1. health and fitness
2. disease
3. cancer

BOR Papers in Press. Published on July 26, 2006 as DOI:10.1095/biolreprod.106.053967
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1. TITLE PAGE
Title
Toll-Like Receptor 4 in Rat Prostate: Modulation by Testosterone and
Acute Bacterial Infection in Epithelial and Stromal Cells1
Short title
Toll-Like Receptor 4 in prostate gland
Summary sentence
Rat prostate expresses and up-modulates TLR4 in response to bacterial infection as well as
after androgen withdrawal, indicating an important role of the prostate gland in the innate
immune responses.
Authors
Amado A. Quintar, Felix D. Roth, Ana Lucía De Paul, Agustín Aoki and Cristina A.
Maldonado2.
Center of Electron Microscopy, School of Medical Sciences. National University of
Córdoba. X5000HRA. Córdoba, Argentina.
Grant support
This work was supported by research grants from Consejo National de Investigaciones
Científicas y Técnicas (CONICET PEI 1923/03), Grant PICT from FONCyT-ANPCyT,
and by a fellowship to AQ from Fundacion Florencio Fiorini.
AA, ADP and CM are members of the scientific career from CONICET.
AQ and FR are fellows from CONICET and FONCyT-ANPCyT respectively.
1
Correspondence and reprint requests
Corresponding author:
Cristina A. Maldonado, PhD.
Centro de Microscopía Electrónica.
Facultad de Ciencias Médicas.
Universidad Nacional de Córdoba.
Pab. Biología Celular 1º piso. Haya de La Torre esq. Enrique Barros.
Ciudad Universitaria.
X5000HRA Córdoba, Argentina.
2
Tel/FAX: 54 0351 4333021.
E-mail: [email protected]
Copyright 2006 by The Society for the Study of Reproduction.
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2. ABSTRACT
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tract. However, little knowledge is available about the immunobiology within this gland.
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Toll-Like Receptor 4 (TLR4) is considered to be a major sensor of dangerous signals and a
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key trigger of the innate immune responses. TLRs have also been implicated in the
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development of different inflammatory diseases in organs where epithelial-stromal
50
interactions are critical for homeostasis. The purpose of this work was to evaluate the
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presence and regulation of TLR4 in the rat prostate. Western blot and immunocytochemical
52
studies revealed a constitutive expression of TLR4 in rat ventral prostate localized in the
53
epithelium, mainly associated with rough endoplasmic reticulum, as well as with smooth
54
muscle cells in the stroma. Additionally, increased concentrations of TLR4 were found in
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castrated rats, predominantly in the hypertrophied smooth cells. On the other hand,
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applying a bacterial prostatitis model, we observed an increment of the TLR4 cytoplasmic
57
content and the migration of this receptor to the apical plasmatic membrane of epithelial
58
cells at 24 and 48 h post-infection.
The prostate gland is the most inflammation-prone organ in the male reproductive
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These findings suggest that the prostate gland is able to recognize pathogens and to
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initiate the immune response. In addition, TLR4 appears to be implicated in the vital
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stromal-epithelial interactions maintaining the prostate homeostasis during prostatitis, as
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well as following androgen deprivation.
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3. INTRODUCTION
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As in the case of many other epithelia exposed continually to external injures, the
66
epithelium lining the reproductive tract is equipped with a variety of antimicrobial, pro-
67
inflammatory and immuno-modulatory compounds, which are key mediators of the innate
68
immune system [1].
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The discovery and characterization of a new host defense protein, the Toll-like
70
receptor (TLR) in invertebrates and mammals, has greatly contributed to understanding
71
how the host organism detects the presence of infectious agents and disposes of invaders,
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without destroying its own tissues [2]. TLRs are transmembrane proteins that distinguish
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specific patterns of microbial components, especially those from pathogens; and regulate
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the activation of both innate and adaptive immunity [3]. Once activated, the TLR
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intracellular signals culminate in the activation of NF-κB as well as MAPK, leading to the
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subsequent induction of various genes that function in host defense responses [4].
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Among the eleven members of the TLR family (TLR1-TLR11) [5], the
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lipopolysaccharide (LPS) receptor Toll-like receptor 4 (TLR4) is a central player in
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signaling pathways of the innate immune response to infection by several pathogens. TLR4
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is activated by the LPS [6] assembled in the wall of Gram-negative bacteria such as E. coli
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or K. pneumoniae. These receptors are widely distributed, not only in immune cells such as
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macrophages [7] and dendritic cells [8], but increasing evidence indicates that they are also
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expressed in the epithelia of respiratory [9], digestive [10] and urinary tracts [11].
