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Pathogenic Consequences in Semen Quality
of an Autoimmune Response against the
Prostate Gland: From Animal Models to
Human Disease
Ruben D. Motrich, Mariana Maccioni, Andres A. Ponce,
Gerardo A. Gatti, Juan P. Mackern Oberti and Virginia E.
Rivero
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
9650 Rockville Pike, Bethesda, MD 20814-3994.
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:957-967; ;
doi: 10.4049/jimmunol.177.2.957
http://www.jimmunol.org/content/177/2/957
The Journal of Immunology
Pathogenic Consequences in Semen Quality of an Autoimmune
Response against the Prostate Gland: From Animal Models to
Human Disease1
Ruben D. Motrich,* Mariana Maccioni,* Andres A. Ponce,† Gerardo A. Gatti,*
Juan P. Mackern Oberti,* and Virginia E. Rivero2*
T
he past decade has seen a resurgence of interest and exciting new research on chronic prostatitis and related syndromes (1). One important reason for this enthusiasm is
the recognition that chronic prostatitis syndromes represent an important worldwide health care problem (2). Chronic bacterial prostatitis is characterized by uropathogenic infections of the prostate
gland that respond well to antimicrobial treatment (3). In contrast,
chronic nonbacterial prostatitis (CNBP)3 (now classified as National Institutes of Health category III), which accounts for 90 –
95% of prostatitis cases, is of unknown etiology and is characterized by the presence of a mixture of pain, urinary, and ejaculatory
symptoms with no uniformly effective therapy (4 – 6). A rationale
treatment is urgently required, because until now the goal therapy
for these patients has not had good results, and it has focused on
*Centro de Investigaciones en Bioquı́mica Clı́nica e Inmunologı́a, Departamento de
Bioquı́mica Clı́nica, Facultad de Ciencias Quı́micas. Universidad Nacional de Córdoba, Córdoba. Argentina; and †Instituto de Fisiologı́a Humana, Facultad de Ciencias
Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina
Received for publication December 5, 2005. Accepted for publication April 25, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Agencia Nacional de Promoción Cientı́fica y Tecnológica Grant PICT 2003 5/13624, Consejo Nacional de Investigaciones Cientı́ficas y
Técnicas de Argentina (CONICET) Grant PEI 6547, and Fundación Antorchas 2002
Grant 14264. R.M. and J.P.M. are Ph.D. fellows of CONICET. G.G. is a Ph.D. fellow
of Fondo para la Investigación Cientifica y Tecnológica, and M.M. and V.R. are
members of the Researcher Career of CONICET.
2
Address correspondence and reprint requests to Dr. Virginia E. Rivero, Haya de la
Torre y Medina Allende, Ciudad Universitaria, Córdoba, 5000 Argentina. E-mail
address: [email protected]
3
Abbreviations used in this paper: CNBP, chronic nonbacterial prostatitis; PAP, prostatic acid phosphatase; EAP, experimental autoimmune prostatitis; PSBP, prostate
steroid-binding protein; SI, stimulation index; HOS, hypoosmotic swelling; PI, propidium iodide; ROS, reactive oxygen species.
Copyright © 2006 by The American Association of Immunologists, Inc.
amelioration of symptoms rather than on a cure (7). One reason for
the lack of significant progress toward the understanding of its
etiology, pathophysiology, and therefore the design of rationale
therapies is the absence of adequate animal models in which to
study the disease process.
Recently, we have analyzed a group of patients with CNBP to
search for the presence of a possible autoimmune response to prostate Ags. We demonstrated the existence of IFN-␥-secreting lymphocytes that proliferate in response to known human prostate Ags
such as prostate-specific Ag and prostatic cancer phosphatase
(PAP) in 34% of patients with CNBP (8). Only this group of patients showed significantly elevated levels of cytokines such as
IFN-␥, IL-1, and TNF-␣ in their seminal plasma, arguing for a
local inflammation of noninfectious cause.
The relationship between chronic prostatitis and infertility has
been controversial for many years with some reports arguing for a
positive relationship between chronic prostatitis and altered semen
quality (9, 10), whereas others showed no alterations (11, 12). One
possible explanation of such contradictory findings could be that
CNBP comprises an heterogeneous group of patients and, as we
and others have described (8, 13, 14), that the presence of autoimmune response against prostate Ags is detected only in a subset
of patients with CNBP. We have also reported major abnormalities
in some of the ejaculate parameters only in CNBP patients who
presented signs of an autoimmune response against the prostate
gland (15). When the comparative analysis of sperm parameters
was done considering all patients enrolled (CNBP patients with
and without an autoimmune response against prostate), nonsignificant
differences were found. These findings allowed us to assume that
these significant differences could have been masked in the other mentioned studies. Therefore, we hypothesize that the autoimmune response to prostate Ags and the inflammatory environment elicited
could have detrimental consequences on sperm quality.
0022-1767/06/$02.00
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We have recently proposed an autoimmune etiology in ⬃35% of chronic nonbacterial prostatitis patients, the most frequent form of
prostatitis observed, because they exhibit IFN-␥-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of
patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality.
In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to
investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a
marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic
spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of
NO, TNF-␣, and IFN-␥ were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating
that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were
further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed.
We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu,
originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels
of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have
consequences on fertility. The Journal of Immunology, 2006, 177: 957–967.
958
AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES
Materials and Methods
Animals and immunization
Sexually mature Wistar rats were bred and maintained under specific
pathogen-free conditions in the vivarium of the Center of Immunology and
Biochemical Chemistry Research of the National University of Cordoba
(Cordoba, Argentina). Fifty-five 2-mo-old male rats were included in our
study. Animals were maintained in a 16-h light, 8-h dark cycle at 20 ⫾ 2°C,
with food and water ad libitum. The experimental protocol and all the
performed tests were carefully reviewed and approved by the institutional
review board.
Animal immunization was achieved by the injection of saline solution
plus CFA (Sigma-Aldrich; group I; n ⫽ 17), 1 mg of prostate extract plus
CFA (group II, n ⫽ 20) or 120 ␮g of purified PSBP plus CFA (group III;
n ⫽ 18), on days 0, 20, and 35. Animals received four intradermal injections in different places: right footpad; left footpad; base of the tail; and
shoulders.
