Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1005597 on March 7, 2006

Published online before print March 7, 2006
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(Journal of Leukocyte Biology. 2006;79:989-998.)
© 2006 by Society for Leukocyte Biology

Prostate epithelial cells can act as early sensors of infection by up-regulating TLR4 expression and proinflammatory mediators upon LPS stimulation

Gerardo Gatti, Virginia Rivero, Rubén Darío Motrich and Mariana Maccioni1

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Argentina

1Correspondence: CIBICI-CONICET, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba, 5000, Argentina. E-mail: mmaccioni{at}bioclin.fcq.unc.edu.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the prevalence of prostate disease, little is known about the immunobiology of the prostate and its contribution to disease. The main goal of this work was to investigate how prostate epithelial cells deal with inflammatory stimuli. To this aim, we stimulated a rat prostate epithelial cell line [metastasis-lung (MAT-LU)] or rat primary epithelial cells with lipopolysaccharide (LPS). Prostate epithelial cells constitutively express significant levels of Toll-like receptor 4 (TLR4) and CD14 mRNA. TLR2 transcription could also be demonstrated, suggesting that these cells could recognize a broader spectrum of microbial molecular patterns. TLR4, TLR2, and CD14 proteins were also detected, although not at the cell surface but intracellularly. Prostate epithelial cells not only express these receptors, but they are also able to respond to LPS, and LPS-stimulated MAT-LU cells activate nuclear factor-{kappa}B transcription factor, induce the expression of inducible nitric oxide (NO) synthase, and secrete NO. Even more, numerous chemokine genes are up-regulated or induced in this response. Our results clearly demonstrate that prostate epithelial cells are fully competent to respond. The fact that they express TLR4 and TLR2 intracellularly suggests the presence of regulatory mechanisms, which once overcome, could turn these cells into active players of the innate immunity, capable of initiating an inflammatory response.

Key Words: innate immunity • Toll-like receptors • inflammation • lipopolysaccharide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epithelial cells have classically been considered important players in innate immune defense mechanisms but merely act as physical barriers capable of secreting some antimicrobial substances when disrupted. At present, there are a compelling number of reports showing that epithelial cells can recognize bacterial products through the family of Toll-like receptors (TLRs), a fact that confers epithelial cells a more protagonist role [1 ]. TLRs conform a family of receptors that mediates the recognition of pathogen-associated molecular patterns (PAMPs), which have been evolutionarily conserved in specific classes of microbes [2 ]. Binding of these PAMPs to TLRs triggers a complicated series of events leading to increased expression of proinflammatory genes. Whereas much has been learned about the function of TLRs on macrophages and dendritic cells (DC), the mechanisms of recognition of microbes by epithelial cells are still poorly understood. Indeed, TLRs have been shown to be expressed in mucosal epithelia, such as intestinal [3 4 5 6 7 ], tracheo-bronchial [8 ], renal [9 , 10 ], bladder [11 ], oral [12 ], and ocular epithelial cells [13 ]. In contrast, little is known about TLR expression in the male genital tract, more specifically, in the prostate gland [14 ]. It is surprising that despite the prevalence of prostate disease, little is known about the immunobiology of the prostate and its contribution to disease. Prostate is the target of many diseases, which affect men of all ages. A wide range of pathological conditions—from infection of the prostate gland (by ascending bacteria from infected urine), chronic pelvic pain syndrome (of an unknown etiology but coursing with lymphocyte infiltration of the gland) to benign hyperplasia and cancer—are all common entities affecting a large proportion of men. Although the vast majority of men with prostatitis has the nonbacterial variant, a small number of men have acute or chronic bacterial prostatitis [15 ]. The causative organism in both of these conditions is usually Enterobacteriaceae, particularly Escherichia coli. Moreover, infection in the form of acute or chronic bacterial prostatitis has also been suggested to be associated with prostate cancer [16 ]. Thus, it seems important to analyze how prostate epithelial cells deal with microorganisms and their products.