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Prostatic inflammation, or prostatitis, represents an important problem for human
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health worldwide. Recommendations from the International Prostatitis Collaborative
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Research Network (initiative funded by the NIH) include further studies on animal models
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towards a better understanding of the immunobiology of the prostate, in order to improve
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our knowledge about this disease [12]. In addition, the prostate gland is the most
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inflammation-prone organ in the male genito-urinary tract, and is frequently a main target
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of venereal diseases. Many peptides with innate immune activity have been found in this
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gland [1]; but neither the presence of TLR4 nor the regulation by inflammatory stimuli
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have been evaluated.
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In the present study, we investigated the expression of TLR4 in the rat prostate
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gland and its response to inflammation and androgens. The prostate morphology was
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assessed by electron microscopy and immunocytochemistry was performed with vimentin
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and smooth muscle alpha actin (ACTA2) antibodies.
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4. MATERIALS AND METHODS
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Animals
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Adult 12-week-old male rats, Wistar strain, weighing 250-350 g, were housed at the
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Animal Research Facility of the National University of Córdoba, in air-conditioned
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quarters, under a controlled photoperiod (14-h light/10-h darkness) with free access to
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commercial rodent food and tap water. The rats were divided into four experimental groups
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(n=10/group in three different experiments): 1) Intact animals (IN group), 2) Acute
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bacterial-induced prostatitis (BP group), 3) Orchidectomized rats (OX group) and 4)
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Testosterone-treated group (TT group). All animal experiments were conducted in
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accordance with the International Guiding Principles for Biomedical Research Involving
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Animals as promulgated by the Society for the Study of Reproduction.
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Bacterial Prostatitis Model (BP group)
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A strain of Uropathogenic Escherichia coli (kindly provided by Dr. Oscar Pessah,
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Department of Microbiology, National University of Córdoba) isolated from patients with a
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complicated urinary tract infection was stored at -20 ºC and grown overnight in tryptic soy
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broth at 37 ºC and then used for inoculations. The bacterial cells were spun down, washed
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three times in sterile PBS and resuspended at a concentration of 108 cells per ml.
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The rats were anesthetized with inspired ether, and subjected to a laparotomy to
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expose the ventral prostate. Acute prostatitis was induced by injection of 200 ul of E. coli
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suspension. Bacterial solution was injected with a 30 gauge needle directly beneath the
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capsule of both ventral lobes. The peritoneum, abdominal muscles and skin were closed
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with a simple continuous suture with a chromic surgical filament. Animals were sacrificed
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at 24 and 48 hours after germ suspension. For controls, sham-operated rats were subjected
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to a laparotomy but were not injected with the bacterium. The ventral prostate was
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harvested and processed for biochemical studies.
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Orchidectomy and testosterone treatment
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The influence of androgens on TLR4 expression in the prostate gland was studied in
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male rats (OX group) orchidectomized via the scrotal route under ether anesthesia; the
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epididymis and epididymal fat were also removed in the operation. Control rats for the OX
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group were submitted to sham surgical manipulation with animals being sacrificed 10 days
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after surgery. In the TT group, castrated rats were injected subcutaneously with testosterone
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(10 mg/kg body weight-day; Sustanon, Organon) in sunflower oil daily for ten days
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beginning one day after castration. For controls in this group, castrated rats were injected
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with the vehicle alone. Animals were sacrificed 24 h after the last injection. Samples of
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ventral prostate were obtained and processed for biochemical studies.
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Serum testosterone levels
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Prior to sacrificing, blood was obtained by intracardiac puncture from IT, OX, and
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TT groups and their controls. Serum total testosterone levels of individual rats were
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determined by electrochemiluminescence (ECL) immunoassay using Roche Elecsys E170
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immunoassay analyzers (Roche Diagnostics).
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Microbiological Study
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For bacterial culture, pieces of prostate from each rat were weighed and cultured
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qualitatively by plating them on a McConkey (Sigma) agar plate and incubating them
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overnight at 37ºC. After 24 h, bacterial isolates were taken and identified using
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conventional Gram staining. The samples were considered sterile if no microorganisms
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were detected 48 h after culture.
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Light and electron microscopy
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Rats were fixed by perfusion with 4% formaldehyde, and the ventral prostate blocks
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were embedded in paraffin; cut into 4 µm thick sections and stained with
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Hematoxilin/Eosin or immunostained with specific antibodies.