Ags and reagents
Prostate extracts were prepared from Wistar rat prostate glands. Pooled
glands from 50 rats were homogenized in 0.01 M PBS, pH 7.2, with protease inhibitors in an Ultra-Turrax homogenizer. The homogenate was centrifuged at 100,000 ⫻ g for 30 min, and the supernatant was used as Ag.
Protein concentration was determined using the Folin phenol reagent. The
preparations were aliquoted and kept frozen at ⫺20°C.
PSBP was purified following the procedure described previously by
Chen et al. (21). Briefly, ventral prostates were removed in a sterile manner
from Wistar male rats (body weight, 250 –300 g) and homogenized in 20
mM Tris-HCl using an Ultra-Turrax homogenizer. The cytosolic fraction
was obtained after 60 min of centrifugation at 100,000 ⫻ g and 4°C. The
resultant supernatant was applied onto a Mono-Q FPLC column. Proteins
were eluted with a linear 0 – 60% NaCl gradient in 20 mM Tris-HCl. Protein concentration of each fraction was evaluated by measuring its OD280.
Each fraction was run under nondenaturing conditions in 15% polyacrylamide gels (SDS-PAGE); then, the gels were stained with Coomassie blue.
Fractions containing two bands of 18 and 20 kDa (corresponding to both
subunits of PSBP) were also run under denaturing conditions to verify their
identity. The purity of the preparation was higher than 95%. Western blotting using a rabbit polyclonal PSBP antiserum or a mouse mAb recognizing the C3 polypeptide (kindly given by Dr. P. Bjork, Pharmacia and Upjohn, Uppsala, Sweden) was performed.
PAP, purified from human seminal plasma, was purchased from SigmaAldrich. Recombinant rat TNF-␣ was purchased from BD Pharmingen, and
recombinant rat IFN-␥ was purchased from BD Biosciences.
Histology
Prostate glands collected 7 days after last immunization (day 42) were
fixed in 4% formalin solution and processed for conventional histology. A
prostatitis score was done in a blinded manner and computed for individual
glands by summing the grade of each section and dividing the total number
of sections examined (usually 4). The degree of inflammation was assessed
using a scale of 0 –3: ⫺, no inflammation; ⫹, mild, but definite perivascular
cuffing with mononuclear cells; ⫹⫹, moderate perivascular cuffing with
mononuclear cells; ⫹⫹⫹, marked perivascular cuffing, hemorrhage, and
numerous mononuclear cells in the parenchyma.
T cell proliferation assay
Single cell suspensions were prepared in HBSS (Sigma-Aldrich) from
pooled draining lymph nodes of individual rats. Lymph node cell viability
was assessed by trypan blue exclusion. Cells were finally resuspended in
DMEM supplemented with Glutamax, sodium pyruvate, nonessential
amino acids, HEPES buffer, penicillin/streptomycin, 50 mM 2-ME, and
10% FCS (Techgen) and were distributed at a concentration of 1,5 ⫻ 105
cells/well in a 0.1-ml volume into flat-bottom 96-well microtiter plates (BD
Biosciences). Syngeneic naive spleen cells irradiated at 1500 rad served as
APCs. These cells were prepulsed for 2 h at 37°C with prostatic Ags:
prostate extract at 130 ␮g/ml; or purified PSBP at 20 ␮g/ml; or PAP at 20
␮g/ml. APCs were then washed twice and added to 0.1 ml of responder
cells at 4 ⫻ 105 cells/well final cell concentration. All cell combinations
were set up in quadruplicate. Plates were incubated for 4 days at 37°C in
water-saturated 7.5% CO2 atmosphere and pulsed for the final 18 h with 1
␮Ci of methyl-[3H]TdR. Labeled material was automatically harvested and
counted in a ␤ plate scanner (Pharmacia). The response was expressed as
stimulation index (SI) and as ⌬cpm. Stimulation indexes were calculated from
cpm incorporated in Ag-pulsed cultures/cpm incorporated in cultures with
nonpulsed APC. Threshold value for positivity for each Ag assayed (prostate
extract, PSBP, and PAP) was set up above the mean SI plus 3 SDs of the SI
observed in the control group. ⌬cpm was calculated from cpm incorporated in
Ag-pulsed cultures minus cpm incorporated in cultures with nonpulsed APC.
Abs against prostate Ags in serum and in semen
Abs against PE, PAP, or PSBP were titrated by conventional ELISA in
multiwell plates (Costar) precoated with 50 ␮l/well prostate extract at 100
␮g/ml, PSBP at 20 ␮g/ml, or PAP at 5 ␮g/ml in 0.05 M carbonate buffer,
pH 9.6. After overnight incubation at 4°C, microwells were washed twice
and blocked with 3% BSA (Sigma-Aldrich) in PBS for 2 h at 37°C, rinsed
once with PBS-Tween 20 at 0.05%, and then filled with 50 ␮l of serum
(obtained after cardiac puncture) or seminal plasma dilutions (1/50 or 1/6
respectively) for 1 h at 37°C. To detect specific IgG, plates were again
washed and incubated with HRP-conjugated goat anti-rat IgG (SigmaAldrich) for 1 h at 37°C. The reaction was developed with o-phenylenediamine-H2O2. OD was measured at 490 nm in a Bio-Rad Plate Reader.
