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Is Produced by Polymorphonuclear Neutrophils in Human Uterine Endometrium and by Cultured Peripheral Blood Polymorphonuclear Neutrophils1
Departments of
*
Microbiology and
Physiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussion References |
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, a Th1 cytokine, in human
endometria. Extracellular IFN-, mostly associated with matrix
components, was located immediately beneath the luminal epithelium and
along the glandular epithelium proximal to the lumen. As evidenced by
intracellular staining, IFN- is produced by both stromal cells and
intraepithelial lymphocytes through all stages of the menstrual cycle.
Surprisingly, the stromal cell containing intracellular IFN- was
identified as a polymorphonuclear neutrophil on the basis of its
reactivity with a panel of mAbs and its nuclear morphology. We further
found that polymorphonuclear neutrophils isolated from normal donors
produce IFN- in response to stimulation with LPS, IL-12, and
TNF-
. Taken together, these findings suggest that polymorphonuclear
neutrophils are capable of producing IFN- both in vitro and in vivo,
indicating that their role in shaping immune responses may be more
extensive than previously thought. Furthermore, these studies strongly
suggest that polymorphonuclear neutrophils play an important role in
determining immune responsiveness within the female reproductive tract. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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found in cultures of endometrial
cells (8, 9).
Roles for IFN- in controlling the growth, differentiation, and
immune responsiveness of normal human uterine endometrium have been
proposed (8, 10, 11, 12). Despite this, only three studies, two looking at
mRNA expression (13, 14) and one staining for protein (15), have shown
evidence of IFN- production in nonpregnant human uterus. Although
these studies concluded that T cells and macrophages were responsible
for IFN- production in the uterus (13, 14, 15), the markers used to
phenotype the IFN-positive cells were not lineage specific.
In the present study we have used a culture system employing viable
vibratome sections of uterine endometrial tissue (EM) from hysterectomy
patients, in conjunction with three-color immunofluorescent staining
and scanning confocal laser microscopy, to investigate the ability of
LA and other uterine endometrial cell types to produce IFN-. We show
that, contrary to previous proposals, LA are not the source of
constitutive IFN- production in uterus. Surprisingly, the majority
of the intracellular IFN- immunoreactivity in this tissue is
associated with polymorphonuclear leukocytes (PMN). Further, we show
that PMN from peripheral blood, which have not previously been shown to
produce IFN-, stain positively for IFN-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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Uterine endometrial tissue was obtained with prior informed
consent and Institutional Review Board approval from patients who were
undergoing hysterectomy (Table I
).
Most patients included in this study were diagnosed as having
leiomyomata, prolapsed uteri, or benign ovarian disease. None had a
postoperative diagnosis of malignant uterine disease. Sections of
tissue were dissected out from sites distal to any gross pathology and
placed immediately in sterile ice cold PBS. The blocks of tissue were
trimmed of excess myometrium, and 30- to 70-µm sections were cut
using a vibratome (V1000, TPI, Energy Beam Sciences, Agawam, MA).
Sections were maintained in ice cold PBS throughout processing. The
stage of the menstrual cycle of the endometria was determined in
accordance with accepted histologic practice using
hematoxylin/eosin-stained paraffin sections. Evaluations were conducted
independently by two pathologists by scoring the degree of stromal
edema and the relative frequency of glandular and stromal mitoses.
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A panel of mAbs (Table II) was
used for direct and indirect immunofluorescent staining. Abs purified
from hybridoma cell culture supernatants (cell lines from ATCC) using
HiTrap protein G-Superose columns (Pharmacia LKB, Piscataway, NJ) were
labeled, where indicated, with Cy3 or Cy5 Fluorolink protein labeling
kits (Amersham, Arlington Heights, IL) according to the manufacturer’s
recommendations. FITC-conjugated mouse mAbs were obtained from
commercial suppliers as denoted in Table II.
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Three-color immunofluorescent staining of tissue sections was conducted immediately after cutting. For direct staining, 2 µg/100 µl each of FITC-, Cy3-, and Cy5-labeled Abs in PBS/1%BSA/0.1% azide (PBA)-containing human Ig (6 mg/ml to block nonspecific binding) were added to sections in 96-well plates and incubated overnight at 4°C in the dark with continuous gentle agitation. Unbound Ab was removed from the sections by aspiration followed by four 20-min washes in PBA. Washed sections were then fixed overnight in the same buffer containing 1% paraformaldehyde. Stained sections were wet-mounted in anti-fade (Molecular Probes, Eugene, OR), sealed with nail varnish, and stored at 4°C in the dark for up to 10 days before confocal imaging.
