Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0604337 on September 30, 2004

Published online before print September 30, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0604337v1
76/6/1220    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McIntire, R. H.
Right arrow Articles by Hunt, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McIntire, R. H.
Right arrow Articles by Hunt, J. S.
(Journal of Leukocyte Biology. 2004;76:1220-1228.)
© 2004 by Society for Leukocyte Biology

Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-ß1

Ramsey H. McIntire*, Pedro J. Morales*, Margaret G. Petroff*, Marco Colonna{dagger} and Joan S. Hunt*,{ddagger},1

* Departments of Anatomy and Cell Biology and
{ddagger} Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas; and
{dagger} Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

1 Correspondence: Department of Anatomy and Cell Biology, Mail Stop 3038, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. E-mail: jhunt{at}kumc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Throughout human pregnancy, activated maternal macrophages producing anti-inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta-derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)-G. Here, we investigated the possibility that two soluble isoforms of HLA-G, HLA-G5 and -G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12-myristate 13-acetate (PMA) and interferon-{gamma} (IFN-{gamma}), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin-like transcript (ILT) receptors for HLA-G, ILT2 and ILT4. Exposure of the PMA/IFN-{gamma}-treated U937 cells to increasing concentrations of recombinant HLA-G5 or -G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)-10 and dramatically increased transforming growth factor-ß1. Differential effectiveness between the isoforms was demonstrated in dose-response studies, as was differential binding to ILT2 and ILT4 in receptor-blocking studies. No effects on production of IL-4, IL-1 receptor antagonist, IL-15, tumor necrosis factor {alpha}, IL-1ß, or IL-6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA-G-producing fetal cytotrophoblast cells.

Key Words: human • macrophage cytokines • pregnancy immunology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During human pregnancy, a delicate immunological balance allows the semiallogeneic fetus and placenta to avoid maternal immune rejection [1 , 2 ]. Production of an array of anti-inflammatory cytokines at the maternal-fetal interface comprises one of the major features of this balance [3 , 4 ]. The cellular sources of the anti-inflammatory cytokines are known to include macrophages [5 ], which consist of a stable population of 10–15% of the cells in the pregnant uterus [6 , 7 ]. The uterine macrophages are well differentiated and are activated, as evidenced by their constitutive expression of human leukocyte antigen (HLA) class II and CD11c antigens [5 ]. Activation is likely to result from their exposure to interferon-{gamma} (IFN-{gamma}), which is a product of uterine natural killer (NK) cells in the pregnant uterus [4 , 8 ].

Factors driving macrophages into their anti-inflammatory profile are unknown, although it seems reasonable to expect that the effector molecules emanate from the fetal cells infiltrating the decidualized uterine endometrium, as it is only following implantation of the blastocyst that anti-inflammatory cytokines begin to predominate [4 ]. One candidate for immune cell programming is HLA-G, a product of infiltrating fetal cytotrophoblast cells. The HLA-G gene has unique features, including generation of multiple isoforms by alternative splicing of a single message giving rise to four membrane-bound isoforms (HLA-G1 through -G4) and two soluble isoforms (HLA-G5 and -G6), generated by the inclusion of intron 4, which encodes a premature stop codon [9 , 10 ] (see Fig. 1 ). Studies now report profound affects on cytolytic T lymphocytes (CTL) with HLA-G driving CTL into functional profiles that prohibit successful attack on foreign cells [11 12 13 14 ]. For the most part, these experiments have focused on the HLA-G1 and HLA-G5 isoforms. However, we recently reported generation of recombinant HLA-G5 (rG5) and HLA-G6 (rG6) and demonstrated that both isoforms inhibit expression of CD8{alpha} by blood mononuclear cells and have no affect on CD3 or cell viability [14 ], thus supporting the idea that HLA-G specifically inhibits CTL function.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Model system. (A) Selected HLA-G mRNA splice variants and their resulting protein products. (Left panel) Schematic depiction of the organization of mRNA splice variants encoding two soluble (HLA-G5 and HLA-G6) isoforms of HLA-G. Asterisks indicate a premature stop sequence encoded within intron 4, which results in the translation of truncated HLA-G proteins that are secreted rather than retained in the plasma membrane. (Right panel) HLA-G5 resembles typical class I structures containing {alpha}1, {alpha}2, and {alpha}3 domains and is associated noncovalently with the light chain [ß2 microglobulin (ß2m)]. HLA-G6 is missing the {alpha}2 domain and may be expressed as homodimerized heavy chains, possibly resembling a class II-like structure; adapted from Fujii et al. [10 ]. (B) Morphology and proliferative activity of U937 cells undergoing differentiation and activation. (Center, bars) 3H-Thymidine incorporation in cells grown in culture medium alone (CM), CM containing 160 nM PMA only (PMA), CM containing PMA followed by 100 U/ml IFN-{gamma} for 24 h (PMA+IFN-{gamma} 24 h), or PMA followed by 100 U/ml IFN-{gamma} for 48 h (PMA+IFN-{gamma} 48 h). Data shown are the mean values obtained in three separate experiments ± SEM. (Inset) Light micrographs of Diff-Quik®-stained U937 cells centrifuged onto glass slides. (Bottom) Viability was measured by trypan blue dye exclusion.