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Other prostate blocks were fixed in Karnovsky mixture containing 1.5% (v/v)
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glutaraldehyde, 4% (w/v) formaldehyde in 0.1M cacodylate buffer, and then treated with
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1% osmium tetroxide, dehydrated and embedded in Araldite. For light microscopy 1 µm
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thick sections were cut serially and stained with a Silver technique following the
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methodology explained below. For ultrastructural studies, thin sections were cut with a
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diamond knife on a Porter-Blum MT2 and JEOL JUM-7 ultramicrotome and examined
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using a Zeiss LEO 906E electron microscope
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For ultrastructural immunocytochemistry, prostate tissue blocks were embedded in
acrylic resin (LR-White, London Resin Corporation) omitting osmium fixation.
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Silver staining
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The silver methenamine technique for polysaccharides according to Sutter and
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Roulet [13] was performed on Araldite semi-thin sections. This procedure applied to plastic
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embedded sections provided similar results to Periodic Acid-Schiff (PAS) staining of
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paraffin sections but with a better resolution making identification easier of tissue structures
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such as collagen fibers, basement membranes and glycoproteic components.
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Immunocytochemistry
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Slides from paraffin-embedded prostates were cleared with xylene and rehydrated in
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a series of descending concentrations of ethanol solutions. Then, a microwave pretreatment
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(antigen retrieval method) was performed. To block the endogenous peroxidase activity,
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slides were treated with H2O2 in methanol for 15 min. Sections were incubated 30 min in
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10% normal rabbit serum (Sigma) to block nonspecific binding, followed by overnight
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incubation with 1/400 diluted polyclonal goat antibody to TLR4 (Santa Cruz) at 4 ºC in a
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humidified chamber. Then the sections were incubated with a biotinylated secondary
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antibody (Santa Cruz) and ABC complex (Vector). Diaminobenzidine (DAB Sigma) was
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used as a chromogen substrate for 10 min at RT, and sections were rinsed in running water.
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Harris hematoxylin was used as a counterstaining solution. Expression of two markers was
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evaluated by immunohistochemistry to characterize the stromal cellular phenotype; the
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procedure was similar to the above mentioned for TLR4, using monoclonal antibodies to
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vimentin (VIM) and ACTA2 from Novocastra, and applying a goat anti-mouse biotinylated
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IgG (Amersham Pharmacia) as a secondary antibody.
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Ultrastructural immunocytochemistry
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LRWhite thin sections mounted on 250 mesh nickel grids were incubated overnight
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on a drop of goat anti-TLR4 (Santa Cruz) diluted 1/500, and immunoreactive sites were
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labelled with 16 nm colloidal gold/anti-goat IgG complex (Santa Cruz). For the controls,
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the primary antibody was replaced with goat normal serum (Sigma), purified Goat IgG
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(Santa Cruz) or PBS–BSA.
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Western blotting
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For immunoblots, prostate tissues were minced and homogenized on ice with a
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teflon-glass Potter-Elvehjem tissue grinder in 2 ml cold PBS containing 1.25% Igepal CA-
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630, 1mM EDTA, 2mM PMSF, 10ug/ml leupeptin and 10ug/ml aprotinin. The lysate was
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centrifuged at 14,000xg for 20 min at 4 ºC to pellet the Igepal CA-630-insoluble material,
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and the supernatant was withdrawn and stored in aliquots frozen at -70 ºC until required.
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Prostatic lysates from duplicate experimental conditions were pooled before loading into
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electrophoresis gels. Total protein concentration was measured with a Bio-Rad kit (Bio-Rad
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Protein Assay, Bio-Rad Laboratories). Denatured protein samples (30µg/lane) were then
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separated on 12% SDS polyacrylamide gel and blotted to a Hybond-C membrane
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(Amersham Pharmacia). To assess the corresponding molecular weight, Full Range
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Rainbow Molecular Weight Marker was used (Amersham Pharmacia). Incubation steps
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were performed in 5% defatted dry milk in PBS/0.1% Tween 20. Blots were incubated with
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1/250 goat anti TLR4 IgG (Santa Cruz) and peroxidase-conjugated bovine antigoat
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antibody (Jackson), and visualized applying the chemiluminescence technique. The
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expression of β-actin (ACTB) (1/5000; monoclonal anti β-actin; Sigma) was used as an
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internal control to confirm the equivalent total protein loading. Semiquantitative signals
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were derived by densitometric analysis using Scion Image (version beta 4.0.2 Scion
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Corporation) and data displayed as area units per mg protein.