Serum reactivity was expressed as a binding index representing the ratio
between the OD given by the test serum and the OD of a preimmune serum
at the same dilution. Minimal threshold value for positivity was set up at
2. Semen reactivity was expressed as OD.
Semen collection
Semen samples were obtained by electroejaculation as described previously by Ponce et al. (22). Briefly, nonanesthetized rats were placed in a
box that covered the cephalic half of the body while the rest laid on a
metallic grid support. After the genital area was cleaned, the prepuce was
retracted, and the penis was introduced into an Eppendorf plastic tube. The
rectal probe was lubricated, and the bronze bipolar electrode was inserted
into the rectum to a depth of 20 –30 mm and held by the technician. The
alternative current (220 V, sinusoidal wave, 50 cycles/s) was applied for 5
of every 10 s. The current was controlled by a rheostat and was calculated
from an oscilloscope reading of the voltage drop across a resister placed in
the circuit. A series of one to five pulses was normally given at the 6- to
6.8-V setting. Immediately after electrostimulation, a white viscous plug
was obtained. Immediately after, a series of 1–10 pulses was given at
13–15 V, and the remaining portion of the ejaculate was collected and
diluted with 150 ␮l of Tyrode’s buffer medium without BSA (pH 7.4, 280
mOsm/kg) to avoid coagulation. Functional parameters of spermatozoa
were determined within 3–10 min by subjective evaluation in fresh semen
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From the results obtained in chronic autoimmune prostatitis patients, some questions arose such as when, where, and how the
sperm cells are being damaged and which are the putative mediators and pathogenic mechanisms involved.
Animal models of many human diseases have been studied for
many years and have provided a great deal of information about
mechanisms involved in the autoimmune response. Our laboratory
has developed over the past decade a rat and a mouse model of
experimental autoimmune prostatitis (EAP) that is characterized
by an organ specific mononuclear infiltration and a cellular and
humoral autoimmune response against prostate Ags (16 –19). Prostate steroid-binding protein (PSBP) has been described as the major autoantigen involved in the autoimmune response (19, 20).
Although very little is known about the immunology of human
disease, most of the features already reported on it, are shared with
those of our animal model. The type of infiltration characterized by
intra-acinar mononuclear cells and the presence of IFN-␥-secreting
T cell response against prostate Ags are the main aspects shared by
human and animal diseases. In contrast, the Ab response, which
reaches important levels in the animal model, is not detected in the
human disease.
Moreover, as further characteristics are being described in human patients, the validity of EAP as an animal model of the disease
is being corroborated. In the present work, we used the animal
model to respond some unanswered questions and to advance on
the underlying mechanisms and pathogenic consequences of autoimmunity against prostate Ags.
The Journal of Immunology
following standard methods in our laboratory. Seminal plasma was obtained by centrifuging semen at 300 ⫻ g for 7 min at room temperature and
kept at ⫺80°C until use.
Fructose, citric acid, and ␣-glucosidase levels in semen
Citrate, fructose, and ␣-glucosidase seminal levels were measured as tests
of function of the prostate, seminal vesicles, and epididymis, respectively.
Neutral ␣-glucosidase, fructose, and citric acid were determined according
to the World Health Organization semen analysis manual (23). Briefly, for
determining neutral ␣-glucosidase, 4-nitrophenyl-␣-D-glucopyranoside
was converted into 4-nitrophenol and ␣-D-glucopyranoside, and 4-nitrophenol was photometrically measured. The acid isoenzyme was inhibited
by addition of 1% SDS and phosphate buffer, pH 6.8, so that only the
neutral isoform was measured. Fructose was determined according to the
hexokinase method, and citric acid was measured with the UV method
using the citrate lyase-catalyzed reaction.
IFN-␥ and TNF-␣ in seminal plasma
Measurement of NO levels in seminal plasma
Nitrite concentration in seminal plasma was measured by using the Griess
reagent. Briefly, 0.10 ml of seminal plasma was mixed with 0.2 ml of
Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid) and incubated for 15 min at room
temperature in the dark. OD was measured at 540 nm, and nitrite concentration was calculated using a calibration curve. Results are expressed as
micromols per milliliter.
Epididymal sperm obtainment
Animals were killed by CO2 asphyxiation followed by cervical dislocation.
The epididymis was removed, trimmed of fat, and clamped at the corpus-cauda
junction. Rat spermatozoa were obtained by chopping cauda epididymidis
with a sharp pair of scissors, allowed to disperse, and homogenized in 5 ml of
Ham’s F-12 medium for 30 min at 37°C in a 5% CO2-saturated atmosphere.
Assays to determine sperm concentration, motility, viability, and
sperm response to the hypoosmotic swelling (HOS) test
Sperm concentration and motility were assessed at 23 ⫾ 2°C in a Makler
counting chamber (Sefi Medical Instruments) under an inverted microscope at ⫻200 magnification. The results are expressed as the percentage
of motile cells (progressive plus nonprogressive spermatozoa). No fewer
than 200 gametes were examined.
The viability parameter was evaluated by supravital staining with
Hoechst 33258 (Calbiochem). Using the appropriate UV fluorescence optics (Axiolab; Zeiss), spermatozoa showing brightly fluorescence nuclei
were scored as dead, and sperm cells that excluded the Hoechst 33258 and
were not fluorescent were scored as viable. The viability of 200 –250 cells
was assessed.
The HOS test evaluates whether an intact membrane is biochemical and
functionally active. The procedure used for the HOS test was similar to the
one described by Jeyendran et al. (24). The sperm suspension was mixed
with a hypoosmotic solution (100 mOsm/L; Fiske Osmometer G-52), pH
7.4, for 45 min at 37°C. Evaluations were made by phase contrast microscopy at a magnification of ⫻400; 200 cells or more were observed, and the
percentage of spermatozoa that showed tail swelling was determined by
dividing the number of reacted cells (⫻100) by the total number of spermatozoa counted in the same area.
Assessment of apoptosis by annexin V staining
Staining for annexin V was performed using an apoptosis detection kit II
(BD Pharmingen) containing annexin V conjugated with FITC. Sperm
samples were resuspended in binding buffer supplemented with 4 mg/ml
BSA (fraction V; A-4503; Sigma-Aldrich) at a concentration of 10 ⫻ 106
spermatozoa/ml. From this solution, an aliquot of 100 ␮l was stained with
5 ␮l of annexin V-FITC solution and 5 ␮l of propidium iodide (PI) solution. Samples were incubated in polypropylene tubes for 15 min in the
dark, and then 400 ␮l of binding buffer were added. Samples were analyzed
within 1 h. The samples were analyzed using a FACS Star Plus-Flow
cytometer (Becton Dickinson Immunochemistry Systems) equipped with
standard optics. An argon ion laser (INNOVA 90; Coherent) tuned at 488
nm and running at 200 mW was used as light source. From each cell,
forward light scatter (FSC), orthogonal light scatter (SSC), A-FITC fluorescence, and PI fluorescence were evaluated using CellQuest version 3.3
(BD Biosciences). A gate was applied in the FSC-SSC dot plot to restrict
the analysis to spermatozoa. For the gated cells, the percentages of annexin
V-negative or -positive (A⫺ or A⫹), and PI-negative or -positive (PI⫺ or
PI⫹), as well as double-positive cells were evaluated, based on quadrants
determined from single-stained and unstained control samples.
