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IFN-γ Is Produced by Polymorphonuclear
Neutrophils in Human Uterine Endometrium and
by Cultured Peripheral Blood
Polymorphonuclear Neutrophils
Grant R. Yeaman, Jane E. Collins, Janet K. Currie, Paul M. Guyre,
Charles R. Wira and Michael W. Fanger
J Immunol 1998; 160:5145-5153; ;
http://www.jimmunol.org/content/160/10/5145
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Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
IFN-g Is Produced by Polymorphonuclear Neutrophils in
Human Uterine Endometrium and by Cultured Peripheral
Blood Polymorphonuclear Neutrophils1
Grant R. Yeaman,2* Jane E. Collins,* Janet K. Currie,* Paul M. Guyre,† Charles R. Wira,†
and Michael W. Fanger*
T
he immune system in the tissues of the female reproductive tract is unique because its capacity to respond to infectious challenge or maintain tolerance to semiallogeneic
Ags is controlled by estradiol and progesterone (1, 2). The human
uterus contains the full range of immune cells that are presumed to
function in a manner analogous to their counterparts in other tissues and in peripheral blood. Endometrial monocytes and macrophages are distributed diffusely throughout the uterine stroma in
relatively high numbers and represent 5% to 15% of the endometrial stromal cells (3– 6). Large granular lymphocytes (NK cells)
are also present in the uterine endometrium and substantially increase in number during the late secretory phase of the menstrual
cycle. Uterine lymphocytes are predominantly T cells that are
found throughout the endometrium and also in discrete lymphoid
aggregates (LA)3 (7). LA, located between the bases of glands in
the basalis region, have a unique and organized structure, consistDepartments of *Microbiology and †Physiology, Dartmouth Medical School, Lebanon, NH 03756
Received for publication November 3, 1997. Accepted for publication January
23, 1998.
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 National Institutes of Health Grants AI34478 (to
C.R.W.) and R03 ESO08589-01. Confocal scanning laser microscopy was performed
in the Herbert C. Englert Cell Analysis Laboratory, which was established with a
grant from the Fannie E. Rippel Foundation and is supported in part by the core grant
of the Norris Cotton Cancer Center (CA-23108).
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.
Copyright © 1998 by The American Association of Immunologists
ing of a core of B cells surrounded by CD81 T cells and an outer
halo of monocytes/macrophages. LA are either small or absent
during the early proliferative stage, significantly larger in size at
mid cycle and during the secretory phase of the menstrual cycle,
and absent in postmenopausal women, indicating that LA are under hormonal control. It has been proposed that LA are the source
of IFN-g found in cultures of endometrial cells (8, 9).
Roles for IFN-g in controlling the growth, differentiation, and
immune responsiveness of normal human uterine endometrium
have been proposed (8, 10 –12). Despite this, only three studies,
two looking at mRNA expression (13, 14) and one staining for
protein (15), have shown evidence of IFN-g production in nonpregnant human uterus. Although these studies concluded that T
cells and macrophages were responsible for IFN-g production in
the uterus (13–15), 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-g. We show that, contrary to previous proposals,
LA are not the source of constitutive IFN-g production in uterus.
Surprisingly, the majority of the intracellular IFN-g 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-g, stain
positively for IFN-g.
Materials and Methods
Preparation of vibratome sections
Uterine endometrial tissue was obtained with prior informed consent and
Institutional Review Board approval from patients who were undergoing
0022-1767/98/$02.00
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Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and
in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require
the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand,
a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh
sections of human tissue, we have investigated the production of IFN-g, a Th1 cytokine, in human endometria. Extracellular
IFN-g, 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-g is produced by both stromal cells and
intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-g
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-g in response to stimulation with
LPS, IL-12, and TNF-a. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing
IFN-g 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. The Journal of Immunology, 1998, 160: 5145–5153.
IFN-g IN UTERINE NEUTROPHILS
5146
Table I. Summary of the patient population and IFN-g in tissue sections of different patient populations
Menstrual
Stage
No. of
Patients
Average
Age (yr)
3
46
Leiomyomata (1)
None (2)
Proliferative
20
40
Secretory
10
40
None (4)
Adenomyosis (6)
Leiomyomata (5)
Prolapse (1)
Hyalin sclerosis myometrium (1)
Hyperplasia/leiomyomata (1)
Leiomyomata/adenomyosis (1)
Adenomyomata (1)
None (2)
Adenomyosis (1)
Leiomyomata (5)
Adenomyomata (1)
Leiomyomata/adenomyomata (1)
Inactive
Uterine Pathology
Monoclonal antibodies
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.
