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International Immunology Advance Access originally published online on December 20, 2007
International Immunology 2008 20(2):235-245; doi:10.1093/intimm/dxm134
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LPS-activated monocytes suppress T-cell immune responses and induce FOXP3+ T cells through a COX-2–PGE2-dependent mechanism

Tone Bryn1,2,4,*, Sheraz Yaqub1,2,*, Milada Mahic1,2, Karen Henjum1,2,3, Einar M. Aandahl1,2 and Kjetil Taskén1,2

1 Biotechnology Centre of Oslo
2 Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, N-0317 Oslo, Norway
3 Department of Gastroenterological Surgery, Ullevaal University Hospital, Oslo, Norway
4 Present address: AstraZeneca, Oslo, Norway

Correspondence to: K. Taskén; E-mail: kjetil.tasken{at}biotek.uio.no


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Monocytes initiate innate immune responses and interact with T cells to induce antigen-specific immune responses by antigen presentation and secretion of humoral factors. We have previously shown that adaptive regulatory T cells inhibit T-cell effector functions in a cyclooxygenase (COX)-2–prostaglandin E2 (PGE2)-dependent manner and that PGE2 converts resting CD4+CD25– T cells into FOXP3+ T cells with a suppressive phenotype. Here, we demonstrate that stimulation of monocytes with LPS leads to suppression of T-cell immune responses by a COX-2–PGE2-dependent mechanism that is reversible with COX-2 inhibitors as well as PGE2-neutralizing antibody and cAMP antagonist. Furthermore, we show that LPS-activated monocytes induce FOXP3 expression in resting CD4+CD25– T cells by the same pathway. These results suggest that monocytes are able to efficiently suppress T-cell immune responses in a regulatory manner and elicit an inhibitory immune profile.

Keywords: COX-2, FOXP3, human, LPS, monocytes, PGE2


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Upon bacterial infection, monocytes are challenged by antigens that initiate an innate immune response by engagement of toll-like receptors (TLRs). In parallel, monocytes initiate and modulate an adaptive immune response through presentation of bacterial antigens by MHC II. Lipopolysaccharide (LPS), a component of the outer wall of gram-negative bacteria, is a prototypical stimulus for an innate immune response through activation of the TLR4/CD14/MD2 receptor complex leading to production of various cytokines and prostaglandins (PGs) (1, 2). High levels of IL-6 and prostaglandin E2 (PGE2) have been reported in serum from patients with sepsis (3, 4). The cytokine profile greatly influences the adaptive immune response and may vary depending on the type of stimulus and the microenvironment. In this report, we have studied how an innate immune response by LPS-activated monocytes influences adaptive immune responses mediated by T cells.

LPS is the main immunostimulatory component of gram-negative bacteria and may elicit an exaggerated systemic inflammatory response in sepsis (5). Peptidoglycan (PepG) and lipoteichoic acid from the outer wall of gram-positive bacteria may provoke similar inflammatory responses as LPS, and the pathophysiological process of sepsis has many similar features irrespective of the bacterial species (5). In sepsis, the dramatic inflammatory response is harmful for the host and the adaptive antigen-specific immune response is inadequate to achieve control of the infection. It is not clear how cytokines and PGs released systemically in response to bacterial wall constituents may modulate the secondary immune response.

PGs are small lipid molecules with diverse immunoregulatory properties that are generated from arachidonic acid by cyclooxygenase (COX) (6). At least two COX isoforms have been identified, COX-1 and COX-2 (7). COX-1 is constitutively expressed in most human tissues and is considered to be the housekeeping isoform, whereas COX-2 is expressed at low levels and is strongly induced by pro-inflammatory agents, such as LPS, IL-1 and tumor necrosis factor-{alpha} (TNF-{alpha}), and leads to production of PGs at sites of immune activation (8). LPS stimulation of human monocytes leads to COX-2 expression and PGE2 production (9). PGE2 has potent immunomodulatory properties and is mainly synthesized by monocytes and macrophages (6, 10). PGE2 acts locally as an autocrine or paracrine mediator by binding to the G-protein-coupled E prostanoid receptors, EP1-4 (11). In human monocytes, binding of PGE2 to EP2 and EP4 receptors leads to inhibition of LPS-induced TNF-{alpha} generation (12). In T-lymphocytes, PGE2 binds to EP2, EP3 and EP4 receptors. While EP3 receptor binding triggers phospholipase C that elicits Ca2+ release and protein kinase C activation that may activate T cells, activation of EP2 and EP4 triggers adenylyl cyclase causing cAMP production and inhibition of T-cell immune responses (11). Cyclic AMP modulates functional activities such as cytokine production and proliferation in a variety of immune cells. In T cells, cAMP effectively suppresses immune responses by eliciting a cAMP–protein kinase A–Csk inhibitory pathway (13).