Analysis of sperm motility in mixture experiments
The working medium for analysis of sperm kinetics was modified KrebsRinger bicarbonate solution containing 94.6 mM NaCl, 4.78 mM KCl, 1.71
mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4, 25.07 mM NaHCO2, 21.58
mM sodium lactate, 0.5 mM sodium pyruvate, 5.56 mM glucose, 4.0 mg/ml
BSA, 50 ␮g/ml streptomycin sulfate, and 75 ␮g/ml potassium penicillin).
They were equilibrated overnight to pH 7.4 at 37°C in a 5% CO2 atmosphere
and kept at 37°C all through the protocol. Sperms were collected from
normal nontreated adult male Wistar rats by electroejaculation as described
in Semen collection and analyzed immediately after collection. Sperm suspensions (40 ␮l) were placed on grease-free slides warmed at 37°C and
covered with 18- ⫻ 18-mm2 coverslips to achieve a chamber depth of
20 –50 ␮m, which does not disturb sperm movements. The slides were
rapidly prepared and transferred to a heated plate of an inverted Olympus
IX 70 phase contrast microscope. The videomicroscopy system consisted
of a charged-coupled device video camera, a time lapse video recorder, and
a Panasonic monitor (Matsushita) connected to the microscope as previously described (25, 26). For each slide, the motility of sperms in no fewer
than 10 fields were recorded for 10 min and analyzed in each experiment.
Recordings were conducted at videotape speed of one image every 0.016
s (continuous running). During the video frame-to-frame playback, cell
tracks (previously selected at random in the pause mode) were drawn by
hand on an acetate sheet attached to the monitor screen during 3 s. Then,
the tracks were scanned and transferred into a computer, and the percentage of sperm motility of each sample was calculated and analyzed
with SigmaScanPro image analysis software (SPSS).
Statistics
Statistical analysis was performed with the Wilcoxon paired test, paired t
test, one-way ANOVA (followed by Dunnet’s test when the ANOVA test
yielded statistical differences ( p ⬍ 0.05) among experimental groups), and
Fisher’s exact test as appropriate. p ⬍0.05 was considered statistically
significant.
Results
Autoimmune response can be detected systemically and locally
in rats bearing experimental autoimmune prostatitis
Autoimmune prostatitis in animals immunized with either prostate
extract or PSBP was evidenced by different methods (Fig. 1). As
expected, histopathological studies revealed lesions of varying intensity in the prostate gland of almost all experimental animals
irrespective of whether the immunizing Ag was prostate extract
(group II) or PSBP (group III) (Fig. 1A). Observed lesions were
mainly characterized by moderate perivascular cuffing, mononuclear infiltration accompanied by hemorrhage, and edema. Histological alterations were not detected either in prostate samples
from animals of group I or in seminal vesicles, testis, and epididymis samples from animals of the three groups under study
(Fig. 1A).
A positive proliferative response against prostate Ags was also detected only in draining LN cells from prostate extract- and PSBPimmunized animals (Fig. 1B). Indeed, lymph nodes cells from control
animals of group I exhibited low cpm values in response to prostate
Ags (group I: ⌬cpm, 118.32 ⫾ 109.76 against prostate extract; ⌬cpm,
177.25 ⫾ 111.89 against PSBP; and ⌬cpm, 165.50 ⫾ 136.95 against
PAP). On the contrary, autoimmune animals from groups II and III
presented high cpm values and therefore positive stimulation indexes:
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TNF-␣ and IFN-␥ quantities present in seminal plasma were determined by
solid phase sandwich ELISA protocol according to the manufacturer’s instructions (BD Biosciences). Briefly, 96-well plates were coated with primary anti-TNF-␣ or anti- IFN-␥ capture Abs and blocked with PBS-5%
BSA. Seminal plasma samples were incubated overnight at 4°C followed
by the addition of biotinylated anti-TNF-␣-detecting mAb or biotinylated
anti-IFN-␥-detecting mAb. Plates were developed by adding avidin peroxidase and its substrate (TMB), and the OD was measured at 492 nm in
a microplate reader (Bio-Rad). The amounts of TNF-␣ or IFN-␥ were
extrapolated from the standard curve, which was generated in 1/2 dilutions.
Results are expressed in picograms per milliliter.
959
960
AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES
group II: ⌬cpm, 3141.66 ⫾ 722.26 against prostate extract; ⌬cpm,
3865 ⫾ 877.70 against PSBP; and ⌬cpm, 2244.66 ⫾ 1148.09 against
PAP; and group III: ⌬cpm, 1493.33 ⫾ 239.89 against prostate extract;
⌬cpm, 2858.33 ⫾ 631.86 against PSBP; and ⌬cpm, 1361.66 ⫾
504.51 against PAP.
Autoantibodies against prostate Ags were also detected in circulating blood (Fig. 1C) and as immune complex depositions in the
prostate gland (16). To investigate whether these autoantibodies
generated against prostate Ags could somehow be part of the prostatic fluid and thus appear in the ejaculate, we searched for its
presence in seminal plasma from animals under study. Indeed,
PSBP-specific IgG could be locally detected in seminal plasma of
animals from groups II and III.
Taken together, our results clearly indicate that male rats immunized with prostate Ags develop inflammatory lesions in prostatic
tissue and produce both a cellular and humoral specific autoimmune
response, which can be detected systemically and locally.
High levels of nitric oxide, TNF-␣, and IFN-␥ are detected in
seminal plasma from autoimmune rats
To analyze the presence of local proinflammatory mediators, the
levels of NO, TNF-␣, and IFN-␥ were studied in seminal plasma
from control and autoimmune rats as a reflection of the prostate
environment.