Three-color immunophenotyping
Three-color immunofluorescent staining of tissue sections was conducted
immediately after cutting. For direct staining, 2 mg/100 ml 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-g staining
Intracellular staining for constitutive production of IFN-g 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 mg/ml), gentamicin (10 mg/ml), and glutamine (0.291 mg/ml), then exposed to brefeldin
A (100 mg/ml) for 4 h at 37°C (all from Sigma, St. Louis, MO). As a
positive control for IFN-g production, matched sections were exposed to
ionomycin (10 mM; Sigma) and PMA (10 mM; Sigma) during the overnight incubation. Sections were washed extensively with PBA and fixed
Endometriosis
Cervical cancer
Prolapse
Benign (14)
Inflammation (3)
Endometriosis (3)
Benign (8)
Inflammation (1)
Endometriosis (1)
3/3
19/20
10/10
overnight in PBA/1% formaldehyde. After washing, sections were incubated for 2 h at room temperature in 200 ml of PBA containing 0.5%
saponin (Sigma) in the presence of human Ig block (6 mg/ml final) and 2
mg/100 ml of unlabeled mouse monoclonal anti-IFN-g Ab (PharMingen) or
a 1:2000 dilution of a polyclonal rabbit anti-human IFN-g. 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-g-producing cells
To phenotype the IFN-g-producing cells, sections were stained with FITC
or Cy5 lineage-specific mouse mAbs before staining for intracellular IFN-g
with a rabbit polyclonal anti-human IFN-g 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-g 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-g 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-g production. For sections
stained with rabbit polyclonal anti-human IFN-g 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-g
(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-g-producing
cells.
IFN-g 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
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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-mm 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.
Diagnosis
IFN-g
Positive/Total
Assayed
The Journal of Immunology
5147
Table II. Specificities and sources of Abs used for immunophenotyping
Specificity
Clone/Designation
Immune Cell
Expressionb
Suppliera
Fluorochrome
CD1a
CD2
CD2
CD3
CD3
CD3
CD4
CD4
CD8
CD8
CD8
CD10
CD11b
CD11c
CD14
CD14
CD15
CD15
CD15
CD16 (FcgRIII)
CD19
CD23 (FceRIII)
OKT6
T11
G11
OKT3
UCHT1
S4.1
OKT4
S3.5
OKT8
DK25
3B5
5-IB4
CR3(Bear-1)
BL-4h4
AML-2-23
TU K4
PM81
PMN6
PMN29
3G8
SJ25-C1
MHM6
ATCC*
Coulter†
Caltag‡
ATCC§
Dako
Caltag
ATCC
Caltag
ATCC
Dako
Caltag
Caltag
Caltag
Caltag
ATCC
Caltag
DC subset, thymocytes
NK cells, T cell
NK cells, T cell
Pan T cells
Pan T cells
Pan T cells
Th cells
Th cells
T cytotoxic/suppressor
T cytotoxic/suppressor
T cytotoxic/suppressor
Pre-B and B cell subsets, PMNs
PMNs, Mo, NK
Mo, PMNs, Mf
Mo, PMNs, DC, Mf
Mo, PMNs, DC, Mf
Cy3, Cy5
FITC
FITC
Cy3, Cy5
FITC
FITC
Cy3, Cy5
FITC
Cy3, Cy5
FITC
FITC
ATCC
Caltag
FITC, Cy3, Cy5
FITC
CD45
CD56
CD56
CD66b
Ber EP4
HLA class II
HLA class II
HLA class II
11C9.13
Mast cell
Eo MBP
Anti-IFN-g
Anti-IFN-g
Anti-IFN-g
Anti-IFN-g
Anti-IFN-g
HI-30
Leu-1
NKI-nbl-1
80H3
Ber EP4
1Va-12
CR3/43
TU 36
Caltag
B-D¶
Caltag
Immunotech\
Dako
ATCC
Dako
Caltag
Guyre Lab
Dako
Serotec[
Serotec
R&D**
PharMingen††
NIH‡‡
R&D
NK, PMNs, Mo
Precursor B and B cells
B cells, Eos, activated
Mo/Mf
All leukocytes
NK cells
NK cells
PMNs
Epithelial cells
HLA class II
HLA class II
HLA class II
PMNs
Mast cell tryptase
Eos, MBP
IFN-g
IFN-g
IFN-g
IFN-g
IFN-g
FITC
PE
FITC
FITC
FITC
Cy3, Cy5
FITC
FITC
Cy3, Cy5
Cy3, Cy5
FITC, PE
PE
a
Abs were purified from cell culture supernatants of *ATCC cell lines (American Type Culture Collection, Rockville, MD); †Coulter, Kennesaw, GA; ‡Caltag Laboratories,
San Francisco, CA; §Dako, Carpinteria, CA; ¶Becton Dickinson, San Jose, CA; \Immunotech, Westbrook, ME; [Serotec, Washington, DC; **R&D, Minneapolis, MN;
††
PharMingen, San Diego, CA; ‡‡NIH, Bethesda, MD.