CD4+ T cells generally initiate an effective antigen-specific immune response upon proper activation by antigen-presenting cells (ACPs). Under certain conditions, naive CD4+ T cells may acquire immunosuppressive properties and develop into FOXP3+ adaptive regulatory T cells (Tregs) (14). Tregs maintain peripheral T-cell tolerance and suppress T cells, B cells, NK cells, macrophages and dendritic cells (DCs) (15–20). Tregs can be generated both in the thymus (naturally occurring Tregs) and in the periphery (adaptive Tregs) during chronic antigen stimulation (21–23). Exposure to PGE2 has been shown to induce FOXP3 expression in CD4+CD25– T cells and a suppressive regulatory phenotype (24, 25), and recently we have observed that adaptive Tregs may use secretion of PGE2 as their mode of immune suppression (23). Here, we demonstrate that monocytes also can act as immunosuppressive cells. LPS activation of monocytes leads to induction of COX-2 and production of PGE2 that severely suppresses the adaptive T-cell immune response. This may be a generic mechanism whereby an exaggerated inflammatory response to LPS leads to suppression of an antigen-specific immune response mediated by T cells and hampers effective immune control in infections with gram-negative bacteria.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Reagents and antibodies
MACS CD25, CD3 and CD14 microbeads and CD4+CD25+ Treg Isolation Kit were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany) and CD19 Positive Isolation Kit from Invitrogen (Oslo, Norway). LPS, derived from Escherichia coli serotype 026:B6, staphylococcal enterotoxin B (SEB) and brefeldin A (BFA) were purchased from Sigma–Aldrich (St Louis, MO, USA). Anti-CD3{epsilon} (OKT3) antibody was affinity purified from a hybridoma cell line from American Type Culture Collection (CRL-8001; Manassas, VA, USA) and mAb toward CD28 (clone CD28.2) was obtained from Immunotech (Marseille, France). Anti-PGE2 (mAB 2B5) was provided by Cayman Chemical (Ann Arbor, MI, USA) and dissolved in PBS at 2 mg ml–1. PGE2, IL-10 and transforming growth factor-β (TGF-β) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies toward CD3, CD14, CD4, CD25, IFN-{gamma}, TNF-{alpha}, IL-2 and propidium iodide were purchased from BD PharMingen. NS 398 and SC 58125 were from Cayman Chemicals, 8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS) was from Biolog Life Science Institute (Bremen, Germany) and indomethacin from Fluka. COX-2 antibody was obtained from Lab Vision (Fremont, CA, USA), COX-1 antibody from Santa Cruz, Erk1/2 antibody from Upstate and polyclonal mouse anti-human FOXP3 from Abcam Ltd (Cambridge, UK). Anti-human FOXP3 APC for flow cytometry was purchased form eBioscience.

The endotoxin content of all reagents was, when not declared endotoxin free from the manufacturer, determined by the Limulus amoebocyte lysate assay (Cambrex Bio Sciences, Walkersville, MD, USA) and was <5 pg ml–1 in any agent used in cell cultures.

Cell isolation and culture
Human PBMCs were isolated from buffy coat or heparinized whole blood by Isopaque-Ficoll (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) density gradient centrifugation. Monocytes were isolated from PBMCs by positive selection using MACS CD14 microbeads, CD3+ T cells were purified from PBMCs using CD3 microbeads and CD4+CD25– and CD4+CD25+ Tregs were isolated using CD4+CD25+ Treg Isolation Kit (Miltenyi Biotec). Cells were cultured in RPMI 1640 containing 2 mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 100 U penicillin/streptomycin supplemented with 10% heat-inactivated human AB serum (RPMI/10% ABS) (BioWhittaker, Walkersville, MD, USA), 5% CO2, 37°C.