 
As yet, no studies have been performed to investigate the effects of the various isoforms of HLA-G on macrophage function. Peripheral blood monocytes, decidual macrophages, andresting U937 myelomonocytic cells all express the two receptors known to bind HLA-G, which are the immunoglobulin (Ig)-like transcripts (ILT)2 and ILT4 [15 , 16 ]. As macrophages in the pregnant uterus have been continuously exposed to HLA-G from the onset of pregnancy, we used the U937 cell line to study the effects of rG5 and rG6 on macrophage production of cytokines that could influence the immunological balance in the pregnant uterus. The U937 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and activated with IFN-{gamma} to simulate the differentiated, activated state of macrophages in the pregnant uterus [5 ]. The results are supportive of the idea that the two soluble isoforms of HLA-G exert profound effects on macrophages by programming their production of cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The human myelomonocytic cell line U937 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, #CRL-1593.2). The cells were maintained in complete medium consisting of RPMI 1640 (Mediatech, Herndon, VA) containing 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 100 U/ml penicillin, 100 µg/ml streptomycin (GibcoTM, Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Mediatech), 1 mM sodium pyruvate (Mediatech), and 50 µM 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO) and were grown at 37°C, 5% CO2. To induce differentiation and activation, 5 x 104 U937 cells in 500 µl complete medium containing 160 nM PMA (Sigma Chemical Co.) were seeded into each well of BD FalconTM 48-well tissue-culture plates (BD Biosciences, San Jose, CA). After 24 h, the culture supernatants were removed, and the cells were treated with complete medium containing 100 U/ml recombinant human (rh)IFN-{gamma} (R&D Systems, Minneapolis, MN) for 48 h. For measuring responses to rG5 and rG6, culture supernatants were removed after PMA differentiation and IFN-{gamma} activation, and the cells were reactivated with 100 U/ml IFN-{gamma} in the presence or absence of increasing doses of rG5 or rG6 diluted in complete medium. In experiments measuring transforming growth factor-ß1 (TGF-ß1) secretion, cells were differentiated and activated in complete medium, the culture supernatants were removed, and rG5 or rG6 was diluted in macrophage serum-free medium (GibcoTM, Invitrogen) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine to avoid erroneous results due to TGF-ß1 in FBS.

Proliferation assays
Cell proliferation was measured by 3H-thymidine incorporation in U937 cells cultured in Costar® 96-well tissue-culture plates (Corning Inc., Corning, NY) in the presence or absence of PMA and/or IFN-{gamma} [17 ]. Eighteen hours prior to harvesting, 1 µCi 3H-thymidine (ICN Biomedicals, Irvine, CA) was added to each well. At the end of the culture period, cells were lysed and harvested using a semiautomated PHDTM cell harvester (Brandel, Gaithersburg, MD) and counted in a scintillation counter.

Morphology
Untreated U937 cells, which were nonadherent, were harvested and placed on glass slides using the Shandon Cytospin® 2 (Thermo Electron Corp., Woburn, MA). PMA-treated and PMA/IFN-{gamma}-treated cells, which were adherent, were harvested from 100 mm culture dishes (Costar, Corning Inc.) by incubation in CellStripperTM cell dissociation solution (Mediatech) for 30 min on ice followed by scraping (Fisher Scientific, Hanover Park, IL) before centrifuging onto glass slides. After drying overnight at room temperature, slides were stained using the Diff-Quik® stain set (Dade Behring Inc., Newark, DE) following the manufacturer’s instructions.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA extraction from cultured U937 cells and RT were performed as described previously [16 ]. Primers for semiquantitative PCR for ILT2 and ILT4 were designed based on accession numbers U82279 and AF000574, respectively, using PrimerSelect software (DNASTAR Inc., Madison, WI). The ILT2 primer sequences synthesized by Integrated DNA Technologies (Coralville, IA) were: sense 5'-TCT GGG GAA TTC CTG GAC ACA AAG-3', located at 1744–1767, and antisense 5'-CAG CTC CCA TGC ATT CCA GAC TCC-3', located at 1985–2008. The ILT4 primer sequences were: sense 5'-CTC ACT CAA CTC CGA CCC CTA CCT-3', located at 1194–1217, and antisense 5'-TGC AGC AGC CCG AGT GTC CAT CT-3', located at 1637–1659. The primers for ß-actin were synthesized by Integrated DNA Technologies and were based on a previously reported sequence [18 ]. After constructing validation curves to determine the exponential range of amplification for each primer set, PCR was carried out for 30 cycles for ILT2 and ILT4 and for 25 cycles for ß-actin. Authenticity of the ILT2 and ILT4 amplicons was confirmed by purification of the products from the PCR reaction using column chromatography (Bio-Rad Laboratories, Hercules, CA) and sequencing by the fluorescent dideoxy-terminator chemistry method using an ABI Prism DNA sequencing kit (PE Applied Biosystems, Foster City, CA). For quantification of amplified products, photographic negatives of the agarose gels were scanned using the Molecular Dynamics Personal DensitometerTM SI and scanner control, Version 4.0 software (Amersham Biosciences, Piscataway, NJ). Signal intensities from ILT2 and ILT4 amplicons were normalized to signal intensities from ß-actin amplicons within each sample.

Flow cytometry
To determine surface expression of ILT2 and ILT4, nonadherent U937 cells were harvested from culture flasks. Adherent cells undergoing PMA differentiation and/or IFN-{gamma} activation were harvested from Costar® 100 mm culture dishes (Corning Inc.) by incubation in CellStripperTM. Fc receptors (FcRs) were blocked using 40% human blood group AB serum (Sigma Chemical Co.). The cells were then incubated with anti-ILT2 [0.5 µg/ml antileukocyte Ig-like receptor 1 (anti-LIR-1), clone M401, Amgen Inc., Thousand Oaks, CA] or anti-ILT4 (1 µg/ml anti-LIR-2, clone M422, Amgen Inc.). The isotype control consisted of normal mouse IgG1, which was used at 1 µg/ml (PharMingen, San Diego, CA). Binding was identified using phycoerythrin (PE)-conjugated sheep anti-mouse IgG F(ab')2 (1:20 dilution, Sigma Chemical Co.), and results were analyzed using a FACSCalibur flow cytometer with BD CellQuestTM software (BD Biosciences).

Preparation of rG5 and rG6
Generation, purification, and characterization of rG5 and rG6 in human embryonic kidney cells 293 (ATCC, CRL-1573) have been reported [14 ]. Each preparation was tested for endotoxin using the Pyrotell detection gel-clot formation Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA). All doses of recombinant proteins used in functional experiments contained industry-acceptable levels (<0.05 ng/ml) of endotoxin.

Cell viability
Viability during PMA differentiation and IFN-{gamma} activation was measured by trypan blue dye exclusion. To determine the cellular toxicity of increasing doses of rG5 or rG6, cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays (CellTiter96, Promega, Madison, WI) following the manufacturer’s instructions.

Cytokine enzyme-linked immunosorbent assays (ELISAs)
After 24 h in the presence of increasing doses of rG5, rG6, or control medium, supernatant culture media were collected and stored at –80°C until analyzed. Interleukin (IL)-10, TGF-ß1, IL-4, IL-1 receptor antagonist (IL-1ra), IL-15, tumor necrosis factor {alpha} (TNF-{alpha}), IL-1ß, and IL-6 were measured using Quantikine® colorimetric sandwich ELISA kits (R&D Systems) following the manufacturer’s instructions. As IL-15 was not detectable in the supernatant media by this ELISA, we measured surface-bound IL-15 by cell-surface ELISA as described [19 ].