High levels of NO were detected in seminal plasma from
autoimmune rats (groups II and III) compared with the levels
observed in the control group (group I; p ⬍ 0.05; Fig. 2A).
Moreover, higher levels of TNF-␣ were also detected in seminal
plasma from autoimmune rats of groups II and III when compared with the levels observed in rats from group I, although
only with a significant increase in samples from group II ( p ⬍
0.05). When the levels of IFN-␥ were evaluated, similar data were
obtained (Fig. 2C), evidencing significantly increased levels of this
cytokine in seminal plasma of autoimmune animals ( p ⬍ 0.05). These
results suggest that an inflammatory process is taking place in the
prostate in prostate extract- and PSBP-immunized animals due to an
autoimmune response against prostate Ags.
Prostate biochemical markers are altered in seminal plasma
from autoimmune rats
Citric acid and zinc are classical biochemical markers of prostate
gland function, as well as fructose for seminal vesicles and neutral
␣-glucosidase for epididymis (23). These markers are usually assayed
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FIGURE 1. Systemic and local autoimmune response in experimental autoimmune prostatitis. A, Histological changes in prostate sections of immunized
animals. Prostate glands, seminal vesicles, testes, and epididymis collected 7 days after last immunization (day 42) were fixed and processed for conventional histology (n ⫽ 5 animals per group). A prostatitis score was computed for individual glands by summing the grade of each section and dividing the
total number of sections examined. B, In vitro proliferative response of inguinal and lumbar draining lymph node cells from immunized rats. Five animals
per group were killed 7 days after the last immunization. Samples from each animal were processed individually. Proliferation was determined by uptake
of [3H]TdR and expressed as a proliferation index calculated from cpm incorporated in Ag-pulsed APC cultures/cpm incorporated in cultures with
nonpulsed APC. The threshold value for positivity was set up above the mean SI plus 3 SDs observed in the control group. The cpm incorporated in
nonpulsed APC cultures were 230.50 ⫾ 98.48 cpm for animals from group I, 1250.45 ⫾ 435.78 cpm for animals from group II, and 650.45 ⫾ 267.44 for
animals from group III. ⴱ, p ⬍ 0.05 vs control group. C, Serum IgG response against prostate Ags in immunized animals. IgG against prostate extract, PSBP,
and PAP Ags was studied by ELISA in serum samples obtained on day 42 (n ⫽ 5 animals per group). Serum reactivity was expressed as a binding index
representing the ratio between the OD given by the test serum and the OD of a preimmune serum at the same dilution. Minimal threshold value for positivity
was 2. ⴱ, p ⬍ 0.05 vs control group. D, Seminal plasma IgG response against prostate Ags in immunized animals. IgG against PSBP was studied by ELISA
in seminal plasma samples obtained on day 42. Five animals per group were assayed individually. ⴱ, p ⬍ 0.05 vs control group.
The Journal of Immunology
961
as sensors of the functionality of the respective glands in human diseases, and many reports show that an inflammatory response can
cause impairment of the functions of these glands. Because the existence of abnormalities in these marker levels has not been demonstrated in our model, we investigated whether the inflammatory process that is taking place in the prostate of autoimmune animals could
affect some biochemical functional markers. For that reason, we measured citric acid, fructose, and neutral ␣-glucosidase levels in seminal
plasma. No differences were found in the levels of fructose and neutral
␣-glucosidase when measured in the different groups under study,
showing that the immunization performed in groups II and III does
not affect the seminal vesicle and epididymal function (Fig. 2D). In
contrast, the levels of citric acid, which mirrors prostate gland functionality, were significantly decreased in samples from autoimmune
rats from groups II and III (Fig. 2E), indicating an altered function of
the gland in autoimmune animals.
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FIGURE 2. NO, TNF-␣, IFN-␥, and biochemical markers of seminal vesicle, epididymis, and prostate levels in seminal plasma
samples from rats bearing autoimmune prostatitis. NO levels (A) and TNF-␣ (B) and
IFN-␥ (C) quantities were determined by
measuring the nitrite (Griess reagent) or cytokine quantities by a solid phase sandwich
ELISA protocol in seminal samples obtained
by electroejaculation of immunized animals.
␣-Glucosidase, fructose (D), and citric acid
(E) levels were determined in seminal
plasma samples of animals from different
groups. In all cases, samples were processed
individually, and five animals per group
were analyzed. ⴱ, p ⬍ 0.05 vs control group.
These results corroborate our previous data on the specificity of
the autoimmune response in this model (19), confirming that only
the prostate gland is affected. In other words, the immunization
procedure generates an immune response that preserves the normal
function of seminal vesicles and epididymis but induces an inflammatory state that to some extent alters the prostate gland function.
Sperm quality parameters are altered in semen from animals
with autoimmune prostatitis whereas epididymal spermatozoa
remain intact
We previously demonstrated that chronic prostatitis patients who
evidenced autoimmune response to prostate Ags have altered
sperm quality (15). To evaluate whether this also occurs in the
experimental autoimmune model, semen samples from animals under study were obtained by electroejaculation, and different parameters such as sperm concentration, total sperm motility, sperm
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AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES
FIGURE 3. Semen quality in samples
from rats with autoimmune prostatitis.
Sperm concentration (A), sperm motility (B),
sperm viability (C), HOS (D), and apoptosis
by annexin V/PI staining (E) were conducted
in semen samples obtained by electroejaculation of immunized animals. Different parameters of spermatozoa were determined
immediately after sperm obtainment by evaluation following standard methods. In all
cases, samples were processed individually,
and five animals per group were analyzed. ⴱ,
p ⬍ 0.01 vs control group.
zation) as well as in a later stage (after the third immunization) of
the autoimmune response against prostate ( p ⬍ 0.01). Finally, the
HOS test, which analyzes whether sperm plasma membrane is intact and also whether it is biochemically and functionally active,
was performed. Very low levels of reactive spermatozoa were detected in both groups of autoimmune animals (groups II and III) compared with the control group (Fig. 3D, p ⬍ 0.05). Our results suggest
that the stronger the autoimmune response against prostate the worse
are the alterations observed in semen. Certainly, animals from group
II, which showed the most severe infiltration and lesions and highest
seminal TNF-␣ and IFN-␥ levels, evidenced the most striking alterations in semen quality.
