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Immunology; |
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223
ABSTRACT
Though gender-based differences in the development of protective or pathological adaptive host responses have been widely noted, it is becoming apparent that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses. These differences may be due to the actions of reproductive hormones, and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions, including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study, we have investigated the acute effects of testosterone on the level of expression of a key trigger for inflammation and innate immunity, Toll-like receptor 4 (TLR4), on isolated mouse macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in TLR4 expression and sensitivity to a TLR4-specific ligand. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression and endotoxin susceptibility. We report that orchidectomized mice were significantly more susceptible to endotoxic shock and show that macrophages isolated from these animals have significantly higher TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, these effects were not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data may represent an important mechanism underlying the immunosuppressive effects of testosterone.
cytokines, immunology, testosterone
Gender has long been known to be a contributing factor in the incidence and progression of disorders associated with immune system dysregulation (as reviewed in [1]). More recently, evidence has accumulated that gender may also play an important role in infectious disease susceptibility (as reviewed in [2]). In general, females generate more robust and potentially protective humoral and cell-mediated immune responses following antigenic challenge than their male counterparts. For example, influenza infection elicits greater severity and hospitalization in male patients [3], and females generate greater humoral and cell-mediated responses to herpes simplex viruses [4] and cytomegalovirus [5]. Women have been found to have higher circulating levels of IgM than men [6], and this difference is most apparent at puberty [7, 8], suggesting a role for reproductive hormones in the development of this gender bias. Furthermore, cell-mediated host responses have also been suggested to exhibit sexual dimorphism, and female mice have been shown to mount more vigorous T-cell responses to exogenous and allogeneic antigens than males [9]. This notion is further supported by the observation that early cell-mediated immune responses to thermal injury are more robust in females than their male counterparts [10].
Differences in immune responses between males and females have generally been assumed to be a consequence of the actions of reproductive hormones. Androgens have been shown to have suppressive effects on immune functions following trauma or trauma-hemorrhage and subsequent sepsis [11–13]. Furthermore, testosterone and other androgens, such as dihydrotestosterone, have been shown to decrease immunoglobulin and cytokine production and to limit lymphocyte proliferation (as reviewed in [13] and [14]). These observations are consistent with the demonstration that exogenous testosterone administration increases female susceptibility to Mycobacterium marinum infection, and castration attenuates such infections in male mice [15]. Although classical androgen receptors do not appear to be expressed by macrophages, recent studies suggest that these important sentinel cells possess nonclassical cell surface receptors for male sex hormones [16]. The presence of such receptors could explain the documented ability of testosterone to down-regulate LPS-induced activation of the pro-inflammatory transcriptional regulators and immune responses in isolated macrophages [17]. To date, it is unclear how testosterone alters the immune responsiveness of macrophages.
The recent discovery of a family of pattern-recognition receptors with a high degree of homology to the Toll family of proteins in Drosophila has shed light on the means by which the innate immune system responds to cellular damage and/or microbial infections [18]. To date, thirteen members of the Toll-like family of receptors (TLR) have been described in mammals, and all of these homologues are thought to initiate cytokines, chemokine, and co-stimulatory molecule expression (as reviewed in [19]). Hence, activation of cells via these receptors initiates the repertoire of defense mechanisms used by the innate immune system. TLR4 has been shown to mediate immune cell responses to bacterial lipopolysaccharides as well as endogenous "danger signals" liberated from injured tissues [19]. As such, an ability of testosterone to influence the level of expression of this molecule could profoundly influence the pathogenesis of a wide range of inflammatory disorders.
In the present study, we demonstrate that testosterone reduces expression of TLR4 on a macrophage cell-line and cultured primary macrophages. Furthermore, we have extended these in vitro findings to an in vivo analysis of TLR4 expression on monocytes/macrophages and report an increase in TLR4 levels on these cells in the absence of endogenous testosterone. Taken together, these data are in agreement with the recent observation that TLR4 expression is increased in the prostate following castration [20] and provides a potential mechanism underlying the immunosuppressive effects of testosterone.
Macrophage-Like Cell Line Culture
RAW 264.7 mouse macrophage-like cells originally derived from male cells (CRL-2278; ATCC, Manassas, VA) were grown on Cellstar culture plates (Greiner Bio-one, Monroe, NC) in RPMI 1640 (Cellgro, Washington, DC) containing 2% NuSerum (BD, Franklin Lakes, NJ) to minimize exposure to reproductive hormones. According to the determinations of NuSerum reproductive hormone content provided by the manufacturer, cells cultured under these conditions are exposed to less than 2.6 x 10–10 M testosterone.