The role of the epithelial cells as innate immune system players differs, depending on their anatomic position. Intestinal epithelial cells have to deal with more than 108 resident bacteria, which confer many benefits to the host. Thus, the role of TLRs on their surface seems to be dual, on one hand, participating in the initiation of host defense against pathogens and on the other, maintaining the intestinal homeostasis upon their interaction with commensal bacterial products [7 ]. In contrast, the upper genitourinary tract or the lower respiratory tract is considered to be sterile. They represent a situation where internal organs are only potentially exposed to pathogenic microorganisms. In these cases, invading bacteria might be detected rapidly by TLRs present on the epithelial cells of the mucosal lining, leading to the production of chemokines and cytokines and facilitating the initiation of the immune response. However, activation of epithelial cells by TLR ligands appears to be highly regulated. Several mechanisms have been described to control that [1 ]. Bladder epithelial cells, for instance, express TLR4, which recognizes lipopolysaccharides (LPS) and hence, Gram-negative bacteria in general. However, they do not express CD14, the accessory molecule, which is presumed to present LPS to TLR4 [2 ]. Thus, they would require a source of soluble CD14 for efficient LPS signaling [17 ]. Also, the subcellular localization of TLR4 would play a role in controlling the activation of epithelial cells by LPS. This is the case of some intestinal epithelial cell lines, which express TLR4 intracellularly [18 ]. Therefore, only upon LPS internalization are intestinal cells activated, possibly representing a regulatory step to avoid inadequate, proinflammatory stimulation by the normal microflora. The ocular surface epithelial cells represent a different situation: although TLR2 and TLR4 are expressed intracellularly in corneal epithelial cells, they are incapable of responding to LPS or proteoglycans, even after they are artificially introduced in the cytoplasm of the cell, probably contributing to the immunosilent condition of the eye [13 ].

The major aim of this study was to elucidate how prostate epithelial cells deal with microorganisms and their products. We chose to analyze the effect of a Gram-negative bacteria component, LPS, on prostate epithelial cells, as LPS recognition is one of the best-studied systems, and at the same time, Gram-negative bacteria (E. coli, Klebsiella, Enterobacteria, Proteus, and Serratia) are the predominant pathogens affecting this organ [15 ]. As in the prostate and in other organs of the genitourinary system, epithelial cells are often the first cells to contact microbial pathogens, we examined whether prostate epithelial cells have a susceptible phenotype to bacterial LPS if they express the LPS receptor, TLR4, and we studied their capability for inducing chemokines and other proinflammatory mediators that would contribute to the initiation of an immune response. Our results clearly demonstrate that prostate epithelial cells are fully competent to respond. The fact that they express TLR4 and TLR2 intracellularly suggests the presence of regulatory mechanisms, which once overcome, could turn these cells into active players of the innate immunity, capable of initiating an inflammatory response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
LPS from E. coli 055:B5 (Sigma-Aldrich, St. Louis, MO; Lot 063K4059; protein content, <3%) was used. The goat polyclonal anti-TLR4 antibody (L-14) was purchased from Santa Cruz Biotechnology (CA). According to the manufacturer’s specifications, this antibody was raised against an extracellular domain of mouse TLR4, which is 92% identical to the same domain of rat TLR4, and reacts with TLR4 of mouse and rat origin. Moreover, Western blotting analysis using this antibody against rat peritoneal cells and prostate tissue extract showed that it recognizes a 90-kD band, which is the expected molecular weight of TLR4 (data not shown).

Goat polyclonal anti-TLR2 (D-17; recognizing a peptide near the N terminus of mouse TLR2 and reacting with rat TLR2), rabbit polyclonal anti-CD14 (M-305; recognizing an internal region of recombinant CD14 of mouse origin and also reacting with rat CD14), goat polyclonal anticalnexin antibody, as well as the mouse monoclonal anti-nuclear factor (NF)-{kappa}B p65 were from Santa Cruz Biotechnology. The monoclonal antibody (mAb) against {alpha}-tubulin was purchased from Sigma-Aldrich. Secondary antibodies conjugated with fluorescein isothiocyanate (FITC; BD-PharMingen, San Diego, CA), Alexa 546, and Alexa 488 (Molecular Probes, Pitchford, OR) were used.

Cell culture and stimulation
Metastasis-lung (MAT-LU) cell line (part of the Johns Hopkins Special Collection) [19 ] is a rat adenocarcinoma cell line with characteristics of prostate epithelial cells obtained from the American Type Culture Collection (Manassas, VA).

MAT-LU cells were grown in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml each penicillin and streptomycin, and 250 nM dexamethasone in 5% CO2 in air at 37°C, as described previously [19 ].