b
DC, dendritic cells; Mo, monocytes; Mf, macrophages; Eos, eosinophils; PMNs, granulocytes; alpha, anti-human.
intracellular IFN-g using Cy5-labeled anti-IFN-g 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-g in peripheral blood PMN
The relative amounts of IFN-g 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-g 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-g 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 3 106 PMN/ml
either without additional stimulation, or with the addition of human rIL-12,
human rTNF-a (Genentec) or LPS (List BiologicLab, Campbell, CA). Supernatants were then harvested for ELISA analysis. IFN-g 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-g (Genentech). Statistical analysis of
mean IFN-g production between treatments was by t test using Statgraphics Plus v3 Software (Manugistics).
Results
IFN-g 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-g 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
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AA1
BMK-13
B-B1
25723.11
45.B3
GO34-501
Goat aHu polyclonal
FITC
FITC
Cy3, Cy5
FITC
5148
IFN-g IN UTERINE NEUTROPHILS
women. In particular, extracellular IFN-g 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-g was often fibrous,
suggesting that the cytokine is associated with extracellular matrix
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FIGURE 1. Location, morphology, and phenotype of IFN-g positivity in uterine endometrium and IFN-g expression in peripheral blood PMN following
IL-12 stimulation. IFN-g immune reactivity (red staining) was localized as a broad band beneath the luminal epithelium and along the glandular epithelium
proximal to the luminal epithelium (a). IFN-g was stained, following brefeldin A treatment and permeabilization, using rabbit polyclonal anti-IFN-g
followed by Cy3-labeled goat anti-rabbit second Ab. Epithelial cells were directly stained with FITC-BerEP4 epithelial cell-specific Ab ( green) before
fixation and permeabilization. After optimizing confocal parameters on the IFN-g Ab-stained sections (a), IFN-g staining specificity was controlled for by
reading a preimmune rabbit antiserum-stained control section at the same confocal settings (b). Intracellular IFN-g-reactive cells in the stroma had a
consistent morphology: irregular shape and large negatively stained areas presumed to be large vacuoles or nuclear shadows (c). IFN-g positivity (red) was
found in cells in two distinct locations in relation to the epithelium (Ber-EP4, green): in stromal cells and in some IEL (d). Stromal cells (lower right)
stained much more intensely than IELs (arrows in d). Panels e to j show three-color immunofluorescent staining for IFN-g-positive cells following brefeldin
A treatment in vibratome sections. IFN-g immunoreactivity (blue) was visualized, following saponin permeabilization, by the addition of directly conjugated Cy5-labeled anti-IFN-g Ab (clone B-B1, Serotec), and their nuclei were visualized by counterstaining with propidium iodide (red). Stromal
IFN-g-positive cells consistently exhibited a polymorphonuclear nucleus (e–j). IFN-g-positive cells were consistently weakly positive for CD11c (e, green);
strongly CD11c positive IFN-g negative cells had a size and nuclear morphology consistent with macrophages. IFN-g-positive cells were consistently
strongly positive for CD11b ( f, green) and CD66b ( g, green). IFN-g-positive cells showed an FcgR receptor profile consistent with peripheral blood PMN
in that they were FcgRIII positive (h, green) and FcgRI low or negative (i, green) (two large FcgRI-positive IFN-g-negative cells below a blue IFN-gpositive polymorphonuclear cell are probably macrophages) and FcaR-positive ( j, green). Highly purified peripheral blood PMN treated with brefeldin A
produced IFN-g in overnight cultures in a donor-variable manner (not shown). No positive staining observed in IL-12 stimulated PMN in the absence of
brefeldin A (k). IFN-g staining was, however, present in brefeldin-treated cells, and staining intensity increased in IL-12-treated cells, indicating that
accumulation of detectable levels of IFN-g was dependent on the presence of toxin (l).