ELISA and cAMP measurements
PBMCs, monocytes or PBMCs depleted of monocytes were activated with LPS (100 ng ml–1). When used, NS 398 (10 µM), SC 58125 (10 µM) or indomethacin (25 µM) were added 90 min prior to LPS activation. Cells were incubated (37°C, 5% CO2) before cell-free supernatants were harvested and PGE2, IL-10 or TGF-β secretion determined using ELISA. The ELISAs were performed in duplicates or triplicates according to the manufacturer’s instruction.

Cyclic AMP radioimmunoassays (cAMP kit from NEN, Boston, MA, USA, catalog no. SP004) were performed in accordance with the manufacturer's instructions. CD3+ T cells were incubated with non-selective phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (200 µM) for 30 min prior to stimulation with cell-free monocyte supernatants for 5 min followed by lysis of cells and subsequent measurements of cAMP.

Intracellular cytokine measurements
Unless otherwise noted human PBMCs were stimulated with LPS (100 ng ml–1) for 20 h prior to SEB activation (3 µg ml–1). The samples were incubated for additional 20 h. BFA (10 µg ml–1) was added to the samples for the last 16–18 h before cells were washed in PBS, fixed (4% PFA, 5 min, 37°C) and permeabilized in FACS permeabilizing buffer (BD Biosciences) for 10 min at room temperature. After washing in PBS with 1% BSA, staining was performed with a mixture of FITC-, PE-, APC- and PerCP-conjugated antibodies for 30 min at 4°C in the dark. The cells were washed before re-suspension in 1% BSA and subsequent sample analysis using a FACSCalibur Instrument (BD PharMingen). The T-cell population was gated for CD3+ cells and analyzed for cytokine expression. When used, NS 398 (10 µM), SC 58125 (10 µM) or indomethacin (25 µM) was added to cell cultures 90 min before addition of LPS. When neutralizing anti-PGE2 mAB 2B5 (100 nM) was used it was added to cell-free monocyte supernatants 30 min prior to transfer to CD4+ T cells.

Human CD3+ T cells were stimulated with cell-free monocyte supernatants for 30 min before being transferred to a 96-well plate coated with OKT3 (2.5 µg ml–1). CD28 antibody (0.5 µg ml–1) was added and the cells were incubated for additional 20 h. The samples were analyzed on FACSCalibur as described above.

Carboxyfluorescein diacetate succinimidyl ester proliferation assay
Human PBMCs (1 x 107 cells ml–1) were labeled with 2 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to stimulation with LPS (100 ng ml–1) for 20 h and further activated with anti-CD3/CD28 or OKT3 (1 µg ml–1) or SEB (2 µg ml–1). The cells were incubated in complete medium for 4 days. The cells were stained for CD3 and CD4 and T-cell division was assessed as CFSE dilution upon analysis on FACSCalibur as described above. When used, indomethacin (25 µM) was added to cell cultures 90 min prior to addition of LPS. When neutralizing anti-PGE2 mAB 2B5 (100 nM) or cAMP antagonist Rp-8-Br cAMPS (1 mM) were used they were added to cell cultures 90 min prior to activation of T cells with anti-CD3/CD28 or OKT3 or SEB.

Induction of FOXP3 in CD4+CD25– T cells
Human CD4+CD25– T cells were stimulated with cell-free monocyte supernatants for the indicated times before analyses of FOXP3 expression with western blotting or by flow cytometry. When stained with anti-human FOXP3 APC, cells were fixed and permeabilized according to the manufacturer's instructions (eBioscience). Supernatants were collected from monocytes that were left untreated, activated with LPS or treated with NS 398 (10 µM) or indomethacin (25 µM) before LPS activation for 20 h.

Western blot analysis
Cells were stimulated as described in Results and expression of proteins was analyzed by immunoblotting for COX-1, COX-2, FOXP3 or Erk1/2. CD4+CD25+ Tregs were used as positive control for FOXP3 and B-cell lysate was used as negative control. Erk1/2 was used as a control for equal protein loading.