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Statistical analysis
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Data from more than two groups were examined using analysis of variance with
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Tukey as a post test. Statistical testing and calculation of western blot data were performed
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using the InStat V2.05 program from GraphPad, Inc.
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5. RESULTS
Characterization of experimental models
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In the rat prostate from the IN group, the epithelium lining the glandular alveoli is
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comprised mainly of cylindric secretory cells; the stroma is scarce, mostly formed by a thin
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periacinar layer of smooth muscle cells and interstitial fibroblasts (Fig. 1A). The periacinar
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stromal layer exhibited a continuous pattern of ACTA2 reactivity, specifically of smooth
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muscle cells (Fig. 1B), and a few VIM-positive fibroblastic cells (fig. 1C).
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Microbiological methods tested positive for E. coli for all prostate samples from the
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BP group at 24 h and 48 h after inoculation; while prostate from controls and the IT group
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were sterile.
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All prostates from the BP group showed acute inflammation, being widespread
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throughout the gland. At 24 h after bacterial inoculation, the epithelium was hypertrophied,
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and displayed an undulating contour. Furthermore, in the stroma, infiltrated by numerous
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inflammatory cells, there was a great development of the ACTA2 positive smooth muscle
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periacinar layer (Fig. 1D and 1E). VIM immunoreactivity formed two interrupted layers
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flanking the smooth muscle cells (Fig. 1F). Infiltrating inflammatory cells in the prostatic
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stroma also showed VIM-immunoreactivity. Similar results were observed at 48 h after
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bacterial inoculation.
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In the OX group, the prostatic epithelium exhibited an important reduction of the
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nucleus/cytoplasm relation as a result of androgen deprivation. In contrast, smooth muscle
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cells in the periacinar layer were significantly enlarged (Fig. 1G); ACTA2 staining showed
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that the periacinar smooth muscle layer was larger in the OX than in BP group (Fig. 1H).
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Isolated fusiform hypertrophied VIM positive cells appeared under the epithelial basal
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membrane as well as external to the smooth cell layer (Fig. 1H).
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In animals treated with testosterone (TT group), the enlarged prostate gland
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exhibited a striking hypertrophy of the glandular alveoli, resulting in large lumen and an
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increased secretory volume while the stroma was comparable to that of intact animals (Fig.
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1J). Antibodies against ACTA2 and VIM provided a staining pattern with similar
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characteristics to the IN group (Fig. 1K and 1L).
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Analysis of TLR4 expression
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To establish whether TLR4 is expressed in rat ventral prostate cells, and to
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characterize its response to Gram-negative bacteria infection, the expression of TLR4
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protein was analyzed by western blot and immunocytochemistry. As shown in Fig. 2,
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prostate gland from intact rats expressed TLR4 constitutively. Western blotting revealed a
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positive band of about 90 kDa for all experimental conditions (Fig. 2).
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As expected, in the BP group there was a significant increase in TLR4 expression
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both 24 h and 48 h after inoculation as compared with their controls and IN rats (Fig. 3).
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The increment was time-dependent, with a greater increase in TLR4 expression at 48h after
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bacterial inoculation compared with 24 h rats.
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Experimental groups exposed to different testosterone levels, i.e.: IN, OX and TT
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groups (Fig. 4A), were used to investigate the influence of androgens on TLR4 expression.
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Surprisingly, TLR4 levels in the ventral prostate increased significantly in OX animals in
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relation to their respective controls and IN rats. In contrast, the TT group exhibited no
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significant differences in relation to the IN group (Fig. 4B).
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Immunocytochemistry of TLR4 expression
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The localization of TLR4 in the prostate gland and the changes in the TLR4
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expression detected by western blot were examined by immunocytochemistry at light and
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electron microscope levels.
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In the IN group, TLR4 immunolabelling was faint in epithelial cells (Fig. 5A) and in
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the stroma. A stronger staining occurred in the wall of arterioles. The ultrastructural
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immunogold technique revealed a labeling associated mainly with rough endoplasmic
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membranes (Fig. 6B) but with the apical plasma membrane being negative (Fig. 6A). Also,
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TLR4 was localized in periacinar smooth muscle cells (Fig. 6C and 7A). When specific
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anti-TLR4 antibody was replaced by goat normal serum or purified goat IgG, no labeling
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occurred (not shown).
