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The Journal of Immunology, 2007, 179, 6305 -6310
Copyright © 2007 by The American Association of Immunologists, Inc.
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HVEM Signaling in Monocytes Is Mediated by Intracellular Calcium Mobilization1

Sook-Kyoung Heo*, Min-A Yoon{dagger}, Sang-Chul Lee{ddagger}, Seong-A Ju{ddagger}, Jang-Hyun Choi§, Pann-Ghill Suh§, Byoung S. Kwon*,§ and Byung-Sam Kim2,*,{dagger},{ddagger}

* Department of Biomedicine, {dagger} Department of Biological Sciences and {ddagger} Immunomodulation Research Center, University of Ulsan, Ulsan, South Korea; § Department of Life Science, Pohang University of Science and Technology, Hyojadong, Pohang, Kyungbuk, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Herpes virus entry mediator (HVEM) is a member of the TNF receptor (TNFR) superfamily and is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Interaction of HVEM with its ligand, LIGHT, costimulates T cells and increases the bactericidal activity of monocytes and neutrophils. The interaction recruits cytoplasmic TNFR-associated factor adaptor proteins to the intracellular domain of HVEM. This leads to NF{kappa}B activation as a result of I{kappa}B{alpha} degradation and/or JNK/AP-1 activation, and ultimately results in the expression of genes required for cell survival, cytokine production, or cell proliferation. In this study, we show that treatment of human monocytes with recombinant human LIGHT (rhLIGHT) induces rapid elevation of intracellular calcium concentration ([Ca2+]i) in a HVEM-specific manner in parallel with TNF-{alpha} production, and enhances the bactericidal activities of monocytes. Immunoprecipitation and Western blotting analyses revealed phosphorylation of phospholipase C{gamma}1 (PLC{gamma}1) but not PLC{gamma}2. rhLIGHT-induced Ca2+response was completely abolished by silencing PLC{gamma}1, or preincubating monocytes with PLC inhibitors, antagonists of the inositol-1,4,5-triphosphate receptor, or [Ca2+]i chelators. Furthermore, these PLC/Ca2+ inhibitors also blocked rhLIGHT-mediated I{kappa}B{alpha} degradation, generation of reactive oxygen species, TNF-{alpha} production and the bactericidal activities of monocytes. Our results indicate that Ca2+is a downstream mediator of the LIGHT/HVEM interaction in monocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes),3 a member of the TNF ligand superfamily, is predominantly expressed on immune cells such as T cells, monocytes, NK cells, and immature dendritic cells (1). LIGHT binds to two membrane receptors, HVEM and lymphotoxin beta receptor (LTbetaR) (2). HVEM is also expressed on immune cells, but LTbetaR is only expressed on stromal cells in the spleen (3). Therefore, LIGHT/HVEM interactions between immune cells are thought to regulate a variety of immune responses. For example, the LIGHT/HVEM interaction costimulates T cell proliferation and cytokine production (4), induces dendritic cell maturation (5), stimulates Ig production in B cells (6), and activates NK cells (7). Previously, we have also shown that this interaction contributes to the antibacterial activity of monocytes and neutrophils by enhancing phagocytosis, the production of ROS, NO, and proinflammatory cytokines, and bactericidal activity (8).

Upon binding of LIGHT, it has been shown adaptor proteins referred to as TNFR-associated factors (TRAFs) are recruited to the cytoplasmic domains of HVEM (9), which leads to NF{kappa}B activation (4). However, the relevance of this signaling pathway to immune responses mediated by HVEM has not been specifically demonstrated. Recently, we observed that inhibitors of NAD(P)H oxidase blocked HVEM-mediated activation of monocytes and neutrophils (8). Because the activity of NAD(P)H oxidase can be regulated by the intracellular calcium ion concentration ([Ca2+]i), we speculated that HVEM signaling in monocytes might involve Ca2+. Monocytes have the highest level of HVEM of any immune cell (8). They play critical roles in clearing bacteria from infection sites and contribute to a wide range of immune and homeostatic functions in which Ca2+ is a major signal mediator.