Statistical analysis
Data are presented as mean ± SEM or SD and were analyzed by paired samples t-test using SPSS for Windows. Differences with two-sided P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
LPS stimulation of PBMCs leads to suppression of T-cell proliferation and cytokine production
To analyze the functional effect of LPS activation of PBMCs on T-cell responses, we performed co-culture experiments. PBMC cultures were stimulated with LPS for 20 h prior to activation of T cells with SEB for an additional 20 h. Subsequently, cytokine production in CD3+ T cells was measured by intracellular cytokine staining and flow cytometry analyses. We observed that LPS pre-treatment of the PBMC cultures suppressed the SEB-induced TNF-{alpha} and IFN-{gamma} production from T cells by 60 and 50%, respectively (Fig. 1a). To investigate whether the inhibitory effect of LPS activation was time dependent, PBMCs were pre-incubated with LPS for 0, 6, 12, 24 and 48 h prior to activation with SEB. As seen in Fig. 1(b), the maximal inhibition of both TNF-{alpha} and IFN-{gamma} production in CD3+ T cells was seen after 12 and 24 h of pre-treatment, whereas IL-2 production was maximally inhibited at 24 h of pre-incubation. To assess the effect of LPS stimulation of PBMCs on T-cell proliferation, PBMCs were stained with CFSE prior to LPS activation. After additional 20 h, the T cells were activated in co-culture with either anti-CD3/CD28 or OKT3 and further incubated for 4 days. As shown in Fig. 1(c), the T-cell proliferation was inhibited in PBMC cultures incubated with LPS.


Figure 1
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  		Fig. 1. LPS stimulation of PBMC suppresses SEB-induced cytokine production in T cells. (a) PBMCs were stimulated with LPS for 20 h prior to SEB activation. After 16–18 h, the samples were subjected to flow cytometry analyses. The data show the frequency of TNF-{alpha} and IFN-{gamma} expressing CD3+ T cells (mean ± SEM, n = 3–4). (b) PBMCs were pre-stimulated with LPS for 0, 6, 12, 24 or 48 h prior to SEB activation and flow cytometry analyses were performed as described in (a). The data show TNF-{alpha}, IFN-{gamma} and IL-2 expression in CD3+ T cells as percent of SEB-activated cells (not treated with LPS) (single experiment in triplicate). (c) PBMCs were stained with CFSE and incubated with LPS for 20 h before the T cells were activated for 4 days in co-culture. The data show CFSE-positive CD3+CD4+ T cells from one experiment in triplicate representative of n = 4 independent experiments.

 
LPS activation of PBMC leads to induction of COX-2 in monocytes followed by production of PGE2
While it is well established that LPS activates monocytes and leads to production of inflammatory mediators such as PGs and cytokines, the overall effect of these mediators on T-cell activation is not known. PGE2 inhibits T-cell immune functions such as production of Th1 cytokines and cell proliferation, and based on of our recent observation of Tregs suppressing effector T cells through a PGE2-dependent mechanism (23), we wanted to investigate whether monocytes could have a regulatory role on T cells. To determine what cell type in the PBMC cultures that produced PGE2 upon LPS activation and the time kinetics of PGE2 secretion, cultures of PBMCs, monocytes and PBMC depleted of monocytes were stimulated with LPS for 0, 6, 12, 24 and 48 h and PGE2 was measured in the supernatants. As shown in Fig. 2(a), maximal production of PGE2 was reached after 24 h of stimulation and sustained to 48 h of stimulation in both monocyte and PBMC cultures. Little or no PGE2 was produced in the cell cultures where CD14+ monocytes were removed, indicating that LPS-activated monocytes are the main source of PGE2 in these experiments.


Figure 2
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  		Fig. 2. LPS activation of PBMC leads to induction of COX-2 and production of PGE2 from monocytes. (a) Human PBMCs, monocytes and PBMCs depleted of monocytes were activated with LPS for 0, 6, 12, 24 or 48 h followed by measurements of PGE2 in the supernatants. (b) Human PBMCs, monocytes and PBMCs depleted of monocytes were left untreated or stimulated with LPS for 20 h. Cell lysates were analyzed for COX-2 and COX-1 levels by western blotting. Erk-1 and -2 was used as protein-loading control. (c) PBMCs were left untreated or treated with NS 398, SC 58125 or indomethacin before LPS stimulation for 20 h prior to quantification of PGE2 in supernatants. (d) CD3+ T cells were incubated with inhibitor 3-isobutyl-1-methylxanthine prior to stimulation with supernatants from monocyte cultures for 5 min and followed by cell lysis and subsequent measurements of cAMP. (e and f) Human PBMCs were left untreated or pre-treated with NS 398, SC 58125 or indomethacin prior to LPS stimulation (20 h) and quantification of IL-10 (e) and TGF-β (f) in supernatants by ELISA. The data show mean ± half range (n = 2) or SEM. (n = 3).