Receptor-blocking studies
Following U937 cell PMA differentiation and IFN-{gamma} activation, supernatant media were aspirated, and FcRs were blocked using 40% human blood group AB serum (Mediatech) for 30 min at 37°C. After aspirating the blocking solution and washing with Hanks’ balanced saline solution (HBSS), monolayers were incubated for 30 min at 37°C in the presence of mouse anti-ILT2 (IgG2b), rat anti-ILT4 (IgM; GHI/75 and 27D6, respectively, gifts from Dr. Marco Colonna, Washington University School of Medicine, St. Louis, MO), mouse IgG2b (PharMingen), or rat IgM (eBioscience, San Diego, CA). After aspirating the antibody solutions and washing with HBSS, cells were treated with 100 U/ml rhIFN-{gamma} (R&D Systems) and 100 nM rG5 or 1000 nM rG6 for 24 h. Preliminary experiments had shown that 100 nM rG5 and 1000 nM rG6 induced TGF-ß1 secretion by approximately threefold compared with control-treated cells (see Fig. 3C and 3D ). Following the 24-h culture period, supernatant media were harvested, and TGF-ß1 production was measured by ELISA. Results were expressed as fold induction of TGF-ß1 secretion after treatment with isotype control antibody or receptor-blocking antibody.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Cytokine production of PMA-differentiated, IFN-{gamma}-activated U937 cells. (A) IL-10 production after 24 h treatment with 100 U/ml IFN-{gamma} and increasing concentrations of rG5 (rHLA-G5). (B) IL-10 production after 24 h treatment with 100 U/ml IFN-{gamma} and increasing concentrations of rG6 (rHLA-G6). (C) TGF-ß1 production after 24 h treatment with 100 U/ml IFN-{gamma} and increasing concentrations of rG5. (D) TGF-ß1 production after 24 h treatment with 100 U/ml IFN-{gamma} and increasing concentrations of rG6. Data shown are the mean values obtained in three separate experiments ± SEM. *, P value <0.05 in comparison with the 0-nM control.

 
Statistical analyses
Receptor expression and cytokine secretion data were subjected to ANOVA using Statistical Analysis Systems (SAS Institute Inc., Cary, NC). Preplanned orthogonal contrast testing was then conducted to determine differences between treatment groups. Receptor blocking data were subjected to ANOVA followed by the Student-Newman-Keuls test using SigmaStat (Jandel Scientific, San Rafael, CA). Data are presented as mean value ± SEM. In all figures, asterisks indicate P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Development of a model system
Our first step toward establishing the biological effects of rG5 and rG6 on human macrophages was to identify an in vitro model system. As decidual macrophages have been exposed previously to trophoblast products, we turned to the U937 myelomonocytic cell line as a model. To mimic the differentiated, activated state of decidual macrophages [5 ], we treated the U937 cells so as to induce these changes. The cells were monitored for evidence of differentiation and activation during culture in the presence of PMA and/or IFN-{gamma}.

Differentiation
As decreased cell division is associated with differentiation, we tested this characteristic of PMA/IFN-{gamma}-treated U937 cells by 3H-thymidine incorporation (Fig. 1B) . Treatment with PMA alone reduced proliferation by 28%. Subsequent treatment with IFN-{gamma} reduced proliferation an additional 35% at 24 h and 66% after 48 h. These reductions were not a result of a loss of cell viability as measured by trypan blue dye exclusion (Fig. 1B , bottom). Thus, PMA arrested proliferation and induced differentiation in U937 cells, and the combination of PMA followed by IFN-{gamma} was even more effective.

Activation
U937 cells cultured in the presence of complete medium, medium plus PMA, or medium plus PMA followed by the addition of 100 U/ml IFN-{gamma} for 24 or 48 h demonstrated profound morphological changes (Fig. 1B , inset). In the presence of PMA, the U937 cells became adherent to plastic tissue-culture dishes but did not undergo any other readily apparent morphological changes. With the addition of IFN-{gamma} to the PMA-treated cultures, cell size increased, nuclear density decreased, the nucleus cytoplasm ratio decreased, cytoplasmic vacuoles were formed, and cytoplasmic blebbing occurred at 24 h, which was more dramatic at 48 h (Fig. 1B , inset). Thus, although PMA and IFN-{gamma} induced differentiation-associated, morphological characteristics, the combination of PMA plus IFN-{gamma} resulted in obvious morphologic changes associated with macrophage activation.

Expression of ILT2 and ILT4 receptors
ILT2 and ILT4 mRNA and protein have been reported in first-trimester decidual macrophages as well as undifferentiated U937 cells [16 ]. It was therefore of prime importance to ascertain whether the differentiated, activated U937 cells maintained expression of these receptors for HLA-G. ILT2 and ILT4 transcripts were monitored throughout differentiation and activation by semiquantitative RT-PCR, and the levels of ILT2 and ILT4 surface expression were monitored by flow cytometry (Fig. 2 ).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Analysis of ILT2 and ILT4 receptor expression in differentiated, activated U937 cells by semiquantitative RT-PCR and flow cytometry. (A) Representative gel from a single experiment conducted in triplicate showing ILT2, ILT4, and ß-actin amplification products. (B) ILT2 and ILT4 signal intensity normalized the ß-actin signal intensity. (C) Representative histogram generated by overlaying the staining profile obtained using anti-ILT2 or anti-ILT4 monoclonal antibodies (mAb; solid lines) with that of isotype control IgG1 (dotted lines). (D) ILT2 and ILT4 median fluorescence intensity (MFI) fold change compared with CM. (B, D) Data shown are means from three independent experiments ± SEM. *, Statistically different from CM (P<0.05); **, statistically different from CM and PMA (P<0.05); ***, statistically different from CM, PMA, and PMA+IFN-{gamma} 24 h. CM, Culture medium alone; PMA, PMA for 24 h followed by CM for 24 h; PMA+IFN-{gamma} 24 h, PMA for 24 h followed by IFN-{gamma} for 24 h; PMA+IFN-{gamma} 48 h, PMA for 24 h followed by IFN-{gamma} for 48 h.