To obtain primary prostate epithelial cells, we followed the protocol described by Ilio et al. [20 ]. Briefly, Wistar rat ventral prostates were minced in phosphate-buffered saline (PBS; pH 7.4) containing 1.0 mM dithiothreitol. After gently stirring at 37°C for 30 min, the supernatant was discarded, and 10 ml dissociation solution containing the enzymes type IV collagenase (200 U/ml) and trypsin (0.25%) in RPMI 1640, supplemented with 10% fetal calf serum (FCS), was added. After incubating at 37°C for 2 h, the tissue pieces were then passed through a tissue sieve. Cells were then washed twice in RPMI supplemented with 10% FCS and 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, and 1 µg/ml epidermal growth factor. Cells were then cultured in 24-well plates after staining with a monoclonal antipan cytokeratin [Sigma-Aldrich, C 2931, Clone C-11, recognizing several cytokeratins (4, 5, 6, 8, 10, 13, and 18)]. Cytokeratin 5 has been described to be a marker of basal epithelial cells and Cytokeratins 8 and 18, for luminal cells [21 ]. The confluent cells were separated and trypsinized with 0.05% trypsin-EDTA to produce single cells, after which they were seeded at 4 x 104 cm–2 and allowed to form subcultures. More than 90% of the cells were positively stained and kept this marker after several passages. In most experiments, cells after three passages were used.

MAT-LU cells and primary prostate epithelial cells were then stimulated with medium or different amounts of LPS (0.010–100 µg/ml) for various time periods (6–72 h). Stimulated and nonstimulated cells as well as supernatants from those cultures were used in different experiments.

Quantitation of nitric oxide (NO) in culture supernatants
One day before stimulation, prostate epithelial cells were plated in 24-well plates (5x105 cells/well). Cells were stimulated with different amounts of LPS ranging from 0.010 to 100 µg/ml for different time periods (24–72 h). After LPS stimulation, production of nitrites was measured by using the Griess reagent. Briefly, Griess reagent was prepared by mixing equal volumes of sulfanylamide (1.5% in HCL) and N-(1-naphtyl) ethylenediamide dihydrochloride (0.13% in H2O). Griess reagent (200 µl) was then mixed with 100 µl supernatant sample and incubated 15 min in the dark. Absorbance was measured at 540 nm, and nitrite concentration was calculated using a calibration curve. Nitrite was not detectable in cell-free medium.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
A standard RT-PCR assay was used in this study. Briefly, total RNA was isolated from MAT-LU cells, primary prostate epithelial cells, or rat prostate tissue using Trizol reagent (Life Technologies, Gaithersburg, MD), according to the manufacturer’s protocol. RT reactions were performed using 2–3 µg total mRNA in a 25-µl mixture. Total RNA was first incubated with 0.5 µg oligo (dT) primer (Promega, Madison, WI) for 10 min at 65°C and allowed to stand at room temperature for 2 min. Samples were then incubated with 1.25 mM deoxy-unspecified nucleoside 5'-triphosphates (Promega), 10 U RNasin inhibitor (Boehringer Mannheim, Germany), and 16 U avian myeloblastosis virus RT (Promega) for 1 h at 42°C in RT buffer. The cDNA obtained was subjected to PCR amplification using the following primers and PCR protocols: rat ß-actin [22 ], rat inducible NO synthase (iNOS) [23 ], rat CXC chemokine ligand 8 (CXCL8) [24 ], rat CC chemokine ligand 5 (CCL5) [25 ], rat CXCL10 [25 ], rat CCL2 [26 ], rat CCL3 [24 ], rat CCL4 [24 ], rat TLR4 [27 ], rat TLR2 [27 ], and rat CD14 [28 ]. The conditions were chosen so that none of the RNAs analyzed reached a plateau at the end of the amplification protocol. (In most reactions, 30 cycles of amplification were used.)

The PCR products were visualized in 2% agarose gels and ethidium bromide staining. To semiquantitate and compare cDNA levels, the gels were photographed, and the intensities of the bands were analyzed using Scion Image software. The relative band intensities in each reaction were normalized to the mean intensity of the ß-actin band. Results are expressed as arbitrary units corresponding to the ratio of sample intensities to the ß-actin band intensity.