 
PGE2 is produced from arachidonic acid after induction of COX-2. COX-2 is not expressed in resting cells, but we observed that LPS-activated PBMC and purified CD14+ monocytes expressed COX-2 whereas PBMCs depleted of monocytes did not (Fig. 2b). Monocytes also expressed COX-1, but it did not appear to be the main source of PGE2 production as no PGE2 was produced when the non-specific COX inhibitor indomethacin or the COX-2-specific inhibitors NS 398 and SC 58125 were added to monocyte cultures prior to LPS activation (Fig. 2c). As PGE2 is known to increase cAMP levels in T cells, we next examined the effect of supernatants from LPS-treated monocytes on cAMP production in T cells. When transferred to T-cell cultures, cell-free supernatants from LPS-activated monocytes increased the cAMP level 2-fold compared with supernatants from unstimulated monocytes (Fig. 2d). Supernatants from monocytes treated with COX-2 inhibitors prior to LPS activation did not induce cAMP in T cells, confirming the observation that cAMP production is induced by PGE2. As IL-10 and TGF-β are known to have immunomodulatory functions on T cells (26–29), we examined IL-10 and TGF-β production upon LPS activation of monocytes with or without the addition of COX-2 inhibitors (Fig. 2e and f). IL-10 secretion was induced by LPS and further elevated with COX-2 inhibitors, while monocytes did not produce TGF-β, indicating that LPS-induced suppression of T cells is independent of these cytokines.

LPS activation of monocytes inhibits T-cell proliferation and cytokine production in a COX-2–PGE2-dependent manner
We next investigated whether the inhibition of T-cell cytokine production and proliferation observed in Fig. 1 was mediated via induction of COX-2 and production of PGE2. PBMC cultures were left untreated, LPS activated or treated with COX inhibitors prior to LPS activation followed by T-cell activation with SEB. As seen in Fig. 3(a), LPS pre-treatment suppressed TNF-{alpha} expression in the T cells whereas inhibition of COX-2 with either NS 398 or indomethacin reversed the suppressive effect of LPS. The same phenomenon was observed when T-cell proliferation was measured in PBMC cultures stimulated with anti-CD3 (Fig. 3b, upper panel) or SEB (Fig. 3b, lower panel) after pre-treatment with LPS. The addition of indomethacin prior to LPS stimulation almost fully reversed LPS-mediated suppression of T-cell proliferation. To assess whether the LPS-mediated inhibition of cytokine production is cell contact dependent or if soluble mediators are sufficient to exert the inhibitory effect, monocytes were left untreated, LPS activated or treated with a COX-2 inhibitor prior to LPS activation, and cell-free supernatants were collected and added to purified CD3+ T cells. The T cells were activated by anti-CD3/anti-CD28 and intracellular cytokine production was measured by flow cytometry. As shown in Fig. 3(c), supernatants from LPS-activated monocytes inhibited production of TNF-{alpha} and IFN-{gamma} in CD3+ T cells by 47 and 24%, respectively. In contrast, supernatants from monocytes treated with a COX-2 inhibitor prior to LPS activation did not have any inhibitory effects on cytokine production. These data indicate that LPS activation of monocytes suppresses cytokine production from T cells by a COX-2-dependent mechanism independently of cell contact. The suppression of T-cell activation by LPS-stimulated monocyte supernatants declined upon serial dilution supporting the notion of a soluble inhibitory factor secreted from monocytes (Fig. 4a, right panel). The inhibitory effect of cell-free supernatants from LPS-treated monocytes was reversed by addition of a specific neutralizing anti-PGE2 mAb (2B5) (30), indicating that the soluble factor is PGE2 (Fig. 4a, left and right panels). Moreover, the suppressive effect on T-cell immune responses is mediated via activation of the cAMP–PKA inhibitory pathway as treatment of PBMC with the cAMP antagonist, Rp-8-Br-cAMPS, was able to reverse the inhibitory effects (Fig. 4b). Similar results were observed when T cells were pre-treated with Rp-8-Br-cAMPS prior to addition of supernatants from LPS-treated monocytes and subsequent T-cell activation (Fig. 4c). To assess whether the same mechanism could be involved in suppression of T-cell proliferation, PMBCs stimulated with LPS were treated with neutralizing anti-PGE2 mAb (2B5) or cAMP antagonist (Rp-8-Br-cAMPS) prior to activation with anti-CD3 or SEB. Both agents reversed the LPS-mediated suppression, demonstrating that the COX-2–PGE2–cAMP is the inhibitory mechanism used by LPS-activated monocytes to suppress adaptive immune responses (Fig. 4d).