IFN- staining
Intracellular staining for constitutive production of IFN- by
cells within the vibratome sections was investigated following
treatment with brefeldin A, which causes accumulation of newly
synthesized proteins within the cells (16). The staining method is an
adaptation of an indirect staining method for flow cytometry described
by Schmitz et al. (17). Briefly, vibratome sections were cultured at
37°C overnight in serum-free, phenol red-free, Excel medium (Medarex
modification, Medarex, Annandale, NJ), supplemented with penicillin (50
U/ml), streptomycin (50 µg/ml), gentamicin (10 µg/ml), and
glutamine (0.291 mg/ml), then exposed to brefeldin A (100 µg/ml) for
4 h at 37°C (all from Sigma, St. Louis, MO). As a positive
control for IFN- production, matched sections were exposed to
ionomycin (10 µM; Sigma) and PMA (10 µM; Sigma) during the
overnight incubation. Sections were washed extensively with PBA and
fixed overnight in PBA/1% formaldehyde. After washing, sections were
incubated for 2 h at room temperature in 200 µl of PBA
containing 0.5% saponin (Sigma) in the presence of human Ig block (6
mg/ml final) and 2 µg/100 µl of unlabeled mouse monoclonal
anti-IFN- Ab (PharMingen) or a 1:2000 dilution of a polyclonal
rabbit anti-human IFN-. Following three 20-min washes in PBA
containing 0.5% saponin, sections were incubated for a further hour
with Cy3-labeled affinity purified anti-mouse Ig Ab.
Immunophenotyping of IFN--producing cells
To phenotype the IFN--producing cells, sections were stained
with FITC or Cy5 lineage-specific mouse mAbs before staining for
intracellular IFN- with a rabbit polyclonal anti-human IFN-
Ab (1/2000 dilution of National Institutes of Health (NIH, Bethesda,
MD) Research reference reagent G034-501-565), followed by a Cy3-labeled
affinity-purified anti-rabbit Ig polyclonal Ab (Amersham). In more
recent experiments, this procedure was replaced by one that utilized a
Cy5-conjugated anti-IFN- Ab (B-B1; Serotec), in conjunction with
FITC- and Cy3-labeled cell surface marker-specific Abs.
Unstained and fluorochrome isotype controls were used to control for autofluorescence and nonspecific Ab binding, respectively. Each set of sections was internally controlled wherever possible by using the same mAb with different fluorochromes attached. For example, sections stained with FITC-CD3, Cy3-CD3, and Cy5-CD3 were used to establish the threshold of detection for channels such that no crossover was seen in the other two channels.
For IFN- staining with mouse mAb, isotype-specific controls were
used to set the threshold of the Cy3 channel. Ionomycin/PMA-treated
sections were used as positive controls for IFN- production. For
sections stained with rabbit polyclonal anti-human IFN- Ab, a
matched preimmune rabbit serum was used (1/2000 dilution of NIH
Research reference reagent G035-501-565). As an additional set of
controls in some sections, staining was blocked by the addition of
excess recombinant human IFN- (Genentech, San Francisco,
CA).
Confocal scanning laser microscopy
Immunofluorescently labeled sections were optically sectioned
using a Bio-Rad MR1000 Confocal Scanning Laser Microscope system
(Bio-Rad Laboratories, Hercules, CA) equipped with a krypton/argon
laser. Laser power, PMT gain, and enhancement factors were then
determined for the FITC, Cy3, and Cy5 channels using the single
fluorochrome-stained sections to ensure effective cross-channel
compensation. Three-color fluorescent sections were then evaluated for
the presence of IFN--producing cells.
IFN- production by peripheral blood PMN
PMN were isolated by a modification of the double layer
Ficoll-Hypaque procedure of English and Anderson (15, 18), as described
previously by Kerr et al. (19). The resulting preparations were
>99.5% pure PMN. PMN were cultured overnight in RPMI 1640/20% FCS in
the presence or absence of G-CSF (Amgen, Thousand Oaks, CA) and with or
without the addition of IL-12 (Peprotec, Rocky Hill, NJ). Cells were
then stained for intracellular IFN- using Cy5-labeled
anti-IFN- either with or without brefeldin A treatment and for
cell surface CD66b using an FITC-labeled Ab. After counterstaining the
nuclei with propidium iodide, cells were examined by confocal
microscopy. Laser power, PMT gains, and confocal thresholds were set
using FITC-IgG1 and Cy5-IgG1 isotype controls and kept constant
throughout the experiment.
Quantitation of intracellular IFN- in peripheral blood PMN
The relative amounts of IFN- in individual cells was
determined using Image Space software (Molecular Dynamics, Irvine, CA)
to analyze image files obtained from peripheral blood staining
experiments. Three-color Image files were given a threshold intensity
of one (three-color images are composed from three gray-scale images,
one for each PMT channel, with each range from 0 to 256 gray levels).
Individual cells were then defined by enclosing each cell in a circle.