Surgical Orchidectomy and Testosterone Replacement
Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), 8 wk of age, underwent bilateral orchidectomy via scrotal incision under inhalant isoflurane anesthesia as previously described [21] to remove the main endogenous source of testosterone. Groups of adult male mice were bilaterally orchidectomized or sham orchidectomized and allowed to recover for 5 wk. This time period ensures that androgens produced by the testis have been metabolized and are no longer present in the blood [21] and is sufficient to allow the turnover of immune cells generated under the influence of this reproductive hormone. Mice in the sham-operated group underwent the same anesthesia and incision procedure, but the testes were not excised. In a third group of animals, orchidectomized mice received subcutaneous injections of testosterone proprionate in corn oil (5 mg/kg) every 3 days for 5 wk. All procedures were approved by the Institutional Animal Care and Use Committee of The University of North Carolina at Charlotte.
In some experiments, orchidectomized, sham-operated, and orchidectomized mice receiving testosterone replacement were given i.p. injections of lipopolysaccharide (LPS; 5 mg/kg) isolated from Escherichia coli (>500 000 EU/mg; Sigma Chemical Co., St. Louis, MO). At 24 h after treatment, animals were studied for behavior and appearance then killed and analyzed for sera cytokine content. The severity of endotoxic shock was scored according to a system modified from that previously employed by Liu et al. [22], in which a score of 1 was given to mice with percolated fur but no detectable behavioral differences compared to untreated mice; a score of 2 was given to mice with percolated fur and a huddle reflex but that were still active; a score of 3 was given to mice that were less active and were relatively passive when handled, a score of 4 was assigned to inactive mice that exhibited only limited response when handled, and a score of 5 was applied to moribund mice.
Isolation of Murine Peritoneal Macrophages
Elicited peritoneal macrophages were isolated as previously described by our laboratory [23–25]. Briefly, mice from each treatment group received i.p. injections of 200 µl incomplete Freunds adjuvant (Sigma-Aldrich, St. Louis, MO). Three days later, the peritoneal cavities were lavaged with RPMI 1640 (7 x 1.5 ml per animal) containing 10% fetal bovine serum (FBS; Atlanta Biologics, Norcross, GA) to remove the peritoneal macrophages. After washing twice in RPMI 1640, adherent macrophages were cultured in RPMI 1640 containing 2% FBS and gentamicin (Fisher Scientific, Pittsburgh, PA).
Immunofluorescence for TLR4/MD-2
Immunofluorescence analysis (FACSCalibur; Becton Dickinson, San Jose, CA) was performed to determine the presence of TLR4 associated with the permissive molecule MD-2 on the surface of macrophages as previously described by our laboratory [23]. Cells were isolated, and a phycoerythrin-conjugated antibody directed against TLR4/MD-2 (Clone MTS510; eBioscience) was added for 45 min at 4°C. Cells were then washed and assayed by FACS analysis for the proportion of TLR4/MD-2 positive cells relative to fluorescence obtained in cells stained with a PE(phycoerythrin)-conjugated antibody directed against an irrelevant peptide. A minimum of 20 000 cells were analyzed from at least three separate cell isolation procedures, and results are presented as the geometric means of the fluorescence intensity. In some experiments, cells were permeabilized during immunofluorescent staining to assess total cellular TLR4 content using a CytoFix/CytoPerm kit according to the directions provided by the manufacturer (BD PharMingen, San Diego, CA).
Quantification of TNF and Interleukin 6 Production
Capture ELISAs were performed to quantify interleukin 6 (IL6) levels as described previously [23, 25] using a commercially available capture antibody against IL6 (clones MP5-20F3; BD PharMingen), a biotinylated anti-mouse IL6 antibody (clones MP5-32C11; BD PharMingen), and streptavidin-horseradish peroxidase (BD PharMingen). A standard curve was constructed using varying dilutions of mouse recombinant IL6 (BD PharMingen). Tumor necrosis factor (TNF) levels were quantified using a commercially available ELISA kit according to the directions provided by the manufacturer (R&D System, Minneapolis, MN). The minimum detectable levels in these assays were 4 pg/ml for IL6 and 16 pg/ml for TNF, and all determinations were made in duplicate from the indicated number of separate cell isolation procedures.