Confocal microscopy
MAT-LU cells were plated onto 12 mm circular coverslips in 24-well plates (5x105 cells/well) and cultured overnight. Then, the cells were incubated, with or without stimulus for different time periods. After the period of stimulation was completed, coverslips were washed twice in PBS and then fixed with 3% formaldehyde in PBS for 10 min at room temperature. Following fixation, coverslips were washed twice in PBS, and cells were then permeabilized by treatment with 0.2% Triton X-100 in PBS for 10 min, washed three times with PBS, and blocked with PBS containing 1% bovine serum albumin (BSA) plus Tween 20 (0.1% v/v; blocking buffer) for 1 h at room temperature in a humid chamber. Cells were then incubated overnight at 4°C with blocking buffer containing the primary antibodies. Cells were then washed with PBS-Tween 20 (0.1% v/v) and incubated for 2 h at room temperature in blocking buffer with the corresponding secondary antibodies conjugated with Alexa 546, Alexa 488, or FITC. Coverslips were washed three times, mounted with Prolong Antifade (Molecular Probes), and visualized on a confocal laser-scanning microscope LSM 510 (Zeiss, Thornwood, NY) using LSM 510 software for image analysis.

To semiquantitate the levels of activation of NF-{kappa}B in LPS-treated MAT-LU cells, 10 fields of each condition were analyzed, and the percentage of cells that presented nuclear p65 staining was calculated according to the number of cells with NF-{kappa}B p65 in the nucleus x 100/number of total cells.

Flow cytometry
Cells were incubated with the primary antibodies stated above and the corresponding secondary antibody for 30 min at 4°C in fluorescein-activated cell sorter (FACS) wash buffer (5 mM EDTA, 0.1% sodium azide, 1% BSA in PBS). For intracellular staining, cells were stained with primary and secondary antibody in FACS wash buffer supplemented with 0.1% saponin for 30 min at 4°C. Stained cells were analyzed with a Cytoron Absolute (Ortho Diagnostic System, Raritan, NJ).

Statistical analysis
Statistical analysis was performed using the least significant difference Fisher test and InfoStat software (developed by Statistics Department, National University of Córdoba, Argentina). Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rat prostate epithelial cells respond to LPS-secreting proinflammatory mediators
Our initial experiments were aimed to elucidate whether a rat adenocarcinoma cell line with characteristics of prostate epithelial cells, the MAT-LU cells, could respond to E. coli LPS, which stimulates different types of cells to initiate a signaling cascade, ultimately leading to activation of NF-{kappa}B and enhanced expression of genes encoding proinflammatory mediators such as iNOS [29 ], interleukin (IL)-8 [30 ], macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, monocyte chemoattractant protein-1 (MCP-1), interferon-inducible protein 10 (IP-10), regulated on activation, normal T expressed and secreted (RANTES) [31 ], and tumor necrosis factor {alpha} [32 ]. NO is a well-known inflammatory factor induced by LPS in other systems, and its measurement looked as a simple approach to begin unraveling the behavior of MAT-LU cells upon LPS stimulation. We measured NO in the supernatants of MAT-LU cells incubated with increasing concentrations of LPS (from 0.010 to 100 µg/ml; Fig. 1A ). Significant NO production was apparent at doses higher than 0.1 µg/ml, reaching maximum levels at ~10 µg/ml. It is interesting that the levels of NO produced by MAT-LU cells stimulated by 1 µg/ml LPS were ~2.5 lower than those secreted by rat normal peritoneal macrophages incubated with the same amount of LPS [33 ]. Nevertheless, these amounts of LPS, added for at least 24 h to the culture, induced the expression of iNOS-specific mRNA, even at concentrations as low as 0.010 µg/ml LPS (Fig. 1B) and as early as 24 h poststimulation (Fig. 1C) , suggesting that iNOS could be responsible, at least in part, for the levels of NO measured [34 , 35 ].


Figure 1
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  		Figure 1. Rat prostate epithelial cells respond to LPS-secreting NO in a dose-dependent manner. (A) Dose-response curve of NO production in MAT-LU cells stimulated with the indicated concentrations of LPS. The stable NO metabolite nitrite present in the medium was analyzed by the Griess method. (B) Induction of iNOS transcript evaluated by RT-PCR at the different doses of LPS assayed. (C) Induction of iNOS transcript evaluated by RT-PCR at the different time-points after stimulation with 1 µg/mL LPS. *, P < 0.05, compared with basal level. Figures are representative of at least three experiments performed.