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In the BP group, the increased TLR4 level found by western blot correlated well
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with the strong immunostaining observed in epithelial cells and the periacinar layer (Fig.
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5B); some inflammatory cells infiltrating the gland were also positive. The immuno-gold
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technique labeled not only intracytoplasmic membranes, as in the IN group (Fig. 6G), but
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also appeared clearly polarized to the apical cytoplasm (Fig. 6F), intercellular junctional
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complexes and microvilli (Fig. 6D and 6E). Abundant secretory granules, increased by
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infection, were negative for TLR4 (Fig. 6D, 6E and 6F). Hypertrophied smooth muscle
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cells also exhibited strong specific TLR4 immunoreactivity for this technique (Fig. 7B).
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In the OX group, correlating with the increased TLR4 expression found by western
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blot, there was an intense immunoreactivity in epithelial and stromal cells (Fig. 5C).
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Surprisingly, TLR4 was strikingly expressed in the scarce cytoplasmic remnant in epithelial
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cells (Fig. 5C). On the electron microscope, TLR4 exhibited a heterogeneous localization in
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the cytoplasm (Fig. 6H) and was often associated with poorly-developed rough
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endosplasmic reticulum, distributed along the lateral membranes (Fig. 6I and 6J) and the
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basal cytoplasm, where gold particles appeared delineating a thin cytoplasmic region in
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contact with the basement membrane (Fig. 6K). In the stromal compartment, an intense
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TLR4 staining was observed in the periacinar layer (Fig. 5C); with immuno-gold labeling
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being constrained to an area containing predominantly smooth muscle cells (Fig. 7C) which
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frequently exhibited intense labelling of the plasmalemma (Inset fig. 7C).
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In the TT group, TLR4 immunoreactivity was weak in the epithelial cytoplasm and
stromal cells, and also in the IN group (Fig. 5D).
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6. DISCUSSION
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Host defense proteins have aroused a great deal of interest in myeloid cells and in
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the respiratory and digestive tracts. Over the last few years, several additional contributions
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demonstrating the importance of innate immune system molecules were reported in the uro-
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genital tract [1, 11, 14-17]. In the male reproductive system, investigations were focused
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particularly on the epididymis [16, 17] and testis [18, 19]. The epididymis was found to
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secrete antimicrobial proteins such as defensins [16], and the testis expresses galectins [18],
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key regulators of the immune system. However, little is known about the innate immune
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response within the prostate gland.
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TLR4, originally detected in mammalian immune cells, is critically involved in the
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innate immune response as a membrane receptor for Gram-negative bacteria whose
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activation triggers an inflammatory cascade mediated by NF-κB [3]. Furthermore, TLR4 is
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expressed by epithelia that interface with the external environment, as in the cornea [20],
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the oral cavity [21], the respiratory tract [22], intestine [23] and urinary tract [11]. In this
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study, we observed constitutive in vivo expression of TLR4 in rat ventral prostate, and up-
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regulation of TLR4 by E. coli and castration, not only in epithelial cells but also in the
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stromal compartment.
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Previous studies have reported that prostatic epithelial cells and their secretory
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products may actively participate as local modulators in response to bacteria [24, 25], but
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the molecules involved in this process have not yet been fully characterized. Recently,
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Takeyama et al. [26] and Gatti et al. [27] have reported in vitro, that prostate cells secrete
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inflammatory cytokines in response to M. hominis and LPS through a TLR2 and TLR4-
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mediated mechanism, suggesting that epithelial cells could act in the first line of host
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defense in the prostate gland. We found in vivo that TLR4 is localized in the prostatic
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epithelium, which exhibited a weak intracytoplasmic staining mainly associated with the
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rough endoplasmic reticulum. This finding contrasts with the classical localization of TLR4
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on the cell surface of macrophages and other immune cells [7]. At variance, Hornef et al.
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[23] have also localized TLR4 in the cytoplasm of epithelial cells in the intestinal mucosa.
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The explanation for this localization seems to be related to cell function: while immune
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cells should be ready to initiate an immune response, epithelial cells exposed to normal
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microflora must try to avoid interaction between normal commensal bacteria and TLR4 and
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the consequent activation of inflammatory signals; although the bacterial flora of the
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prostate is not well defined, the occurrence of normal microflora has been suggested [28].