To investigate which is the sperm death process taking place,
sperm apoptosis in ejaculate samples was studied. The Annexin
V-PI assay, which measures early and late apoptosis, was performed in semen samples obtained on day 42 of the experimental
protocol. The annexin V-PI assay identified four categories of cells
(Fig. 3E). Higher levels of apoptotic spermatozoa were detected in
semen from both autoimmune animal groups (groups II and III)
compared with the control group ( p ⬍ 0.05). The frequency of
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viability, and the HOS test were analyzed. For a complete overview of the sperm quality during the development of the prostatespecific autoimmune response, the tests were performed after the
second immunization (on day 27; data not shown) and after the
third immunization (on day 42; data shown in Fig. 3) of the experimental protocol. In general, all these evaluated parameters
were impaired in the autoimmune animals, and they worsened as
long as the autoimmune response had progressed and become established. For example, group II (animals immunized with prostate
extract that develop a florid autoimmune prostatitis) was the first to
show a decrease in sperm concentration on day 27, but, as the
immune response progressed (day 42), both groups of autoimmune
animals (groups II and III) demonstrated a marked reduction in
sperm concentration compared with control animals ( p ⬍ 0.01).
At the same time, sperm motility and viability in semen samples
from animals under study were assayed. Almost null sperm motility and viability levels were detected in both autoimmune animal
groups (groups II and III) when compared with the control group
(Fig. 3, B and C). These huge alterations in sperm motility and
viability were seen in an earlier stage (after the second immuni-
The Journal of Immunology
FIGURE 4. Epididymal sperm functionality in rats with autoimmune prostatitis.
Sperm concentration (A), sperm motility (B),
sperm viability (C), HOS (D), and apoptosis
by annexin V staining (E) were conducted in
epididymal sperm samples of immunized animals. Different parameters of spermatozoa
were determined immediately after epididymal sperm obtainment by evaluation following standard methods. In all cases, samples
were processed individually, and five animals per group were analyzed. ⴱ, p ⬍ 0.01 vs
control group.
Animals were sacrificed on days 27 and 42, and the same sperm
analyses previously performed in semen samples were done in
epididymal sperm. Epididymal sperm concentration, motility, and
viability revealed no differences between autoimmune animals and
control animals either on day 27 (data not shown) or on day 42
(Fig. 4, A–C). In accordance with these results, when the HOS
assay was performed, no alterations were observed, revealing normal epididymal sperm cell membrane integrity and functionality in
both autoimmune groups compared with the control group (Fig.
4D). As shown in Fig. 4E, when epididymal sperm apoptosis was
analyzed, no increase in the percentage of apoptotic or necrotic
spermatozoa was found in samples from both autoimmune animal
groups (groups II and III) when compared with the control group.
These findings evidenced that the striking alterations observed
in semen originated after sperm genesis in testis and its storage in
epididymis, suggesting that the inflammation in the prostate, the
consequence of an autoimmune response against prostate Ags,
could be involved in sperm cell damage. The kinetics of this damage appeared to be quick, and its detrimental effect could seriously
compromise the seminal quality of an individual.
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early apoptotic cells (A⫹/PI⫺) was significantly higher in group
III than in group II. However, seminal spermatozoa from group II
showed significantly higher percentages of late apoptotic, early
necrotic (A⫹/PI⫹), and necrotic (A⫺/PI⫹) spermatozoa than
those in group III ( p ⬍ 0.05) (Fig. 3E).
Physiologically, spermatozoa genesis and differentiation occur
in the testis. In the last steps, spermatozoa mature and are stored in
the epididymis. The most mature spermatozoa are kept in the caudal region of the epididymis until the ejaculation, when they join
the sex accessory gland fluids to form the semen/ejaculate. Regarding this fact, we wished to investigate where the sperm alterations observed in semen originated. There were two possibilities:
1) that these alterations were the result of epididymal damage as a
result of a satellite effect of the prostate inflammation (although we
had not previously detected any histological alteration in epididymis or autoantibodies against epididymal or testicular Ags, (data
not shown)); 2) that these alterations were a result of damage originated during the encounter of sperm with the prostate fluids during ejaculation. To answer these questions, we also analyzed epididymal sperm quality parameters in immunized animals.
963
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AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES
Sperm alterations observed in autoimmune rats occur in a short
time and are caused at least in part by cytokines locally
produced by the autoimmune response
FIGURE 5. Effect of the addition of seminal plasma
from autoimmune animals on normal spermatozoa. A,
Sperm kinetics analysis of samples from autoimmune
rats during the 10 min after ejaculation. B, Effects of
autoimmune seminal plasma (SP) on the motility of
normal spermatozoa. ⴱ, p ⬍ 0.01 vs control group. In
all cases, samples were processed individually, and five
animals per group were analyzed.
Discussion
Witebsky et al. (27) proposed rationales for the autoimmune basis
of clinical disease. They were consciously modeled on Koch’s
postulates and required an autoimmune response to be recognized
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The results presented here supported the hypothesis that the alterations observed in the sperm cells occurred once the prostate secretions, loaded with the inflammatory mediators produced as a
consequence of the autoimmune response, meet the spermatozoa
during ejaculation. However, one can wonder what mechanism is
involved in the production of these alterations and whether the
damage could occur in such a short time.
To answer this question, the time course of sperm motility was
evaluated in semen samples from different groups. As shown in
Fig. 5A, sperm motility diminished slowly in semen samples from
animals of group I, showing a sperm motility of 60% immediately
after ejaculation with a slow drop after 8 –9 min. However, marked
loss of sperm motility was observed in semen samples from autoimmune animals (groups II and III); this effect was more drastic in
samples from group II. This loss in motility was evident both immediately after sperm collection and during the time course of the
analysis.