Geometric means of immunofluorescence histogram plots were obtained using commercially available software (CellQuest; Becton Dickinson). These values and mean cytokine levels were statistically compared using SAS v9.1.3 software (SAS Institute Inc., Cary, NC). Data from each treatment group were compared with the appropriate control group using an unpaired Student t-test, except those experiments in which the treated and untreated cells were derived from the same animal, in which case data were compared statistically using a paired Student t-test. In addition, individual immunofluorescence histograms were directly compared using a Kolmogorov-Smirnov test (using CellQuest software) when the treated and untreated cells were derived from the same animal. In all experiments, results are presented as the mean ± SEM. A P-value of less than 0.05 was considered statistically significant.
Testosterone Decreases TLR4 Expression in RAW 264.7 Cells
To begin to determine the in vitro effects of testosterone on cell surface TLR4 expression, we have utilized a murine macrophage-like cell line, RAW 264.7. This cell line was cultured in media containing 2% NuSerum, which served to minimize exposure to reproductive hormones. These cells were exposed to varying concentrations of testosterone propionate (1–1000 nM) for various time periods prior to analysis of cell surface TLR4 by flow cytometry. As shown in Figure 1, A and B, testosterone elicited an approximately 10% reduction in fluorescence attributable to cell surface TLR4 at doses as low as 100 nM (final ethanol concentration of 0.0002%), and this effect was significant (n = 16 per group, P < 0.05) at a dose of 1 µM (final ethanol concentration of 0.002%). This effect was most apparent at 24 h following testosterone administration (Fig. 1C).
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To determine whether the reduction in cell surface TLR4 expression was due to receptor internalization, we have assessed the effect of testosterone on relative TLR4 protein content in permeabilized cells by flow cytometry. In these studies, 24-h treatment of RAW 264.7 cells with 1 µM testosterone elicited a statistically significant 7.3% decrease in cellular TLR4-asssociated fluorescence (fluorescence geometric means of 7.37 ± 0.14 versus 6.83 ± 0.13 in untreated and testosterone-treated cells, respectively; n = 12 per group, P < 0.05), indicating that receptor internalization does not account for the apparent decrease in cell surface TLR4 expression in this cell line.
To begin to assess the functional significance of testosterone-mediated decreases in TLR4 expression in this macrophage-like cell line, we have determined whether testosterone alters the sensitivity of these cells to the TLR4 ligand, LPS. RAW 264.7 cells were exposed to testosterone (1 µM) for 24 h prior to challenge with LPS (250 ng/ml), and the production of the inflammatory cytokine TNF was assessed after 12 h by specific capture ELISA. We report that testosterone significantly decreased LPS-induced TNF production from 703 ± 15 pg/ml to 620 ± 20 pg/ml (n = 12 per group, P < 0.05), consistent with a decreased functional responsiveness of these cells to this TLR4 ligand.
Testosterone Decreases TLR4 Expression in Primary Macrophages Derived from Animals Largely Devoid of Endogenous Androgens
Having determined optimal doses and kinetics using a cell line, we have confirmed the effect of testosterone on TLR4 expression in primary murine peritoneal macrophages. Macrophages were elicited from orchidectomized animals that are largely devoid of endogenous androgens [21], sham orchidectomized mice, and orchidectomized mice with testosterone replacement. Cells were cultured either in the presence or absence of 1 µM testosterone, and TLR4 cell surface expression was assessed at 24 h after testosterone treatment by flow cytometry. As shown in Figure 2, testosterone treatment again elicited a modest but significant decrease in the cell surface expression of TLR4 on macrophages derived from orchidectomized mice (n = 16 per group, P < 0.05, paired Student t-test). In contrast, cells derived from orchidectomized mice that received in vivo testosterone replacement or sham orchidectomized mice failed to show sensitivity to in vitro testosterone addition, suggesting that the effects of this hormone are long lasting and maximal at even endogenous levels.
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To determine whether the reduction in cell surface TLR4 expression was due to receptor internalization or decreased TLR4 protein levels, we have assessed the effect of testosterone on relative TLR4 protein content in permeabilized cells by flow cytometry. In these studies, 24-h treatment of primary macrophages with 1 µM testosterone decreased TLR4-associated fluorescence in cells from five of seven orchidectomized animals, with an average decrease of 13.8% (fluorescence geometric means of 7.60 ± 0.62 versus 6.55 ± 0.58 in untreated and testosterone treated cells, respectively; n = 5, P < 0.05), indicating that testosterone-induced decreases in cell surface TLR4 expression cannot be explained on the basis of receptor internalization.