 
ILT2 expression
The abundance of ILT2 transcripts relative to ß-actin transcripts did not change as a result of PMA treatment alone (Fig. 2B) . Although ILT2 mRNA increased after 24 h in the presence of IFN-{gamma} compared with untreated cells, this increase was not maintained at 48 h (Fig. 2B) .

Figure 2C (upper panels) shows a representative histogram illustrating the staining pattern of immunoreactive ILT2 (solid lines) on the cell surface at different stages of differentiation and activation. PMA alone induced a minor but statistically significant change (1.39±0.15-fold increase) in ILT2, but the changes as a result of IFN-{gamma} treatments at 24 h and 48 h were not statistically significant (Fig. 2D) .

ILT4 expression
RT-PCR identified no change in the abundance of ILT4 transcripts relative to ß-actin transcripts with PMA treatment alone (Fig. 2B) . However, ILT4 mRNA was significantly increased as a result of IFN-{gamma} treatment at 24 h and 48 h compared with untreated cells or cells treated with PMA alone (Fig. 2B) .

Figure 2C (lower panels) shows a representative histogram illustrating the staining pattern of immunoreactive ILT4 (solid lines) on the cell surface at different stages of differentiation and activation. Expression of ILT4 was not elevated significantly by PMA alone compared with untreated cells, but IFN-{gamma} increased ILT4 surface expression at 24 h and further increased expression at 48 h. PMA treatment with 24 h of IFN-{gamma} activation caused a statistically significant change in ILT4 (1.44±0.05-fold increase) compared with PMA alone, and 48 h of IFN-{gamma} activation caused a statistically significant change in ILT4 (2.19±0.02) compared with PMA alone and PMA with 24 h IFN-{gamma} treatment (Fig. 2D) .

rG5 and rG6 glycoproteins do not affect viability of differentiated/activated U937 cells
To be certain that our measurements of cytokine production in the U937 cells, differentiated and activated as described above, did not represent changes in cell number, we measured their MTT activity after 24 h in culture with increasing concentrations of rG5 and rG6. Neither rG5 nor rG6 influenced cell number at any concentration (data not shown).

Cytokine production in response to rG5 and rG6
As decidual macrophages are constantly exposed to a gradient of soluble isoforms of HLA-G from implantation until parturition and are known to produce cytokines, we explored a potential causal relationship between these two aspects of decidual macrophage behavior using the U937 model. Following the differentiation/activation protocol described in Materials and Methods, the cells were treated with increasing doses of rG5 or rG6 (or not, controls) and were simultaneously reactivated with 100 U/ml IFN-{gamma}. After 24 h, culture supernatants were collected.

Modulation of IL-10
Figure 3A and 3B , shows that levels of IL-10 were altered by exposing the cells to rG5 or rG6. rG5 (10 nM) slightly, but reproducibly and significantly (P<0.05), increased IL-10 by an average of 14%. This was a unique feature of rG5; rG6 had no stimulatory effect. By contrast, high concentrations of rG5 and rG6 glycoproteins dramatically decreased IL-10 in supernatant culture media, and rG6 had a greater suppressive affect than rG5 (reduction of 55% compared with 27% at 1000 nM).

Modulation of TGF-ß1
Figure 3C and 3D , shows that rG5 and rG6 promoted production of TGF-ß1. Differences between the stimulatory abilities of the two isoforms were again observed. rG5 was effective at low doses, and reproducible increases in TGF-ß1 in the supernatant media occurred with treatment of only 10 nM. Dose-dependent increases were observed with all higher doses of rG5. By contrast, rG6 had no affect until the concentration of the potential modulator was increased to 1000 nM. rG5 at 1000 nM increased TGF-ß1 levels 14-fold, whereas rG6 at 1000 nM increased TGF-ß1 by less than threefold. rG6 had a slight low-dose, inhibitory affect on levels of TGF-ß1, which was not investigated further.

Other cytokines
Secretion of other cytokines produced by macrophages at the maternal-fetal interface, IL-4, IL-1ra, IL-15, TNF-{alpha}, IL-1ß, and IL-6, was also tested. Neither rG5 nor rG6 had any affect on the levels of these cytokines (data not shown). TNF-{alpha} was entirely absent from U937 cell culture supernatant media, and the lack of TNF-{alpha} production in response to rG5 or rG6 treatment supplied further evidence that stocks of rG5 and rG6 did not contain stimulatory levels of endotoxin, as these cells are known to be capable of producing TNF-{alpha} in response to lipopolysaccharide [20 ].

TGF-ß1 production following antibody blocking of ILT2 or ILT4
ILT2 and ILT4 have been shown to bind HLA-G [21 , 22 ], but no studies have compared the receptor preferences of the different isoforms of HLA-G. As 100 nM rG5 and 1000 nM rG6 consistently induced TGF-ß1 secretion by approximately threefold compared with control-treated cells (see Fig. 3C and 3D ), we tested receptor involvement in this response by blocking each receptor’s ability to bind HLA-G using the mAb GHI/75 (anti-ILT2) and 27D6 (anti-ILT4). Differentiated, activated U937 cells were treated with 100 U/ml IFN-{gamma} and 100 nM rG5 or 1000 nM rG6 for 24 h immediately following receptor-blocking treatments.

Figure 4A shows that rG5-stimulated production of TGF-ß1 was inhibited by anti-ILT2 (GHI/75) but that the 25.2% inhibition was not significant. By contrast, rG6-stimulated production of TGF-ß1 was significantly (P<0.05) blocked, demonstrating a reduction of 56.7% by anti-ILT2.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. TGF-ß1 production following antibody blocking of ILT2 or ILT4. (A) Fold induction of TGF-ß1 production by differentiated, activated U937 cells treated with 100 U/ml IFN-{gamma} and 100 nM rG5 (rHLA-G5) or 1000 nM rG6 (rHLA-G6) for 24 h following antibody blocking of ILT2 using GHI/75 or control antibody [mouse IgG2b (mIgG2b)]. (B) Fold induction of TGF-ß1 production by differentiated, activated U937 cells treated with 100 U/ml IFN-{gamma} and 100 nM rG5 or 1000 nM rG6 for 24 h following antibody blocking of ILT4 using 27D6 or control antibody [rat IgM (rIgM)]. Data shown are the mean values obtained in four separate experiments conducted in triplicate ± SEM. *, P value <0.05 in comparison with the control antibody treatment.