Software algorithms were then used to determine the total IFN-
staining intensity in each cell, which may be defined as the sum of the
intensities of all pixels in the 256 gray-scale image representing the
Cy5 channel that were greater or equal to one. This value is referred
to as total pixel intensity per cell. Statistical analysis between
treatments was conducted by nonparametric Mann-Whitney U
test using Statgraphics Plus v3 Software (Manugistics, Rockville,
MD).
ELISA quantitation of IFN- in peripheral blood PMN culture
supernatants
Peripheral blood PMN from normal male donors were prepared by
double layer Ficoll-Hypaque separation as described above. Differential
counts of the resulting PMN preparations showed a contaminating
nongranulocyte count of less than 1%. PMN were cultured in AIM-V
medium (Life Technologies, Grand Island, NY)/5% FCS at a cell density
of 7 x 106 PMN/ml either without additional
stimulation, or with the addition of human rIL-12, human rTNF-
(Genentec) or LPS (List BiologicLab, Campbell, CA). Supernatants were
then harvested for ELISA analysis. IFN- ELISAs were conducted using
paired capture and biotinylated detection antibodies from R&D Systems
following the manufacturer’s recommendations. Concentrations were
determined against a standard curve constructed by serially diluting
recombinant human IFN- (Genentech). Statistical analysis of mean
IFN- production between treatments was by t test using
Statgraphics Plus v3 Software (Manugistics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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expression in human uterine endometrium
In studies designed to evaluate the localization of cytokine
production and its influence on the immune responsiveness of cells in
the female reproductive tract, we stained uterine vibratome sections
with fluorescently labeled anti-cytokine Abs. Of particular
interest, we found that IFN- was present in vibratome sections of
fresh uterine tissue from all patients (Table I
) in the absence of
exogenous stimuli. Although the intensity of staining varied from
patient to patient, no discernible correlation was found with the stage
of the menstrual cycle or between pre- and postmenopausal women. In
particular, extracellular IFN- was evident throughout the stroma of
the tissues but was mostly concentrated as a broad band immediately
below the luminal epithelium and adjacent to the glandular epithelium
proximal to the lumen (Fig. 1,
a and b). The staining pattern of the
extracellular IFN- was often fibrous, suggesting that the cytokine
is associated with extracellular matrix components. Such an association
is consistent with the reported binding of IFN- to heparin sulfate
on endothelial cells described by others (20, 21, 22). Specificity of
staining was confirmed by the ability of polyclonal goat
anti-IFN- to block staining with mouse monoclonal
anti-IFN- Ab and by the ability of excess rIFN- to block
staining (Figs. 2 and
3). In addition, we labeled the B-B1 mAb
(Serotec) with Cy5 and used it to stain sections in conjunction with
two other mAbs labeled with FITC and phycoerythrin (PE) (see Table II)
or a polyclonal Ab stained indirectly with an FITC second Ab and
propidium iodide (PI) nuclear counterstaining. The results showed
uniform dual staining of cells with all Ab pairings, indicating that
all three monoclonals and the polyclonal recognized Ag expressed in the
same cells (data not shown).
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LA represent the major concentration of organized lymphoid cells
in human endometrium (7, 15, 23). Since T cells are the
predominant cell type present in LA and since T cells are known to
produce IFN-, it has been proposed that these structures are
responsible for uterine IFN- production (8, 9). Based on staining
with FITC-CD19, Cy3-CD14, and Cy5-CD3 (for B cells, macrophages, and T
cells, respectively), LA were present in five of the seven
proliferative phase and all secretory phase tissues, but LA were absent
from the postmenopausal tissue. IFN- was not found associated with
the cells in the LA, but intracellular periepithelial IFN- staining
of discrete cells in the matrix around the LA was observed. In
contrast, when uterine vibratome sections were incubated in the
presence of ionomycin/PMA, IFN--positive cells were observed in LA
in addition to the periepithelial population seen in unstimulated
sections. Periepithelial cells were large and irregular in shape, while
IFN--positive cells in LA after treatment with ionomycin/PMA had a
morphology consistent in size and location with T cells. In summary, LA
showed no IFN- positivity in the absence of exogenous stimuli. These
results, taken together with the observation that stromal IFN-
staining is evident in postmenopausal tissues when LA are absent,
suggests that LA T cells are not the source of constitutive uterine
IFN-.
In an attempt to define the source of constitutive IFN- production,
we examined the morphology and immunophenotype of cells staining
positively for intracellular IFN-. Cells containing intracellular
IFN- immunoreactivity were evident in all patient samples. Some
IFN--positive cells were intraepithelial lymphocytes (IELs), as
defined by their location, morphology, and reactivity with anti-CD3
and anti-CD8, although both the proportion of IELs positive for
IFN- and the number of IELs varied greatly between patients. Another
population of positive stromal cells was evident immediately below the
luminal epithelium, adjacent to the glandular epithelium, and these
appeared to have large vacuoles in their cytoplasm (Fig. 1c). The relative intensity of staining differed
between these two cell types in that the IEL stained much less
intensely (about fivefold less as calculated from mean pixel intensity
in the two cell types) than the stromal cells (Fig. 1d).