Figure 3
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  		Fig. 3. LPS activation of monocytes leads to suppression of T cells in a COX-2–PGE2-dependent manner. (a) Human PBMCs were left untreated or stimulated with LPS with or without pre-incubation with NS 398 or indomethacin prior to SEB activation for 20 h followed by flow cytometry analyses. Data from one representative experiment are shown as dot plots and the frequency of TNF-{alpha} expressing CD3+ T cells as a histogram (mean ± SEM, n = 4–5). (b) PBMCs were stained with CFSE and left untreated or treated with indomethacin for 90 min before LPS stimulation for 20 h. Subsequently the PBMC cultures were activated for 4 days with anti-CD3 (upper panels) or SEB (lower panels). The data show CFSE-positive CD4+ T cells from one experiment performed in duplicate and are representative of n = 3 independent experiments. The numbers indicate percentage of non-proliferated cells. (c) Human CD3+ T cells were left untreated or stimulated with monocyte supernatants prior to activation with plate-bound anti-CD3 and soluble anti-CD28 for 20 h before fixation, permeabilization, staining with antibodies and subsequent flow cytometry analyses. The data show the frequency of TNF-{alpha} and IFN-{gamma} expressing CD3+ T cells (mean ± SEM, n = 3 or mean ± half range). Supernatants were collected from monocytes that were left untreated, activated with LPS or treated with NS 398, SC 58125 or indomethacin before LPS activation for 20 h. *P < 0.05, no LPS versus LPS; #P < 0.05 LPS versus LPS + NS 398, LPS + SC 58125 and LPS + indomethacin; determined by paired samples t-test.

 

Figure 4
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  		Fig. 4. LPS activation of monocytes mediates suppression of T-cell responses by secretion of PGE2 and activation of the cAMP–PKA inhibitory pathway. (a) Human monocytes were incubated for 20 h with LPS and cell-free supernatants were collected and incubated with PGE2-neutralizing antibody 2B5 for 30 min. Subsequently, the supernatants were added to CD4+ T cells followed by activation with plate-bound anti-CD3 for 20 h before flow cytometry analyses. The data show the frequency of TNF-{alpha} expressing CD4+ T cells in unstimulated T cells and stimulated in the presence or absence of LPS-stimulated monocyte supernatant (M-sup) or M-sup pre-incubated with 2B5 (left panel). The LPS-stimulated monocyte supernatants suppress T-cell activation in a concentration-dependent manner that is reversible with PGE2-neutralizing antibody 2B5 (right panel) (mean ± half range). (b) LPS-mediated suppression of TNF-{alpha} production from T cells was reversed by addition of Rp-8-Br-cAMPS prior to LPS activation and subsequent SEB stimulation. Data shown are mean ± s.e.m.(n = 3). (c) CD3+ T cells were pre-treated with Rp-8-Br-cAMPS prior to addition of supernatants from monocytes and activation of T cells. Data represent mean ± s.e.m. of three independent cultures. *P < 0.05, SEB versus SEB + LPS; #P < 0.05, SEB + LPS versus SEB + LPS + Rp-8-Br; determined by paired samples t-test. (d) PBMCs were stained with CFSE and stimulated with LPS for 20 h, after which cells were treated with PGE2-neutralizing antibody 2B5 or Rp-8-Br-cAMPS for 90 min prior to stimulation of T cells in co-culture for 4 days with anti-CD3 (upper panels) or SEB (lower panels). The data show CFSE-positive CD4+ T cells from one experiment performed in duplicate representative of n = 3 independent experiments. The numbers indicate percentage of non-proliferated cells.