 
Figure 4B shows that anti-ILT4 (27D6) did not interfere with rG5- and rG6-stimulated production of TGF-ß1. Although both recombinant isoforms reduced levels of TGF-ß1 in the culture supernatant, (rG5, 33.9%; rG6, 18%), neither result was statistically significant.

Proposed pathways
Figure 5 illustrates a potential network that might operate in the pregnant uterus. It incorporates previous findings and the observations made in this study. In this network, IFN-{gamma} from uterine NK cells activates macrophages, which are then receptive to modulation of function by soluble HLA-G emanating from migrating cytotrophoblast cells. The end result of TGF-ß1 production could be facilitation of immune privilege in the pregnant uterus with concurrent promotion of placental development.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 5. Schematic illustration of local interaction between invading cytotrophoblast cells and macrophages. Cytotrophoblast cells leave the fetal placenta and invade the maternal decidual tissue. Maternal uterine NK (uNK) cells secrete IFN-{gamma}, which activates newly arriving monocytes and resident macrophages. As macrophages come into close association with the cytotrophoblast cells, they are exposed to high concentrations of soluble HLA-G (sHLA-G). Binding of HLA-G to inhibitory receptors stimulates macrophage production of TGF-ß1, which targets maternal immune cells and placenta-derived cells, resulting in lymphocyte tolerance, suppression of inflammation, placental development, and recruitment of monocytes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first to report the responses of macrophage lineage cells to the two soluble isoforms of HLA-G. The results demonstrate dose-dependent effects, with high concentrations of rG5 and rG6 suppressing production of IL-10 and promoting production of TGF-ß1. Although preferences for rG5 remained unclear, we here report that mononuclear phagocytes, as represented by U937 cells, bind rG6 via ILT2.

U937 cells have been used widely for in vitro studies on the macrophage lineage [20 , 23 , 24 ]. This study reports the precise effects of PMA and IFN-{gamma} and demonstrates that PMA/IFN-{gamma}-treated cells resemble tissue macrophages in their morphological and growth characteristics. Throughout PMA differentiation and IFN-{gamma} activation, the U937 cells maintained expression of ILT2 and ILT4, with doubling of the levels of ILT4 surface expression following IFN-{gamma} activation. This observation has not been reported previously and could comprise an important feature of decidual macrophages responding to IFN-{gamma} produced by uterine NK cells (Fig. 5) .

Soluble HLA-G concentrations in sera from pregnant women are on the order of 25–50 ng/ml (1–2 nM) [25 26 27 28 ]. The concentrations that might exist at the maternal-fetal interface are unknown, although they are undoubtedly much higher as a result of the proximity of the placenta. We therefore conducted our studies using a 1 nM to 1000 nM range. This range is similar to those used in previous studies (10 ng/ml–10 µg/ml; 0.5–400 nM), where the sources of soluble HLA-G were placentas, cytotrophoblast cultures, or transfected cells [12 , 13 , 29 , 30 ]. Concentration is clearly of critical importance, as the effects we observed were highly dose-dependent and suggest that the anatomic location of the decidual macrophage relative to the placenta will determine its response to G5 and G6.

It is important that neither rG5 nor rG6 affected the viability of the differentiated/activated U937 cells, a finding that is consistent with a previous report from our laboratory showing that our recombinant soluble HLA-G proteins do not induce apoptosis in IFN-{gamma}-activated blood mononuclear cells [14 ]. Although Fournel et al. [13 ] reported that HLA-G5 kills phytohemagglutinin-stimulated lymphocytes, others have documented no change in CTL viability or other types of immune cells treated with HLA-G [11 , 12 , 31 32 33 34 ]. Relevant to the present investigation, Le Friec et al. [30 ] showed that dendritic cells, which are of myeloid lineage, are also impervious to killing by HLA-G. The discrepancy between the Fournel et al. [13 ] results and those of ours and others suggests that differing responses to HLA-G may result from differences in the responder cell type and the mechanism of activation.

Investigation of isoform-specific effects is critical, as HLA-G5 and -G6 are differentially expressed by placental trophoblast cells [14 ]. This is only the second study to investigate potential isoform-specific effects. The first study by Morales et al. [14 ] showed definitively that the two soluble isoforms function similarly, i.e., both reduce CD8{alpha} expression by blood mononuclear cells and have no affect on CD3 [14 ]. We report similar results here; overall, rG5 and rG6 drove U937 cells into similar patterns of cytokine expression. At high concentrations, which would likely be found locally and in close proximity to migrating cytotrophoblast cells, both isoforms reduced IL-10 and stimulated TGF-ß1.

Although common patterns were the rule, differences were observed in the magnitude of the responses to rG5 and rG6 treatment. rG5 appeared overall to be more stimulatory, demonstrating a slight low-dose stimulation of IL-10 and TGF-ß1, while rG6 had none, and a more profound stimulation of TGF-ß1 at high doses (14-fold increase by rG5; threefold increase by rG6, Fig. 3 ). Instead, the G6 isoform appeared overall to be more suppressive, with a greater inhibition of IL-10 production (rG5, 27%; rG6, 55%) and slight low-dose inhibition of TGF-ß1 production. Thus, the two isoforms differ in their ability to regulate macrophage cytokines. It is possible that this apparent difference in function might be related to their entirely different secondary structures [14 ], a testable hypothesis. Alternatively, the individual isoforms may have evolved to perform individual functions, as appears to be the case with CD45 [35 , 36 ]. Relevant to this possibility, immunohistochemical stains indicate that the two soluble isoforms are localized to different cells in the placenta and extraplacental membranes [14 ].

The cytokines whose levels were altered by rG5 and rG6, IL-10 and TGF-ß1, are believed to be critical to the maintenance of immune privilege at the uteroplacental interface. Regarding IL-10, suppression by rG5 and rG6 was an entirely unexpected result. Decidual macrophages spontaneously produce high levels of IL-10 [37 ], a cytokine known for its ability to counter T helper cell type 1 cytokines. It therefore seemed reasonable to postulate that HLA-G is an inducer. Yet, this was not the case in our model system. The possibility remains that soluble HLA-G might affect production of IL-10 by migrating cytotrophoblast cells [38 ], altering invasion via an impact on matrix metalloproteinase-9 [39 ].