Phenotypic analysis of the stromal IFN--producing cell
T cells and NK cells are regarded as the major producers of
IFN-. In an effort to identify whether T cells or NK cells were the
stromal IFN--producing cells, sections were stained with different
lineage-specific mAbs in conjunction with an anti-IFN- Ab. No
dual positive staining was observed either for the T cell markers CD2,
CD3, CD4 and CD8, or using two different NK-specific anti-CD56 mAbs
(data not shown). The IFN--positive cells were CD45 weakly positive.
Anti-CD14 and anti-HLA class II Abs consistently showed no
reactivity with the cytokine-positive cells, indicating that the
cytokine-producing cells were not monocytes or macrophages.
In a further effort to identify the IFN--reactive stromal cell, we
stained intracellularly with monoclonal anti-mast cell tryptase and
anti-eosinophil major basic protein. Both of these cell types have
been reported to produce IFN- (24, 25). Since we had already
concluded that these cells were not macrophages, T cells, or NK cells,
we also included a panel of granulocyte-specific Abs, since PMNs also
express CD11c. Of the five patients studied, no mast cells were evident
in the uterus of one patient, whereas, in agreement with previous
findings (26), a few scattered mast cells were observed in the other
four patients, all of which were IFN- negative. Eosinophils were
present as scattered stromal cells, and a very occasional
IFN--positive eosinophil was observed. Most of the stromal
IFN--reactive cells were weakly positive for CD11c, whereas CD11c
bright cells (presumably macrophages) were negative (Fig. 1e).
IFN--positive cells were intensely positive for CD11b (Fig. 1f) and for CD66b, a marker expressed only on
granulocytes (Fig. 1g). IFN--positive cells were
also positive for CD15 with two mAbs (PMN6 and PMN 29) but negative
using another (PM 81). Stromal IFN--positive cells were positive for
FcRIII (CD16) (Fig. 1h), negative for Fc
RII,
which is highly expressed on eosinophils and on activated monocytes
(not shown), and positive for FcR (CD89) (Fig. 1j). To confirm the identity of the stromal
IFN--positive cell as a PMN, sections were stained with Abs specific
for the markers described above and treated with propidium iodide to
counterstain the nucleus. The results confirmed the presence in all of
the stromal IFN--producing cells of a polymorphonuclear nucleus
usually with at least three distinct lobes (Fig. 1e to
1j).
In summary, in the absence of exogenous stimuli, most of the
IFN--positive cells in the stroma of human endometrium have the
classic multilobed polymorphous nucleus and a CD expression profile
most consistent with a PMN (Table III).
Occasional IFN--positive cells were seen that were positive for
eosinophil major basic protein (MBP) or had a nucleus with eosinophil
morphology, suggesting that these cells may contribute to the pool of
stromal IFN--positive cells.
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by cultured peripheral blood PMN
Production of IFN- by PMN has not previously been described.
Therefore, experiments were conducted to determine whether peripheral
blood PMN were able to produce IFN- following stimulation with
IL-12, a cytokine known to induce IFN- production by NK cells.
Highly purified PMN, with no detectable monocytes or lymphocytes on
Wright’s/Giemsa staining, were cultured for 18 h in the presence
or absence of G-CSF (added to maintain cell viability) and with or
without the addition of IL-12. Following incubation, some cells were
treated with brefeldin A for 4 h. PMN were then stained with FITC
anti-CD66b and intracellularly with Cy5 anti-IFN-. Following
PI counterstaining, the cells were examined by confocal microscopy. The
results show an accumulation of IFN- immunoreactivity in the PMN
population either in the presence (Fig. 1l) or in the
absence of G-CSF (not shown). The intensity of IFN- was greater in
the IL-12 cells. IFN- was barely detectable in cells that received
no brefeldin A (Fig. 1k), suggesting that IFN-
accumulates intracellularly in cells treated with brefeldin. In
brefeldin-treated cells, IFN- positivity revealed a heterogeneity
within the PMN population, in that only one third of PMN were positive.
IFN- staining in brefeldin-treated cells was inhibitable by excess
rIFN- in both PMN (Fig. 3; W = 1368, p =
2.1 x 10-12 Mann-Whitney U test) and in
PHA-stimulated PMNC from the same donor (Fig. 3; W = 1353,
p = 2.3 x 10-7 Mann-Whitney
U test). Staining was also inhibited by preincubation with
polyclonal anti-IFN- (not shown). U937 cells cultured under
identical conditions showed no IFN- staining (not shown). These
results show that peripheral blood PMN produce IFN- and that the
pattern of staining seen in the vibratome sections is consistent with
production of IFN-, rather than accumulation of IFN- from
extracellular sources.