The Journal of Immunology
5149
components. Such an association is consistent with the reported
binding of IFN-g to heparin sulfate on endothelial cells described
by others (20 –22). Specificity of staining was confirmed by the
ability of polyclonal goat anti-IFN-g to block staining with mouse
monoclonal anti-IFN-g Ab and by the ability of excess rIFN-g 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).
Cells in uterine LA do not constitutively produce IFN-g
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-g, it has been proposed that these structures are responsible for uterine IFN-g 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-g was not found as-
sociated with the cells in the LA, but intracellular periepithelial
IFN-g 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-g-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-g-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-g positivity
in the absence of exogenous stimuli. These results, taken together
with the observation that stromal IFN-g staining is evident in postmenopausal tissues when LA are absent, suggests that LA T cells
are not the source of constitutive uterine IFN-g.
In an attempt to define the source of constitutive IFN-g production, we examined the morphology and immunophenotype of cells
staining positively for intracellular IFN-g. Cells containing intracellular IFN-g immunoreactivity were evident in all patient samples. Some IFN-g-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-g 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
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FIGURE 2. IFN-g staining in vibratome sections is increased by brefeldin A treatment and
can be inhibited by preincubation with polyclonal
anti-IFN-g. This figure shows four color images.
Each color image has been split into its red (a, d,
g, and j) green (b, e, h, and k) and blue (c, f, i, l)
components to facilitate comparison between images. Specificity of staining for IFN-g was determined by setting confocal parameters at a lower
limit of sensitivity on the same vibratome section
(a to c) stained with PI nuclear counterstain (a),
FITC-IgG1 isotype (b), and Cy5-IgG1 isotype
(c). Using the same settings, images were obtained using PI nuclear counterstain (d, g, and i),
FITC anti-CD66b (e, h, and k), and Cy5 antiIFN-g in sections either untreated ( f), treated
with brefeldin A (i), or brefeldin A treated, incubated with unlabeled goat anti-human antiIFN-g specific polyclonal Ig, following permeabilization but before exposure to mouse antihuman Cy5 anti-IFN-g (clone B-B1, Serotec) (l).
If sections were not brefeldin treated ( f), or were
preincubated with a goat anti-human IFN-g antiserum (l), staining intensity in the Cy5 channel
was markedly reduced.
5150
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-g-producing cell
T cells and NK cells are regarded as the major producers of IFN-g.
In an effort to identify whether T cells or NK cells were the stromal
IFN-g-producing cells, sections were stained with different lineage-specific mAbs in conjunction with an anti-IFN-g 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-g-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-g-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-g (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-g negative. Eosinophils were present as scattered stromal cells, and a
very occasional IFN-g-positive eosinophil was observed. Most of
the stromal IFN-g-reactive cells were weakly positive for CD11c,
whereas CD11c bright cells (presumably macrophages) were negative (Fig. 1e).
IFN-g-positive cells were intensely positive for CD11b (Fig. 1f )
and for CD66b, a marker expressed only on granulocytes (Fig. 1g).
IFN-g-positive cells were also positive for CD15 with two mAbs
(PMN6 and PMN 29) but negative using another (PM 81). Stromal
IFN-g-positive cells were positive for FcgRIII (CD16) (Fig. 1h),
negative for FceRII, which is highly expressed on eosinophils and
on activated monocytes (not shown), and positive for FcaR
(CD89) (Fig. 1j). To confirm the identity of the stromal IFN-gpositive 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-g-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-g-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 IFNg-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-g-positive cells.
IFN-g by cultured peripheral blood PMN
Production of IFN-g by PMN has not previously been described.