 
Rat prostate epithelial cells respond to LPS up-regulating the expression of several chemokine genes
We then investigated if prostate epithelial cells could up-regulate the expression of different chemokine genes. Again, we first performed a dose-response curve, stimulating MAT-LU cells with growing concentrations of LPS. As shown in Figure 2A and 2B , minute levels of LPS (0.010 µg/ml) were able to induce the expression of CCL5 (RANTES)-specific mRNA, otherwise undetectable. Although some basal expression of CXCL8 (IL-8)-specific mRNA could be observed, LPS induced a 0.5-fold increase in its expression at concentrations as low as 0.1 µg/ml (Fig. 2A and 2B) . To see if other chemokine genes were induced or up-regulated in these cells upon LPS stimulation, we incubated MAT-LU cells with an intermediate LPS concentration (1 µg/ml) and performed RT-PCR. CCL5-, CXCL10 (IP-10)-, and CCL3 (MIP-1{alpha})-specific mRNAs in MAT-LU cells were readily visible 6 h after LPS stimulation (Fig. 2C and 2D) . Although some basal expression of CCL2 (MCP-1) and CXCL8 could be observed in nonstimulated cells, there was at least a onefold increase in their mRNA levels, with peaks at 24 and 48 h of LPS stimulation, respectively (Fig. 2C and 2D) . Transcription levels of CCL4 (MIP-1ß) were kept unchanged. Comparable results were obtained when a rat primary prostate epithelial cell culture was stimulated in the same way (Fig. 2E and 2F) . Again, an increased expression of specific mRNA levels was seen for CXCL8, CCL2, and CCL3 after 48 h of LPS stimulation, clearly indicating that the LPS-susceptible phenotype is not a particularity of MAT-LU cells.


Figure 2
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  		Figure 2. Rat prostate epithelial cells respond to LPS by up-regulating the expression of several chemokine genes. (A) mRNA expression levels of CXCL8, CCL5, and ß-actin evaluated by RT-PCR, following stimulation of MAT-LU cells with increasing doses of LPS. (B) Semiquantitation of CXCL8- and CCL5-specific mRNA following stimulation of MAT-LU cells with increasing doses of LPS. The relative band intensities in each reaction were normalized to the mean intensity of the ß-actin band (see Materials and Methods). Results are expressed as densitometric arbitrary values corresponding to the ratio of sample intensities to the ß-actin band intensity and are representative of two different experiments. (C) mRNA expression levels of CXCL8, CCL5, CXCL10, CCL2, CCL3, CCL4, and ß-actin evaluated by RT-PCR, following stimulation of MAT-LU cells for different time periods (LPS 1 µg/ml). (D) Semiquantitation of chemokine-specific mRNA at different time-points after stimulation. (E) mRNA expression levels of CXCL8, CCL2, CCL3, CCL4, and ß-actin were evaluated by RT-PCR, following stimulation of rat primary prostate epithelial cells for 48 h with 1 µg/mL LPS. (F) Semiquantitation of chemokine-specific mRNA in rat primary prostate epithelial cells stimulated with LPS for 48 h. Figures are representative of at least three experiments performed.

 
Taken together, these findings strongly demonstrate that prostate epithelial cells can respond to LPS stimulation, secreting inflammatory mediators such as NO and up-regulating the expression of chemokine genes.

Rat prostate epithelial cells respond to LPS by activating nuclear translocation of NF-{kappa}B
The responsiveness of MAT-LU cells to LPS shown above led us to examine the activation state of the transcription factor NF-{kappa}B. We cultured MAT-LU cells on coverslips, subjected them to LPS for various time periods, performed indirect immunofluorescent staining for the p65 subunit of NF-{kappa}B, and analyzed nuclear localization of the transcription factor by confocal microscopy. As shown in Figure 3 , unstimulated MAT-LU cells did not present nuclear localization of NF-{kappa}B (3% of cells presented nuclear staining). In contrast, p65 nuclear translocation began to be evident after 15 min of exposure to LPS, remaining in the nucleus after 30 min of stimulation (75% of the cells with nuclear staining). Two hours after LPS stimulation, nuclear NF-{kappa}B levels appear to wane, and a colocalization with calnexin (used as a cytoplasmic marker) was observed. Thus, LPS treatment of MAT-LU cells induces NF-{kappa}B activation, which explains the numerous proinflammatory genes being activated.