TGF-ß1 is a versatile cytokine known to induce tolerance in peripheral lymphocytes [40 , 41 ], resolve inflammatory responses [42 43 44 ], and promote placental cell growth and differentiation [45 , 46 ]. TGF-ß1 accomplishes these functions by activating fibroblasts, supporting tissue remodeling and angiogenesis [47 , 48 ], and attracting monocytes from the blood circulation [42 , 49 ], all of which are crucial to normal pregnancy. Following recruitment of monocytes to organs and tissues, TGF-ß1 suppresses their cytotoxic functions related to production of IFN-{gamma}, nitric oxide, and reactive oxygen intermediates [42 , 49 ]. Collectively, our data support the concept of placental programming of macrophage production of TGF-ß1, which acts through multiple pathways to generate uteroplacental immune privilege and support placental development.

ILT2 and ILT4 bind a broad range of major histocompatibility complex class I molecules, including HLA-G, and are expressed on many types of leukocytes [15 , 50 51 52 53 ]. As isoform-specific binding has not been previously reported, we were surprised when our receptor-blocking experiments revealed an isoform-specific preference; rG6 clearly used ILT2 to signal production of TGF-ß1. The differential binding of rG5 and rG6 to ILT2 could not be a result of receptor recognition of the {alpha}3 domain [22 , 54 ], as this is present in both isoforms. Possibly, the absence of ß2m in association with G6 heavy chains [14 ] facilitates the interaction of rG6 to ILT2. Furthermore, our expectations had been that ILT4, not ILT2, would be the primary receptor, as reported previously in experiments that used artificial tetramers [21 ]. Yet, although we observed a trend for rG5 to bind ILT4, our results were not statistically significant. More experiments need to be done to determine whether HLA-G5 might signal through an as-yet unidentified receptor. Clearly, much remains to be learned about how HLA-G might signal through these two receptors to obtain cell type-specific outcomes.

In summary, these studies introduce new concepts relative to the immunology of pregnancy. We here provide experimental data in support of the idea that immune privilege in the pregnant uterus rests on programming maternal uterine immune cells by HLA-G, a product of the fetal placenta.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institute of Child Health and Human Development to J. S. H. (HD26429, HD35859, HD39878) and by a fellowship from the KUMC Biomedical Training Program to R. H. M. The contributions of the U54 Reproductive Sciences Center (HD33994) and the Kansas Biomedical Research Infrastructure Network (P20 RR16475) to the performance of these studies are greatly appreciated. The authors appreciate the gifts of mAb M401 (anti-LIR-1) and M422 (anti-LIR-2) from Amgen Inc. and collection of flow cytometry data by J. Pace (University of Kansas Medical Center, Kansas City, KS).