To begin to assess the functional significance of testosterone-mediated decreases in TLR4 expression in primary macrophages, we have determined whether testosterone alters the sensitivity of these cells to LPS. Peritoneal macrophages were again isolated from orchidectomized and sham orchidectomized mice and orchidectomized mice with testosterone replacement, and cells were cultured either in the presence or absence of 1 µM testosterone. At 24 h after testosterone treatment, cells were challenged with LPS (250 ng/ml), and the production of the inflammatory cytokine TNF was assessed by specific capture ELISA. As shown in Figure 3, testosterone significantly decreased LPS-induced TNF production by cells derived from orchidectomized mice (n = 16 per group, P < 0.05, paired Student t-test), consistent with a decreased functional responsiveness of these cells to this TLR4 ligand. In agreement with the inability of testosterone to reduce TLR4 expression on cells derived from sham orchidectomized mice or orchidectomized mice that received testosterone replacement (Fig. 2), testosterone failed to elicit significant decreases in LPS sensitivity in these cells (Fig. 3).
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Removal of Endogenous Testosterone Increases Cell Surface TLR4 Expression on Macrophages/Monocytes In Vivo and Increases Susceptibility to Endotoxic Shock
To confirm that testosterone suppresses cell surface TLR4 expression on these immune cells in vivo, we have examined TLR4 expression on monocytes/macrophages acutely isolated from orchidectomized, sham orchidectomized, and orchidectomized mice that received testosterone replacement. As shown in Figure 4A, orchidectomy elicits a significant decrease (n = 16 animals per group, P < 0.05) in animal body weight that is prevented by exogenous testosterone replacement as previously reported [26]. Peritoneal monocytes/macrophages from sham orchidectomized animals express very low levels of cell surface TLR4 (Fig. 4, B and C). Importantly, cells from orchidectomized mice express approximately 25% greater TLR4 expression (n = 16 animals per group, P < 0.05) than sham operated animals. This effect was largely abolished in cells derived from animals that received exogenous testosterone replacement (Fig. 4, B and C). These data indicate that removal of endogenous testosterone, and hence elimination of its suppressive effects, elevates in vivo TLR4 expression on this key sentinel immune cell type.
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To begin to test the physiological relevance of these changes in TLR4 expression, we have investigated the susceptibility of male mice to endotoxic shock following removal of endogenous testosterone. Orchidectomized, sham orchidectomized, and orchidectomized mice that received testosterone replacement were challenged with a sub-lethal dose of LPS (5 mg/kg, i.p.). At 24 h after treatment, the severity of endotoxic shock was assessed according to appearance and behavior using a scoring system modified from that employed by Liu et al. [22]. Animals were then killed, and sera were isolated for inflammatory cytokine content. As shown in Figure 5, orchidectomized mice exhibited a markedly elevated susceptibility to endotoxin compared to that seen in sham treated animals, with a close correlation between severity scores and sera levels of the inflammatory cytokine IL6 (n = 6–7 animals per group, P < 0.05). Importantly, this effect was abolished in animals that received exogenous testosterone replacement (Fig. 5, A and B). These data indicate removal of endogenous testosterone elevates susceptibility to endotoxic shock and is consistent with the observed in vivo increase in TLR4 expression on immune cells.
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It has recently been recognized that gender may influence host responses to infectious organisms. Examples include the observation that female deer have lower parasite loads than males and the finding that helminth infections are generally more severe in males than females [27, 28]. These phenomena appear to correlate with clinical and laboratory studies demonstrating that females generally exhibit greater adaptive immune responses following antigenic challenge than males (as reviewed in [2]). Importantly, many of these differences become apparent at puberty [7, 8], suggesting a role for reproductive hormones in their development, and this hypothesis has been supported by the finding that receptors for reproductive hormones have been found in a variety of immune cell types (as reviewed in [13]). Androgens have been shown to have suppressive effects on immune functions following trauma or trauma-hemorrhage and subsequent sepsis [11–13]. Furthermore, male reproductive hormones have been shown to decrease immunoglobulin and cytokine production, and to limit lymphocyte proliferation (as reviewed in [13] and [14]). These observations are consistent with the demonstration that exogenous testosterone administration increases female susceptibility to M. marinum infection, and castration attenuates such infections in male mice [14]. Recent studies suggest that macrophages, a key sentinel immune cell type, possess nonclassical cell surface receptors for androgens [16] and that testosterone can decrease LPS-induced activation of transcription factors that regulate inflammatory responses [17]. However, it is currently unclear how testosterone alters the immune responsiveness of macrophages.
