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Granulysin, a Cytolytic Molecule, Is Also a Chemoattractant and Proinflammatory Activator ¹

Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305

	Abstract

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Granulysin, a cationic protein produced by activated human CTL and NK cells, is cytolytic against microbial and tumor targets. In this study we show that granulysin also functions as a chemoattractant and activates monocytes to produce cytokines/chemokines. Although granulysin-mediated cytotoxicity occurs at micromolar concentrations, chemoattraction occurs in the nanomolar range, and immune activation occurs over a wide range of concentrations (nanomolar to micromolar). Granulysin causes a 2- to 7-fold increase in chemotaxis of monocytes, CD4⁺, and CD8⁺ memory (CD45RO) but not naive (CD45RA) T cells, NK cells, and mature, but not immature, monocyte-derived dendritic cells. Pertussis toxin treatment abrogates chemoattraction by granulysin, indicating involvement of G-protein-coupled receptor(s). At low concentrations (10 nM), granulysin promotes a 3- to 10-fold increase in MCP-1 and RANTES produced by monocytes and U937 cells, while a 2-fold increase in TNF- production by LPS-stimulated monocytes requires higher concentrations of granulysin (micromolar). Taken together, these data indicate that the local concentration of granulysin is critical for the biologic activity, with high concentrations resulting in cytotoxicity while lower concentrations, presumably further from the site of granulysin release, actively recruit immune cells to sites of inflammation.

	Introduction

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Granulysin is a human antimicrobial peptide with broad activity against both Gram-positive and -negative bacteria, fungi, and parasites (1, 2, 3, 4). Granulysin also kills human tumor cells, and expression of granulysin in peripheral NK cells from cancer patients has been associated with good clinical outcomes (5). Granulysin is a member of the saposin-like protein family of lipid binding proteins and colocalizes in cytolytic granules with perforin and granzymes (2). Granulysin is expressed in CTL and NK cells as both 15- and 9-kDa proteins, with the 9-kDa form derived by proteolytic cleavage at both the N and C termini of the 15-kDa form (1). Recombinant 9-kDa granulysin disrupts artificial liposomes and cell membranes, damages mitochondria, and activates caspase 9 to induce apoptosis in nucleated cells (6).

It has become increasingly clear that some antimicrobial peptides, exemplified by defensins, function in local innate immunity not only as lytic agents but also as modifiers of immune cell migration and activation, further promoting their protective effects (7, 8, 9, 10). We show in this study that granulysin not only contributes to innate immunity but also to adaptive immunity as it attracts and activates human monocytes, dendritic, NK, and T cells.

	Materials and Methods

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Reagents

RANTES, TNF-, GM-CSF, and IL-4 were purchased from R&D Systems. fMLP and chemicals, unless otherwise specified, were purchased from Sigma-Aldrich. Salmonella typhimurium LPS, Escherichia coli 0111:B4 LPS, and Staphylococcus aureus lipoteichoic acid (LTA) ⁶ were purchased from Sigma-Aldrich. Contamination of granulysin and LTA with endotoxin was assessed using the Limulus amebocyte lysate assay (Sigma-Aldrich). Endotoxin levels were <1 ng/ml for LTA and <1.0 EU/µg granulysin.

Recombinant granulysin was prepared as previously described (2). Briefly, a cDNA encoding the 9-kDa form of granulysin was cloned into the pET28a vector (Novagen) and expressed in E. coli BL21. Cells were induced with isopropylthio--D-galactoside for 3 h, the cells were lysed, and material was purified under denaturing conditions on a Ni²⁺ column (Novagen) according to the manufacturer’s instructions. After purification, the material was reduced by the addition of DTT, refolded, and dialyzed against Tris-saline buffer. After concentration and dialysis, the polyhistidine tag was removed by thrombin digestion. Granulysin was purified by reverse-phase HPLC and stored as a lyophilized powder at –80°C. Anti-granulysin Ab was prepared by immunizing rabbits with recombinant 9-kDa granulysin (1). The IgG fraction was isolated using a protein G column.