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In addition to LPS from Gram-negative bacteria, other products such as hyaluronan,
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heparan and fibrinogen have been shown to be able to activate the TLR4 system [29]. Some
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of these molecules, the so-called endogenous ligands of TLRs, are normally present in the
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seminal plasma [30-32] and could trigger unwanted inflammatory reactions since semen is
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often in contact with the prostate epithelium surface [33]. Consequently, the intracellular
336
distribution of TLR4 could serve to prevent a permanent activation of TLR4 cascades in
337
prostatic epithelial cells.
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In view of the increment in epithelial TLR4 content described in the digestive and
339
respiratory tracts due to bacterial infection [3], we were interested in determining if E. coli
340
could achieve the same effect in the prostate. With this aim, in the present study we applied
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a novel experimental model of bacterial prostatitis induced by a direct injection of E. coli
342
into the ventral prostate. In contrast with other bacterial prostatitis models in which E. coli
343
is introduced through transurethral instillation, in our model the delivery of the bacteria to
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the prostate was controlled, and contamination with urine or pathogens from the bladder
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and the urethra was avoided. These experimental conditions induced a strong local immune
346
response. Interestingly, in our model, prostatic epithelial cells exhibited an early
347
hypertrophy, an increase in TLR4 cytoplasmic content and the migration of this receptor to
348
the apical plasma membrane. In this way, the prostatic epithelium mimicked the cells of the
349
innate immune system; moreover, we found an increment of epithelial secretory granules
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containing antimicrobial proteins such as Surfactant protein-D after bacterial infection
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(Oberley and Quintar, unpublished data). This modulation of innate immunity within the
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prostate gland could be a key mechanism in guaranteeing an effective clearing of
353
microorganisms, thus avoiding the progression of infections towards the restricted sites of
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the male reproductive tract such as the epididymis and the testis.
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It is well established that testosterone is required for the structural and functional
356
integrity of the prostate. Androgen deprivation caused by castration leads to a marked
357
involution of this gland, with severe epithelial alterations that include a decrease in
358
secretion activity and loss of epithelial cells by apoptosis. Expression of TLR4 is
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modulated by a variety of environmental factors such as microbial invasion, microbial
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components, and cytokines [3]; however little information is available about the influence
361
of sexual hormones on TLR expression. In our experimental models, prostatic TLR4 was
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significantly increased in the castrated group as quantified by western blot. From
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immunocytochemistry, this increase could be ascribed to two different sources, the
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epithelium and the stroma. Epithelial cells not only maintained their TLR4 expression after
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castration, but often showed more intense immunostaining in comparison to intact rats.
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TLR4 molecules appeared associated with basal and basolateral plasma membranes, in
367
addition to the cytoplasmic localization. It has been described that remnants of the
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epithelial population are mostly comprised of basal cells after castration [34, 35]; hence,
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TLR4-highly stained cells could correspond to basal cells that probably weaken the TLR4
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expression when they differentiate into mature secretory cells after testosterone
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replacement therapy. On the other hand, testosterone deprivation caused hypertrophy of the
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stromal compartment, contributing significantly to increase TLR4 content in the prostatic
373
gland.
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TLRs are considered a key factor in the stimulation of the immune system; hence
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the increment of TLR4 in the prostate gland after castration described here, supports the
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concept that testosterone has suppressive effects on the immune responses [36]. Androgens
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appear to be responsible for the immunosuppressive profile of normal prostatic cells,
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resulting in the low expression of proinflammatory compounds and the generation of high
379
levels of immunosuppressive factors. In this matter, Desai et al. [37] have described that
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several genes which encode the cytokines involved in the immune response such as IL-15
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and IL-18 are specifically up-regulated in the ventral prostate after androgen deprivation.
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This regulation by testosterone contributes to the immune privileged status of the prostate
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gland, where harmful inflammatory responses are usually suppressed [38].
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The prostatic stroma is generally implicated in the paracrine regulation of the
385
epithelial structure and function, producing critical regulatory factors responsible for organ
386
homeostasis and mutual crosstalk [39]. In castrated rats, increased concentrations of TLR4
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were detected in the basal aspect of the basement lamina and on the plasmalemma of
388
smooth muscle cells. This observation could implicate TLR4 as a crucial player in
389
epithelial-stromal interactions following androgen deprivation.
- 15 390
Smooth muscle cells of the periacinar layer not only underwent hypertrophy but
391
also became a great TLR4-positive mass, probably acting as a pivotal component in the
392
response to the two different stimuli evaluated here. It is well recognized that smooth
393
muscle cells are responsible for the stromal reorganization after androgen withdrawal [40].
