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Regular Articles |
From the Departments of Pathology*
and Cell
Biology and Anatomy,
University of Virginia,
Charlottesville, Virginia
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-hydroxylase and side-chain cleavage
enzyme of steroid-producing cells have been characterized, they are
detected only in POF patients associated with autoimmune polyglandular
syndrome type I or type II.7,8 Studies on rodent models of AOD have added some insights into the pathology and mechanisms of immune-mediated ovarian injury. Neonatal thymectomy in susceptible mouse strains causes ovarian inflammation and autoantibodies resulting in ovarian atrophy and ovarian dysfunction.9 This model has been used to understand regulation of autoimmune disease in general and AOD in particular.10,11 The second rodent model of AOD is elicited by immunization with a self-peptide of murine zona pellucida (ZP)3 (330 to 342).12 Murine pZP3 immunization causes activation of autoreactive T cells and generation of anti-ZP autoantibody. The autoreactive T cells mediate autoimmune oophoritis characterized by focal or diffuse inflammation in the ovarian interstitium and organized monocytic granulomata. Although oophoritis alone does not affect ovarian function, a high level of ZP autoantibody reduces fertility in female mice.13 On the other hand, combined ZP antibody and T-cell-mediated inflammation are required for the induction of ovarian atrophy and infertility.14 Chimeric peptides that contain foreign T-cell epitope and native ZP3 B-cell epitope induce ZP antibody without inducing ovarian inflammation, and they are potential candidate antigens for contraceptive vaccine development.15
In primates, long-term immunization with zona pellucida proteins for >1 year resulted in antibodies to zona pellucida with contraceptive effect. However, the infertility was irreversible and the monkeys developed POF.16,17 The ovaries from immunized animals show few oocytes or developing follicles but increased numbers of aberrant granulosa cell nests. Ovarian inflammation was not reported in the atrophic ovaries of animals after long-term immunization. To investigate AOD as a basis for POF and a complication of ZP vaccination, we have developed a primate model of AOD. In this study, we demonstrate that cynomolgus macaques are indeed susceptible to AOD using human and macaque homologues of murine pZP3 (330 to 342). The resulting ovarian histopathology is compared to murine AOD, and with the reported findings in human AOD.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The ZP3 peptides from different species used in this study are as follows: human 328 to 341 [GTPSHSRRQPHVMS pZP3(hu)]; macaque 328 to 341 [GTPSHSRRQPHVVS pZP3(mac)], and mouse 330 to 342 [NSSSSQFQIHGPR pZP3 (mou)].18 The peptides were synthesized in an automated peptide synthesizer (Beckman Instruments, Fullerton, CA) using F-moc chemistry. Deprotection was achieved by 20% piperidine in dimethyl formamide, and the peptides were chemically cleaved from the resin with 85% trifluoroacetic acid. The peptides were purified by HPLC eluting as a single peak on PRLP-S columns (Waters, Milford, MA) and were of >90% purity.
Monkeys: Immunization, Cyclicity, Surgery, Sacrifice
All animal housing and procedures were performed in accordance with the National Institutes of Health guidelines. Adult female cynomolgus macaques (Macaca fascicularis) were anesthetized with ketamine hydrochloride (10 to 20 mg/kg) for immunization and bleeding. Experimental groups included immunization with peptides [pZP3(mac) or pZP3(hu): two animals per group] or controls (untreated or adjuvant-treated: two animals per group). Immunizations were done on days 0, 14, and 28 intramuscularly with 50 nmol/L of peptide and 0.1 mg of nor-muramylated dipeptide (a gift from Dr. Vernon Stevens, Ohio State University, Columbus, OH) in phosphate-buffered saline (PBS) emulsified in equal volume of squalene:arlacel A (4:1, v:v; Sigma Chemical Co., St. Louis, MO). Animals were given booster immunizations at times indicated. Blood collected from the femoral vein was used for serum antibody analyses, T-cell responses, and circulating steroid levels. All of the animals were monitored daily for menstrual bleeding. Ovaries were surgically removed under general anesthesia through a midline abdominal incision. To study ovarian immunopathology at multiple time points, the first ovary was removed between 1.5 to 6 months and the second between 11 to 16.5 months. Each ovary was cut into 4 to 5 pieces collected in buffered formalin for histopathology. One piece was snap-frozen in liquid nitrogen for detection of ZP-bound IgG. The animals were euthanized at the end of the study and organs collected for histopathological analysis.