Cell lines

Cell lines were obtained from American Type Culture Collection and maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Cells growing in log phase were washed three times in RPMI 1640 before the chemotaxis assay.

Isolation and purification of PBMC

Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation from leukopacs obtained from the Stanford University Blood Bank.

Monocytes, CD4⁺ T cells, CD8⁺ T cells, CD4⁺/CD45RA⁺ T cells, CD4⁺/CD45RO⁺ T cells, CD8⁺/CD45RA⁺ T cells, and CD8⁺/CD45RO⁺ T cells were purified from human PBMC using MACS isolation kits (Miltenyi Biotec) following the manufacturer’s instructions. The purity of each subset population was determined by FACS analysis and only preparations with >90% purity were used.

Immature monocyte-derived dendritic cells (iDC) were generated by culturing adherent PBMC with GM-CSF (100 ng/ml) and IL-4 (25 ng/ml) for 7 days. Mature dendritic cells (mDC) were obtained by incubating iDCs in the same medium supplemented with 50 ng/ml TNF- for an additional 2 days.

Chemotaxis assays

Migration of purified populations was assessed using a 96-well microchemotaxis chamber (NeuroProbe). Cells were washed three times and resuspended in RPMI 1640 supplemented with 1% BSA (chemotaxis medium). Different concentrations of chemotactic factors were placed in the lower compartment of the chambers, and 100 µl of cell suspension (1 x 10⁶ cells/ml) was added to the upper compartment. A 5- or 8-µm polycarbonate filter separated the upper and lower compartments. For T cell chemotaxis, 5-µm filters were coated overnight at 4°C in RPMI 1640 containing 10 µg/ml fibronectin (Sigma-Aldrich) and air-dried just before use. Cells were allowed to migrate at 37°C for 1.5 h (monocytes and dendritic cells (DC)) or 3 h (T cells). Filters were then washed with PBS, and the filter and the lower chamber were centrifuged. Migrated cells were collected and quantitated using CyQuant GR dye (Molecular Probes). The chemotaxis index is defined as the fold increase in the number of migrated cells in the presence of test factors over the spontaneous cell migration (in the absence of test factors). Statistical significance was determined by t test. A chemotiaxis index 2 is associated with p values <0.05.

NK cell chemotaxis was assessed by immunophenotyping and FACS analysis as described (11). Briefly, 5 x 10⁵ PBMC in 100 µl of RPMI 1640 medium supplemented with 0.5% BSA were added to the upper chambers of Costar Transwells (6.5 mm diameter, 5-µm pore size, polycarbonate membrane), and test agent (10 nM) was added to the lower chamber. Wells were incubated at 37°C for 3 h, and migrated cells were collected. Adherent cells were detached from the lower chamber by incubation with 0.5 ml of 5 mM EDTA for 15 min at 37°C and added to the migrated cells. The total number of cells that migrated to each agent was determined for each well, and these cells, as well as the input PBMC, were then immunophenotyped with FITC-anti-CD3, PE-anti-CD56, and PECy5-anti-CD16 (BD Pharmingen) and analyzed by FACS. To calculate specific migration for each subset, the mean number of migrated cells in the medium control wells was subtracted from the total number of cells per well that migrated to each experimental agent and then multiplied by the percent of each subset in the experimental well. All chemotaxis experiments were performed in triplicate.

LPS-stimulated TNF- production by U937 cells

The human monocyte-like cell line U937 was maintained in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 5 mM HEPES, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a 5% CO₂ atmosphere at 37°C. Two days before each experiment, 100 ng/ml PMA was added to differentiate the U937 cells. After washing, LPS, LTA, and/or granulysin were added. Supernatants were removed after 4 h and tested for TNF- by ELISA (R&D Systems).

MCP-1 and RANTES production by U937 or primary monocytes

U937 cells or freshly purified human monocytes were cultured with granulysin at 37°C for 4 h. Supernatants were collected and analyzed for MCP-1 and RANTES by ELISA (R&D Systems).