Therefore, experiments were conducted to determine whether peripheral blood PMN were able to produce IFN-g following stimulation with IL-12, a cytokine known to induce IFN-g 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-g. Following PI counterstaining, the cells were examined by confocal microscopy. The results show an accumulation
of IFN-g immunoreactivity in the PMN population either in the
presence (Fig. 1l) or in the absence of G-CSF (not shown). The
intensity of IFN-g was greater in the IL-12 cells. IFN-g was barely
detectable in cells that received no brefeldin A (Fig. 1k), suggesting that IFN-g accumulates intracellularly in cells treated with
brefeldin. In brefeldin-treated cells, IFN-g positivity revealed a
heterogeneity within the PMN population, in that only one third of
PMN were positive. IFN-g staining in brefeldin-treated cells was
inhibitable by excess rIFN-g in both PMN (Fig. 3; W 5 1368, p 5
2.1 3 10212 Mann-Whitney U test) and in PHA-stimulated PMNC
from the same donor (Fig. 3; W 5 1353, p 5 2.3 3 1027 MannWhitney U test). Staining was also inhibited by preincubation with
polyclonal anti-IFN-g (not shown). U937 cells cultured under
identical conditions showed no IFN-g staining (not shown). These
results show that peripheral blood PMN produce IFN-g and that
the pattern of staining seen in the vibratome sections is consistent
with production of IFN-g, rather than accumulation of IFN-g from
extracellular sources.
Detection of IFN-g by ELISA in PMN culture supernatants
To confirm that IFN-g was being secreted by PMN, culture supernatants were assayed following treatment with IL-12, TNF-a,
IL-12 and TNF-a, or LPS (Fig. 4). The results show a marginal
increase in detected IFN-g following IL-12 treatment, more increase following TNF-a treatment, and a greater increase with
IL-12 and TNF-a or LPS. The level of mononuclear cell contamination in this experiment was 1% or 7 3 104 cells/ml. Levels of
IFN-g production by PHA-stimulated PMNC in this culture system was 75 pg/106 cells (not shown). Therefore, the maximal
IFN-g production attributable to contaminating PBMC is likely to
be less than 6 pg/ml. The detected levels of IFN-g are therefore
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. IFN-g staining in cultured purified peripheral blood PMN
and PBMC is inhibited by the addition of excess rIFN-g. PMN and PBMC
were purified from peripheral blood on Ficoll density gradients. After culturing without for 24 h (PMN without exogenous stimuli, PBMC with
PHA) and Brefeldin A treatment, cells were stained with FITC anti-CD66b
(PMN) or with FITC anti-CD45 (PBMC). Cells were then fixed/permeabilized and stained with Cy5 anti-IFN-g (1 mg/100 ml) either in the presence or absence of a 20 M excess of rIFN-g. After counterstaining with PI,
cells were mounted on slides in anti-fade. Confocal parameters were set to
just below saturation levels on the unblocked PMN. Images were then
captured to file of three random fields from each of three replicate slides for
each incubation. Each cell was then counted for intensity of blue staining
using Image Space software. Staining intensity was reduced to baseline
levels in both PMN (W 5 1368, p 5 2.1 3 10212 Mann-Whitney U test)
and in PBMC (W 5 1353, p 5 2.3 3 1027 Mann-Whitney U test).
IFN-g IN UTERINE NEUTROPHILS
The Journal of Immunology
5151
Table III. Phenotypic comparison of stromal IFN-g-positive cells with the published phenotypes of potential IFN-g-producing cells a
Ag
2
2
2
2
2
1
1
1
2
1
1
1
2
1b
2
1
1
1
1weak
PMN
Eosinophil
Uterine
Mast
Cell
2
2
2
2
2
1
1
1
1weak
1
1
1
2
2/1
2
1
1
1
2
1
1
2
2
2
2
1
1
1
1
1
1
1
1
?*
2
2
1
1
2
1
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
?
1
1
Basophil
Macrophage
T
Cell
NK
Cell
1 weak
2
2
2
2
2
1
1
2
2
2
1
2
2
2
?
1
?
1
1
2
2
2
1
2
2
1
1
1
1/2
1
1
1/2
1
2
2
1
1
2
1
2
2
1
1/2
1/2
2
1/2
1/2
2
2
2
1
2
2
2/1
2
1
2
2
1
2
2
2
2
2
2
1
1
2
2
1
1
2
2
1
2
2
2
2
1
a
The first data column summarizes the current findings, which are compared to the published phenotypes of the other cells. Expression profiles were drawn from a number
of sources (6, 35).
b
Tissues from approximately 30% of patients are positive.