Mice: Immunization and Sacrifice
(C57BL/6 X A/J)F1 females were obtained from the National Cancer Institute, Bethesda, MD and housed as per National Institutes of Health guidelines. As previously described, for induction of AOD, mice were anesthetized by intraperitoneal injection of tribromoethanol (0.5 mg/mouse) and immunized subcutaneously with 50 nmol/L of pZP3(mou) emulsified in CFA (Mycobacterium tuberculosis H37RA strain, 0.16 mg/mouse) (Difco Laboratories, Detroit, MI) and sacrificed 14 days later.12 Control mice were injected with CFA alone. One ovary was collected in Bouin’s fixative for histopathological grading of AOD and the other ovary was collected in 4% paraformaldehyde in PBS (w/v), pH 7.4, for immunohistochemical staining.
Detection of Antibody to Peptides by Enzyme-Linked Immunosorbent Assay
Serum IgG to pZP3(hu) or pZP3(mac) were detected by enzyme-linked immunosorbent assay.18 Ninety-six well plates (Corning Glass Works, Corning, NY) were coated overnight at 4°C with 25 nmol/L of peptide in 0.1 mol/L bicarbonate buffer, pH 9.0. All subsequent incubations were performed for 2 hours at room temperature. The plates were washed with 0.05% Tween 20 in PBS, pH 7.4, and blocked with 3% bovine serum albumin (BSA) in PBS. The plates were incubated with serial dilutions of monkey sera. The plates were then washed and incubated with horseradish peroxidase-labeled goat anti-monkey IgG (Nordic Immunological Laboratories, Capistrano Beach, CA) (1:3000 dilution). After washing, the reaction was developed by o-phenylene diamine (0.5 mg/ml; Sigma Chemical Co.) with freshly added 0.06% H2O2 in 0.1 mol/L citrate buffer, pH 5.0. The color intensity was read at 490 nmol/L by an enzyme-linked immunosorbent assay reader (Molecular Devices, Menlo Park, CA). The end-point titer was calculated as dilution of serum giving an absorbance of 2 SD above the background reading using preimmune monkey serum.
Detection of Anti-ZP Antibody by Direct and Indirect Immunofluorescence
In vivo binding of ZP antibody was detected by direct immunofluorescence. Frozen monkey ovary sections from peptide-immunized macaques or adjuvant-treated controls were fixed in 95% ethanol for 10 minutes and rinsed with PBS. The sections were then blocked with normal goat serum (1:10 v/v) diluted in PBS with 3% BSA for 20 minutes in a humid chamber at room temperature. After rinsing with PBS, the bound antibody was detected by fluorescein isothiocyanate conjugated to anti-monkey IgG (1:100; Nordic Immunological Laboratories) for 30 minutes. The slides were rinsed and mounted in Vectashield medium (Vector Laboratories, Burlingame, CA).
Antibody to zona pellucida in serum was detected by indirect immunofluorescence. The procedure followed was same as above with some differences. Five µm sections of snap-frozen normal monkey ovary were fixed in 95% ethanol, blocked with normal goat serum in PBS with 3% BSA (1:10 v:v). The sections were then incubated with immune serum or adjuvant control serum diluted in 3% BSA in PBS for 1 hour. IgG antibody bound to ZP was detected by fluorescein isothiocyanate conjugated to anti-monkey IgG (Nordic Immunological Laboratories).
Lymphocyte Proliferative Responses in Monkeys
Estimation of T-cell proliferative responses was done as previously described.18 Monkey peripheral blood was collected in heparinized tubes and centrifuged to separate supernatant plasma. The cell pellet was resuspended in sterile PBS, layered over Histopaque 1083 (Sigma), and centrifuged at 400 x g for 20 minutes. Mononuclear cells at the interphase were collected, washed, and resuspended at 2 x 106/ml in Dulbecco’s minimum essential medium supplemented with 1% sodium pyruvate, 1% of 200 mmol/L glutamine, 1% nonessential amino acids, 5 x 10-5 mol/L ß-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum. The assay was set up in triplicates in which cells (0.1 ml/well) were cultured with an equal volume of peptide (0 to 30 µmol/L) in 96-well flat-bottom plates for 4 days at 37°C in 5% CO2. [3H]-thymidine (0.5 µCi/well; Dupont NEN Products, Boston, MA) was added to each well 16 hours before harvesting the cells (Skatron Instruments, Sterling, VA), and the cell-associated thymidine measured by scintillation counting (Beckman Instruments). Results are presented as stimulation index (experimental counts per minute with peptide/background counts per minute without peptide). The monkeys Babs and Harriette gave high background counts when cultured in media with 10% fetal calf serum. For these animals, fetal calf serum was substituted with 10% autologus heat-inactivated plasma.