In this study, we report that in human monocytes, HVEM stimulation by rhLIGHT induces rapid [Ca2+]i rise in parallel with tyrosine phosphorylation of phospholipase C{gamma}1 (PLC{gamma}1). Silencing PLC{gamma}1 or HVEM by small interfering RNA (siRNA) abrogated rhLIGHT-induced calcium responses and incubation of cells with PLC/[Ca2+]i inhibitors such as, U73122, genistein, xestospongin C (XeC) and BAPTA-AM abolished recombinant human LIGHT (rhLIGHT)-induced NF{kappa}B activation, production of TNF-{alpha} and ROS, and bactericidal activity, as well as the Ca2+responses of monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

rhLIGHT was purchased from R & D Systems, and diluted in 0.1% BSA-PBS buffer. According to the manufacturer, the rhLIGHT contains <0.1 ng of endotoxin contamination per µg of protein, as determined by the low density lipoprotein method. The antihuman HVEM mAb (clone 108, 1D4, 4G2, 3G06) were developed in this laboratory (8, 10). Herpes simplex virus-1 glycoprotein D (HSV-1gD) was purchased from Advanced Biotechnologies. Anti-PLC{gamma}1, anti-PLC{gamma}2, and anti-phosphotyrosine mAb were provided by Dr. S. H. Ryu (11) (Pohang University, Pohang, South Korea). All other chemicals were purchased from Sigma-Aldrich. Heat-inactivated (HI) Abs and HI-rhLIGHT were prepared by incubation at 80°C for 20 min, and neutralized rhLIGHT was prepared by incubating rhLIGHT with anti-LIGHT mAb (1 µg/ml) at 0°C for 30 min.

Preparation of human monocytes and cell lines

Mononuclear cells were isolated from adult peripheral blood obtained from local routine blood donation by centrifugation over Ficoll Hypaque-1077 gradients. After two washes with HBSS without Ca2+ and Mg2+, monocytes (CD14+ cells) were purified from the PBMCs using an Ab-magnetic bead isolation system (VarioMACS; Miltenyi Biotec). Monocytic THP-1 cells obtained from the American Type Culture Collection (ATCC), were cultured in RPMI 1640 containing 10% FBS.

Measurement of [Ca2+]i

The rhLIGHT-induced rise in [Ca2+]i was determined using fluo-3/AM and monitored by confocal microscopy (12). In brief, freshly prepared monocytes were incubated in serum-free RPMI 1640 medium with 5 µM of fluo-3/AM at 37°C for 30 min with continuous stirring. After washing with serum-free RPMI 1640 medium, the cells were suspended in PBS containing 0.1% BSA, bound to a poly-L-lysine coated slide for 10 min, and placed in the confocal microscope chamber. PBS containing rhLIGHT was added, and changes in fluorescence every 1.1 s were analyzed with a confocal microscope (Olympus). [Ca2+]i was calculated from the equation [Ca2+]i = Kd(F/Fo)/{Kd/[Ca2+]rest + 1 – (F/Fo)}, where Fo is the mean fluorescence over the 10 s period before addition of rhLIGHT, Kd is the dissociation constant of fluo-3 (1100 nM), and [Ca2+]rest is the resting Ca2+ concentration. HI-rhLIGHT and 0.1% BSA were used as negative controls.

Measurement of intracellular inositol-1,4,5-trisphosphate (IP3)

Intracellular IP3 levels were measured as described (13). Cells were plated at 5 x 105 cells/well in 6-well plates and incubated for 48 h in inositol-free medium supplemented with 5 µCi/ml [3H] inositol (Amersham Bioscience). They were washed twice with PBS and stimulated for the indicated times with rhLIGHT and ice-cold 10% trichloroacetic acid was added. The cells were lysed by sonication for 30 s, extracted three times with water-saturated ether, and filtered, and [3H] inositol polyphosphates were analyzed by anion-exchange HPLC, using a Partisphere SAX column as described previously (14).

siRNA for HVEM and PLC{gamma}1

siRNA for HVEM and PLC{gamma}1 were purchased from Dharmacon. The sequences for the siRNA for luciferase (control siRNA) were: sense, 5'-CUUACGCUGAGUACUUCGAdTdT-3'; antisense, 5'-UCGAAGUAC UCAGCGUA AGdTdT-3'. Double-stranded RNA was produced using the conditions described previously (15). THP-1 cells were transfected with siRNA by electroporation (Bio-Rad), cultured for 48 h, and used for measurement of calcium influx.

Measurement of reactive oxygen species (ROS)

ROS generated by monocytes was assayed with the ROS-sensitive fluorescent dye, 2,7-dichlorofluorescein diacetate (DCF-DA). Cells were stained with 5 µM DCF-DA for 15 min at 37°C in the dark, washed twice, and incubated in culture medium containing 1 ng/ml rhLIGHT for 45 min. After washing, the cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences) (16).

Bacterial killing assay

Listeria monocytogenes (ATCC 15313) was grown in brain-heart infusion broth (BD Biosciences). Aliquots were frozen at –80°C and thawed immediately before use. Killing of bacteria was measured as previously described (8). In brief, an overnight culture of L. monocytogenes (1 x 107cells/ml) was opsonized by incubation with 0.1% gelatin (weight-to-volume ratio) and 10% (v/v) human Ab serum in HBSS. One ml of HBSS containing 1 x 107 opsonized bacteria was added to 1 x 107 monocytes/ml in 100 µl of HBSS, and the mixture incubated at 37°C for 3 min with continuous rotation to promote phagocytosis. Noningested bacteria were removed by differential centrifugation for 5 min at 1200 rpm, and the cells containing ingested bacteria were cultured at 37 °C for 10 min with slow rotation in the presence of rhLIGHT. Killing was stopped by spinning the cells onto ice after adding 1 ml of distilled water containing 0.01% BSA. The cells were disrupted by vigorous vortexing, and the number of viable bacteria released was determined by plating serial dilutions on agar plates. The percent killing was calculated as [1-(the number of viable bacteria at time t/the number of viable bacteria at time 0)] x 100.

Immunoprecipitation and immunoblot analysis

THP-1 cells (1 x 107 cells/ml) were incubated with rhLIGHT (1 ng/ml) or HI-rhLIGHT (1 ng/ml) for 10 s and immediately lysed in 500 µl of lysis buffer (20 mM Tris-Cl (pH 8.0); 137 mM NaCl; 10% v/v glycerol; 1% v/v Triton X-100; 1 mM Na3VO4; 2 mM EDTA; 1 mM PMSF; 20 µM leupeptin) for 30 min at 4°C. The extracts were precleared with protein A-Sepharose, followed by immunoprecipitation with 10 µg of anti-PLC{gamma}1 or anti-PLC{gamma}2. Protein A-Sepharose (40 µg) was added to the cell extracts, which were then washed by centrifugation. Protein content was determined by the standard Bradford method. Equal quantities of solubilized proteins were resolved on 10% SDS-PAGE at 20 mA. The separated proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-phosphotyrosine mAb (clone BY2), anti-pTyr771, anti-pTyr783, or anti-pTyr1254 mAb. The blots were then incubated with antimouse IgG conjugated to HRP and proteins were detected by ECL (Amersham Biosciences).

Statistics

Differences between groups were evaluated by Student’s t test. Results presented are representatives of at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
rhLIGHT induces calcium flux in an HVEM-specific manner

First, we investigated whether signaling resulting from the rhLIGHT/HVEM interaction in monocytes was mediated by Ca2+. Monocytes were purified from adult human blood by density gradient centrifugation and isolated with anti-CD14 mAb-magnetic beads. The cells were labeled with Fluo-3/AM and placed into the confocal microscope chamber. PBS containing rhLIGHT was added, and the fluorescence changes in cells were monitored by confocal microscopy. The addition of rhLIGHT caused a rapid increase of [Ca2+]i in primary monocytes (Fig. 1A) and in cells of the monocytic cell line, THP-1 (Fig. 1B). More than 90% of the primary monocytes and THP-1 cells responded to rhLIGHT, which was maximally effective in elevating [Ca2+]i at 10 ng/ml in monocytes, and at 1 ng/ml in THP-1 cells (Fig. 1, C and D). [Ca2+]i increased to 868 ± 201 and 886 ± 243 nM, in the monocytes and THP-1 cells, respectively, and returned to basal levels ~30 s after rhLIGHT addition. Oscillation of [Ca2+]i was observed in the THP-1 cells (Fig. 1B).


Figure 1
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  		FIGURE 1. rhLIGHT stimulates [Ca2+]i rise in monocytes and THP-1 cells. A and B, Freshly isolated human monocytes (A), or log-phase THP-1 cells (B) were stained with fluo-3/AM, attached to glass slides, and placed in a confocal microscope. PBS buffer containing rhLIGHT was added, and changes in fluorescence were monitored every 1.1 s for 100 s. Results are representative of 10 randomly chosen cells. C and D, Mean of the maximum [Ca2+]i of ten monocytes (C) and THP-1 cells (D) in response to rhLIGHT. E, THP-1 cells were stimulated with the indicated clonal anti-HVEM mAbs (1 µg/ml), and [Ca2+]i was determined as described for B. F, rhLIGHT induces the bactericidal activities of THP-1 cells. Cells were fed Listeria monocytogens and treated with rhLIGHT for 10 min. Viable bacteria were determined by plating cell lysates on agar. G, THP-1 cells (2 x 105 cells/ml) were cultured with the indicated concentrations of rhLIGHT for 48 h, and TNF-{alpha} in culture supernatants was determined by ELISA. Data are means ± SEM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001 compared with controls. HI, heat inactivated rhLIGHT (1 ng/ml for THP-1 and 10 ng/ml for primary monocytes); BSA, 0.1% BSA-PBS.

 
To investigate whether the rhLIGHT-induced Ca2+ response was HVEM-specific, we used anti-HVEM mAb instead of rhLIGHT to stimulate the HVEM. Of four anti-HVEM mAb clones tested, only clone 108 induced as much Ca2+ flux as rhLIGHT (Fig. 1E). Because the anti-HVEM clones 1D4, 4G2, or 3G06 did not induce a Ca2+ response (although they bound HVEM, as shown by FACS and Western blotting (data not shown)), we used them to block HVEM in additional experiments. HI-rhLIGHT did not induce Ca2+ response (Fig. 1, C and D), indicating that there was no contamination with endotoxin in our system. THP-1 cells treated with rhLIGHT had enhanced bactericidal activity (Fig. 1F) and produced more TNF-{alpha} (Fig. 1G), indicating that the rhLIGHT/HVEM interaction altered the activity of the THP-1 cells as well as induced [Ca2+]i. We previously demonstrated rhLIGHT-induced bactericidal activity and TNF-{alpha} production in primary human monocytes (8).

Using Abs to LIGHT and HVEM, and HSV-1gD proteins, we confirmed that rhLIGHT bound specifically to HVEM. First, neutralizing rhLIGHT with anti-LIGHT mAb completely abolished the [Ca2+]i response (Fig. 2A). LIGHT can bind to LTbetaR as well as to HVEM (2). Although we have previously shown that LTbetaR is not detectible in monocytes (8), it remained possible that very low levels were present and contributed to the rhLIGHT-induced Ca2+ flux. To exclude this possibility, we used HVEM-specific Abs and HSV-1gD to interfere with the binding of rhLIGHT to HVEM. Preincubation of THP-1 cells with anti-HVEM mAb or HSV-1gD, but not with anti-LTbetaR mAb, completely blocked the rise in [Ca2+]i, indicating that the rhLIGHT-induced Ca2+ response was mediated only by HVEM, not LTbetaR (Fig. 2B). Neither HSV-1gD nor anti-LTbetaR mAb on their own stimulated [Ca2+]i rise in THP-1 cells (Fig. 2B).


Figure 2
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  		FIGURE 2. The rhLIGHT-induced rise of [Ca2+]i is mediated by the rhLIGHT/HVEM interaction. A, rhLIGHT(1 ng/ml) was neutralized by incubation with anti-LIGHT mAb (1 µg/ml) at 0°C, for 30 min. The calcium responses of THP-1 cells to control rhLIGHT, neutralized rhLIGHT, HI-rhLIGHT, or BSA were determined. B, THP-1 cells were preincubated with anti-HVEM mAb (3G06), HSV-1gD (gD), or anti-LTbetaR mAb ({alpha}-LT) for 30 min, and rhLIGHT-induced Ca2+ responses were measured. Control cells were preincubated with PBS, and gD, {alpha}-LT or BSA-induced Ca2+ responses were determined. C, THP-1 cells transfected with control siRNA (left panel) or siRNA to HVEM (right panel) were stained with fluo-3/AM, and placed in a confocal microscope. PBS buffer containing 1 ng/ml rhLIGHT was added, and changes in fluorescence were monitored. After 1 min of rhLIGHT addition, PBS containing 1 µg/ml fMLP was added. Results are representative of 10 cells and three independent experiments. Data are means of the maximum [Ca2+]i ± SEM of 10 cells. ***, p < 0.001 compared with group of rhLIGHT alone.

 
To further show that the rhLIGHT-induced Ca2+ response was mediated by HVEM, we specifically knocked down HVEM mRNA in THP-1 cells by siRNA, and performed an rhLIGHT-induced calcium response experiment. As shown in Fig. 2C, silencing HVEM significantly attenuated rhLIGHT-induced [Ca2+]i rise while calcium responses to fMLP were intact.

The rhLIGHT/HVEM interaction induces tyrosine phosphorylation of PLC{gamma}1

To investigate the mechanism of the rise in [Ca2+]i, we tested whether the Ca2+ was derived from the medium or intracellular calcium stores. Preincubation of cells with the intracellular calcium chelator, BAPTA-AM, but not with the extracellular calcium chelator, EGTA, blocked rhLIGHT-induced [Ca2+]i rise, indicating that the increase in [Ca2+]i derived from intracellular stores (Fig. 3A). The rhLIGHT-mediated Ca2+ spikes in the presence of extracellular Ca2+ were more sustained than those in the absence of extracellular Ca2+ (data not shown).


Figure 3
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  		FIGURE 3. rhLIGHT increases [Ca2+]i in THP-1 cells via PLC activation. A, rhLIGHT-induced [Ca2+]i is derived from intracellular Ca2+ stores. THP-1 cells were preincubated with PBS containing BAPTA-AM (1, 2.5, 10 µµ) or EGTA (2 mµ) for 30 min, stained with fluo-3, attached to a glass slide, placed in a confocal microscope and the rhLIGHT-induced Ca2+ response was determined. B, PLC inhibitors inhibit rhLIGHT-induced Ca2+ response. THP-1 cells were preincubated with U73122 (0.1, 1, and 3 µM), U73343 (3 µM), or genistein (100 µM) for 30 min, or with pertussis toxin (PTX, 100 ng/ml) for 120 min, and rhLIGHT-induced rise of [Ca2+]i was determined by confocal microscopy. C, IP3 receptor antagonists inhibit rhLIGHT-induced Ca2+ response. THP-1 cells were preincubated with XeC (2 and 4 µM) or 2-APB (10 and 100 µM) for 30 min, and rhLIGHT-induced rise of [Ca2+]i was determined as described in A. The mean of the maximum [Ca2+]i of ten cells is shown. D, Dose- and time-dependent rhLIGHT-mediated IP3 productions. THP-1 cells were stimulated with the indicated concentrations of rhLIGHT for 1 min (left panel) or with 1 ng/ml rhLIGHT for the indicated times (right panel). Intracellular IP3 was measured as described in Materials and Methods.

 
These findings prompted us to investigate the rhLIGHT-induced activation of PLC enzymes using pharmacological inhibitors. As expected, incubation of cells with U73122 or genistein completely abolished rhLIGHT-induced Ca2+ response, while incubation with the U73122 congenor U73343 or pertussis toxin did not, suggesting that the rhLIGHT-induced Ca2+response was mediated by PLC (Fig. 3B). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycrol and 1,4,5-inositol triphosphate (IP3); the latter binds to the IP3 receptor and regulates the release of Ca2+ from intracellular stores. To confirm that IP3 is produced and is involved in the rhLIGHT-mediated Ca2+ response, we determined whether XeC and 2-APB, antagonists of the IP3 receptor, blocked the response. Fig. 3C shows that this was indeed the case. To further confirm the rhLIGHT-mediated PLC induction, we measured rhLIGHT-induced IP3 production by HPLC: IP3 levels increased dose-dependently in response to rhLIGHT, with peaks 1 min after rhLIGHT addition (Fig. 3D).

Eleven isoforms of PLC, grouped into four subfamilies (beta, {gamma}, {delta}, and {epsilon}), have been identified in mammals. Two isoforms of PLC{gamma} ({gamma}1 and {gamma}2) are phosphorylated and activated after ligands bind to nonreceptor protein tyrosine kinases, such as membrane IgM in B cells, the TCR in T cells, Fc{epsilon}R in basophils, and Fc{gamma}R in monocytes (17). To see whether the rhLIGHT/HVEM interaction also activated and phosphorylated PLC{gamma} in monocytes, THP-1 cells were treated with rhLIGHT, and lysates were immunoprecipated with anti-PLC{gamma}1 or anti-PLC{gamma}2 mAb and blotted with anti-phosphotyrosine mAb. rhLIGHT induced tyrosine phosphorylation of PLC{gamma}1, but not PLC{gamma}2 (Fig. 4A). PLC-{gamma}1 can be phosphorylated on residues Tyr771, Tyr783, and Tyr1254 (18). Lysates of rhLIGHT-treated THP-1 cells were immunoprecipated with anti-PLC{gamma}1 and blotted with site-specific anti-phosphotyrosine mAbs. Only Tyr771 of PLC{gamma}1 was phosphorylated by HVEM activation (Fig. 4B).


Figure 4
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  		FIGURE 4. rhLIGHT induces phosphorylation of PLC{gamma}1 in THP-1 cells. A, Cells were preincubated with anti-HVEM mAb (3G06) or control mouse IgG1 for 30 min, and stimulated with rhLIGHT (1 ng/ml) or HI-rhLIGHT (1 ng/ml) for 10 s. Cell lysates were prepared, immunoprecipitated with anti-PLC{gamma}1 or anti-PLC{gamma}2 mAb, resolved on SDS-PAGE, and immunoblotted with antiphosphotyrosine mAb. B, left, Samples from A were immunoblotted with site-specific anti-pTyr771, anti-pTyr783 and anti-pTyr1254 mAb. Right, COS-7 cells were stimulated with 0, 1, 10, or 100 ng of recombinant human epidermal growth factor (rhEGF) for 1 min, immunoprecipitated with anti-PLC{gamma}1 mAb, and blotted with anti-Tyr771, Tyr783, or Tyr1254 mAb. C, THP-1 cells transfected with PLC{gamma}1 siRNA are impaired in rhLIGHT-induced calcium responses. THP-1 cells transfected with control siRNA (top panel) or siRNA to PLC{gamma}1 (middle and bottom panel) were attached to glass slides, incubated with fluo-3/AM, and placed in a confocal microscope. PBS buffer containing 1 ng/ml rhLIGHT was added, and changes in fluorescence were monitored. After 1 min of rhLIGHT addition, PBS containing 1 µM of A23187 or 1 µg/ml fMLP was added. Results are representative of 10 cells and three independent experiments.

 
To further establish PLC{gamma}1 as a downstream signaling molecule of the rhLIGHT/HVEM interaction, we knocked down PLC{gamma}1 mRNA in THP-1 cells with siRNA. As shown in Fig. 4C, silencing PLC{gamma}1 completely abolished rhLIGHT-induced [Ca2+]i rise while responses to A23187 or fMLP remained intact.

The rhLIGHT/HVEM interaction induces I{kappa}B degradation, which is blocked by pharmacological PLC/[Ca2+]i inhibitors

rhLIGHT-induced NF{kappa}B activation and cytokine production have been demonstrated in T cells (4). We tested for rhLIGHT-induced NF{kappa}B activation and examined its relation to PLC/Ca2+ signaling by measuring I{kappa}B{alpha} in THP-1 cells. Cells were stimulated with rhLIGHT and lysates were blotted with anti-I{kappa}B{alpha} mAb. Fig. 5A shows that rhLIGHT treatment led to degradation of I{kappa}B{alpha}. Moreover, preincubation of THP-1 cells with U73122, XeC, or BAPTA-AM completely blocked this rhLIGHT-induced I{kappa}B{alpha} degradation (Fig. 5B), indicating that the PLC/Ca2+ signaling lies up-stream of NF{kappa}B activation.


Figure 5
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  		FIGURE 5. rhLIGHT induces I{kappa}B{alpha} degradation. A, THP-1 cells were stimulated with rhLIGHT (0, 1, 10, or 100 ng/ml) for 30 min. Cell lysates were prepared, resolved on SDS-PAGE, and immunoblotted with anti-I{kappa}B{alpha} mAb. I{kappa}B{alpha} levels were quantified and normalized to actin in three independent experiments and the graph shows mean ± SDs. B, THP-1 cells were pretreated with U73122, BAPTA-AM, or XeC (4 µM) for 30 min and stimulated with 1 ng/ml rhLIGHT for 30 min. Cell lysates were prepared, and immunoblotted with anti-I{kappa}B{alpha} mAb as in A.

 
Inhibitors of PLC/[Ca2+]i signaling block rhLIGHT-mediated TNF-{alpha} and ROS production, and the bactericidal activity of monocytes

Monocytes are the primary lines of defense against bacterial pathogens. They phagocytose and kill bacteria by producing ROS and inflammatory cytokines such as TNF-{alpha}. We have shown that the rhLIGHT/HVEM interaction causes the production of inflammatory cytokines and ROS, and enhances bactericidal activities in human primary monocytes (8). In the present study, we showed that inhibitors of PLC/[Ca2+]i blocked these responses; preincubation of purified monocytes with genistein, U73122, XeC, or BAPTA-AM dose-dependently suppressed the production of TNF-{alpha} (Fig. 6A) and ROS (Fig. 6B), and the increase of bacterial killing activity (Fig. 6C).


Figure 6
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  		FIGURE 6. Inhibitors of the PLC/[Ca2+]i signaling cascade abolish rhLIGHT-induced monocyte activation. A, Freshly isolated human monocytes were preincubated with genistein, U73122, U73343 (3 µM), XeC, or BAPTA-AM for 30 min, washed, and cultured with 10 ng/ml rhLIGHT for 24 h. TNF-{alpha} in the culture supernatants was determined by ELISA. B, Monocytes were preincubated with inhibitors as in A, washed, stained with DCF-DA, treated with 10 ng/ml rhLIGHT for 45 min, and analyzed by flow cytometry for ROS-positive cells. C, Monocytes were preincubated with inhibitors as in A, allowed to phagocytose Listeria monocytogens and treated with 10 ng/ml rhLIGHT for 10 min. The number of viable bacteria remaining in the cells was determined by serial plating of cell lysates on agar plates. Data are means ± SEM, and are representative of three similar experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with group of rhLIGHT alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recently we reported that HVEM stimulation by rhLIGHT enhanced the bactericidal activities of human monocytes and neutrophils against L. monocytogenes and S. aureus (8). In this study, we investigated the signaling pathways evoked by the LIGHT/HVEM interaction. rhLIGHT as well as anti-HVEM mAb produced a rapid [Ca2+]i rise in both primary monocytes and THP-1 cells (Fig. 1). Treatment of THP-1 cells with pharmacological inhibitors of the PLC/[Ca2+]i pathway and with siRNA to PLC{gamma}1 completely abolished the [Ca2+]i elevation (Fig. 3), and the rhLIGHT stimulus resulted in IP3 production and specific phosphorylation of PLC{gamma}1 on Tyr771. Further observations showed that the LIGHT/HVEM interaction acts via a PLC/[Ca2+]i signaling cascade.

LIGHT binds to three molecules, HVEM, LTbetaR, and DcR3 (2). However, there is good evidence that the events considered in this study result from specific binding of rhLIGHT to HVEM. First, HVEM is the only receptor for LIGHT in monocytes; LTbetaR is not expressed in monocytes (8) and DcR3 is a soluble protein not present on membranes. Second, preincubation of cells with HSV-1gD, which specifically binds to HVEM, not to LTbetaR or DcR3, abolished rhLIGHT-induced Ca2+response (Fig. 2B). Third, rhLIGHT-induced Ca2+response was blocked by preincubation of cells with antagonistic anti-HVEM mAb, but not anti-LTbetaR mAb (Fig. 2B). Moreover, agonistic Abs specific for HVEM also induced Ca2+influx (Fig. 1E) and silencing HVEM mRNA abolished the rhLIGHT-induced calcium responses (Fig. 2C).

Both PLC{gamma}1 and PLC{gamma}2 are present in immune cells and play critical roles in the generation of calcium signals in response to stimulation of immune receptors. The signaling mechanisms of PLC{gamma}1 and PLC{gamma}2 are similar, in that both isoforms are phosphorylated and are targets of the same protein tyrosine kinases (19). However, most immune cells use only one isoform for specific immune receptor activation. For example, only PLC{gamma}1 is activated by TCR activation in T cells, while PLC{gamma}2 is activated by the BCR complex in B cells and by collagen receptor glycoprotein VI in platelets (19). We showed in this study that HVEM activation only leads to phosphorylation of PLC{gamma}1 not of PLC{gamma}2 (Fig. 4A). It has been shown that phosphorylation of only a small fraction of the expressed PLC{gamma} is enough to induce normal Ca2+ responses (20). Therefore, the preference of PLC{gamma}1 in HVEM signaling in monocytes may not be due to the greater expression of PLC{gamma}1 but to differential affinities of the HVEM signaling molecules for PLC{gamma}1 or for the protein tyrosine kinases that phosphorylate PLC{gamma}1. It is known that Src, Syk, and Tec protein tyrosine kinases are present in monocytes and phosphorylate PLC{gamma} upon activation of specific immune receptors (21, 22, 23).

PLC{gamma}1 contains many protein binding domains: two Src-homology domain (SH)2, one SH3, and two pleckstrin homology domains, as well as two catalytic domains, and binds to many proteins via these domains. For example, the pleckstrin homology domains of PLC{gamma}1 bind the neurofilament L chain (24), as well as EF-1{alpha} (25), the beta{gamma}-subunit of a small G-protein (26), and beta-tubulin (27). The SH3 domain binds the GTPase, dynamin (28), the Ras GEF, son-of-sevenless (29), Akt (30), CAIR-1 (31), and phospholipase D2 (32). Activated HVEM or downstream signaling molecules such as the TRAFs may bind to one of the PLC{gamma}1 domains and modulate PLC{gamma}1 activity.

Recently, we observed that the introduction of dominant negative forms of TRAF2 into monocytes resulted in reduced rhLIGHT-mediated Ca2+responses and ROS generation (data not shown), suggesting that TRAF2 is involved in generating the Ca2+influx. Although the precise mechanism of PLC{gamma}1 activation is unknown, the results of this study indicate that rhLIGHT/HVEM signaling in monocytes mobilizes Ca2+via PLC{gamma}1.


   Acknowledgments
 
We thank members of the Ulsan Red Cross Blood Center for supplying human peripheral blood samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Science Research Center Fund to the Immunomodulation Research Center at the University of Ulsan from KOSEF, the Ministry of Korean Science and Technology. S.-K.H., M.-A.Y., and S.-A.J. were supported by the Second Project of BK21, the Ministry of Korean Education and Human Resources Development. Back

2 Address correspondence and reprint requests to Dr. Byung-Sam Kim, Department of Biological Sciences and Immunomodulation Research Center, University of Ulsan, Ulsan, South Korea. E-mail address: bskim{at}mail.ulsan.ac.kr Back

3 Abbreviations used in this paper: LIGHT, homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM/TR2); PLC{gamma}, phospholipase C{gamma}; IP3, inositol-1,4,5-trisphosphate; rhLIGHT, recombinant human LIGHT; gD, glycoprotein D; LTbetaR, lymphotoxin beta receptor; XeC, xestospongin C; TRAF, TNFR-associated factor; siRNA, small interfering RNA; HI, heat inactivated; ROS, reactive oxygen species; DCF-DA, 2,7-dichlorofluorescein diacetate; SH, Src-homology domain. Back

Received for publication November 30, 2006. Accepted for publication August 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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