394
However, these are the first data defining a specific stromal reaction in response to bacterial
395
infection, and therefore, it is an active component of the innate immune system. A lot of
396
evidence suggests that smooth muscle cells are metabolically dynamic cells with the
397
potential to express and secrete numerous highly active signaling proteins [41]. In the
398
prostate gland, the enhancement of TLR4 expression could provide the smooth muscle cells
399
with the necessary sensors to detect a wide range of harmful signals from the environment,
400
and thereby quickly produce an innate immune response similar to that of cells of the
401
immune system.
402
Expression of TLRs in stromal cells has been reported at some sites, including the
403
female genital tract [42], respiratory system [43] and in vascular smooth muscle cells [44].
404
At these locations, the stromal compartment has an important role in inflammatory
405
diseases, and it is possible that TLR4 system could greatly contribute by amplifying
406
inflammatory signals in conditions such as atherosclerosis and asthma. In the prostate
407
gland, modifications of the fibromuscular stroma have been involved in the development of
408
benign and malignant cell growth. Alternatively, an emerging body of evidence supports a
409
possible link between chronic intraprostatic inflammation and prostate cancer. Therefore, it
410
is conceivable that TLR4 expressed in modified prostatic stromal cells could participate in
411
the extensive signaling pathway involved in prostate carcinogenesis. In fact, an association
412
between polymorphisms of the TLR4 gene and prostate cancer risk has been reported [45].
413
The data presented here have a wide range of implications for the physiopathology
414
of the prostate gland. In part, prostatic cells appear to provide the most important
415
membrane safeguard in sensing the microenvironment of the gland. Simultaneously, the
416
intracellular localization of TLR4 emerges as a defensive mechanism that could protect the
417
gland from unnecessary activation of TLR4 signaling. Finally, these results provide new
418
strong evidence that the smooth muscle might play a major role in controlling situations
419
that could compromise homeostasis of the prostate gland.
420
- 16 420
421
7. ACKNOWLEDGMENTS
422
The authors thank Mrs. Elena Pereyra, Mrs. Lucia Artino, Ms. Mercedes Guevara,
423
and Mr. Cristian Giacomelli for their expert technical assistance and Gabriel Balabanian for
424
his valuable assistance in the use of the electron microscope. We are principally indebted to
425
the entire Team-18 for help with the biochemical analysis and for their permanent advice.
426
427
428
429
We would like to thank Dr. Paul Hobson, native speaker, for revising the
manuscript.
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9. FIGURE LEGENDS
539
Figure 1
540
541
Photomicrographs of prostate sections from IN (A-C), BP 24 h after inoculation (DF), OX (G-I) and TT (J-L) groups.
542
Silver technique (A, D, G and J) on Araldite semithin (1µm) sections was
543
performed to verify modifications in the periacianar layer underlying the basement
544
membrane. Silver technique stains dark brown all basement membranes and delineates the
545
periaciar stromal layer surrounding the alveoli. It can be observed that this layer is very thin
546
in the IN group (A) while in the BP (D) and OX (G) groups, stromal cells are highly
547
hypertrophied and exhibit several layers (arrows in D and G). In the TT group, the prostatic
548
stroma is similar to IN, but the alveoli are wider.
549
Immunohistochemistry for ACTA2 (B, E, H, K) and VIM (C, F, I, L) on paraffin
550
prostate sections. In intact animals (B), ACTA2 immunostaining pattern forms a continuous
551
ring around the alveoli, which was significantly thickened in the BP group (arrows and
552
Inset in E) as well as in the OX group (H). VIM-positive fibroblast-like cells are scarce in
553
intact animals (C) while in the BP group they describe two uniform layers enclosing the
554
smooth muscle band (arrows and Inset in F). By contrast, OX rats show isolated VIM-
555
positive cells within the enlarged periacinar layer (arrows in I).
556
In the TT group, immunostaining confirmed that the stroma was slightly modified by
557
testosterone treatment compared with the IN group.
558
All photomicrographs were taken at the same magnification. Bar= 100 µm
559
560
Figure 2
561
Western blot illustrating membrane bands stained with TLR4 antibody. Lanes 1 and
562
2: Intact rats; lane 3: BP group, lanes 4 and 5: OX and TT groups respectively. In lane 6,
563
prostatic homogenate from OX rats was run and the anti-TLR4 primary antibody was
564
replaced by goat purified IgG. MW: Molecular Weight.