 
LPS-activated monocytes induce FOXP3+ in CD4+CD25– T cells by a COX-2–PGE2-dependent mechanism
It has recently been demonstrated that PGE2 induces FOXP3 in CD4+CD25– T cells (23, 24). We therefore wanted to examine whether supernatants from LPS-activated monocytes could exert the same effect as they contain high levels of PGE2. Resting CD4+CD25– cells were stimulated with cell-free supernatants from resting or LPS-activated monocytes and as shown in Fig. 5, supernatants from LPS-activated monocytes induced FOXP3 expression in these cells. Furthermore, the FOXP3 induction is mediated in a COX-2–PGE2-dependent manner as the COX-2 inhibitor NS 398 reversed the induction of the transcription factor (Fig. 5a). Flow cytometry analyses revealed an increase in the fraction of T cells expressing FOXP3 from 0.3 to 3% after 3 days whereas 6 days of stimulation induced 5% FOXP3+ T cells (Fig. 5b and data not shown). All FOXP3+ T cells also expressed the CD25 activation marker (Fig. 5b).


Figure 5
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  		Fig. 5. LPS stimulation of monocytes induces CD4+ FOXP3+ T cells by a COX-2–PGE2-dependent mechanism. (a) Human CD4+CD25– T cells were stimulated with monocyte supernatants for 20 h before cell lysates were analyzed for FOXP3 by western blotting. CD4+CD25+ T cells were used as positive control and B-cell lysate as negative control. Monocyte supernatants were collected from monocytes that were left untreated, activated with LPS or treated with NS 398 before LPS activation for 20 h. (b) Human CD4+CD25– T cells were left untreated, stimulated with monocyte supernatants for 6 days before flow cytometry analyses of FOXP3 and CD25 expression. Cell-free supernatants were collected from resting or LPS-activated monocytes (20 h). Data from one representative experiment are shown (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Monocytes are critical in the initial acute inflammatory phase of an immune response to an infectious agent and trigger and modulate the adaptive immune system by cytokine secretion and antigen presentation to T cells. The adaptive immune response complements the innate immune response, and these two processes act in parallel to clear the pathogen. In most circumstances, monocytes act to initiate and reinforce the immune response. However, here we show that LPS activation of monocytes may also suppress T-cell immune responses and induce expression of FOXP3 in resting CD4+CD25– T cells by a PGE2-dependent mechanism. This demonstrates a direct immunosuppressive role of monocytes.

Several studies have demonstrated that gram-negative sepsis leads to a status of relative immune suppression (reviewed in ref. 5). The pathogenesis of sepsis is complex, and several opposing processes occur. In the early stage of sepsis, there is a dramatic pro-inflammatory phase that may contribute to capillary leakage and the systemic inflammatory response syndrome. Later, septic patients may develop a relative immune dysfunction which is associated with poor clinical outcome and mortality (5, 31–34). The immunosuppression of monocytes and macrophages results in decreased phagocytic activity and antigen presentation, reduced bactericidal activity, attenuated cytokine production and increased apoptosis (34–36). Furthermore, sepsis is associated with a monocyte hyporesponsiveness to LPS that appears to be proportional to the severity of sepsis and release of cytokines like PGE2, TGF-β and IL-10, which further leads to decreased HLA-DR expression (4). The associated T-cell dysfunction involves suppressed proliferative responses and cytokine production that may result in inadequate antigen-specific adaptive immune responses (31, 33, 35). The link between LPS stimulation of monocytes and direct inhibition of T-cell immune function has not been clearly established. Here, we show that the overall effect of LPS on monocytes is suppression of T-cell function. The suppressive activity is consistent over time and appears at 6 h after LPS stimulation of monocytes and is present for at least 48 h. Add-back experiments of purified monocytes stimulated with LPS in the absence and presence of COX-2 inhibitors clearly demonstrate that the suppressive activity is attributable to monocytes and is dependent on COX-2 activity in these cells. Addition of COX-2 inhibitors suppressed the production of PGE2 and fully reversed T-cell inhibition. Furthermore, the T-cell inhibition was also reversed when a PGE2-neutralizing mAB or a cAMP antagonist were added to supernatants from LPS-activated monocytes.

