized mice (3) and rats (4) are highly susceptible to bacterial endotoxins (lipopolysaccharide, LPS) and that animals with compromised HPA function show a more severe, and frequently lethal, course of inflammatory disease after induction of e.g. experimental arthritis or autoimmune encephalomyelitis (1, 2). Communication between the neuroendocrine and immune systems is effected in part by signaling molecules that are released and recognized by both systems (reviewed in Ref. 5). The common usage of these molecules has blurred the distinction between cytokines and hormones because it has become apparent, for instance, that the receptors for the pituitary hormones, growth hormone and prolactin, belong to the cytokine receptor superfamily and that proinflammatory cytokines such as IL-1 and IL-6 are produced in the neuroendocrine system and act at multiple levels to stimulate glucocorticoid secretion (2). In particular, IL-6 has been shown to enhance and prolong the HPA response to stress (6).

The positive effects of peripherally produced cytokines (e.g. IL-1, IL-6, tumor necrosis factor-{alpha}, TNF{alpha}) on ACTH release are largely dependent on the secretion of CRH and vasopressin by the hypothalamus (reviewed in Refs. 1 and 2). However, cytokines produced within the pituitary and adrenal glands themselves also play a role in activating the HPA axis. Within the anterior pituitary gland, the folliculo-stellate (FS) cells, first identified by Rhinehart and Farquar (7), are an important source of cytokines (e.g. IL-1, IL-6, leukemia inhibitory factor) and growth or angiogenic factors (e.g. vascular endothelial growth factor and basic fibroblast growth factor, reviewed in Ref. 8), which they use to communicate with the surrounding endocrine and vascular cells. FS cells are also a target for endotoxin (9, 10). Reports that LPS administration in vivo increases pituitary IL-6 expression (9) together with evidence that pituitary reaggregate cultures respond to LPS with an increase in ACTH release that is dependent upon the release of IL-6 from FS cells (10), point to an important role of FS cells in the manifestation of the HPA responses to endotoxemia.

The biology of the FS cells is poorly understood. The origin of the cells is disputed and may differ from that of the surrounding endocrine cells, which are derived during development from the oral ectoderm. FS cells stain for S100, a neuroectodermal marker; however, reports that FS cells show characteristics such as phagocytotic activity (11), cytokine production (12, 13, 14, 15), and CD14 positivity (16) have fueled a premise that FS cells are of lymphoid rather than neuroectodermal origin. On the other hand, whereas FS cells undoubtedly show some characteristics of macrophages, no cells have been identified in the anterior pituitary gland that stain positively for both FS (S100) and macrophage (OX-42) markers (17). However, Allaerts et al. (18) demonstrated cells staining positively for both FS (S100) and the dendritic cell marker, OX-6, suggesting a relationship between these cell types. Interestingly, peripheral cells of a dendritic lineage are also S100 positive (19).

Macrophage migration inhibitory factor (MIF) is a potentially interesting protein with regard to neuroendocrine-immune interactions because it is produced by both immune cells and endocrine tissues (20). MIF was first described as a T cell-derived cytokine that inhibits the random migration of macrophages (21). It has since also been shown to be produced by monocytes and macrophages and to have an important role in the development of Gram-negative sepsis (22). Administration of MIF thus potentiates the lethal effects of LPS (23), whereas neutralizing anti-MIF antibodies have a protective effect (24) and MIF knockout mice are relatively resistant to LPS (25). Interestingly, low levels of glucocorticoids promote MIF release from monocytes and macrophages (26), an apparent paradox given that MIF is a proinflammatory cytokine and glucocorticoids usually exert powerful anti-inflammatory actions. MIF then acts in an autocrine or paracrine manner within the host-defense system to block the effects of glucocorticoids (another paradox) on LPS-induced cytokine release (26). The interplay between the pro- and anti-inflammatory actions of MIF and glucocorticoids thus appears to act as a counterregulatory system that aids the maintenance of homeostasis (20).

The anterior pituitary gland is another important source of MIF. Immunohistochemical analysis at the electron microscopic level has localized MIF to granules in the corticotrophs and thyrotrophs and shown that in corticotrophs some granules contain MIF only, whereas others contain both MIF and ACTH (27). Functional studies on the AtT20 murine corticotroph cell line showed that MIF is secreted in response to LPS and CRH, with a peak response occurring at 3 h (27), compared with 20 h in monocytes, macrophages, and T cells (22). Interestingly, these studies also revealed that the concentration of CRH required to release MIF is lower than that needed to release ACTH, indicating the presence of a mechanism for differential release.