To assay whether some metabolite present in seminal plasma
from autoimmune rats could produce these alterations, mixture experiments were performed. As can be seen in Fig. 5B, when sperm
cells from normal nontreated animals were mixed with seminal
plasma from autoimmune rats, a marked drop in sperm motility
was observed. However, when seminal plasma from animals of
group I (control group) was added to normal sperm cells, the same
slow drop on sperm motility seen in animals from group I was
observed, indicating that the immediate contact between sperm
cells and an inflammatory seminal plasma can reproduce the detected abnormalities. To investigate the mechanisms involved in
the observed alterations, we performed experiments adding
rTNF-␣ to normal sperm at the concentrations detected in seminal
plasma from autoimmune rats. The addition of recombinant
TNF-␣ was able to significantly diminish the sperm motility and
increase sperm apoptosis of normal spermatozoa (Fig. 6, A and B).
Moreover, when TNF-␣-neutralizing Abs were added, this effect
was partially blocked (Fig. 6A). Because IFN-␥ is a crucial mediator of an inflammatory process, and its presence had been detected in seminal plasma samples from autoimmune CNBP patients (8) and autoimmune animals (Fig. 2C), its direct effect on
sperm motility was also investigated. The addition of rIFN-␥ was
able to significantly reduce the sperm motility and enhance sperm
apoptosis of normal spermatozoa (Fig. 6, C and D). Moreover,
when IFN-␥- neutralizing Abs were added, this effect was partially
blocked (Fig. 6C).
These results allow us to affirm that an inflammatory environment,
the result of an autoimmune response directed against the prostate
gland, can seriously affect the sperm, having a negative impact in the
semen quality of an individual and compromising his fertility.
The Journal of Immunology
965
as either autoantibody- or cell-mediated immunity, the corresponding Ag to be identified, and an analogous autoimmune response to
be induced in an experimental animal. In addition, the immunized
animal must also develop a similar disease. Some years later, Rose
and Bona (28) proposed novel criteria to consider a disease as an
autoimmune disease, based on new information gained from the
use of molecular biology and hybridoma techniques; these include
direct evidence of the transfer of a pathogenic Ab or pathogenic T
cells, indirect evidence based on the reproduction of the autoimmune disease in experimental animals, and circumstantial evidence
from clinical clues. These authors emphasized the importance of
the animal models to corroborate the autoimmune nature of a given
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FIGURE 6. Effect of the addition of
TNF-␣ and IFN-␥ on normal spermatozoa. Sperm kinetics analysis of normal
spermatozoa during the 10 min after
ejaculation in the presence of inflammatory cytokines. A, Effects of recombinant TNF-␣ on sperm motility in normal spermatozoa. B, Effects of
recombinant TNF-␣ on apoptosis
marker expression in normal spermatozoa. C, Effects of recombinant IFN-␥ on
sperm motility of normal spermatozoa.
D, Effects of recombinant IFN-␥ on apoptosis marker expression of normal
spermatozoa. ⴱ, p ⬍ 0.01 vs control
group. Values are representative of at
least two experiments performed. SP,
Seminal plasma.
pathology. Indeed, animal models of numerous human diseases
have been studied for many years and have provided a great deal
of information about mechanisms involved in autoimmune responses (29).
The etiology of the most frequent form of chronic prostatitis,
CNBP (National Institutes of Health category III), has proved elusive
despite years of investigation (7). Recently, we have proposed that
CNBP is a heterogeneous syndrome and that the etiology of this disease, in some cases, involves an autoimmune response against prostate components. This hypothesis is based on the fact that a significant
percentage of these patients exhibited IFN-␥-secreting lymphocytes
against prostate Ags such as PAP, prostate-specific Ag, and other Ags
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AUTOIMMUNITY AGAINST PROSTATE AND ITS CONSEQUENCES
Our findings suggest that mediators present in seminal plasma
from autoimmune animals were responsible for these alterations.
In fact, when seminal plasma from autoimmune rats was added to
normal ejaculated spermatozoa, reduction of sperm motility was
evident, suggesting that humoral mediator(s) present in seminal
plasma from autoimmune animals was/were responsible for the
observed effects. ROS may be involved in the effects observed, and
indeed, high levels of NO were detected in the seminal plasma of
autoimmune rats. Other putative candidates to produce these effects are TNF-␣ and IFN-␥; certainly, seminal plasma from autoimmune animals contained high levels of these cytokines. When
rTNF-␣ or rIFN-␥ were added to normal seminal plasma and then
to normal sperm cells, they were able to mirror the observed effects, diminishing motility and enhancing apoptosis. In concordance, their effects could be blocked, at least in part, by neutralizing Abs. However, the decrease in sperm quality observed after
recombinant cytokine addition (at doses measured in seminal
plasma from EAP animals) does not reach the levels observed
when total EAP seminal plasma was added. When both cytokines
were put together simultaneously, at doses measured in seminal
plasma from EAP animals, neither additive nor synergic effects were
observed compared with the single addition (data not shown). Therefore, TNF-␣, IFN-␥, NO, and probably other proinflammatory mediators play a role in the induction of the damage described.
TNF-␣ exerts its effects via two TNF-␣-specific membranebound receptors, TNFR1 and TNFR2, which are coexpressed in
most tissues (37, 38). TNFR1 activation is associated with the
activation of kinases and NF-␬B and the induction of apoptosis
(38). Spermatozoa may be exposed to abnormal levels of TNF-␣ in
the male reproductive tract or during their passage into the female
reproductive tract in pathological conditions (39, 40). Some researchers have demonstrated that exposing human spermatozoa to
pathological concentrations of TNF-␣ and IFN-␥ can result in a
significant loss of their functional and genomic integrity (41). Apoptosis may be a potential mechanism for the occurrence of TNF-␣
toxic effects. Upon engagement with its receptor, TNF-␣ activates
the TNF-␣ receptor-associated factor, TNFR-associated death domain, and receptor-interacting protein kinase 1. In turn, caspase-2
and caspase-8 become activated with the end result of effector
caspase-3 activation followed by cell death (42). Infliximab, a humanized mAb that blocks specifically TNF-␣, reverses the toxic
effects induced by TNF-␣ in human spermatozoa (43). These toxic
effects that TNF-␣ exerts on spermatozoa could possibly be mediated by ROS because TNF-␣ has the potential to stimulate spermatozoa to generate ROS. In turn, ROS-related sperm membrane
peroxidation may occur (44).