Immunohistology: Monkey and Mouse Ovaries
Serial sections (5 µm) were cut through the entire formalin-fixed, paraffin-embedded, monkey ovaries. Every 10th section was stained with hematoxylin and eosin. Adjacent sections were stained with the following antibodies at indicated dilutions. Polyclonal rabbit antibody to human CD3 and mouse monoclonal antibodies to HLA (CR3/43), CD20 (L26), CD 68 (KP1), and myeloid/histiocyte antigen (Mac 387) obtained from DAKO Corporation, Carpinteria, CA. A rabbit polyclonal antibody to HLA DR (a gift from Dr. S. M. Fu, University of Virginia, Charlottesville, VA) was also used in some of the studies. Optimal dilutions of primary antibody (1:20 for rabbit anti-HLA polyclonal; 1:50 for L26, Mac387, CR3/43; and 1:100 for anti-CD3 and KP1) were determined using control monkey spleen sections.
The tissues were deparaffinized in xylene, transferred to absolute alcohol, rehydrated with decreasing grades of alcohol, and rinsed in PBS. Endogenous peroxidase was inactivated with 0.6% hydrogen peroxide in methanol PBS (4:1, v:v). Because some antibodies (CD3, CD68, and Mac 387) did not recognize their cognate antigens, unmasking of epitopes was performed by digestion with pepsin (4 mg/ml) in 0.1% HCl at 37°C for 7 minutes. For HLA monoclonal and polyclonal antibodies, antigen retrieval was done by heating slides in 10 mmol/L of citrate buffer, pH 6.0, for 20 minutes in a microwave. Evaporated fluid was replaced with water every 4 minutes. The slides were allowed to cool to room temperature before use. Sections were rinsed in PBS after antigen retrieval and blocked with normal goat or rabbit serum followed by primary antibody diluted in 3% BSA PBS. After washing, the slides were incubated with biotinylated goat anti-mouse or anti-rabbit Ig (Vector Laboratories) (1:100 dilution). Detection of bound antibody was done by avidin-biotinylated enzyme complex (Vectastain ABC kit; Vector Laboratories) and visualized by incubation with diaminobenzidine substrate (Biogenex Corp., San Ramon, CA). The sections were counterstained with methylene blue, rinsed with water, and dehydrated with ascending grades of alcohol. The slides were then rinsed with xylene and mounted with Cytoseal60 (Stephens Scientific, Kalamazoo, MI). Monkey spleen sections concomitantly processed were positive controls and ovaries without primary antibody served as negative controls.
Immunochemical staining of mouse ovaries was done as previously described.13 Briefly, 5-µm sections from mouse ovaries fixed in 4% paraformaldehyde were incubated with primary antibody to CD5 (Lyt1 53-7.313), major histocompatibility complex (MHC) class II (M5/114.15.2), B220 (RA3-3A1/61), or macrophage (F4/80). After washing in PBS, the sections were incubated with biotinylated rabbit anti-rat antibody. Bound antibody was visualized by the ABC detection system followed by diaminobenzidine and counterstained with methylene blue as described above.
Steroid Assays
For analysis of ovarian function, monkeys were bled every 3 days for 1 to 3 consecutive months at two phases of the study, with the first early phase starting 1 to 2 months before immunization until 2 months after immunization and the second late phase between 3 to 11 months when the antibody titers were high. Endocrine function was monitored by estimation of circulating steroid hormone levels. One hundred µl of serum was used in competitive radioimmunoassays for progesterone and estradiol estimations, as per the manufacturer’s instructions (ICN Pharmaceuticals, Costa Mesa, CA, for progesterone and Diagnostic Products Corporation, Los Angeles, CA, for estradiol). The detection limit for progesterone was 0.27 ng/ml with an interassay coefficient of variation (CV) of 11.7% and intra-assay CV of 8.9%. For estradiol, the sensitivity limit was 27 pg/ml with an interassay and intra-assay CV being 7.4% and 4.5%, respectively.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Two normal, cycling, adult female cynomolgus macaques were
immunized with 50 nmol/L of pZP3(mac) in a squalene arlacel A emulsion.