TLRs bind a variety of molecular components of microbes that include LPS, PepG, bacterial DNA and outer membrane bacterial lipopeptides (1). Most TLR ligands lead to APC activation that supports Th1 differentiation through the MyD88-dependent pathway (37). Interestingly, TLR signaling may block the action of Tregs as TLR stimulation of DCs leads to secretion of IL-6 as well as other cytokines that protect naive and effector T cells from Treg-mediated immunosuppression (38). TLRs may also modulate T-cell immune function directly in the absence of APCs. LPS stimulation of Tregs has been reported to increase their suppressive activity in addition to enhancing proliferation and survival (39). However, whereas activated CD4+ T cells express TLR4, they are generally not responsive to LPS (40–44). This is consistent with our results as we did not observe any direct effect of LPS on CD4+CD25– T cells.

It has been observed an increased frequency of Tregs in septic patients (45). Recently, it was demonstrated that Tregs inhibit LPS-induced monocyte survival through a Fas/Fas ligand-dependent mechanism that augments the immunosuppression (36). Adaptive Tregs inhibit effector T cells by a contact-independent mechanism (26, 46), and we have recently shown that adaptive Tregs express COX-2 and produce PGE2 that inhibits T-cell responses in a paracrine manner (23). Furthermore, tumor-derived PGE2 and supernatants from COX-2-expressing lung cancer cells that secrete high levels of PGE2 enhance Treg function and induce FOXP3 and a regulatory phenotype in CD4+CD25– T cells (24, 25). Thus, PGE2 may inhibit T-cell immune function by several modes of action, both directly as an inhibitory paracrine cytokine and by inducing and enhancing Treg function. Our results demonstrate that PGE2 produced by LPS-stimulated monocytes directly suppresses T-cell function and may further modulate the phenotype of a sub-population of CD4+ T cells into FOXP3+ T cells. These findings support the notion that LPS-activated monocytes may suppress adaptive immune responses by both (i) secretion of inhibitory cytokines such as PGE2 and (ii) induction of Tregs. Further studies with septic patients will be required to explore the clinical relevance of these findings.

IL-10 is important for the immunomodulatory function of monocytes. IL-10 and TGF-β contribute to an inhibitory cytokine profile that affects both T-cell function and promotes the generation of tolerogenic DCs (47). However, neither cytokine appeared to be directly involved in the suppressive activity of LPS-activated monocytes on T-cell immune responses. The production of IL-10 increased when the cells were treated with COX-2 inhibitors in contrast to the reversal of the suppressive activity. We did not observe any production of TGF-β upon LPS stimulation of monocytes.

Our results demonstrate that monocytes acquire a strong suppressive function upon LPS activation, which is reversed by COX-2 inhibitors. Thus, the PGE2-mediated immunosuppressive activity that develops in parallel with an ongoing immune response may balance the immune reactivity to prevent an exaggerated inflammatory response and ultimately contribute to terminate the immune response once the antigen is cleared. However, in situations where the immune system is unable to establish a proper immune response and control the infection, the compensatory immune suppression may be a double-edged sword that contributes to T-cell dysfunction and inadequate adaptive immune responsiveness.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Norwegian Functional Genomics Programme (158835/S10, 175261/S10, 183675/S10); Research Council of Norway (164023, 164295); Norwegian Cancer Society (C05010); Novo Nordic Foundation Committee; European Union (037189, thera-cAMP).


   Acknowledgements
 
We are grateful for the technical assistance of Gladys M. Tjorhom. S.Y. and M.M. are fellows of the Norwegian Cancer Society.

Funding to pay the Open Access publication charges for this article was provided by the University of Oslo.


   Abbreviations
 
APC, antigen presenting cell
BFA, brefeldin A
CFSE, carboxyfluorescein diacetate succinimidyl ester
COX, cyclooxygenase
DC, dendritic cell
LPS, lipopolysaccharide
PepG, peptidoglycan
PG, prostaglandin
PGE2, prostaglandin E2
SEB, staphylococcal enterotoxin B
TGF-β, growth factor-β
TLR, toll-like receptor
TNF-{alpha}, tumor necrosis factor-{alpha}
Treg, regulatory T cell

   Notes
 
* These authors contributed equally to the study. Back

Transmitting editor: A. Falus Back

Received 25 April 2007, accepted 22 November 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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