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by ELISA in PMN culture supernatants
To confirm that IFN- was being secreted by PMN, culture
supernatants were assayed following treatment with IL-12, TNF-,
IL-12 and TNF-, or LPS (Fig. 4). The
results show a marginal increase in detected IFN- following IL-12
treatment, more increase following TNF- treatment, and a greater
increase with IL-12 and TNF- or LPS. The level of mononuclear cell
contamination in this experiment was 1% or 7 x
104 cells/ml. Levels of IFN- production by
PHA-stimulated PMNC in this culture system was 75 pg/106
cells (not shown). Therefore, the maximal IFN- production
attributable to contaminating PBMC is likely to be less than 6 pg/ml.
The detected levels of IFN- are therefore considerably lower than
those observed for optimally stimulated T cells and NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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in uterine endometrium
The findings reported here show that most human uterine
endometrial samples have some level of IFN- immune reactivity. These
findings are in agreement with those shown previously for IFN- (15)
and for IFN- mRNA (13). Our finding that IEL are positive for
IFN- is also in agreement with the findings of Stewart et al. (15),
who reported IFN- in IELs in frozen sections, whereas Klein et al.
(13) did not specifically comment on the location of IFN-
mRNA-positive lymphocytes. There are, however, significant differences
between the results presented here and those previously reported for
IFN- staining, particularly with regard to LA reactivity. Stewart et
al. concluded, based on single color immunohistochemistry of sequential
sections, that LA were often positive for IFN- (15). Our results
consistently show no reactivity with LA T cells in freshly isolated
tissues, a finding that is supported by the presence of similar levels
of stromal IFN--positive cells in postmenopausal patients when LA
are absent (7). Although the reasons for these discrepancies are
unclear, they may arise from differential sensitivity between
immunohistochemistry in frozen sections vs the in situ
immunofluorescence in unfixed brefeldin-treated sections reported here
and by the different markers used to identify different cell types. It
is interesting that these authors conclude, as do we, that NK cells
were negative for IFN-, since NK cells are known to be able to
produce large amounts of IFN- (27, 28).
Phenotype of the stromal IFN--positive cell
Our findings show clearly that a high proportion of the stromal
IFN- positive cells are of the granulocyte lineage. The staining
profile and the nuclear morphology strongly suggest that these cells
are PMN (Table III). The possibility that some of these cells are
mature basophils with well-segmented nuclei cannot entirely be ruled
out. This appears unlikely based on the reported low frequency of
basophils in uterine tissue, relative to the number of IFN--positive
cells seen in our study. Whether these IFN--positive granulocytes
are producing IFN- or taking it up from the surrounding tissue
remains to be formally proved by the demonstration of IFN- mRNA in
these cells. That some of the intracellular IFN- may be due to
endocytosis or phagocytosis from the surrounding extracellular matrix
cannot be entirely ruled out. However, a number of arguments can be
made in support of our hypothesis that IFN- is synthesized by
polymorphonuclear granulocytes. First, our results show that the
majority of IFN--positive stromal cells are CD11c positive; it is
therefore likely that this is the same population of cells reported by
Klein et al. (13). These authors concluded that many of the
stromal IFN- mRNA-positive cells were macrophages solely on the
basis of their CD11c staining. CD11c, although highly expressed on
macrophages, is also expressed on granulocytes. Second, these stromal
cells were most positive after treatment with the toxin brefeldin A,
which inhibits translocation of proteins from endoplasmic reticulum to
golgi, leading to an intracellular accumulation of newly synthesized
protein. Thus, the low reactivity seen in the absence of brefeldin A
treatment may have been missed in other studies. Third, the IFN-
positivity of these cells was far greater than the intensity of
staining in IELs or in extracellular locations; if these cells were
taking up IFN- they would have to be actively accumulating it from
the surrounding tissue. Fourth, we show that IFN- production can be
induced in peripheral blood PMN following exposure to IL-12 and that
our ability to detect intracellular IFN- is dependent on brefeldin A
treatment.