It has been reported that sperm cells from human, rabbit, and pig
species express IFN-␥ receptors, which seem to develop during
spermatogenesis in the testes (45). Moreover, signaling members
of the IFN-␥ pathway like the JAK/STAT proteins, TYK 2,
STAT1, and STAT4, are present and active in human sperm (46).
Dimitrov et al. (47) studied the effects of recombinant cytokines on
spontaneous and ionophore-induced acrosome reaction and demonstrated that spermatozoa that underwent capacitation in medium
with rIFN-␥ showed a significant increase in spontaneous and induced acrosome reaction compared with the control. They postulated that some cases of infertility might result from a defective
acrosome reaction caused by products of activated lymphocytes
and macrophages that are released into the male and female reproductive tracts. Indeed, increased levels of IFN-␥ have been reported in seminal plasma of infertile men and CNBP patients (8,
15, 48). In the present work, we demonstrate the detrimental effect
of IFN-␥ in sperm motility and viability, evidencing its capability
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present in prostate homogenates and seminal plasma (8, 13, 14, 30).
High levels of proinflammatory cytokines and striking abnormalities
in semen parameters were also described in CNBP that exhibited this
autoimmune component (15). We consider that Witebsky’s postulates
can be readily applied in this particular group of patients, arguing for
an autoimmune etiology of their disease: they have cellular autoimmune response against prostate Ags; and the disease can be reproduced in rodents. Indeed, our animal model of autoimmune prostatitis
shows almost all the previously mentioned characteristics of human
chronic autoimmune prostatitis: specific lymphocytes against prostate
Ags; the presence of Th1 cytokines in the lymphocyte supernatants;
and the type of infiltration and histological lesions seen in the gland
(observed in both the animal models and the autoimmune prostatitis
patients; Refs. 8, 18, and 31). In this work we demonstrate that many
of the abnormalities seen in the semen of chronic autoimmune prostatitis patients are also reproduced in the animal model: markers of
local inflammation (increased proinflammatory cytokines in seminal
plasma); reduction of biochemical markers of prostate gland; and
marked alterations of sperm quality, abnormalities detected in both
the animal model and in this subset of CNBP patients that evidenced
an autoimmune response to prostate gland.
The use of our animal model of autoimmune prostatitis also
allowed us to address questions otherwise difficult to answer in the
human patients. Among them, it was important to determine when,
where, and how sperm cells from autoimmune prostatitis individuals were being damaged. Two possibilities were evaluated: either
these alterations were a result of an epididymal damage as a consequence of a satellite effect of the prostate inflammation; or they
were the result of a rapid damage originated during the joining of
sperm cells with the prostate fluids at the moment of ejaculation.
Our findings demonstrate that only ejaculated spermatozoa are being damaged and that apoptosis is involved.
Numerous studies have now shown the presence of nuclear
DNA strand breaks and apoptosis in human ejaculated spermatozoa from infertile patients and the abnormal persistence of apoptotic marker proteins (32). Why human spermatozoa possess
these abnormalities is still not clear (33). Two processes that have
been linked to the presence of DNA strand breaks in spermatozoa
are anomalies in apoptosis during spermatogenesis or abnormalities after spermatogenesis.
Many recent studies indicate that oxygen-derived free radicals
induce apoptosis to spermatozoa. The excessive generation of
these reactive oxygen species (ROS; superoxide, hydroxyl, NO,
peroxide, peroxynitrile) by spermatozoa and by contaminating leukocytes associated with genitourinary tract inflammation has been
identified in idiopathic male infertility (34). Henkel et al. (35) reported that DNA fragmentation was strongly correlated with ROS
production. However, oxidative stress is the result of elevated
ROS, depressed total antioxidant capacity, or both. Mammalian
spermatozoan membranes are rich in polyunsaturated fatty acids,
making them very susceptible to oxygen-induced damage, which is
mediated by lipid peroxidation. In a normal situation, the antioxidant mechanisms present in the reproductive tissues and their secretions are likely to quench these ROS and protect against oxidative damage to gonadal cells and mature spermatozoa. The
prostate gland contribute with its secretion to the antioxidant capacity present in semen (34), and significant oxidative stress in the
semen or prostatic fluid from patients with chronic noninfectious
prostatitis has been reported, comparable with levels reported in
men with recognized infertility (36). In concordance with these previous reports, we evidenced elevated levels of NO in semen from
animals with autoimmune prostatitis. This finding clearly shows that
spermatozoa from these animals are exposed to an oxidative stress
that could be a cause of the many alterations observed in semen.
The Journal of Immunology
of inducing sperm apoptosis. Further work is needed to elucidate
the mechanisms involved in these phenomena.
Thus, sperm damage in experimental autoimmune prostatitis can
be the consequence of several causes: an inflammatory milieu,
originally produced by an autoimmune response in the prostate
gland, characterized by an enhancement of ROS levels and proinflammatory cytokines in seminal plasma; and a diminished antioxidant capacity caused by an impairment of prostate function; or
both mechanisms simultaneously.
Our results argue in favor of the importance of prostate functionality and suggest that besides the clinical manifestations in chronic
prostatitis patients (pelvic pain, irritative voiding symptoms, effects on
sexual function), a group of them who have an autoimmune response
against prostate could also have diminished fertility.
Utilization of animal models of autoimmune prostatitis will provide important approaches, which may help to identify the pathogenic mechanisms and also possible therapies for human disease.
Acknowledgments
Disclosures
The authors have no financial conflict of interest.
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Downloaded from http://www.jimmunol.org/ by guest on June 15, 2014
We thank Paula Icely for technical assistance and Drs. Laura Giojalas and
Hector Guidobaldi from Centro de Biologı́a Molecular y Celular, Facultad
de Ciencias Exactas, Fı́sicas y Naturales, Universidad Nacional de Córdoba, for assistance in the sperm motility experiments.
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