Received June 11, 2004; revised August 20, 2004; accepted September 8, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hunt, J., Johnson, P. M. (1999) Immunology of reproduction Knobil, E. Neill, J. D. eds. Encyclopedia of Reproduction 2,798-806 Academic London, UK.
  2. Hunt, J., Petroff, M. G. (2003) Placental immunology Henry, H. Norman, A. W. eds. Encyclopedia of Hormones 3,224-231 Academic London, UK.
  3. Marzi, M., Vigano, A., Trabattoni, D., Villa, M. L., Salvaggio, A., Clerici, E., Clerici, M. (1996) Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy Clin. Exp. Immunol. 106,127-133[CrossRef][Medline]
  4. Bowen, J. M., Chamley, L., Mitchell, M. D., Keelan, J. A. (2002) Cytokines of the placenta and extra-placental membranes: biosynthesis, secretion and roles in establishment of pregnancy in women Placenta 23,239-256[CrossRef][Medline]
  5. Hunt, J. S., Robertson, S. A. (1996) Uterine macrophages and environmental programming for pregnancy success J. Reprod. Immunol. 32,1-25[CrossRef][Medline]
  6. Vince, G. S., Starkey, P. M., Jackson, M. C., Sargent, I. L., Redman, C. W. (1990) Flow cytometric characterisation of cell populations in human pregnancy decidua and isolation of decidual macrophages J. Immunol. Methods 132,181-189[CrossRef][Medline]
  7. Hunt, J. S., Petroff, M. G., Burnett, T. G. (2000) Uterine leukocytes: key players in pregnancy Semin. Cell Dev. Biol. 11,127-137[CrossRef][Medline]
  8. Veith, G. L., Rice, G. E. (1999) Interferon {gamma} expression during human pregnancy and in association with labour Gynecol. Obstet. Invest. 48,163-167[CrossRef][Medline]
  9. Ishitani, A., Geraghty, D. E. (1992) Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens Proc. Natl. Acad. Sci. USA 89,3947-3951[Abstract/Free Full Text]
  10. Fujii, T., Ishitani, A., Geraghty, D. E. (1994) A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4 J. Immunol. 153,5516-5524[Abstract]
  11. Le Gal, F. A., Riteau, B., Sedlik, C., Khalil-Daher, I., Menier, C., Dausset, J., Guillet, J. G., Carosella, E. D., Rouas-Freiss, N. (1999) HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes Int. Immunol. 11,1351-1356[Abstract/Free Full Text]
  12. Kapasi, K., Albert, S. E., Yie, S., Zavazava, N., Librach, C. L. (2000) HLA-G has a concentration-dependent effect on the generation of an allo-CTL response Immunology 101,191-200[CrossRef][Medline]
  13. Fournel, S., Aguerre-Girr, M., Huc, X., Lenfant, F., Alam, A., Toubert, A., Bensussan, A., Le Bouteiller, P. (2000) Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8 J. Immunol. 164,6100-6104[Abstract/Free Full Text]
  14. Morales, P. J., Pace, J. L., Platt, J. S., Phillips, T. A., Morgan, K., Fazleabas, A. T., Hunt, J. S. (2003) Placental cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype J. Immunol. 171,6215-6224[Abstract/Free Full Text]
  15. Borges, L., Hsu, M. L., Fanger, N., Kubin, M., Cosman, D. (1997) A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules J. Immunol. 159,5192-5196[Abstract]
  16. Petroff, M. G., Sedlmayr, P., Azzola, D., Hunt, J. S. (2002) Decidual macrophages are potentially susceptible to inhibition by class Ia and class Ib HLA molecules J. Reprod. Immunol. 56,3-17[CrossRef][Medline]
  17. James, S. P. (1994) Measurement of proliferative responses of cultured lymphocytes Coligan, J. E. Kruisbeek, A. M. Margulies, D. H. Shevach, E. M. Strober, W. eds. Current Protocols in Immunology 1,7.10.5 John Wiley & Sons Hoboken, NJ.
  18. Nandan, D., Reiner, N. E. (1997) TGF-ß attenuates the class II transactivator and reveals an accessory pathway of IFN-{gamma} action J. Immunol. 158,1095-1101[Abstract]
  19. Musso, T., Calosso, L., Zucca, M., Millesimo, M., Ravarino, D., Giovarelli, M., Malavasi, F., Ponzi, A. N., Paus, R., Bulfone-Paus, S. (1999) Human monocytes constitutively express membrane-bound, biologically active, and interferon-{gamma}-upregulated interleukin-15 Blood 93,3531-3539[Abstract/Free Full Text]
  20. Ishizuka, T., Hirata, I., Adachi, M., Kurimoto, F., Hisada, T., Dobashi, K., Mori, M. (1995) Effects of interferon-{gamma} on cell differentiation and cytokine production of a human monoblast cell line, U937 Inflammation 19,627-636[CrossRef][Medline]
  21. Allan, D. S., Colonna, M., Lanier, L. L., Churakova, T. D., Abrams, J. S., Ellis, S. A., McMichael, A. J., Braud, V. M. (1999) Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells J. Exp. Med. 189,1149-1156[Abstract/Free Full Text]
  22. Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Braud, V. M., Allan, D. S., Makadzange, A., Rowland-Jones, S., Willcox, B., Jones, E. Y., van der Merwe, P. A., Kumagai, I., Maenaka, K. (2003) Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G Proc. Natl. Acad. Sci. USA 100,8856-8861[Abstract/Free Full Text]
  23. Vrana, J. A., Saunders, A. M., Chellappan, S. P., Grant, S. (1998) Divergent effects of bryostatin 1 and phorbol myristate acetate on cell cycle arrest and maturation in human myelomonocytic leukemia cells (U937) Differentiation 63,33-42[CrossRef][Medline]
  24. Chabot, S., Charlet, D., Wilson, T. L., Yong, V. W. (2001) Cytokine production consequent to T cell–microglia interaction: the PMA/IFN {gamma}-treated U937 cells display similarities to human microglia J. Neurosci. Methods 105,111-120[CrossRef][Medline]
  25. Rebmann, V., Pfeiffer, K., Passler, M., Ferrone, S., Maier, S., Weiss, E., Grosse-Wilde, H. (1999) Detection of soluble HLA-G molecules in plasma and amniotic fluid Tissue Antigens 53,14-22[CrossRef][Medline]
  26. Hunt, J. S., Jadhav, L., Chu, W., Geraghty, D. E., Ober, C. (2000) Soluble HLA-G circulates in maternal blood during pregnancy Am. J. Obstet. Gynecol. 183,682-688[CrossRef][Medline]
  27. Pfeiffer, K. A., Rebmann, V., Passler, M., van der Ven, K., van der Ven, H., Krebs, D., Grosse-Wilde, H. (2000) Soluble HLA levels in early pregnancy after in vitro fertilization Hum. Immunol. 61,559-564[CrossRef][Medline]
  28. Rebmann, V., van der Ven, K., Passler, M., Pfeiffer, K., Krebs, D., Grosse-Wilde, H. (2001) Association of soluble HLA-G plasma levels with HLA-G alleles Tissue Antigens 57,15-21[CrossRef][Medline]
  29. Contini, P., Ghio, M., Poggi, A., Filaci, G., Indiveri, F., Ferrone, S., Puppo, F. (2003) Soluble HLA-A, -B, -C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation Eur. J. Immunol. 33,125-134[CrossRef][Medline]
  30. Le Friec, G., Laupeze, B., Fardel, O., Sebti, Y., Pangault, C., Guilloux, V., Beauplet, A., Fauchet, R., Amiot, L. (2003) Soluble HLA-G inhibits human dendritic cell-triggered allogeneic T-cell proliferation without altering dendritic differentiation and maturation processes Hum. Immunol. 64,752-761[CrossRef][Medline]
  31. Riteau, B., Menier, C., Khalil-Daher, I., Sedlik, C., Dausset, J., Rouas-Freiss, N., Carosella, E. D. (1999) HLA-G inhibits the allogeneic proliferative response J. Reprod. Immunol. 43,203-211[CrossRef][Medline]
  32. Bainbridge, D. R., Ellis, S. A., Sargent, I. L. (2000) HLA-G suppresses proliferation of CD4(+) T-lymphocytes J. Reprod. Immunol. 48,17-26[CrossRef][Medline]
  33. Lila, N., Rouas-Freiss, N., Dausset, J., Carpentier, A., Carosella, E. D. (2001) Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism Proc. Natl. Acad. Sci. USA 98,12150-12155[Abstract/Free Full Text]
  34. Wiendl, H., Mitsdoerffer, M., Hofmeister, V., Wischhusen, J., Weiss, E. H., Dichgans, J., Lochmuller, H., Hohlfeld, R., Melms, A., Weller, M. (2003) The non-classical MHC molecule HLA-G protects human muscle cells from immune-mediated lysis: implications for myoblast transplantation and gene therapy Brain 126,176-185[Abstract/Free Full Text]
  35. Clement, L. T. (1992) Isoforms of the CD45 common leukocyte antigen family: markers for human T-cell differentiation J. Clin. Immunol. 12,1-10[CrossRef][Medline]
  36. Trowbridge, I. S., Thomas, M. L. (1994) CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development Annu. Rev. Immunol. 12,85-116[CrossRef][Medline]
  37. Heikkinen, J., Mottonen, M., Komi, J., Alanen, A., Lassila, O. (2003) Phenotypic characterization of human decidual macrophages Clin. Exp. Immunol. 131,498-505[CrossRef][Medline]
  38. Roth, I., Corry, D. B., Locksley, R. M., Abrams, J. S., Litton, M. J., Fisher, S. J. (1996) Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10 J. Exp. Med. 184,539-548[Abstract/Free Full Text]
  39. Roth, I., Fisher, S. J. (1999) IL-10 is an autocrine inhibitor of human placental cytotrophoblast MMP-9 production and invasion Dev. Biol. 205,194-204[CrossRef][Medline]
  40. Nakamura, K., Kitani, A., Fuss, I., Pedersen, A., Harada, N., Nawata, H., Strober, W. (2004) TGF-ß 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice J. Immunol. 172,834-842[Abstract/Free Full Text]
  41. Stassen, M., Schmitt, E., Jonuleit, H. (2004) Human CD(4+)CD(25+) regulatory T cells and infectious tolerance Transplantation 77,S23-S25[CrossRef][Medline]
  42. Letterio, J. J., Roberts, A. B. (1998) Regulation of immune responses by TGF-ß Annu. Rev. Immunol. 16,137-161[CrossRef][Medline]
  43. Prud’homme, G. J., Piccirillo, C. A. (2000) The inhibitory effects of transforming growth factor-ß-1 (TGF-ß1) in autoimmune diseases J. Autoimmun. 14,23-42[CrossRef][Medline]
  44. Huynh, M. L., Fadok, V. A., Henson, P. M. (2002) Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-ß1 secretion and the resolution of inflammation J. Clin. Invest. 109,41-50[CrossRef][Medline]
  45. Meisser, A., Chardonnens, D., Campana, A., Bischof, P. (1999) Effects of tumour necrosis factor-{alpha}, interleukin-1 {alpha}, macrophage colony stimulating factor and transforming growth factor ß on trophoblastic matrix metalloproteinases Mol. Hum. Reprod. 5,252-260[Abstract/Free Full Text]
  46. Ingman, W. V., Robertson, S. A. (2002) Defining the actions of transforming growth factor ß in reproduction Bioessays 24,904-914[CrossRef][Medline]
  47. Watson, A. L., Palmer, M. E., Burton, G. J. (1996) An in vitro model for the study of wound healing in first trimester human placenta Cell Tissue Res 286,431-438[CrossRef][Medline]
  48. Cox, D. A., Maurer, T. (1997) Transforming growth factor-ß Clin. Immunol. Immunopathol. 83,25-30[CrossRef][Medline]
  49. Ashcroft, G. S. (1999) Bidirectional regulation of macrophage function by TGF-ß Microbes Infect 1,1275-1282[CrossRef][Medline]
  50. Colonna, M., Navarro, F., Bellon, T., Llano, M., Garcia, P., Samaridis, J., Angman, L., Cella, M., Lopez-Botet, M. (1997) A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells J. Exp. Med. 186,1809-1818[Abstract/Free Full Text]
  51. Colonna, M., Samaridis, J., Cella, M., Angman, L., Allen, R. L., O’Callaghan, C. A., Dunbar, R., Ogg, G. S., Cerundolo, V., Rolink, A. (1998) Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules J. Immunol. 160,3096-3100[Abstract/Free Full Text]
  52. Fanger, N. A., Cosman, D., Peterson, L., Braddy, S. C., Maliszewski, C. R., Borges, L. (1998) The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes Eur. J. Immunol. 28,3423-3434[CrossRef][Medline]
  53. Navarro, F., Llano, M., Bellon, T., Colonna, M., Geraghty, D. E., Lopez-Botet, M. (1999) The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells Eur. J. Immunol. 29,277-283[CrossRef][Medline]
  54. Chapman, T. L., Heikeman, A. P., Bjorkman, P. J. (1999) The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18 Immunity 11,603-613[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Reproductive SciencesHome page
R. H. McIntire, K. G. Ganacias, and J. S. Hunt
Programming of Human Monocytes by the Uteroplacental Environment
Reproductive Sciences, May 1, 2008; 15(5): 437 - 447.
[Abstract] [PDF]