In this study we have detected IFN- protein by intracellular and
extracellular immunofluorescence. The validity of these findings is
dependent on the specificity of the staining methods used. That the
staining is specific for IFN- was confirmed by a number of lines of
evidence. First, the same cells were positive in these tissues using
three different IFN--specific mouse mAbs (Table II), a rabbit
IFN--specific polyclonal antiserum (NIH Research reference reagent
G034-501-565), and a goat affinity-purified anti-IFN--specific
polyclonal Ab (R&D). Furthermore, these results are consistent using
either direct staining with different fluorochromes (FITC, PE, or Cy5)
or using indirect staining. In contrast, none of the other Abs used in
this study (anti-MBP, anti-mast cell tryptase, or the isotype
controls) or numerous other mAbs used in studies of uterine endometrium
in this laboratory showed a similar reactivity with PMN. Second,
immunofluorescent staining of vibratome sections using two different
IFN--specific mAbs, each labeled with a different fluorochrome,
colocalized in exactly the same cells. Third, the intracellular
staining with a mAb was inhibited by preincubation with either rabbit
or goat polyclonal anti-IFN- but not with control sera. Fourth,
IFN- staining was inhibited by the addition of excess rIFN-.
Fifth, while our conclusions as to the identity of the
IFN--producing cell type is different from those of Klein et
al.(13), the CD11c positivity of the IFN- mRNA-positive cell
described by these authors is in agreement with our findings.
To our knowledge, this is the first report of IFN- production by a
polymorphonuclear leukocyte. Typically lymphocytes, in the form of T
cells (including IELs) and NK cells, are thought of as
IFN--producing cells (27, 28). Evidence has recently been published
that, in addition to these cell types, cells of the myelocytic lineage
can synthesize IFN-. In addition to human eosinophils and rodent
mast cells (24, 25), it has recently been shown that murine macrophages
produce IFN- response to LPS and IL-12 stimulation (29, 30, 31). PMNs
are traditionally thought of as short-lived terminally differentiated
cells. However, recent studies showing MHC class II expression and
presentation of superantigen suggest that, given certain stimuli in the
form of cytokines and/or adhesion molecule interactions, these cells
may exhibit a much greater range of immunologic function. PMNs are
capable of producing a number of cytokines in response to IFN-
stimulation, including macrophage inflammatory protein (MIP)-1,
MIP-1ß, and IL-12 (32, 33, 34). Induction of IFN- production in PMN
may occur following LPS-triggered IL-12 production in a manner
analogous to that described for murine macrophages (31). The results
presented in this study suggest that tissue PMNs may be capable of
biasing immune responses to a Th1 response by altering the local
cytokine environment. In the particular case of the uterus, Wegmann
proposed that establishment and maintenance of pregnancy requires a Th2
cytokine profile (35). Here, we have shown that IFN- is produced
irrespective of menstrual status, perhaps suggesting that a Th1 bias
represents the default status and that a Th2 switch must occur to
establish pregnancy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Grant R. Yeaman, Department of Microbiology, Dartmouth Medical School, HB7556, 1 Medical Center Drive, Lebanon, NH 03756.
3 Abbreviations used in this paper: LA, lymphoid aggregates; PMN, polymorphonuclear leukocyte; Cy, cyanine; PBA, PBS/1%BSA/0.1% azide; NIH, National Institutes of Health; PI, propidium iodide; IEL, intraepithelial lymphocyte; G-CSF, granulocyte-CSF; MBP, major basic protein; PE, phycoerythrin; PMT, photomultiplier tube.
Received for publication November 3, 1997. Accepted for publication January 23, 1998.
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Y. Hirota, Y. Osuga, K. Koga, O. Yoshino, T. Hirata, C. Morimoto, M. Harada, Y. Takemura, E. Nose, T. Yano, et al. The Expression and Possible Roles of Chemokine CXCL11 and Its Receptor CXCR3 in the Human Endometrium J. Immunol., December 15, 2006; 177(12): 8813 - 8821. [Abstract] [Full Text] [PDF]
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 T. Hirata, Y. Osuga, Y. Hirota, K. Koga, O. Yoshino, M. Harada, C. Morimoto, T. Yano, O. Nishii, O. Tsutsumi, et al. Evidence for the Presence of Toll-Like Receptor 4 System in the Human Endometrium J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 548 - 556. [Abstract] [Full Text] [PDF]
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D. Fairweather, S. Frisancho-Kiss, S. A. Yusung, M. A. Barrett, S. E. Davis, R. A. Steele, S. J. L. Gatewood, and N. R. Rose IL-12 Protects against Coxsackievirus B3-Induced Myocarditis by Increasing IFN-{gamma} and Macrophage and Neutrophil Populations in the Heart J. Immunol., January 1, 2005; 174(1): 261 - 269. [Abstract] [Full Text] [PDF]