Much attention has focused on the release of MIF from endocrine cells, and the possibility that MIF is also produced by FS cells has not been addressed. Because FS cells have much in common with immune cells, they represent a potential source of and also a possible target for MIF. Our review of the literature has led us to propose that 1) FS cells contribute to the pituitary-derived pool of MIF, 2) MIF is released from FS cells in response to an LPS challenge, and 3) MIF modulates the release of cytokines (e.g. IL-6) from FS cells, which normally play a critical role in effecting the neuroendocrine responses to endotoxemia, in particular the activation of the HPA axis. In the present study, we addressed this hypothesis by using 1) immunohistochemistry to determine whether MIF is expressed by primary FS cells and 2) well-characterized FS [TtT/GF (28)], corticotroph [AtT20 D16:16 (29)], and macrophage/monocyte [RAW 264.7 (30)] cell lines to explore the effects of CRH, endotoxin, and dexamethasone on MIF release and to examine the effects of MIF on IL-6 release.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Double immunofluorescence staining for S100 and MIF
Pituitary glands were removed postmortem from adult male Sprague-Dawley rats (200–250 g), fixed overnight in 4% paraformaldehyde (Sigma, Poole, UK) in PBS (0.1 M PBS, pH 7.4; Sigma), washed in PBS, and cryopreserved for 24 h in 20% sucrose (VWR International, Poole, UK) in PBS. Cryostat sections (15 µm) were cut with a rotary microtome with a fixed blade (Bright, Cambridge, UK) and mounted on gelatin-coated slides. The sections were air-dried before immunofluorescence staining for S100 (a marker of FS cells) and MIF, using a modification of the method of Chapman et al. (31). Briefly, the sections were subjected sequentially to the following procedures with PBS washes between each stage: antigen retrieval [1 mg/ml trypsin (Sigma), 1 mg/ml CaCl2 (VWR International) in PBS for 15 min at 37 C], permeabilization [0.2% Triton X-100 (Sigma) for 5 min], reduction of autofluorescence [75 mM NH4Cl (VWR International), 20 mM glycine (Sigma) for 10 min], and blocking of nonspecific binding [3% BSA (Sigma) for 1 h]. The sections were then stained for S100 or MIF using the following antibody systems (all made up in 3% BSA in PBS): 1) rabbit anticow S100 (1:100, 4 C, overnight; DakoCytomation Ltd., Ely, Cambridgeshire, UK) followed by biotinylated swine antirabbit IgG (1:200, room temperature, 30 min; DakoCytomation Ltd.) and streptavidin-Texas Red (10 µg/ml, room temperature, 30 min; Pierce, Rockford, IL) and 2) affinity-purified goat antihuman MIF (1:100, 4 C, overnight; R&D Systems, Abington, Oxon, UK) followed by fluorescein isothiocyanate-conjugated rabbit antigoat IgG (1:200, room temperature, 30 min; Sigma). Antibody dilutions were selected on the basis of published observations and the supplier’s data sheet (31, 32). Negative controls were performed by replacing the primary antibody with nonimmune rabbit or goat serum at the same concentration as the primary antibody. (Further details of antibody specificity are provided in Refs. 31 and 32 .) The slides were mounted in Mowiol (Sigma) and visualized using a LSM5 Pascal laser-scanning confocal microscope (Zeiss, Thornwood, NY).

Cell lines
Murine FS-like (TtT/GF; a gift from Prof. John Morris, University of Oxford, UK), corticotroph (AtT20 D16:16; a gift from Dr. Simon Guild, University of St. Andrews, Fife, UK), and macrophage (RAW 264.7; purchased from American Type Culture Collection, Manassas, VA, purchased via LGC Promochem, Teddington, Middlesex, UK) cell lines were maintained as described previously (33) at 37 C in DMEM (Invitrogen Ltd., Paisley, UK) enriched with 10% fetal calf serum (FCS; PAA Laboratories Ltd., Yeovil, UK), penicillin (100 U/ml, Sigma), and streptomycin (0.1 mg/ml, Sigma) in a 5% CO2 humidified atmosphere.

Cell line extracts
Total cell lysates of TtT/GF, AtT20 and RAW cells were prepared by extraction in lysis buffer [1ml/T-75 flask: 250 mM Tris (Sigma), 1 mM EDTA (Sigma), 1 mM EGTA (Sigma) 0.1% Triton X-100 (Sigma), pH 7.5 + protease inhibitors (Complete EDTA free; Roche Diagnostics, Lewes, UK)]. The lysates were sonicated on ice, subjected to one freeze-thaw cycle before being cleared by centrifugation (13,000 x g, 5 min), and stored at –20 C. The total protein content of the extracts was determined by the Bradford assay (34).

Cell line incubations
Cells were plated at 250,000 (TtT/GF) or 500,000 (AtT20 and RAW) cells per well in 24-well plates and maintained for 2 d in DMEM and 1% FCS before treatments. They were then incubated for 24 h in DMEM + 1% FCS in the presence and absence of graded concentrations of CRH (0.1–1000 nM; Bachem, St. Helen’s, Merseyside, UK), LPS (0.1–1000ng/ml, Escherichia coli 0.111.B4, activity not <500,000 EU/mg; Sigma), or dexamethasone (10 fM to 1 µM; Faulding Pharmaceutical plc, Leamington Spa, UK). In some experiments, the TtT/GF cells were preincubated with dexamethasone (0.1–1000 nM) and/or murine recombinant MIF (rMIF, 100 ng/ml) for 1 h before the addition of LPS (100 ng/ml). At the end of the 24-h incubation period, the medium was collected and centrifuged at 10,000 x g for 5 min to pellet any floating cells. The supernatant fluid was removed and either assayed immediately for MIF or IL-6 or stored at –20 C for subsequent assay.

MIF and IL-6 ELISA
MIF and IL-6 were quantified by commercial ELISAs (R&D Systems) in accordance with the manufacturer’s instructions. Briefly, 96-well high-binding plates (VWR International, Lutterworth, UK) were coated overnight with capture antibody (2 µg/ml monoclonal anti-MIF or monoclonal anti-IL-6 in PBS). After washing three times with wash buffer [Dulbecco’s PBS (Oxoid Ltd., Basingstoke, UK) plus 0.05% Tween 20 (Sigma)], the plates were blocked for 1 h [5% sucrose (VWR International), 1% BSA (Sigma), and 0.05% sodium azide (VWR International)] in PBS. After further washes, standards (8–2000 pg/ml MIF or 4–1000 pg/ml IL-6) or samples were added to the wells in duplicate and incubated at room temperature for 2 h. With further washes between each stage, the plates were incubated sequentially with the detection antibody [2 h; 100 µl 200ng/ml biotinylated goat anti-MIF or biotinylated goat anti-IL-6 in reagent diluent (PBS + 1% BSA)], 100 µl streptavidin-HRP (20 min; 1:200 in reagent diluent) and substrate solution (100 µl, 20 min; R&D Systems). The color reaction was stopped by addition of 50 µl 2 N H2SO4 and the intensity read at 450 nm with background correction at 540 nm. Samples from each experiment were always analyzed in single assays to avoid interassay variance.

Statistical analysis
The data are presented as mean ± SEM of triplicate wells. Because there was some variation between experiments in basal MIF and IL-6 release and in the magnitude (but not the pattern) of the secretory responses to stimulation, statistical analysis was applied to data from individual experiments rather than pooled data from two or more experiments. The analysis was carried out using one-way ANOVA followed by Dunnett’s post hoc test (Instat 3; GraphPad Software Inc., San Diego, CA) or two-way ANOVA followed by Holm-Sidak post hoc test (SigmaStat 3.0; SPSS, Chicago, IL). Data were considered statistically significant when P < 0.05. The quantitative data shown are representative of at least two separate experiments.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go shows immunofluorescence staining of S100 and MIF in rat pituitary sections, both individually (A and B) and overlaid (C). Cells staining positively for S100 (red, A) and MIF (green, B) were abundant in the tissue. There was a high degree of colocalization of S100 and MIF (yellow staining, C), indicating that rat FS cells contain MIF protein. Other areas stained for MIF only, an observation that is consistent with reports that MIF is also expressed by endocrine cells in the anterior pituitary gland (27). No staining was evident when the primary antibodies were replaced with nonimmune serum (Fig. 1Go, D and E), indicating the staining was specific.



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  		FIG. 1. Confocal images of 15-µm sections of adult male rat pituitary tissue stained for S100 (marker of FS cells, Texas Red; A), MIF (fluorescein isothiocyanate; B), and S100 and MIF (C). Double-positive cells (orange) in panel C are indicated with arrows. Note that not all cells stain positively for both antigens, suggesting that MIF is also expressed by cells other than FS cells. Negative controls (secondary antibody alone) for S100 and MIF are shown in panels D and E, respectively.

 
To study the regulation of MIF release from FS cells, we used the murine TtT/GF FS cell line as a model and compared the responses of these cells with those of murine corticotroph (AtT20) and macrophage (RAW) cell lines. Preliminary experiments on protein extracts of the cells demonstrated by ELISA that all three cell lines express MIF protein, with a concentration of approximately 700 pg/mg protein in the TtT/GF cells, compared with 200 and 275 pg/mg in AtT20 and RAW cells, respectively.

Figures 2–4GoGoGo demonstrate the effects of graded concentrations of CRH, dexamethasone, and LPS on the secretion of MIF from the three cell lines. The AtT20 cells responded to CRH with a concentration-dependent increase in MIF release. Maximal effects were observed at 10 nM (P < 0.01 vs. basal), and at higher concentrations, the effects were reduced (Fig. 2AGo). In contrast, CRH had no significant effects (P > 0.05 vs. basal) on the release of MIF from TtT/GF (Fig. 2BGo) or RAW (Fig. 2CGo) cells. Unlike CRH, dexamethasone stimulated the release of MIF from all three cell lines (Fig. 3Go). Thus, at a concentration of 100 pM, dexamethasone had a highly significant effect in AtT20 cells (P < 0.01 vs. basal, Fig. 3AGo), although lower (10 fM and 1 pM) and higher (10 nM and 1 µM) concentrations of the steroid were without effect (P > 0.05 vs. basal), whereas in the TtT/GF (Fig. 3BGo) and RAW (Fig. 3CGo) cells, dexamethasone produced significant increases in MIF release at 100 pM and 10 nM (P < 0.01 or P < 0.05), but not at the other concentrations tested. LPS also stimulated the release of MIF from all three cell lines (Fig. 4Go). In the AtT20 cells, a maximal response (P < 0.01 vs. basal) was attained at 10 ng/ml LPS (Fig. 4AGo), and the response to a higher concentration appeared less pronounced. By contrast, in TtT/GF (Fig. 4BGo) and RAW (Fig. 4CGo) cells, a maximal response was evident at 1000 ng/ml LPS. The magnitude of the response of the AtT20 cells to LPS was considerably greater than that of either the TtT/GF or RAW cells. In addition, the magnitude of response of AtT20 cells to LPS far exceeded that of the responses to CRH (see Fig. 2AGo) or dexamethasone (see Fig. 3AGo).



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  		FIG. 2. Effects of graded concentrations of CRH on MIF release over 24 h from AtT20 cells (A), TtT/GF cells (B), and RAW 264.7 cells (C). *, P < 0.05, and **, P < 0.01, CRH-stimulated vs. control. Statistical analysis was carried out by ANOVA and Dunnett’s post hoc test. The data shown (mean ± SEM, n = 3) are derived from a single experiment and are representative of two separate experiments.

 


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  		FIG. 3. Effects of graded concentrations of dexamethasone on MIF release over 24 h from AtT20 cells (A), TtT/GF cells (B), and RAW 264.7 cells (C). *, P < 0.05, and **, P < 0.01, dexamethasone-stimulated vs. control. Statistical analysis was carried out by ANOVA and Dunnett’s post hoc test. The data shown (mean ± SEM, n = 3, from a single experiment) are representative of two separate experiments.

 


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  		FIG. 4. Effects of graded concentrations of LPS on MIF release over 24 h from AtT20 cells (A), TtT/GF cells (B), and RAW 264.7cells (C). *, P < 0.05, and **, P < 0.01 LPS-stimulated vs. control. Statistical analysis was carried out by ANOVA and Dunnett’s post hoc test. The data shown (mean ± SEM, n = 3) are derived from a single experiment and are representative of two separate experiments.

 
Subsequent experiments aimed to determine whether TtT/GF cells, as well as being a source of MIF, are a target for MIF. This was done by assessing the ability of human rMIF to reverse the inhibitory actions of dexamethasone on LPS-stimulated IL-6 release. Figure 5AGo shows that LPS caused a concentration-dependent increase in IL-6 release over 24 h, with significant elevations in IL-6 release evident at concentrations of 10 ng/ml LPS and above (P < 0.01 vs. basal). A separate experiment showed that the ability of LPS (100 ng/ml) to induce the release of IL-6 release is reduced in a concentration-dependent manner by preincubation of the cells with dexamethasone (0.1–1000 nM, Fig. 5BGo). In the absence of dexamethasone, preincubation of the cells with rMIF (100 ng/ml) had no significant effect on LPS-stimulated IL-6 release (Fig. 6Go). However, rMIF (10 ng/ml) partially reversed the inhibitory actions of dexamethasone (1 nM) on LPS-stimulated (100 ng/ml) MIF release (P < 0.01), although it failed to modify the inhibitory effects of a higher concentration of the steroid (100 nM, P > 0.05). Neither dexamethasone nor rMIF had any effect on basal IL-6 release (data not shown).



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  		FIG. 5. Effects of LPS on the release of IL-6 from TtT/GF cells over 24 h. A, Concentration response curve. B, Effects of graded concentrations of dexamethasone (added to the medium 1 h before LPS) on the release of MIF induced by LPS (100 ng/ml). **, P < 0.01 LPS-stimulated vs. control. {dagger}{dagger}, P < 0.01 LPS alone vs. LPS + dexamethasone. Statistical analysis was carried out by ANOVA and Dunnett’s post hoc test. The data shown (mean ± SEM, n = 3, from a single experiment) are representative of two separate experiments.

 


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  		FIG. 6. Effects of rMIF on the inhibitory effects of dexamethasone on the release of IL-6 induced over 24 h by LPS (100ng/ml) from TtT/GF cells. Dexamethasone and rMIF were added to the medium 1 h before LPS. Closed columns, Controls; open columns, rMIF. **, P < 0.01, LPS-stimulated vs. control. In the absence of LPS, basal IL-6 release was <5.0 pg/ml and near the limit of detection of the assay. Neither dexamethasone nor rMIF affects basal IL-6 release. {dagger}{dagger}, P < 0.01 LPS + dexamethasone vs. corresponding LPS alone; ##, P < 0.01 LPS + dexamethasone + rMIF vs. LPS + dexamethasone. Statistical analysis was carried out by two-way ANOVA and Holm-Sidak post hoc test. The data shown (mean ± SEM, n = 3, from a single experiment) are representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows for the first time that MIF, a proinflammatory cytokine, is found in abundance in the S100-positive FS cells of the anterior pituitary gland. It thus suggests that the FS cells are an important source of MIF and raises the possibility that FS cell-derived MIF plays a role in mediating the pituitary responses to endotoxemia and other immune insults. Interestingly, a small number of S100-positive FS cells appeared not to stain for MIF (red cells, Fig. 3CGo). This could be because some FS cells express relatively low levels of the protein, possibly because of their stage of the cell cycle, which defy detection with our staining methodology; alternatively, it is feasible that a subpopulation of FS cells exists that does not stain positively for the protein. Our immunohistochemical analysis also revealed other MIF-positive cells in the pituitary gland; as MIF protein has been identified previously in corticotrophs and thyrotrophs (27), it seems likely that these were endocrine cells.

To provide some insight into the physiological relevance of FS-derived MIF, we compared the effects of three putative MIF secretagogues on MIF release from FS cells and corticotrophs to determine whether the two cell types respond similarly to different stimuli. Because purified populations of primary FS cells and corticotrophs are difficult to prepare and require substantive amounts of pituitary tissue, we based our study on mouse pituitary FS (TtT/GF) and corticotroph (AtT20 D16:16) cell lines after first confirming by ELISA that both cell lines, like their parent cells, express MIF. In addition, as FS cells have been described as "glial-like" and show some characteristics of macrophages (11), we also performed parallel studies on a MIF-expressing murine macrophage/monocyte cell line, RAW 264.7. Although we appreciate that cell lines are not necessarily representative of their parent cell types, we reasoned that these clonal populations would provide useful models in which to explore fundamental mechanisms.

Our data show clearly that CRH stimulates the release of MIF from AtT20 cells but not from TtT/GF and RAW cells. In line with these findings, Nishino et al. (27) also demonstrated positive concentration-dependent effects of CRH on MIF release from AtT20 cells, although in their study maximal effects of CRH were observed at only 0.1 vs. 10 nM in the present study. This potency difference may reflect differences in contact time (16 vs. 24 h), culture conditions (particularly the presence or absence of FCS) or, possibly, phenotypic drift of the cell lines. Irrespective of this difference, both studies showed bell-shaped concentration-response curves, with maximal effects occurring at CRH concentrations well below those required for maximal release of ACTH (27, 33), a phenomenon that has been attributed to the presence of two populations of secretory granules, viz. those containing MIF only and those containing both MIF and ACTH (27). The failure of CRH to cause MIF release from the TtT/GF and RAW cells is interesting and supports the hypothesis that FS cells behave more like immune cells than endocrine cells. To the best of our knowledge, CRH receptor expression has not been examined in either primary FS cells or the TtT/GF cell line and, thus, we cannot exclude the possibility that the apparent inactivity of CRH in the TtT/GF cells reflects a lack of functional CRH receptors. CRH receptors are, however, expressed by RAW 264.7 cells and, when activated, they augment LPS-induced TNF{alpha}, IL-1ß, and IL-6 release (35). Because TNF{alpha} stimulates MIF release from macrophages (22), these findings raise the possibility that the reported proinflammatory effects of CRH (2) are mediated in part by potentiation of LPS-stimulated MIF release. If a similar synergistic mechanism operates in FS cells, the LPS-induced release of proinflammatory cytokines would be enhanced; as these cytokines act locally on the corticotrophs to increase ACTH release (reviewed in Ref. 2), this would provide a mechanism for enhancing the HPA response to endotoxemia.

Previous studies have shown that LPS induces MIF release from RAW (22) and AtT20 (23) cells and from primary pituitary tissue (27). In accord with these findings, we also demonstrated positive, concentration-dependent effects of LPS on MIF release from both cell lines; in addition, we showed for the first time that LPS produces comparable effects on TtT/GF cells. LPS signaling is enhanced by a se-rum LPS-binding protein (36) and transduced, in a cell-specific manner, by complex interactions between various membrane-bound and intracellular proteins; these proteins include CD-14, MD-2, and the Toll-like receptors, Tlr-4 and, possibly, Tlr-2 (reviewed in Ref. 37). Although the roles of these proteins are well defined in immune cells (37), their expression and function in the pituitary gland is less well understood, although there is evidence that TtT/GF cells express CD14 and Tlr4 (16). Our laboratory has recently shown by RT-PCR and Western blot analysis that the cell lines used in this study express CD14, Tlr4, Tlr2, and MD-2 mRNAs and protein (Mehet, D., E. Solito, C. John, and J. Buckingham, unpublished data), observations that support a role for the Toll-like receptor family in LPS signal transduction in these cells. Although it is not yet known whether the primary corticotrophs are targets for LPS, several lines of evidence suggest that primary FS cells are. Indeed, LPS-induced ACTH release in vivo appears to be effected in part via paracrine actions of IL-6 (10) and possibly other glycoprotein 130 cytokines (2) that are released from the FS cells in response to the toxin.

Like LPS, dexamethasone also stimulated the release of MIF from all three cell lines studied. In the case of the RAW cells, our data confirm those of Calandra et al. (26), who also showed a bell-shaped concentration-response curve to the steroid. However, whereas Calandra et al. (26) reported maximal effects at steroid concentrations of 10 fM to 100 pM, we required concentrations of 100 pM to 10 nM to evoke maximal release. This discrepancy may be attributable to differences between 1) batches of cell lines that are prone to phenotypic drift, 2) culture conditions, or 3) steroid preparations (free steroid vs. sodium salt). Although the magnitude of response was lower, the profile of LPS-induced MIF release from TtT/GF cells bore a striking resemblance to that of the RAW cells, with peak responses occurring at concentrations of 100 pM to 10 nM dexamethasone. As in classical immune cells (26), this finding poses a paradox as the release of proinflammatory cytokine from FS cells is normally inhibited by glucocorticoids (e.g. Ref. 38). The finding that dexamethasone stimulates the release of MIF from AtT20 cells was particularly surprising, although glucocorticoids have recently been shown to augment MIF protein expression in endocrine tissues, apparently via a posttranslational mechanism (39). CRH and glucocorticoids typically have opposing actions on corticotrophs, and the steroids exert powerful inhibitory effects on ACTH secretion in vivo (40) and in vitro by both primary pituitary tissue (41) and AtT20 cells (42). However, there are examples of CRH and glucocorticoids acting in concert on AtT20 cells, for example in the regulation of type 2 iodothyronine deiodinase (43). Furthermore, although the colocalization of ACTH and MIF in some corticotroph secretory granules predicts that dexamethasone would suppress MIF release, it is possible that the steroid promotes exocytosis of MIF-only-containing granules; such a view is supported by our recent observation (unpublished) that the prosecretory effect of dexamethasone on MIF is dependent upon the presence of extracellular Ca2+.

The functional relevance of our finding that MIF is localized to FS cells as well as endocrine cells (27) in the anterior pituitary gland requires further investigation. In general, substances secreted from the endocrine cells (e.g. corticotrophs) are destined for the circulation and act as endocrine factors. In accord with this premise, previous studies have shown that pituitary-derived MIF has important roles in the periphery (23), hence its classification as a hormone as well as a cytokine. However, unlike substances derived from the endocrine cells, FS cell-derived factors (e.g. cytokines and growth factors) generally have more local actions and are likely to be present in high concentrations in the extracellular fluid surrounding the pituitary cells vs. the systemic circulation (8). FS cell-derived MIF may thus act as a local paracrine or autocrine factor. Because macrophages are both a source of and a target for MIF (22) and FS cells show macrophage-like characteristics (11), we explored the possibility that FS cells may be a target for MIF, using the TtT/GF cells as a model. Our data show clearly that, like RAW cells (22), TtT/GF cells respond to LPS with a dexamethasone-reversible increase in IL-6 release. They also show that the inhibitory effects of a low, but not a higher, concentration of dexamethasone, on IL-6 release are attenuated by rMIF and, thus, demonstrate for the first time that TtT/GF cells are a target for MIF. In human peripheral blood mononuclear cells, MIF inhibits glucocorticoid action by inhibiting the steroid-induced synthesis of inhibitory protein {kappa}B (I{kappa}B) and thereby preventing I{kappa}B binding to nuclear factor (NF)-{kappa}B. This facilitates the translocation of NF-{kappa}B to the nucleus and, thereby, allows LPS-stimulated cytokine production to continue in the presence of glucocorticoids (44). Because the LPS-induced IL-6 production by TtT/GF cells is also mediated by the NF-{kappa}B pathway and subject to glucocorticoid inhibition (16), it is possible that effects of MIF on TtT/GF cells reported here are effected via a similar mechanism.

Increasing evidence supports the view that direct actions of endotoxin on the pituitary gland play an important part in mediating the HPA responses to endotoxemia. Particular attention has focused on the role of FS cell-derived IL-6 (reviewed in Ref. 2). If the present data can be translated to primary cells, they raise the possibility that MIF may also be important in this regard. Our data predict that MIF would be released from pituitary cells in endotoxemia through the combined actions of LPS, glucocorticoids, and CRH on both FS cells and corticotrophs. In addition to acting systemically, MIF would act locally within the pituitary gland to augment and sustain ACTH release by opposing the suppressive effects of endogenous glucocorticoids on IL-6 release. The bell-shaped concentration-response curves of the steroid predict that this effect would be self-limiting and that once the concentration of endogenous glucocorticoids reached a threshold, both MIF release and the ability of MIF to overcome the inhibitory effects of the steroids on IL-6 release would be diminished. This "escape mechanism" would allow the negative feedback regulation of the release of IL-6, and possibly other pituitary cytokines, to continue. It would thereby check the ACTH response to ensure that glucocorticoid levels were maintained within appropriate limits.

In conclusion, the present data suggest that FS cell-derived MIF is an important intrapituitary mediator of neuroendocrine communication in conditions of infection and inflammatory disease. Such a view is supported by the recent discovery of a functional polymorphism in the promoter region of the human Mif gene, especially given that this polymorphism lies within the transcription binding site for the pituitary-specific Pit-1 transcription factor (45, 46), and warrants further investigation.


   Acknowledgments
 
We are grateful to Mr. Colin Rantle and Mr. Simon McArthur for their help in the laboratory.


   Footnotes
 
We acknowledge the Wellcome Trust (Grant 69234/B/02/Z, to J.C.B.) and National Institutes of Health (Grants A142310 and AR050498, to R.B.) for financial support.

First Published Online September 23, 2004

Abbreviations: FCS, Fetal calf serum; FS, folliculo-stellate; HPA, hypothalamo-pituitary-adrenocortical; LPS, lipopolysaccharide; MIF, macrophage migration inhibitory factor; NF, nuclear factor; rMIF, recombinant MIF.

Received July 22, 2004.

Accepted for publication September 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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