In the present study, we demonstrate that in vitro exposure to testosterone elicits significant decreases in the expression of cell surface TLR4 in a macrophage-like cell line, and we have confirmed this effect in primary murine macrophages. Interestingly, we show that the ability of testosterone to decrease TLR4 expression on primary macrophages is only apparent on cells derived in the absence of endogenous gonadal androgens, because cells isolated from sham orchidectomized animals or orchidectomized animals that receive testosterone replacement fail to demonstrate such sensitivity. This finding suggests that endogenous testosterone exerts sustained effects on cell surface TLR4 molecule expression and is characteristic of the long-lasting actions of reproductive steroid hormones on cellular machinery. The testosterone-mediated decrease in cell surface TLR4 expression does not appear to be due to receptor internalization, as significant decreases in TLR4 expression were also observed in permeabilized cells.
Furthermore, we have demonstrated the ability of endogenous testosterone to decrease TLR4 expression on immune cells in vivo by showing that levels of this microbial pattern recognition receptor on acutely isolated peritoneal monocytes/macrophages derived from orchidectomized mice are significantly higher than those seen on cells obtained from sham orchidectomized animals or orchidectomized animals that received testosterone replacement. Importantly, this elevation in TLR4 expression in vivo correlates with a dramatic increase in endotoxin susceptibility in orchidectomized animals. Taken together, the present findings demonstrate that the presence of testosterone in vitro or in vivo significantly decreases the cell surface expression of a critical receptor for microbial components and inflammatory signals liberated from injured tissues on an important sentinel immune cell type.
In our in vitro studies, acute administration of exogenous testosterone evoked maximal reductions of 7%–10% in TLR4 expression on macrophages, and one might be tempted to question the functional significance of such an effect. However, it is important to note that the number of these receptor molecules on the surface of immune cells is relatively low, so even modest changes in the level of expression may have marked effects on cellular responsiveness. In this study, we have begun to assess the functional relevancy of testosterone-mediated reductions in TLR4 expression by measuring inflammatory cytokine production elicited by a TLR4-specific ligand. We show that testosterone significantly decreases LPS-induced TNF production by primary macrophages generated in the absence of endogenous androgens and a macrophage-like cell line, and though we cannot rule out possible effects of testosterone on the signal transduction pathway, its effects on inflammatory cytokine production correlate well with the observed changes in TLR4 expression. If these in vitro findings were reproduced in vivo, then a 10% difference in inflammatory mediator production would be anticipated to have profound effects on disease outcome. However, it is quite possible that our in vitro studies underestimate the chronic influence of testosterone on immune cell function. This notion is supported by our in vivo studies showing that the removal of endogenous testosterone elicits a more marked effect on TLR4 expression on in situ monocytes/macrophages than that seen in vitro. Importantly, this effect correlates with a profound increase in susceptibility to in vivo endotoxin challenge.
Our results are in agreement with a recent study demonstrating that the androgen dihydrotestosterone can inhibit the expression of mRNA encoding TLR4 in human endothelial cells derived from neonatal tissue and can reduce LPS-mediated inflammatory mediator production by this cell type [29]. In addition, the present in vivo studies also support the findings of Quintar et al. [19] showing that prostate TLR4 protein expression is elevated in castrated rats. Furthermore, the present study could describe a mechanism underlying the ability of testosterone to down-regulate LPS-induced activation of the pro-inflammatory transcriptional regulator p38 MAP kinase and reduce nitric oxide production in macrophage-like cell lines [17]. However, it is important to note that the cellular effects of reproductive hormones are complex, so ascribing specific roles to each hormone is fraught with peril. For example, though a number of studies indicate that testosterone alters inflammatory cytokine release by macrophages following bacterial endotoxin exposure [30, 31], others have provided contrary evidence, and this has led to the suggestion that male reproductive hormones only exert such effects in immunocompromised hosts [12]. As such, though it is apparent that the precise role of testosterone in the regulation of immune function remains contentious, the present demonstration that testosterone can modulate the expression of a key receptor for danger signals in vitro and in immune-competent animals may represent an important mechanism underlying the immunosuppressive effects of this androgen.
Correspondence: 1Ian Marriott, Department of Biology, 9201 University City Blvd., University of North Carolina at Charlotte, Charlotte, NC 28223. FAX: 704 687 3128; e-mail: imarriot{at}uncc.edu
Received: 20 June 2007.
First decision: 8 July 2007.
Accepted: 3 October 2007.
REFERENCES
This article has been cited by other articles:
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  A. T. MASI, R. T. CHATTERTON, and J. C. ALDAG Hypothalamic-Pituitary-Gonadal Axis Hormones and Male Rheumatoid Arthritis: Novel Perspectives J Rheumatol, May 1, 2009; 36(5): 859 - 862. [Full Text] [PDF]
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