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 S. P. Matzer, F. Rodel, R. M. Strieter, M. Rollinghoff, and H. U. Beuscher Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes Int. Immunol., November 1, 2004; 16(11): 1675 - 1683. [Abstract] [Full Text] [PDF]
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 M. Eriksson, S. K. Meadows, C. R. Wira, and C. L. Sentman Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-{beta} J. Leukoc. Biol., September 1, 2004; 76(3): 667 - 675. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Maegawa, T. Kondo, A. Kimura, Y. Iwakura, S. Nakamura, and N. Mukaida Essential Involvement of IFN-{gamma} in Clostridium difficile Toxin A-Induced Enteritis J. Immunol., March 1, 2004; 172(5): 3018 - 3025. [Abstract] [Full Text] [PDF]
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 M. Lessard, M. Lepine, J. J. Matte, M. F. Palin, and J. P. Laforest Uterine immune reaction and reproductive performance of sows inseminated with extended semen and infused with pooled whole dead semen J Anim Sci, November 1, 2003; 81(11): 2818 - 2825. [Abstract] [Full Text] [PDF]
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 A. A. Ashkar and K. L. Rosenthal Interleukin-15 and Natural Killer and NKT Cells Play a Critical Role in Innate Protection against Genital Herpes Simplex Virus Type 2 Infection J. Virol., September 15, 2003; 77(18): 10168 - 10171. [Abstract] [Full Text] [PDF]
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 K. C. Malcolm, P. G. Arndt, E. J. Manos, D. A. Jones, and G. S. Worthen Microarray analysis of lipopolysaccharide-treated human neutrophils Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L663 - L670. [Abstract] [Full Text] [PDF]
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 L. Massai, P. Carbotti, C. Cambiaggi, M. Mencarelli, P. Migliaccio, M. Muscettola, and G. Grasso Prepro-endothelin-1 mRNA and its mature peptide in human appendix Am J Physiol Gastrointest Liver Physiol, February 1, 2003; 284(2): G340 - G348. [Abstract] [Full Text] [PDF]
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P. Luppi, C. Haluszczak, D. Betters, C. A. H. Richard, M. Trucco, and J. A. DeLoia Monocytes are progressively activated in the circulation of pregnant women J. Leukoc. Biol., November 1, 2002; 72(5): 874 - 884. [Abstract] [Full Text] [PDF]
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A. C. Kirby, U. Yrlid, and M. J. Wick The Innate Immune Response Differs in Primary and Secondary Salmonella Infection J. Immunol., October 15, 2002; 169(8): 4450 - 4459. [Abstract] [Full Text] [PDF]
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T. N. Ellis and B. L. Beaman Murine polymorphonuclear neutrophils produce interferon-{gamma} in response to pulmonary infection with Nocardia asteroides J. Leukoc. Biol., August 1, 2002; 72(2): 373 - 381. [Abstract] [Full Text] [PDF]
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K. Venuprasad, P. P. Banerjee, S. Chattopadhyay, S. Sharma, S. Pal, P. B. Parab, D. Mitra, and B. Saha Human Neutrophil-Expressed CD28 Interacts with Macrophage B7 to Induce Phosphatidylinositol 3-Kinase-Dependent IFN-{gamma} Secretion and Restriction of Leishmania Growth J. Immunol., July 15, 2002; 169(2): 920 - 928. [Abstract] [Full Text] [PDF]
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 T. H. Stumpf, R. Case, C. Shimeld, D. L. Easty, and T. J. Hill Primary herpes simplex virus type 1 infection of the eye triggers similar immune responses in the cornea and the skin of the eyelids J. Gen. Virol., June 1, 2002; 83(7): 1579 - 1590. [Abstract] [Full Text] [PDF]
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 A. E. King, D. C. Fleming, H. O.D. Critchley, and R. W. Kelly Regulation of natural antibiotic expression by inflammatory mediators and mimics of infection in human endometrial epithelial cells Mol. Hum. Reprod., April 1, 2002; 8(4): 341 - 349. [Abstract] [Full Text] [PDF]
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 J.-Y. Kwak, S.-W. Park, K.-H. Kim, Y.-J. Na, and K.-S. Lee Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis Hum. Reprod., March 1, 2002; 17(3): 595 - 600. [Abstract] [Full Text] [PDF]
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F. Ethuin, C. Delarche, S. Benslama, M.-A. Gougerot-Pocidalo, L. Jacob, and S. Chollet-Martin Interleukin-12 increases interleukin 8 production and release by human polymorphonuclear neutrophils J. Leukoc. Biol., September 1, 2001; 70(3): 439 - 446. [Abstract] [Full Text] [PDF]
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 M. Christian, P. Marangos, I. Mak, J. McVey, F. Barker, J. White, and J. J. Brosens Interferon-{{gamma}} Modulates Prolactin and Tissue Factor Expression in Differentiating Human Endometrial Stromal Cells Endocrinology, July 1, 2001; 142(7): 3142 - 3151. [Abstract] [Full Text] [PDF]