Both animals (Babs and Harriette) had detectable serum anti-peptide
antibody on day 24 after immunization (Figure 1A)
. The titers were maintained at
103
to 105
by subsequent
booster immunizations until sacrifice. Their serum IgG reacted with
macaque ZP by indirect immunofluorescence (Figure 1A
, inset). Antibody
bound to ovarian ZP was also detected by direct immunofluorescence at
time of sacrifice (data not shown). Thus pZP3(mac) includes a native
B-cell epitope. The antibody detected was of the IgG isotype suggesting
a helper T-cell response and the presence of a T-cell epitope in
pZP3(mac). T-cell proliferative response was detected in
vitro by days 28 to 32 in both animals (data not shown). The
T-cell proliferative response could also be recalled 10 days after the
final booster immunization (Figure 1B)
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. Antibody bound to ovarian ZP was
detected by direct immunofluorescence at the time of sacrifice (Figure 1C
, inset). Circulating antibody to ZP was also detectable by indirect
immunofluorescence (data not shown). T cells from pZP3(hu)-immunized
animals proliferated strongly in response to the immunogen and
cross-reacted to a lesser extent with the homologous pZP3(mac) but not
at all with pZP3(mou) (Figure 1D)
. These experiments demonstrate that
the macaque and human peptides cross-react at the B-cell and T-cell
levels even in outbred macaques. Our subsequent investigations showed
that immunization with these immunologically cross-reactive peptides
have similar effects on the ovary and on ovarian function. pZP3(mac) and pZP3(hu) Immunization Is Associated with Ovarian Infiltration of T Cells and Activated Macrophages without Detectable Oophoritis
The ovary has many leukocytes that play a critical role in normal
ovarian function including follicular atresia, ovulation, and
involution of the corpus luteum.20-22
Thus we first
examined the distribution of T cells and macrophages in the ovaries
from two untreated and two adjuvant-treated cycling adult macaques. As
described in Materials and Methods, serial sections were cut through
the entire ovary for each animal, and every 10th section was studied
histologically. Adjacent sections were immunostained with antibodies to
CD3, MHC II, macrophages (CD68, Mac 387) and B cells. As reported in
humans,23
T cells and macrophages expressing MHC II were
detected in normal or adjuvant-treated monkey ovaries. In addition,
atretic follicles and corpora lutea were infiltrated by small numbers
of T cells and MHC II-positive macrophages (Figure 2, A and B)
. Occasional clusters of T
cells were also located in the ovarian cortex and medulla (Figure 2C)
. However, these T-cell clusters did not contain any detectable MHC
II-positive macrophages (Figure 2D)
.
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; A to D). These clusters of
inflammatory cells were found in the interstitial or perifollicular
locations. Because these typical leukocyte infiltrates were detected in
pZP3-immunized monkeys but not in monkeys injected with adjuvant alone,
they were considered to be pathologically relevant. Although
macrophages (Mac 387 or CD68 positives) were detected in these T-cell-
and MHC II-positive cellular infiltrates, they did not account for all
of the MHC II staining in the inflammatory foci (data not shown). The
additional MHC II-expressing cells may represent ovarian dendritic
cells or granulosa cells of adjacent follicles, as described in human
AOD.24
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. Immunostaining of
adjacent sections showed invasion of macrophages in the region (Figure 3F)
. The granuloma showed characteristic features of central MHC
II-expressing cells surrounded by a ring of CD3-positive T lymphocytes
(Figure 3, G and H)
. Histopathological evaluation of other organs
collected at sacrifice failed to show any detectable inflammation. Ovarian Function Is Retained in the Presence of Oophoritis
Menstrual bleeding in the pZP3-immunized and adjuvant-treated
animals was monitored. The cycles in all of the animals except Eenie
(the animal with granulomatous oophoritis) were found to be regular and
comparable to adjuvant-treated controls (Figure 4)
. Eenie stopped menstruating for 7
months after ovariectomy after which menstrual bleeding resumed
spontaneously. Sera were collected at two phases of the study to
monitor endocrine function as described in Materials and Methods. The
mean peak estradiol and progesterone levels studied early (when
antibody titers were low) and late (when ZP antibody titers remained
high) were comparable in all of the animals except Eenie (Table 1)
. Although the data collected is during
a limited period of study, the pattern showed functioning ovaries with
an estradiol peak preceding the progesterone peak (data not shown).
Steroid analysis after ovariectomy in Eenie showed loss of cyclicity
with estradiol <20 pg/ml and peak progesterone reaching only 2.3
ng/ml.
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AOD is induced by injection of pZP3(mou) in adult B6AF1 female mice,12 but a detailed analysis of the immunopathology of the oophoritic lesion has not been described. To compare murine and macaque AOD, we evaluated mouse ovaries for the distribution of T cells (CD5), macrophages (F4/80), MHC II, and B cells (B220).