Figure 3
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  		Figure 3. Rat prostate epithelial cells respond to LPS by activating nuclear translocation of NF-{kappa}B. MAT-LU cells were cultured with LPS for the indicated time periods. Cells were subsequently stained with anti-p65 mAb and FITC-labeled secondary antibodies. To visualize cytoplasm localization, a polyclonal anticalnexin antibody and an Alexa 546-labeled secondary antibody were used. Cytoplasmic localization results in colocalization of red and green signals resulting in yellow fluorescence. Cells were visualized by confocal microscopy. The experiment was performed twice, and the pictures observed correspond to a representative field for each time period studied.

 
Rat prostate epithelial cells constitutively express CD14- and TLR4-specific mRNA
Given that MAT-LU cells and prostate epithelial cells responded to LPS, the next step was to elucidate whether prostate epithelial cells harbor the main receptor, which has been involved so far in E. coli LPS recognition—TLR4.

TLR4-specific mRNA was present in MAT-LU cells (Fig. 4A ), in normal prostate epithelial cells (Fig. 4B) , and in rat prostate gland (ex vivo; Fig. 4C ). Its expression as well as the expression of CD14 were constitutive, although an up-regulation in the level of TLR4 mRNA was seen after 24 h of stimulation with LPS in MAT-LU cells (Fig. 4A) and after 48 h of stimulation in prostate epithelial cells. Transient expression of TLR2-specific mRNA was seen in MAT-LU cells after 6 h of LPS stimulation, probably reflecting the presence of some contaminants such as lipoprotein in the commercial preparation of LPS [36 , 37 ] (Fig. 4A) .


Figure 4
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  		Figure 4. Rat prostate epithelial cells express TLR4, TLR2, and CD14-specific mRNA. (A) mRNA expression levels of TLR4, TLR2, CD14, and ß-actin evaluated by RT-PCR, following stimulation of MAT-LU cells with 1 µg/ml LPS for different time periods. (B) mRNA expression levels of TLR4, CD14, and ß-actin evaluated by RT-PCR following stimulation of rat primary prostate epithelial cells (P.E.C) for 48 h with 1 µg/ml LPS. (C) mRNA expression levels of TLR4 and ß-actin evaluated by RT-PCR in rat prostate ex vivo samples. Figures are representative of at least three experiments performed.

 
MAT-LU cells express TLR4 and TLR2 intracellularly but not at the cell surface
To investigate if MAT-LU cells express TLR4 and TLR2 at their cell surface, we first performed FACS analysis using rat-specific antibodies.

No surface expression of TLR4, TLR2, or CD14 was detected on the cell line, neither on basal conditions nor upon stimulation with LPS (Fig. 5 ). Although these molecules were easily detected at the surface of rat-activated peritoneal cells (data not shown), expression of TLR4 protein was only detected when MAT-LU cells were permeabilized, indicating an intracellular localization of TLR4. Stimulation of the cells with LPS did not modify its subcellular localization; however, an increase in the percentage of cells expressing TLR4 as well as enhanced levels of expression of this receptor was seen after 24 h of treatment with LPS (basal: 34.3%, mean fluorescence channel: 16.80; 24 h: 46.6%, mean fluorescence channel: 22.44; 48 h: 35.8%, mean fluorescence channel: 18.24). Also, the percentage of cells expressing TLR2 raised intracellularly (basal: 8.2%; LPS 24 h: 23.4%; LPS 48 h: 13.1%), and an intracellular expression of CD14 protein was also detected. To directly demonstrate the intracellular expression of TLR4 by MAT-LU cells, immunohistochemistry examination using confocal image analysis was performed. MAT-LU cells were stained for tubulin (Fig. 6 ) to delineate the cytoplasmic compartment. A diffuse cytoplasmic distribution of TLR4 protein without showing a specific pattern was apparent in basal conditions. LPS treatment of the cells did not induce any significant redistribution of cytoplasmic TLR4.


Figure 5
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  		Figure 5. MAT-LU cells express TLR4, TLR2, and CD14 intracellularly but not at the cell surface. MAT-LU cells were stimulated with 1 µg/ml LPS, washed, and permeabilized or not, as was described in Materials and Methods. Cells were incubated with anti-TLR4, anti-TLR2, anti-CD14, or isotype control antibodies followed by secondary FITC-labeled antibody. Fluorescence tracing is representative of three different experiments. Figures are representative of at least three experiments performed.