565
566
Figure 3
- 21 567
Relative amounts of TLR4 measured by densitometric study of western blot in
568
ventral prostate homogenates. TLR4 expression was significantly increased at 24 h and 48
569
h after bacterial inoculation in comparison with its controls (*P< 0.05) as well as with the
570
IN group (♦ P < 0.05). The values represent TLR4 expression after normalization with
571
ACTB expression. Note that TLR4 expression at 48 h was higher than at 24 h (● P < 0.05).
572
573
Figure 4
574
Effects of androgen manipulation on prostatic TLR4 expression. A) Serum levels of
575
total testosterone assayed by ECL. B) Semiquantitative analysis of TLR4 expression by
576
western blot, showing a significant increment of TLR4 in the OX group compared to its
577
controls and intact rats. Testosterone treatment immediately after castration avoided the
578
TLR4 increment observed in orchidectomized rats inoculated with the vehicle alone.
579
Results are shown as the mean ± SEM. The values represent TLR4 expression after
580
normalization with ACTB expression. Data present results from three independent
581
experiments of three to five rats in each group. * P< 0.05 vs. controls for the same
582
treatment group, ♦ P< 0.05 vs. IN group.
583
584
Figure 5
585
Immunocytochemical analysis of TLR4 protein in ventral prostate. A) Weak TLR4
586
expression on the prostatic epithelium from IN rats is observed; while a strong
587
immunoreactivity can be seen in an arteriole (arrow). B) Prostate from BP group, 24 h after
588
inoculation, shows mildly to moderately increased TLR4 expression in epithelial cells.
589
Periacianar stromal cells (arrows) and inflammatory cells (arrowheads) are also positive. C)
590
A strong labeling for TLR4 is seen in the epithelial cells as well as in the thicker periacinar
591
layer from OX rats. D) Prostate sections from TT group showing TLR4 expression similar
592
to IN rats. Bar = 75 µm.
593
594
595
596
Figure 6
TLR4 immunogold labelling of epithelial cells on thin sections of prostate glands
embedded in LR-White.
- 22 597
A-C: IN group. A) Apical region of an epithelial cell exhibiting weak TLR4
598
labelling. Secretory granules (Gr) are negative. B) Paranuclear region showing gold
599
particles mainly associated with the rough endoplasmic reticulum (RER) membranes.
600
Mitochondria (Mi) appear negative. C) Epithelial-stromal region: this micrograph exhibits
601
positive staining for TLR4 in the infranuclear location of epithelial cells and in smooth
602
muscle cells (SMC). Meanwhile, the epithelial basal membrane (arrowheads) and the
603
basement membrane (BM) are negative.
604
D-G: BP group. D-F) Apical region of the epithelial cells: gold labelling is
605
localized on the microvilli and junction complex (arrowheads in D and E). TLR4
606
immunoreaction is also concentrated on the cortex region (arrowheads in F). Secretory
607
granules at different stages of maturation appear negative. G) Gold particles appear
608
associated with dilated RER membranes containing abundant secretory TLR4-negative
609
content.
610
H-K: OX group. The epithelial apical compartment exhibits labelling often
611
associated with lateral membranes (arrowheads in H) or in the cytoplasm, probably related
612
to poor-developed RER cisternae. The lateral membrane also shows an intense
613
immunostaining in interdigitated regions (I and J). In the basal surface of the prostatic
614
epithelium, gold particles delineate the epithelial basal membrane (arrowheads in K),
615
touching the TLR4-negative basement membrane. N: nucleus. Bar = 1 µm.
616
617
618
619
Figure 7
TLR4 immunogold labelling of periacinar stroma on thin sections of prostate glands
embedded in LR-White.
620
A) IN group. Micrograph shows the sheath of interstitial tissue surrounding the
621
epithelial alveoli. Smooth muscle cells present low TLR4 specific labelling. Basement
622
membranes (*) appear negative.
623
624
B) In the BP group, a portion of a smooth muscle cell exhibits intense TLR4
immunolabelling that contrasts with the negative extracellular matrix in the surroundings.
625
C) In the OX group, the smooth muscle cells look hypertrophied and have intense
626
TLR4 labelling. In these cells, gold particles are often found decorating the plasmalemma
- 23 627
(arrowheads in C and Inset). EC: Epithelial Cell; SMC: Smooth Muscle Cell; *: Basement
628
membranes; EM: Extracellular Matrix. Bar = 1 µm.