*?, expression uncertain.
considerably lower than those observed for optimally stimulated T
cells and NK cells.
Discussion
Incidence and location of IFN-g in uterine endometrium
The findings reported here show that most human uterine endometrial samples have some level of IFN-g immune reactivity.
These findings are in agreement with those shown previously for
IFN-g (15) and for IFN-g mRNA (13). Our finding that IEL are
positive for IFN-g is also in agreement with the findings of Stewart
et al. (15), who reported IFN-g in IELs in frozen sections, whereas
FIGURE 4. Detection of IFN-g by
ELISA in PMN culture supernatants
following cytokine and LPS treatment. Representative experiment using PMN from a single healthy male
donor. Isolated peripheral blood 7 3
106 PMN/ml were cultured for 24 h in
AIM-V/5% FCS with either no treatment (untreated), IL-12 (20 U/ml),
TNF-a (300 U/ml), a combination of
IL-12 (20 U/ml) and TNF-a (300
U/ml) or LPS (50 ng/ml). Triplicate
100-ml aliquots of culture supernatants were then assayed by ELISA for
the presence of IFN-g. Results are
shown as the mean IFN-g concentration/106 cells. Error bars represent the
SE of the mean (medium alone
showed no reaction; not shown). Significant increases in IFN-g production at the 95% level (t test) compared with untreated are indicated
by *.
Klein et al. (13) did not specifically comment on the location of
IFN-g mRNA-positive lymphocytes. There are, however, significant differences between the results presented here and those previously reported for IFN-g 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-g (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-g-positive
cells in postmenopausal patients when LA are absent (7). Although
the reasons for these discrepancies are unclear, they may arise
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MBP
tryptase
CD3
CD4
CD8
CD10
CD11b
CD11c
CD14
CD15
CD16 (FcgRIII)
CD18
CD23 (FceRII)
CD64 (FcgRI)
CD56
CD66b
CD32 (FcgRII)
CD89 (FcaR)
FceRI
CD45
Stromal
IFN-g-Positive
IFN-g IN UTERINE NEUTROPHILS
5152
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-g, since
NK cells are known to be able to produce large amounts of IFN-g
(27, 28).
Phenotype of the stromal IFN-g-positive cell
Acknowledgments
We thank the following individuals for their technical and clinical and
scientific support: Dr. Alice Given, Kenneth Orndorf, Dr. Vincent Memoli,
Dr. John Currie, Dr. Stephen Andrews, Dr. Joan Barthold, Dr. Jackson
Beecham, Dr. John Ketterer, Dr. Eileen Kirk, Dr. Benjamin Mahlab, Dr.
Paul Manganiello, Dr. Eric Sailer, Dr. Barry Smith, Dr. William Young,
Jaclyn Logren, Fran Reinfrank, Jeannette Sawyer, Tracy Stokes, Joanne
Lavin, Nancy Leonard, Kris Ramsey, Tamara Krivit, Laura Wolf, Peter
Seery, Maryalice Achbach, Judy Rook, and Esther Colby.
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IFN-g positive cells are of the granulocyte lineage. The staining
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treatment may have been missed in other studies. Third, the IFN-g
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-g they would have to be actively accumulating it from the
surrounding tissue. Fourth, we show that IFN-g production can be
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that our ability to detect intracellular IFN-g is dependent on brefeldin A treatment.
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extracellular immunofluorescence. The validity of these findings is
dependent on the specificity of the staining methods used. That the
staining is specific for IFN-g was confirmed by a number of lines
of evidence. First, the same cells were positive in these tissues
using three different IFN-g-specific mouse mAbs (Table II), a rabbit IFN-g-specific polyclonal antiserum (NIH Research reference
reagent G034-501-565), and a goat affinity-purified anti-IFN-gspecific 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
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uterine endometrium in this laboratory showed a similar reactivity
with PMN. Second, immunofluorescent staining of vibratome sections using two different IFN-g-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-g but not
with control sera. Fourth, IFN-g staining was inhibited by the addition of excess rIFN-g. Fifth, while our conclusions as to the
identity of the IFN-g-producing cell type is different from those of
Klein et al.(13), the CD11c positivity of the IFN-g mRNA-positive
cell described by these authors is in agreement with our findings.
To our knowledge, this is the first report of IFN-g production by
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