 

Figure 6
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  		Figure 6. MAT-LU cells express TLR4 intracellularly but not at the cell surface. MAT-LU cells were cultured with 1 µg/ml LPS for different time periods. Cells were subsequently stained: first, with primary antibodies against TLR4 and {alpha}-tubulin ({alpha}-Tub) and then, with the correspondent secondary antibody labeled with Alexa-546 and Alexa-488, respectively. Note that the type of fixation used does not allow the visualization of microtubules but only soluble tubuline. Cytoplasmic localization results in colocalization of red and green signals resulting in yellow fluorescence. Cells were visualized by confocal microscopy. The experiment was performed twice, and the pictures observed correspond to a representative field for each of the times studied.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Whereas much has been learned during recent years about the importance of TLRs in the process of immune recognition by myeloid cells such as macrophages or DC, the role of TLR expression by epithelial cells is still poorly understood. Moreover, most of what is presently known about TLR expression by the epithelial layers concerns the intestinal mucosa, which has attracted the attention of immunologists for many years, as it represents the paradigmatic situation in which discrimination between harmless commensal bacteria and pathogens represents a difficult task to overcome [1 ].

In contrast to the gastrointestinal tract, the upper genitourinary tract is considered to be sterile. The epithelial layer here is not acting as a barrier but probably contributes significantly to the initiation of the immune response. Although there are some reports regarding TLR expression by bladder and kidney epithelial cells [9 10 11 ], to our knowledge, there are few reports concerning TLR expression by the prostate [14 ], which is an organ usually neglected by immunologists, in spite of the many pathologies that affect it and the tremendous health impact that they have on human population. Although only a small percentage of men suffers of infectious chronic or acute prostatitis [National Institutes of Health (NIH) Categories I and II], chronic, noninfectious prostatitis (NIH Category III) is a highly prevalent disease among young adults [15 ]. Chronic, nonbacterial prostatitis is an inflammatory state of the prostate with direct impact on the quality of life of the patients. Yet, its etiology is unknown. Furthermore, prostate cancer is one of the major causes of death in Western population, being the most commonly diagnosed cancer in men in industrialized countries. There is an expanding body of literature suggesting a link between chronic inflammation and cancer [16 , 38 39 40 ], and recently, an association between a TLR4 sequence variant in the 3'-untranslated region of the gene and prostate cancer risk has been reported [41 ]. Therefore, efforts in improving the current knowledge about how prostate epithelial cells respond to inflammatory stimuli such as microorganisms or their products are necessary.

Prostate epithelial cells are able to respond to LPS, and LPS-stimulated MAT-LU cells activate NF-{kappa}B, induce the expression of iNOS, and secrete NO in a dose-dependent manner. Even more, numerous chemokine genes are up-regulated or induced in the cell line as well as in normal prostate epithelial cells.

The experimental data from prostate tissue or rat prostate primary cells demonstrate that the results obtained with the cell line were not a particularity of the cell line but that they could also be reproduced to some extent in primary cells. However, although all the chemokine primers were tested in the primary cell culture, we could not detect significant differences for CXCL5 and CXCL10 before and after LPS stimulus under the assayed conditions. Therefore, they do not respond exactly in the same way, but they do respond up-regulating several chemokine genes. Moreover, the levels of NO produced are lower than those measured routinely in our laboratory in rat normal peritoneal macrophage supernatants incubated with LPS. In any case, our results clearly demonstrate that prostate epithelial cells are well-equipped to respond to LPS, which is a poor stimulator of epithelial cytokine production [11 , 42 ], and sometimes to induce a response, it acts in synergy with other bacterial compounds. For example, bladder epithelial cells augment the IL-6 response to uropathogenic bacteria through a mechanism that seems to involve Type 1 pili (necessary for bacterial invasion) and also LPS recognition. Other reports have shown that in vitro, pretreatment of two distinct intestinal epithelial cell lines with other cytokines before the addition of LPS augments their capacity of response, measured as IL-8 secretion [43 ]. Thus, other bacterial-secreted factors or cytokines secreted by endothelial cells or by some of the few innate immune cells present in the lumen of the gland could lower the activation threshold of these cells. In any case, MAT-LU cells or normal rat prostate epithelial cells are capable of mounting a robust response to LPS as seen by any of the proinflammatory mediators studied.