Home page
 J. Immunol.Home page
G. I. Bondarenko, D. W. Burleigh, M. Durning, E. E. Breburda, R. L. Grendell, and T. G. Golos
Passive Immunization against the MHC Class I Molecule Mamu-AG Disrupts Rhesus Placental Development and Endometrial Responses
J. Immunol., December 15, 2007; 179(12): 8042 - 8050.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Naji, S. Le Rond, A. Durrbach, I. Krawice-Radanne, C. Creput, M. Daouya, J. Caumartin, J. LeMaoult, E. D. Carosella, and N. Rouas-Freiss
CD3+CD4low and CD3+CD8low are induced by HLA-G: novel human peripheral blood suppressor T-cell subsets involved in transplant acceptance
Blood, December 1, 2007; 110(12): 3936 - 3948.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
T. V. F. Hviid
HLA-G in human reproduction: aspects of genetics, function and pregnancy complications
Hum. Reprod. Update, May 1, 2006; 12(3): 209 - 232.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. Lafon, C. Prehaud, F. Megret, M. Lafage, G. Mouillot, M. Roa, P. Moreau, N. Rouas-Freiss, and E. D. Carosella
Modulation of HLA-G Expression in Human Neural Cells after Neurotropic Viral Infections
J. Virol., December 15, 2005; 79(24): 15226 - 15237.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
H. Wiendl, U. Feger, M. Mittelbronn, C. Jack, B. Schreiner, C. Stadelmann, J. Antel, W. Brueck, R. Meyermann, A. Bar-Or, et al.
Expression of the immune-tolerogenic major histocompatibility molecule HLA-G in multiple sclerosis: implications for CNS immunity
Brain, November 1, 2005; 128(11): 2689 - 2704.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Le Discorde, C. Le Danff, P. Moreau, N. Rouas-Freiss, and E. D. Carosella
HLA-G*0105N Null Allele Encodes Functional HLA-G Isoforms
Biol Reprod, August 1, 2005; 73(2): 280 - 288.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
J. S. Hunt, M. G. Petroff, R. H. McIntire, and C. Ober
HLA-G and immune tolerance in pregnancy
FASEB J, May 1, 2005; 19(7): 681 - 693.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0604337v1
76/6/1220    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire