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Originally published online as doi:10.1189/jlb.1104694 on November 7, 2005

Published online before print November 7, 2005
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(Journal of Leukocyte Biology. 2006;79:330-338.)
© 2006 by Society for Leukocyte Biology

LIGHT enhances the bactericidal activity of human monocytes and neutrophils via HVEM

Sook-Kyoung Heo*, Seong-A Ju*, Sang-Chul Lee*, Sang-Min Park*, Suck-Young Choe{dagger}, Byungsuk Kwon*, Byoung S. Kwon*,{ddagger} and Byung-Sam Kim*,1

* Immunomodulation Research Center and
{dagger} Department of Food Science and Nutrition, University of Ulsan, Korea; and
{ddagger} Louisiana State University Eye Center, New Orleans

1Correspondence: Immunomodulation Research Center, University of Ulsan, San 29, Mookeo-dong, Nam-ku, Ulsan, Republic of Korea, 680-749. E-mail: bskim{at}mail.ulsan.ac.kr


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ABSTRACT
 
Human monocytes and neutrophils play major roles in clearing bacteria from human blood and tissues. We found that the herpes virus entry mediator (HVEM) was highly expressed in monocytes and neutrophils, and its interaction with "homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM/tumor necrosis factor (TNF)-related 2" (LIGHT) enhanced bactericidal activity against Listeria monocytogenes and Staphylococcus aureus. The LIGHT-HVEM interaction increased levels of phagocytosis, interleukin (IL)-8, TNF-{alpha}, nitric oxide (NO), and reactive oxygen species (ROS) in monocytes and neutrophils. Anti-HVEM monoclonal antibody was able to block LIGHT-induced bactericidal activity, cytokine production (IL-8 and TNF-{alpha}), and ROS generation. Moreover, inhibition of ROS and NO production blocked LIGHT-induced bactericidal activity. Our results indicate that the LIGHT/HVEM interaction in monocytes and neutrophils contributes to antibacterial activity.

Key Words: innate immunity • inflammatory response


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INTRODUCTION
 
Monocytes and neutrophils play critical roles in clearing bacteria from infection sites and contribute to a wide range of immune and homeostatic functions. Many crucial processes such as phagocytosis, inflammation, and microbial killing are mediated by the vast repertoire of leukocyte cell-surface receptors [1 ].

Homologous to lymphotoxins (LTs), shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM)/tumor necrosis factor (TNF)-related 2 (LIGHT) [2 3 4 5 ] is a member of the TNF family [6 , 7 ], which binds to three distinct TNF receptors (TNFRs) [6 , 8 ]: HVEM [9 10 11 ], LTß receptor (LTßR) [12 13 14 ], and soluble receptor 3 (DcR3) [15 , 16 ]. It is expressed in activated T cells, natural killer cells (NK), and immature dendritic cells (DC) [3 , 10 , 13 , 17 ]. HVEM is a member of the TNFR superfamily and has two more ligands: HSV surface envelope gD and LT{alpha} [18 ]. It is expressed on T cells, B cells, NK cells, monocytes, neutrophils, and DC [3 , 10 , 17 ]. It has been reported that LTßR is not expressed on primary T cells or monocytes but is prominently expressed on the stromal cells of lymph nodes in the red pulp and along the borders of red and white pulp [14 ]. LTßR is also expressed on a human follicular DC line, follicular DC-1, and on leukemia cell lines such as K562, U937, and HL60 [12 , 14 ].

The LIGHT-HVEM interaction has been shown to be involved in cell survival, inflammation [19 ], and up-regulation of intracellular adhesion molecule-1 [20 ]; it promotes the maturation of DC from myelodysplastic syndrome patients [21 ] and induces apoptosis in A375 melanoma cells [22 ] and morphological changes in the rhabdonmyosarcoma cell line RD [5 ]. LIGHT/HVEM signaling also mediates a number of T cell responses, and antibodies to HVEM block T cell proliferation, expression of activation markers, and the production of cytokines [11 ]. It also enhances cytolytic T lymphocyte-mediated tumor immunity and mediates allograft rejection, and blockade of LIGHT/HVEM signaling prevents graft-versus-host disease [3 , 13 ]. Recently, we reported high levels of soluble HVEM in the sera of patients with allergic asthma, atopic dermatitis, and rheumatoid arthritis, indicating that HVEM may also be involved in autoimmune diseases [10 ]. However, little is known about the role of LIGHT/HVEM signaling in innate immune responses, particularly in antibacterial activity, in spite of the high and constitutive expression of HVEM on monocytes and neutrophils.

In this study, we report that the LIGHT-HVEM interaction in monocytes and neutrophils increases their bactericidal and phagocytic activity and promotes the production of the inflammatory cytokines, interleukin (IL)-8 and TNF-{alpha}. We also present evidence that LIGHT/HVEM-mediated induction of reactive oxygen species (ROS) and nitric oxide (NO) contributes to the enhanced bactericidal activity of these cells.


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MATERIALS AND METHODS
 
Reagents
Recombinant human LIGHT (rhLIGHT) was purchased from R&D Systems (Minneapolis, MN) and diluted in 0.1% bovine serum albumin (BSA)-phosphate-buffered saline (PBS) buffer. According to the manufacturer, the rhLIGHT contains less than 0.1 ng endotoxin contamination per µg protein, as determined by the low-density lipoprotein method. Anti-human CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD19 (HIB19), and CD14 (M5E2) monoclonal antibodies (mAb) were purchased from PharMingen (San Diego, CA). Anti-human HVEM (clone, 3G06) mAb was from Immunomics (Ulsan, Korea) [10 ]. Biotinylated anti-human LTßR mAb was purchased from R&D Systems. Mouse immunoglobulin G1 (mIgG1)-fluorescein isothiocyanate (FITC) and streptavidin-FITC were purchased from PharMingen. Histopaque-1077 was purchased from Sigma Chemical Co. (St. Louis, MO), and PolymorphprepTM for purification of neutrophils [1 ] was from Nycomed Pharma (Oslo, Norway). Greiss reagent, fetal bovine serum (FBS), and 2',7'-dichlorofluorescin diacetate (DCF-DA) were from Sigma Chemical Co., and RPMI 1640 and Hanks’ balanced salt solution (HBSS) were from Gibco-BRL (Grand Island, NY). FITC was purchased from Pierce Biotechnology (Rockford, IL), and nitrite (NO2–) ion standard solution was from Fluka (St. Quentin Fallavier, France). Red blood cell lysis buffer was obtained from Boehringer Mannheim (Indianapolis, IN), and enzyme-linked immunosorbent assay (ELISA) kits for IL-8 were from Pierce Biotechnology and for TNF-{alpha}, from Endogen (Woburn, MA). Polymyxin B came from Sigma Chemical Co., and Detoxi-Gel endotoxin removal gel was from Pierce Biotechnology. Formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), N{omega}-nitro-L-arginine methyl ester (L-NAME), and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) were purchased from Sigma Chemical Co. Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) was purchased from Peptron (Pohang, Korea). To heat-inactivate the anti-rhLIGHT (HI-rhLIGHT) and anti-HVEM antibodies (3G06), they were incubated for 20 min at 80°C.

Preparation of monocytes and neutrophils
Adult peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from local routine blood donations. Mononuclear cells were obtained from adult peripheral blood by centrifugation on a Ficoll Hypaque-1077 gradient. After two washes with HBSS without Ca2+ and Mg2+, PBMCs were suspended in RPMI 1640 containing 10% FBS and incubated for 60 min at 37°C to allow monocytes to attach to the dishes. The cells were harvested by vigorous pipetting, and CD14+ monocytes were purified using an antibody-magnetic bead isolation system (Vario MACS, Miltenyi Biotec, Auburn, CA). Neutrophils were purified from adult peripheral blood by centrifugation on a PolymorphprepTM gradient [1 ]. After two washes with HBSS without Ca2+ and Mg2+, the cells were suspended in the appropriate medium. Purities of isolated monocytes and neutrophils were more than 95% when analyzed by flow cytometry and Giemsa staining of cytospin preparations.

Bacterial strains
Listeria monocytogenes [ATCC 15313, American Type Culture Collection (ATCC), Manassas, VA] was grown in brain-heart infusion broth (Becton Dickinson, Sparks, MD), and Staphylococcus aureus (ATCC 56389, ATCC) was grown in nutrient broth (Becton Dickinson). Aliquots were frozen at –80°C and thawed prior to use [23 ].

Flow cytometric analysis
PBMCs and neutrophils were incubated with 20% AB serum in PBS at 4°C for 30 min. Subsequently, the cells were washed twice with fluorescein-activated cell sorter (FACS) buffer (PBS containing 0.3% bovine serum albumin and 0.1% NaN3) and finally, incubated with appropriate fluorochrome-labeled mAb such as anti-CD3-phycoerythrin (PE), anti-CD4-PE, anti-CD8-PE, anti-CD14-PE, anti-CD19-PE, and anti-HVEM-FITC mAb or isotype control mAb at 4°C for 30 min [10 ]. Monocytes and neutrophils were immunostained with biotinylated anti-human LTßR mAb, followed by streptavidin-FITC. Samples were analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer and CELL-Quest software (BD Biosciences, San Jose, CA). The percentage of positive cells and mean fluorescence intensity (MFI) as well as the fluorescence histogram were recorded for each sample.

Phagocytosis
L. monocytogenes were labeled with 0.1 mg/ml FITC and incubated for 60 min at 25°C. They were washed by centrifugation and resuspended in ice-cold PBS, and this was repeated until the supernatant was clear of FITC. Monocytes and neutrophils were cultured with rhLIGHT or HI-rhLIGHT for 18 h, and their phagocytic activity was determined as described previously [24 25 26 ]. Briefly, cells (1x105 cells/ml) were incubated with 1 x 106 cells/ml FITC-labeled L. monocytogenes in 500 µl HBSS containing 0.1% gelatin (w/v) and 10% (v/v) human serum at 37°C for the indicated periods of time. They were washed three times with ice-cold PBS and analyzed with a confocal laser microscope (Fluoview-500, Olympus, Melville, NY) and flow cytometer (FACScan, Becton Dickinson). To exclude an effect of endotoxin, cells were pretreated with 5 µM lipopolysaccharide (LPS) receptor antagonist polymyxin B at 37°C for 1 h before measuring phagocytosis.

Bactericidal activity
Killing of bacteria was measured as described previously [25, 27 ]. Briefly, overnight cultures of L. monocytogenes (1x107cells/ml) and S. aureus (1x107 cells/ml) were opsonized by incubation with 0.1% gelatin (w/v) and 10% (v/v) human AB serum in HBSS, where 1 ml containing 1 x 107 opsonized bacteria was added to 1 x 107 cells/ml in 100 µl HBSS and incubated at 37°C for 3 min with continuous rotation to promote phagocytosis. Noningested bacteria were discarded by differential centrifugation for 5 min at 1200 rpm, and 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 addition of 1 ml distilled water containing 0.01% BSA. Cells were disrupted by vigorous vortexing, and the number of viable bacteria was determined by plating tenfold serial dilutions. The percent killing was calculated as [1–(number of viable bacteria at Time t/number of viable bacteria in Time 0)] x 100. To block the LIGHT-HVEM interaction, cells were preincubated with 1 µg/ml anti-HVEM mAb (clone 3G06) for 15 min at 4°C before addition of rhLIGHT.

ELISAs for cytokines
Purified monocytes and neutrophils were resuspended in RPMI 1640 (Gibco-BRL) with 10% FBS (Gibco-BRL) and treated with rhLIGHT for the indicated times. Cell-free supernatants were collected, and cytokines were assayed with ELISA kits for IL-8 (Pierce Biotechnology) and TNF-{alpha} (Endogen) [22 , 24 ].

NO production
NO production was estimated from the accumulation of NO2–, the end product of NO metabolism, in the medium using the Greiss reagent as described previously [28 ]. Briefly, monocytes and neutrophils were cultured with rhLIGHT for the indicated times. Equal volumes (50 µl) of supernatants and Greiss reagent were mixed and incubated at room temperature for 15 min, and the absorbance at 540 nm was measured with a spectrophotometer. A standard solution of NO2– was used for calibration. To prevent NO production, cells were preincubated with the NO synthase (NOS) inhibitor L-NAME for 30 min at 37°C before addition of rhLIGHT.

ROS generation
ROS generated by monocytes and neutrophils were assayed with the ROS-sensitive fluorescent dye, DCF-DA [29 ]. The cells were stained with 5 µM DCF-DA for 15 min at 37°C in the dark. After incubation with DCF-DA, they were washed twice with PBS buffer and resuspended in FACS buffer. The DCF-DA-stained leukocytes were then treated with rhLIGHT for the indicated times. After washing, the cells were analyzed by flow cytometry. To prevent ROS production, cells were preincubated with the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor AEBSF for 30 min at 37°C before addition of rhLIGHT.

Statistics
Differences between groups were evaluated by Student’s t-test. The results presented are representatives of at least three independent experiments from different blood donors.


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RESULTS
 
HVEM is highly expressed in monocytes and neutrophils
Previously, we and other investigators have shown that HVEM is expressed on subpopulations of PBMCs [3 , 10 , 17 ]. In this study, we stained PBMCs with FITC-conjugated anti-HVEM mAb and PE-conjugated cell-surface markers and examined HVEM expression on the PBMC by measuring MFI by flow cytometry. HVEM was expressed on all lymphoid and myeloid subpopulations, including CD3+, CD4+, and CD8+ T cells, CD19+ B cells, and CD14+ monocytes and neutrophils (Fig. 1A ). It was most highly expressed on monocytes, B cells, and neutrophils (Fig. 1B ; MFI value; monocytes, 1038±66; B cells, 892±124; neutrophils, 839±109). LTßR is another membrane protein that binds LIGHT. We determined whether LTßR is expressed on monocytes and neutrophils by flow cytometry. LTßR was not detected on neutrophils, and as other investigators have reported, it was also not found on CD3+ T cells and monocytes [30 ] (Fig. 1C) .


Figure 1
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  		Figure 1. HVEM is highly expressed on the monocytes (Mo) and neutrophils (Neu) from PBMCs, and LTßR is not. (A) HVEM expression on CD3+, CD4+, CD8+, CD19+, cells, monocytes and neutrophils. PBMCs were stained with FITC-conjugated anti-HVEM antibody and PE-labeled anti-CD3+, -CD4+, -CD8+, -CD19+, or -CD14+ antibody. Expression of HVEM was analyzed by flow cytometry of cells gated on each cell-surface marker. Purified neutrophils were used to assess HVEM expression on neutrophils. Results are representative of 10 independent experiments with different donors. Filled histograms are isotype controls (mIgG1), and open histograms are HVEM-positive cells. (B) The magnitude of HVEM expression on each cell subtype expressed as MFI. The flow cytometric data in A were analyzed in terms of MFI. Data are means ± SEM of 10 individuals. (C) LTßR expression on monocytes and neutrophils. Cells were purified and stained with biotin-conjugated anti-LTßR followed by FITC-streptavidin. Results are representative of 10 independent experiments from different blood donors. The filled histograms are isotype controls (mIgG1), and open histograms are LTßR-positive cells.

LIGHT stimulates phagocytosis of monocytes and neutrophils
The high constitutive expression of HVEM on monocytes and neutrophils prompted us to investigate its role in these cells. First, we tested whether HVEM ligation by rhLIGHT treatment increased the phagocytic activity of the monocytes and neutrophils. The cells were cultured with rhLIGHT for 18 h, and phagocytic activity against L. monocytogenes was determined. We found that rhLIGHT stimulated phagocytosis by monocytes and neutrophils in a dose (Fig. 2A and 2B )- and time-dependent (data not shown) manner. The rhLIGHT was maximally effective in stimulating phagocytosis at 10 ng/ml for monocytes and neutrophils. rhLIGHT treatment enhanced phagocytosis more than fourfold over the basal level (0 ng/ml rhLIGHT, Fig. 2B ). HI-rhLIGHT had no effect on the phagocytic activity of monocytes and neutrophils (Fig. 2B) nor did pretreatment of cells with polymyxin B. Both results showed there was no contamination with endotoxin in our system (Fig. 2B) .


Figure 2
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  		Figure 2. Effects of rhLIGHT on phagocytic activity of monocytes and neutrophils. Purified monocytes and neutrophils were cultured with 0, 1, 10, or 100 ng/ml rhLIGHT. After 18 h, the cells were mixed with FITC-labeled L. monocytogenes and analyzed by confocal microscopy and flow cytometry. (A) Analysis of phagocytosis by confocal laser microscopy. Cells ingesting bacteria were transferred to microscope slides, and FITC-positive cells were determined (x200). (B) Analysis of phagocytosis by flow cytometry. FITC-positive cells were analyzed by flow cytometry, and the percent positive cells were calculated. Solid bars refer to rhLIGHT treatment; hatched bars refer to HI-rhLIGHT treatment; and open bars refer to rhLIGHT treatment following incubation with polymyxin B (see Materials and Methods). Results shown are representatives of at least three independent experiments from different donors and are means ± SEM. ***, P < 0.001, compared with control group. poly.B, Incubation with polymyxin B.

The LIGHT/HVEM interaction increases the bactericidal activity of monocytes and neutrophils against L. monocytogenes and S. aureus
To assess the role of LIGHT, we tested whether it would stimulate the bactericidal activity of monocytes and neutrophils. Cells were allowed to ingest L. monocytogenes or S. aureus, treated with 1, 10, and 100 ng/ml rhLIGHT for 10 min, and the number of viable bacteria remaining in the cells was determined by plating serial tenfold dilutions of cell lysates on agar plates. As shown in Figure 3A (1 and 10 ng/ml rhLIGHT), the killing activity of monocytes and neutrophils against L. monocytgenes significantly increased, but 100 ng/ml rhLIGHT had less effect on monocytes and neutrophils than 10 ng/ml rhLIGHT. 10 ng/ml of rhLIGHT was maximally effective in promoting killing of L. monocytogenes by monocytes and neutrophils. The killing activity of monocytes and neutrophils against S. aureus was also increased by rhLIGHT, but in this case, 100 ng/ml rhLIGHT had the greatest stimulatory effect (Fig. 3A) . rhLIGHT enhanced the bactericidal activity of monocytes and neutrophils two- to threefold against each bacterium. The enhancement of bacterial killing by rhLIGHT was also time-dependent (Fig. 3B) . Incubation for more than 60 min for L. monocytogenes or more than 30 min for S. aureus did not increase the effect of rhLIGHT. The potency of rhLIGHT in enhancing bactericidal activity was comparable with that of the well-known positive control peptides, fMLP and WKYMVm, which were assayed in the same conditions (Fig. 3C ; percent of killing, control, 30.7±3.9%; LIGHT, 10 ng/ml, 65.0±3.3%; fMLP, 5 ng/ml, 87.1±2.3%; WKYMVm, 10 ng/ml, 69.8±1.6%) [27 ].


Figure 3
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  		Figure 3. LIGHT/HVEM interaction increases the bactericidal activity of monocytes and neutrophils. Opsonized L. monocytogenes or S. aureus were phagocytosed by monocytes or neutrophils. After washing away noningested bacteria, the cells were incubated with rhLIGHT for the indicated times. They were lysed with distilled water, and the numbers of viable bacteria per 1 million cells were determined by plating the lysates on agar. (A) Dose-dependent increase of bactericidal activity against L. monocytogenes or S. aureus in response to rhLIGHT. Bactericidal activity was determined following treatment with 0, 1, 10, or 100 ng/ml rhLIGHT for 10 min. (B) Time-dependent increase of bactericidal activity against L. monocytogenes or S. aureus in response to rhLIGHT. Bactericidal activity was determined in response to 10 ng/ml rhLIGHT for 10, 20, 30, or 60 min. (C) Increase of bactericidal activity induced by the positive-control peptides fMLP or WKYMVm. Monocytes ingesting L. monocytogens were incubated with the indicated concentrations of fMLP or WKYMVm for 10 min, and numbers of viable bacteria were determined. (D) Anti-HVEM antibody (3G06) blocks the rhLIGHT-induced increase of bactericidal activity. Monocytes ingesting L. monocytogenes were incubated with 1 µg/ml anti-HVEM mAb or heat-inactivated anti-HVEM mAb for 15 min at 4°C. After washing, the cells were incubated with 10 ng/ml rhLIGHT for 10 min at 37°C. They were lysed by distilled water, and the number of remaining viable bacteria per 1 million cells was determined by plating cell lysates on agar plates. Results shown are representatives of at least three independent experiments from different donors. **, P < 0.01; and ***, P < 0.001, compared with the control group. HI, heat inactivated.

There are two cell-surface receptors for LIGHT, HVEM, and LTßR. HVEM is highly expressed on monocytes and neutrophils, whereas flow cytometric analysis showed that LTßR was not expressed on these cells (Fig. 1C) . However, it seemed possible that low numbers of LTßR, not detectable by flow cytometry, played a role in the rhLIGHT-induced responses in our system. To verify that the rhLIGHT-induced enhancement of bacterial killing was only dependent on HVEM, we tested the effect of anti-HVEM mAb. Monocytes and neutrophils were preincubated with anti-HVEM mAb, and rhLIGHT-induced bactericidal activity was determined as described in Materials and Methods. The rhLIGHT-induced bactericidal activity of monocytes was completely blocked by pretreatment with anti-HVEM antibody (Fig. 3D , left panel), and the same was true of the rhLIGHT-induced bactericidal activity of neutrophils (Fig. 3D , right panel). Heat-inactivated rhLIGHT had no effect on bactericidal activity (Fig. 3A) , and heat-inactivated anti-HVEM mAb did not inhibit the rhLIGHT-induced increase of bacterial killing activity (Fig. 3D) .

LIGHT/HVEM signaling induces the production of IL-8 and TNF-{alpha} by monocytes and neutrophils
At infection sites, activated monocytes and neutrophils produce IL-8 and TNF-{alpha}, which are major proinflammatory cytokines/chemokines [31 ]. We determined whether rhLIGHT treatment increased the production of these cytokines. Monocytes and neutrophils were cultured with rhLIGHT for the indicated times, and culture supernatants were analyzed for IL-8 and TNF-{alpha}. rhLIGHT stimulated the production of IL-8 and TNF-{alpha} in a dose- and time-dependent manner in monocytes and neutrophils (Fig. 4A and 4B ). In each case, 100 ng/ml rhLIGHT was maximally stimulatory for cytokine production after 18 h incubation (Fig. 4A and 4B) .


Figure 4
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  		Figure 4. LIGHT/HVEM interaction increases IL-8 and TNF-{alpha} production by monocytes and neutrophils, which were cultured with rhLIGHT for the indicated times, and IL-8 and TNF-{alpha} were determined in the culture supernatants by ELISA. (A) Increase of IL-8 production in response to rhLIGHT treatment. Monocytes and neutrophils were cultured with 0, 1, 10, or 100 ng/ml rhLIGHT for 18 h (left panel) or with 100 ng/ml rhLIGHT for 6, 12, 18, and 24 h (right panel). IL-8 was determined in culture supernatants. (B) Increase of TNF-{alpha} production caused by rhLIGHT. Monocytes and neutrophils were cultured with 1, 10, and 100 ng/ml rhLIGHT for 18 h (left panel) or with 100 ng/ml rhLIGHT for 0, 6, 12, 18, and 24 h (right panel). TNF-{alpha} in culture supernatants was determined by ELISA. (C) Anti-HVEM antibody (3G06) blocks LIGHT-induced increases of IL-8 and TNF-{alpha} production. Monocytes were incubated with 1 µg/ml anti-HVEM mAb or HI-anti-HVEM mAb for 15 min at 4°C. After washing, the cells were cultured with 10 ng/ml rhLIGHT for 18 h. The results shown are representative of three independent experiments from different donors and are means ± SEM. ***, P < 0.001, compared with control group.

To block the LIGHT/HVEM interaction, we preincubated monocytes with anti-HVEM mAb and measured IL-8 and TNF-{alpha} in the culture medium. As shown in Figure 4C , preincubation with anti-HVEM mAb completely blocked the IL-8 and TNF-{alpha} production induced by rhLIGHT, indicating that rhLIGHT-induced HVEM signaling is responsible for cytokine production.

LIGHT induces NO production by monocytes and neutrophils
NO, produced by inducible NOS, is a major component of the bactericidal activity of monocytes and neutrophils [32 ]. To identify the mechanism of the LIGHT-induced enhancement of bactericidal activity, we tested whether rhLIGHT stimulated NO production. rhLIGHT treatment indeed induced NO production in both cell types (Fig. 5A and 5B ). It was maximally effective at 100 ng/ml and over a 24-h period in monocytes (Fig. 5A) and over 72 h in neutrophils (Fig. 5B) . rhLIGHT stimulated NO production 33-fold in monocytes (Fig. 5A) and 25-fold in neutrophils (Fig. 5B) compared with the controls. The level of rhLIGHT-mediated induction was comparable with that of LPS-mediated induction in monocytes (Fig. 5C ; 36-fold) and neutrophils (Fig. 5C ; 27-fold).


Figure 5
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  		Figure 5. Effects of rhLIGHT treatment on NO production by monocytes and neutrophils. Cells were cultured with rhLIGHT for the indicated times, and NO in the culture supernatants was determined with the Greiss reagent. (A) Effects of rhLIGHT on NO production by monocytes. Cells were cultured with 0, 1, 10, and 100 ng/ml rhLIGHT for 24 h (left panel) or 100 ng/ml rhLIGHT for 0, 18, 24, 48, and 72 h (right panel). (B) Effects of rhLIGHT on NO production by neutrophils. Cells were cultured with 0, 1, 10, and 100 ng/ml rhLIGHT for 48 h (left panel) or 100 ng/ml rhLIGHT for 0, 18, 24, 48, and 72 h (right panel). (C) Effects of LPS on NO production by monocytes and neutrophils. Cells were cultured with 100 ng/ml LPS for 0, 18, 24, 48, and 72 h. Results shown are representatives of at least three independent experiments from different blood donors and are means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with the control group.

LIGHT increases ROS generation in monocytes and neutrophils via HVEM
ROS, which are produced by an activated, phagosome-bound NADPH oxidase, constitute another major component of the bactericidal activity of monocytes and neutrophils [33 34 35 ]. We tested if rhLIGHT treatment increased levels of ROS in monocytes and neutrophils. Cells were stained with the ROS-sensitive fluorescent dye DCF-DA and incubated with rhLIGHT. They were then harvested, and fluorescent cells were detected by flow cytometry at the indicated times. Treatment of rhLIGHT did stimulate ROS production in a dose- and time-dependent manner in monocytes (Fig. 6A ) and neutrophils (Fig. 6B) and was maximally effective at 10 ng/ml over a 45-min incubation (Fig. 6A and 6B) . The percent of ROS-positive cells of both types was increased 2.5-fold by rhLIGHT relative to the control group. The positive controls, 100 nM PMA, and 100 ng/ml WKYMVm increased the percent of ROS-positive cells six- and sevenfold compared with the control, respectively (Fig. 6C) . Pretreatment of cells with anti-HVEM mAb also completely inhibited the rhLIGHT-induced increase of ROS generation in monocytes (Fig. 6D) , indicating that the LIGHT/HVEM interaction was responsible for the rhLIGHT-mediated ROS production. rhLIGHT-stimulated ROS production in neutrophils was also inhibited by pretreatment with anti-HVEM antibody (data not shown).


Figure 6
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  		Figure 6. LIGHT/HVEM interaction induces the production of ROS in monocytes and neutrophils. (A) Effects of rhLIGHT treatment on ROS production by monocytes. Cells were stained with the ROS-sensitive fluorescent dye DCF-DA and cultured with 0, 1, 10, or 100 ng/ml rhLIGHT for 45 min (left panel) or with 10 ng/ml rhLIGHT for 0, 15, 45, and 90 min (right panel). After incubation, the cells were harvested, and the percentages of ROS-positive cells were assessed by flow cytometry. (B) Effects of rhLIGHT on ROS production by neutrophils. Cells were stained with DCF-DA and assayed as described in A for monocytes. (C) Preincubation with anti-HVEM antibody (3G06) blocks the LIGHT-induced increase of ROS. Monocytes were stained with DCF-DA and incubated with 1 µg/ml anti-HVEM mAb or heat-inactivated anti-HVEM mAb for 15 min at 4°C. After washing, the cells were cultured with 10 ng/ml rhLIGHT for 45 min. ROS-positive, fluorescent cells were counted by flow cytometry. (D) WKYMVm and PMA were used as positive controls for the ROS analysis. Monocytes were stained with DCF-DA and cultured with 10 and 100 ng/ml WKYMVm or 10 and 100 nM PMA for 45 min. ROS-positive cells were assayed as described in A. The results shown are representative of at least three independent experiments from different blood donors and are means ± SEM. **, P < 0.01; and *** P < 0.001, compared with control group.

Blocking the production of ROS and NO inhibits LIGHT-induced bacterial killing
We used inhibitors of ROS and NO production to verify that rhLIGHT-induced bacterial killing by monocytes and neutrophils is the result of ROS and/or NO production. Cells were preincubated with AEBSF, a NADPH oxidase inhibitor, or L-NAME, NOS inhibitor before stimulation with rhLIGHT, and bactericidal activity was determined. rhLIGHT-induced bactericidal activity was partially blocked by these inhibitors in both cells. NADPH oxidase inhibitor AEBSF inhibited rhLIGHT-induced bacterial killing activity up to 65% in monocytes (Fig. 7A , left panel; percent of killing; control, 32.7±3.9%; LIGHT alone, 65.7±3.3%; with 0.3 mM AEBSF, 44.2±2.2%) and up to 55% in neutrophils (Fig. 7A , right panel; percent of killing; control, 33.7±3.6%; LIGHT alone, 67.9±2.2%; with 0.3 mM AEBSF, 48.6±6.13%). NOS inhibitor L-NAME also inhibited rhLIGHT-induced bacterial killing activity up to 58% in monocytes (Fig. 7B , left panel; percent of killing; control, 15.3±2.2%; LIGHT alone, 46.7±8.3%; with 5 mM L-NAME, 28.9±3.8%) and up to 80% in neutrophils (Fig. 7B , right panel; percent of killing; control, 16.7±5.5%; LIGHT alone, 58.4±3.7%; with 5 mM L-NAME, 24.69±6.08%). This indicates that LIGHT-induced bactericidal activity is in part regulated by the production of ROS in monocytes and neutrophils.


Figure 7
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  		Figure 7. Inhibition of ROS and NO production blocks rhLIGHT induction of bactericidal activity against L. monocytogens in monocytes and neutrophils. (A) Effect of NADPH oxidase inhibitor on rhLIGHT-induced bacterial killing activity. Monocytes (left panel) and neutrophils (right panel) were preincubated with the NADPH oxidase inhibitor AEBSF (0.1 and 0.3 mM) for 30 min before stimulation with rhLIGHT (10 ng/ml). (B) Effect of NADPH oxidase inhibitor on rhLIGHT-induced bacterial killing activity. Monocytes (left panel) and neutrophils (right panel) were preincubated with the NOS inhibitor L-NAME (0.1 and 0.3 mM) for 30 min. Cells were stimulated with rhLIGHT (10 ng/ml) for 24 h, and bacterial killing was determined as described in Materials and Methods. Results shown are representatives of at least three independent experiments from different donors and are means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with LIGHT alone.


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DISCUSSION
 
We have shown that the LIGHT-HVEM interaction in monocytes and neutrophils increases phagocytosis (Fig. 2) , bactericidal activity (Fig. 3) , and production of IL-8 and TNF-{alpha} (Fig. 4) as well as of NO (Fig. 5) and ROS (Fig. 6) . We have also presented evidence that LIGHT/HVEM-mediated induction of ROS contributes directly to the enhanced bactericidal activity (Fig. 7) . Our hypothesis is that the LIGHT/HVEM interaction in monocytes and neutrophils activates these innate immune cells, promoting phagocytosis, bactericidal activity, and production of IL-8 and TNF-{alpha}, as well as of NO and ROS. LIGHT-induced ROS and NO production contributes, in particular, to the enhanced bactericidal activity of these cells, as shown by our observation that LIGHT-induced bactericidal activity is inhibited by the ROS inhibitor AEBSF and NO inhibitor L-NAME (Fig. 7) .

It has been reported that LIGHT can bind to three known receptors: HVEM, LTßR, and DcR3. However, our observations indicate that LIGHT binds only to HVEM in monocytes and neutrophils. HVEM was highly expressed in these cells (Fig. 1A and 1B) , whereas LTßR was not detected by flow cytometry (Fig. 1C) [12 , 14 , 30 ], and DcR3 is a soluble receptor, not present on the cell membrane. Moreover, blocking HVEM with anti-HVEM mAb completely inhibited rhLIGHT-induced ROS generation (Fig. 6C) , production of proinflammatory cytokines (Fig. 4C ; IL-8 and TNF-{alpha}), and bactericidal activity (Fig. 3D) . rhLIGHT-induced ROS generation and the bactericidal activity (Fig. 3D) of neutrophils were also inhibited by anti-HVEM antibody. Hence, the signal system involved is similar to that of monocytes. Moreover, anti-HVEM also inhibited rhLIGHT-induced IL-8, TNF-{alpha}, and NO production by neutrophils (data not shown), indicating that all the LIGHT-induced signals involve HVEM in monocytes and neutrophils. This interpretation is reinforced by the recent observation that preincubation of cells with HSV gD, which competes with LIGHT for HVEM binding, also inhibits LIGHT-induced responses (data not shown).

Leukocytes such as monocytes and neutrophils ingest microorganisms by phagocytosis, and the ingested bacteria are killed by ROS derived from superoxide, which is produced by an activated, phagosome-bound NADPH oxidase and/or by NO produced by NOS [33 34 35 ]. rhLIGHT treatment enhanced ROS and NO production by monocytes and neutrophils, and inhibiting ROS and NO production decreased rhLIGHT-induced bactericidal activity (Fig. 7) . The amplitude of the rhLIGHT-induced signals was comparable with well-known positive controls, such LPS, PMA, and WKYMVm (Figs. 3C 5C and 6D) . rhLIGHT-stimulated phagocytosis was stronger and more rapid in neutrophils than monocytes (data not shown), whereas induction of the inflammatory cytokines, IL-8 and TNF-{alpha}, and NO was more effective in monocytes (Figs. 4 and 5) . It is noteworthy that HVEM expression is generally higher on monocytes than neutrophils (Fig. 1B) .

We observed consistently that LIGHT-induced bactericidal effects and phagocytic activities declined at dosages of rhLIGHT exceeding some value, for example, more than 10 ng/ml for L. monocytogenes killing and more than 100 ng/ml for S. aureus killing (Fig. 3A) . The basis of such decreased responses at higher concentration of rhLIGHT remains to be determined. When we measured the bactericidal activity of monocytes and neutrophils after 5-min incubations rather than the 10-min incubations used above, these decreased responses at higher LIGHT concentrations were not observed (data not shown).

Gram-positive bacteria such as L. monocytogenes and S. aureus cause severe infections in immune-compromised patients [36 ]. Leukocytes, such as the monocytes and neutrophils, are the first line of defense against bacterial infection. Monocytes and neutrophils are rapidly recruited to sites of bacterial infection, phagocytose the bacteria, and process bacterial antigens for presentation to the T cells, which orchestrate the adaptive immune response. Activated T lymphocytes are also recruited to sites of bacterial infection, and LIGHT is strongly expressed on their surfaces [17 ]. As HVEM is constitutively and highly expressed on monocytes and neutrophils (Fig. 1) , it is possibile that an activated T cell-monocyte/neutrophil interaction takes place via LIGHT-HVEM-binding at infection sites. The antibacterial activities of monocytes and neutrophils would be enhanced by this interaction, which would thus accelerate the removal of bacteria.

In conclusion, a LIGHT-HVEM interaction in monocytes and neutrophils induces phagocytosis (Fig. 2) , bactericidal activity (Fig. 3) , and production of IL-8 and TNF-{alpha} (Fig. 4) , as well as of NO (Fig. 5) and ROS (Fig. 6) . LIGHT/HVEM-mediated induction of ROS and NO contributes to bactericidal activity (Fig. 7) . To our knowledge, this is the first report that a TNF/TNFR interaction is directly involved in the bactericidal activity of monocytes and neutrophils.


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ACKNOWLEDGEMENTS
 
This work was supported by the SRC fund to the IRC at the University of Ulsan from Kosef, the Ministry of Korean Science and Technology. The authors thank members of the Red Cross Ulsan Blood Center for supplying human peripheral blood samples.

Received November 28, 2004; revised July 12, 2005; accepted September 26, 2005.


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