The production of proinflammatory mediators by epithelial cells in response to bacterial compounds has largely been known. The system studied most extensively has been the intestinal epithelial cells, whose ability to up-regulate proinflammatory mediators in response to LPS appears to vary with the cell line tested [43 , 44 ]. Airway epithelial cells have demonstrated sensitivity to LPS, activating NF-{kappa}B, producing cytokines, and up-regulating various defense mechanisms [8 , 45 ]. In contrast, much of what is known about the capability of prostate epithelial cells to secrete chemokines is related to cancer, tumor growth, vascularization, and metastasis. In our study, we demonstrate for the first time that MAT-LU cells, a rat adenocarcinoma cell line with characteristics of prostate epithelial cells, react against an inflammatory stimuli such as LPS. Moreover, similar results were obtained with primary prostate epithelial cells, supporting the hypothesis that the normal prostate epithelium could eventually initiate a series of events that will ultimately culminate in inflammation or in an immune response.

Prostate epithelial cells constitutively express significant levels of TLR4 and CD14 mRNA. TLR2 transcription could also be demonstrated in MAT-LU cells, suggesting that these cells could recognize a broader spectrum of microbial molecular patterns. It is interesting that TLR2 expression (at mRNA or protein level) showed a marked, temporary up-regulation similar to the situation described in macrophages [46 ]. TLR4 and CD14 proteins were also detected, although not on the cell surface but intracellularly, in a diffuse pattern that did not seem to change upon LPS stimulation. The expression of TLR4 in the cytoplasmic compartment has been demonstrated by several studies performed in monocytes, polymorphonuclear cells, or epithelial cells [47 48 49 50 ]. Indeed, there are consistent data showing that TLR4 is expressed primarily in two different subcellular localization—the plasma membrane and the Golgi apparatus—and that only the heavily glycosylated, mature forms of TLR4 are expressed on the cell surface [47 ]. Actually, TLR4 appears to be a highly mobile protein, which recycles rapidly and continuously between its two major pools—the Golgi complex and the plasma membrane—in a process with little immunological significance [47 ]. However, an exclusively intracellular expression of TLR4, with almost undetectable levels of membrane protein, has also been reported in numerous epithelial cell lines [6 , 13 , 18 , 42 , 43 ]. It has been suggested that this subcellular localization could imply an immunoregulatory mechanism that would prevent unnecessary activation of the epithelial layer. Activation of epithelial cells by TLR ligands seems to be highly regulated. Indeed, there exists myriad mechanisms, which control, for example, TLR4 down-regulation [51 ]; expression of a nonsignaling, truncated form of myeloid differentiation primary-response protein 88 (the adaptor molecule involved in TLR signaling) [52 ]; activation of IL-1 receptor-associated kinase (IRAK)-M, a negative regulatory member of the IRAK family [53 ]; and induction of A20 expression, an endogenous inhibitor of NF-{kappa}B [54 ]. Intracellularly expressed TLR4 and TLR2 in the ocular epithelium are completely refractory to LPS stimulation [13 ]. Nevertheless, intracellular TLRs have been shown to signal from inside the cell. A paradigmatic case would be the m-ICcl2 cell line, a murine cell line derived from the intestinal epithelial crypts, which express TLR4 exclusively in the Golgi apparatus, and that is highly responsive to LPS [6 , 18 ]. Thus, these cells could only be activated upon internalization of LPS, in other words, after invasion by pathogenic bacteria. A LPS recognition event appears to occur in the cytoplasmic compartment and not at the cell surface in these cells. A similar situation might be occurring in LPS-stimulated MAT-LU cells, as they express TLR4 intracellularly, but they are fully capable of responding. It has been demonstrated recently that TLR4 is necessary to initiate the cascade of signaling events, but it is not the LPS up-take receptor. Internalization of LPS in MAT-LU cells might be accomplished by the scavenger receptor-mediated pathways [55 ], as these cells do not express CD14 at the cell surface either. Further experiments are necessary to investigate the mechanisms by which LPS is internalized in these cells and the principal intracellular site where LPS meets TLR4, and the recognition events occur.

In conclusion, our results strongly demonstrate that prostate epithelial cells could actively participate in initiating an inflammatory process. The intracellular localization of TLR4 and CD14 suggest that this process is highly regulated. Unraveling the physiological conditions in which this situation could occur will provide new clues in understanding prostate pathology.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT) PICT 2002 11944, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET) PEI No. 6557, and Fundación Antorchas 2004 (to M. M.). G. G. is a Ph.D. fellow of FONCyT, and R. D. M. is a Ph.D. fellow of CONICET. M. M. and V. R. are members of the Researcher Career of CONICET.

Received October 17, 2005; revised January 11, 2006; accepted January 12, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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