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C. E. Gargett, F. Lederman, B. Heryanto, L. S. Gambino, and P. A.W. Rogers Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils Hum. Reprod., June 1, 2001; 16(6): 1065 - 1075. [Abstract] [Full Text] [PDF]
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 T. H. Stumpf, C. Shimeld, D. L. Easty, and T. J. Hill Cytokine Production in a Murine Model of Recurrent Herpetic Stromal Keratitis Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 372 - 378. [Abstract] [Full Text]
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I. A. Khan, P. M. Murphy, L. Casciotti, J. D. Schwartzman, J. Collins, J.-L. Gao, and G. R. Yeaman Mice Lacking the Chemokine Receptor CCR1 Show Increased Susceptibility to Toxoplasma gondii Infection J. Immunol., February 1, 2001; 166(3): 1930 - 1937. [Abstract] [Full Text] [PDF]
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A. E. King, R. W. Kelly, H. O. D. Critchley, A. Malmstrom, M. Sennstrom, and R. P. Phipps CD40 Expression in Uterine Tissues: A Key Regulator of Cytokine Expression by Fibroblasts J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 405 - 412. [Abstract] [Full Text]
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 A. Haque, H. Echchannaoui, R. Seguin, J. Schwartzman, L. H. Kasper, and S. Haque Cerebral Malaria in Mice : Interleukin-2 Treatment Induces Accumulation of {{gamma}}{{delta}} T Cells in the Brain and Alters Resistant Mice to Susceptible-Like Phenotype Am. J. Pathol., January 1, 2001; 158(1): 163 - 172. [Abstract] [Full Text] [PDF]
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L. J. Lathbury and L. A. Salamonsen In-vitro studies of the potential role of neutrophils in the process of menstruation Mol. Hum. Reprod., October 1, 2000; 6(10): 899 - 906. [Abstract] [Full Text] [PDF]
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 A. A. Ashkar, J. P. Di Santo, and B. A. Croy Interferon {gamma} Contributes to Initiation of Uterine Vascular Modification, Decidual Integrity, and Uterine Natural Killer Cell Maturation during Normal Murine Pregnancy J. Exp. Med., July 17, 2000; 192(2): 259 - 270. [Abstract] [Full Text] [PDF]
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E. Brandt, G. Woerly, A. B. Younes, S. Loiseau, and M. Capron IL-4 production by human polymorphonuclear neutrophils J. Leukoc. Biol., July 1, 2000; 68(1): 125 - 130. [Abstract] [Full Text]
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 J. A. Gollob, J. W. Mier, K. Veenstra, D. F. McDermott, D. Clancy, M. Clancy, and M. B. Atkins Phase I Trial of Twice-Weekly Intravenous Interleukin 12 in Patients with Metastatic Renal Cell Cancer or Malignant Melanoma: Ability to Maintain IFN-{{gamma}} Induction Is Associated with Clinical Response Clin. Cancer Res., May 1, 2000; 6(5): 1678 - 1692. [Abstract] [Full Text] [PDF]
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 R. M. de Oca, A. J. Buendia, L. Del Rio, J. Sanchez, J. Salinas, and J. A. Navarro Polymorphonuclear Neutrophils Are Necessary for the Recruitment of CD8+ T Cells in the Liver in a Pregnant Mouse Model of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection Infect. Immun., March 1, 2000; 68(3): 1746 - 1751. [Abstract] [Full Text] [PDF]
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C. Bebington, F.J. Doherty, and S.D. Fleming Ubiquitin cross-reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy Mol. Hum. Reprod., October 1, 1999; 5(10): 966 - 972. [Abstract] [Full Text] [PDF]
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G. N. Milligan Neutrophils Aid in Protection of the Vaginal Mucosae of Immune Mice against Challenge with Herpes Simplex Virus Type 2 J. Virol., August 1, 1999; 73(8): 6380 - 6386. [Abstract] [Full Text]
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 Ali. A Ashkar and B. A. Croy Interferon-{gamma} Contributes to the Normalcy of Murine Pregnancy Biol Reprod, August 1, 1999; 61(2): 493 - 502. [Abstract] [Full Text]
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A. J. Buendia, R. Montes de Oca, J. A. Navarro, J. Sanchez, F. Cuello, and J. Salinas Role of Polymorphonuclear Neutrophils in a Murine Model of Chlamydia psittaci-Induced Abortion Infect. Immun., May 1, 1999; 67(5): 2110 - 2116. [Abstract] [Full Text] [PDF]
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A.J. Thomson, M.R. Greer, A. Young, F. Boswell, J.F. Telfer, I.T. Cameron, J.E. Norman, and S. Campbell Expression of intercellular adhesion molecules ICAM-1 and ICAM-2 in human endometrium: regulation by interferon-{gamma} Mol. Hum. Reprod., January 1, 1999; 5(1): 64 - 70. [Abstract] [Full Text] [PDF]
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