Immunostaining of the ovarian inflammatory infiltrates in murine
AOD and the primate AOD are similar. In murine AOD, the infiltrating
lymphocytes were predominantly T cells (Figure 5A)
, with only occasional B cells (data
not shown). Like the lesion in macaque AOD, T cells were found to form
clusters with F4/80-positive macrophages that expressed MHC II (Figures 5, A and C)
. Macrophages were normally present in large numbers in the
normal mouse ovary. Adjuvant treatment resulted in the increase in
ovarian macrophages that expressed MHC class II (Figure 5D)
, but there
was no increase in T-cell infiltration (Figure 5B)
.
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. The inflammation was focal or diffuse, frequently
appearing as monocytic granulomata and in some cases, multinucleated
giant cells. No inflammatory lesions were seen in animals immunized
with adjuvant alone (Figure 5F)
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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by infiltrating T
cells has been implicated in the aberrant expression of MHC II on
granulosa cells at the site of inflammation.24
These
features are similar to our findings in the macaque and mouse
ZP-induced AOD. Although typical granulomatous oophoritis resembling
the inflammation seen in Eenie, has been reported in patients their
immunohistological findings were not studied.5
B cells and
plasma cells are identified in the human AOD as well as murine AOD
induced by neonatal thymectomy (preliminary data) but are rarely found
in monkey or murine AOD induced by pZP3. Another type of human
oophoritis contains mainly eosinophils,28
and similar
pathology has been elicited in mice by adoptive transfer of Th2
(interleukin-4 and interleukin-5 producing), pZP3-specific T cells (P
Alard and KSK Tung, unpublished), and in pinworm-infected
neonatal mice injected with pZP3(mou) without adjuvant.29 Immunization with zona pellucida proteins and peptides has also been investigated as immunocontraceptive vaccine. Vaccines based on the whole porcine zona pellucida proteins have been applied effectively to control feral horse and elephant populations.30,31 As a potential vaccine for human population control, primates (marmoset, baboons, squirrel monkeys, and macaques) have been immunized with heterologous zona pellucida proteins. Although this approach resulted in infertility,16,17,32 varying extents of irreversible ovarian dysfunction were also observed. Antibody to ZP was postulated to interfere with junctional complexes between granulosa cells and oocytes leading to the loss of developing follicles, and accelerated recruitment of the primordial follicles. However, other studies on squirrel monkeys and bonnet monkeys immunized with porcine ZP proteins with alum and muramyl di-peptide (MDP) adjuvants were found to develop high levels of circulating ZP antibodies. These studies showed a reversible contraceptive effect without any loss of ovarian function.33,34 These contradictory data suggest that the high titers of ZP antibody alone are not sufficient to explain irreversible loss of ovarian function after ZP protein immunization in primates. Because no obvious inflammation was detected, autoimmune oophoritis was not considered a basis for ovarian failure in ZP-immunized primates.
The ZP3 peptide with well-defined T- and B-cell epitopes capable of
eliciting a restricted immune response has been used to dissect the
mechanism of AOD12
in the mouse model. Transfer of ZP3
peptide-specific T cells into naïve recipient mice resulted in
granulomatous oophoritis and enhanced ovarian expression of
interleukin-1, tumor necrosis factor-, and interferon-. However,
the ovarian function of cell recipients was normal and the mice
remained fertile.13
Antibody to ZP3 also does not cause
any ovarian pathology. These results indicate that T-cell-mediated
ovarian inflammation alone or ZP antibody alone does not affect ovarian
function. However, co-transfer of pathogenic T cells and ZP antibody
targets the inflammation into developing follicles leading to their
destruction and the development of ovarian atrophy.14
Thus
combined action of both antibody and proinflammatory T cells is
required for severe ovarian injury leading to POF. In this study we
show that ZP3 peptide immunization in primates can elicit a T-cell
response and cause ovarian immunopathology that is similar to murine
AOD. These data suggest a role for T cells in macaque AOD. It will be
important to determine in the future whether the combined effects of
antibody and proinflammatory T cells are responsible for the long-term
ovarian changes in monkeys injected with ZP3.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant U54 HD 29099) and a Fogarty Foundation fellowship (to H. B.).
Accepted for publication September 28, 2001.
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This article has been cited by other articles:
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  Y. Y. Setiady, E. T. Samy, and K. S. K. Tung Maternal Autoantibody Triggers De Novo T Cell-Mediated Neonatal Autoimmune Disease J. Immunol., May 1, 2003; 170(9): 4656 - 4664. [Abstract] [Full Text] [PDF]
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