Quantitative real-time PCR

RNA was isolated using RNeasy mini kits (Qiagen) according to the manufacturer’s instructions. For cDNA synthesis, 1 µg total RNA was transcribed with cDNA transcription reagents (Applied Biosystems) using random hexamers, according to the manufacturer’s instructions. Gene expression was measured in real-time with the GeneAmp 7900 Sequence Detection System (Applied Biosystems) using primers and other reagents purchased from Applied Biosystems. The expression level of a gene in a given sample was represented as 2^–Ct where CT = [CT_{(experimental)}] – [CT_(medium)] and CT = [CT_{(experimental)}] – [CT_{(housekeeping)}]. Value represents fold increase of target gene expression from treated well over untreated well. All PCR assays were performed in triplicate.

	Results

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Granulysin attracts T cells, monocytes, and NK cell lines but not B cell lines

To determine which cell types might be attracted by granulysin, 15 cell lines were tested in a 96-well microchemotaxis assay (see Materials and Methods). Granulysin attracted some T, myelomonocytic, and NK cell lines (Fig. 1, A–C) with peak responses at 10 nM. This is much lower than the concentration of granulysin required to cause lysis (10–50 µM) (6). B cell lines did not migrate in response to granulysin (Fig. 1D). To demonstrate that chemotaxis was specific, anti-granulysin Ab (5 µg/ml) was added to the chemotaxis assay in the presence of 10 nM granulysin (Fig. 1E). No chemotaxis was observed under this condition. None of the murine cell lines tested (EL-4, RAW, P388D1, and A20) was responsive to granulysin. This is notable because the granulysin gene is not present in mice.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Migration of cell lines in response to granulysin. Chemotaxis of T cell lines (A), monocytic cell lines (B), NK cell lines (C), and B cell lines (D) to the indicated concentration of granulysin. Results are expressed as the chemotaxis index, defined as the number of cells migrating to wells containing granulysin/the number of cells migrating to wells containing medium ± the SD. The p value is <0.05 for a chemotaxis index >2. E, Addition of anti-granulysin polyclonal rabbit Ig blocks migration to granulysin. Black bar, 10 nM granulysin; light gray bar, 10 nM granulysin plus 5 µg/ml anti-granulysin Ab; dark gray bar, medium. Similar results were obtained in three independent experiments.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Migration of freshly isolated subpopulations of PBMC to granulysin. A, Human CD4+ T cells, CD8+ T cells, and monocytes were purified from PBMC and tested for chemotaxis to granulysin. Each population was >90% pure, as judged by FACS. Results are expressed as the chemotaxis index, defined as the number of cells migrating to wells containing granulysin/the number of cells migrating to wells containing medium ± the SD. B, Serum does not interfere with granulysin-induced chemotaxis of purified monocytes. , no added serum; , 10% human AB serum. The p value is <0.05 for a chemotaxis index >2.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Granulysin chemoattracts memory but not naive T cells. CD45RO or CD45RA cells were isolated from PBMC using magnetic beads. Each population was >90% pure, as judged by FACS. Results are representative of three similar experiments. The p value is <0.05 for a chemotaxis index >2.

To examine the effect of granulysin on chemotaxis by NK cell subsets, PBMC were added to the upper chambers of Costar Transwells, and medium or 10 nM granulysin, IL-8, MIP-1, or stromal cell-derived factor 1 (SDF-1) were added to the lower chamber. Wells were incubated at 37°C for 3 h, and the migrated cells were collected, stained with anti-CD3, -CD56, and -CD16, and analyzed by FACS (Tables I and II). The percent of each NK subpopulation that migrated in response to each agent was determined using the method described by Butcher and coworkers (11) as described in Materials and Methods. Granulysin, IL-8, and MIP-1 all induced migration of CD56⁺ cells while SDF-1 had no effect. Within the CD56⁺ population, granulysin caused migration of CD56⁺CD16⁺ and CD56⁺CD16^– but not NKT cells (CD56⁺CD3⁺). In contrast, IL-8 attracted CD56⁺CD16⁺ cells but not CD56⁺CD16^– or NKT cells; MIP-1 attracted all three subsets; and SDF-1 did not attract any NK subset.

Because DC are potent APCs and essential for the initial induction of Ag-specific adaptive immunity (14, 15, 16), we evaluated the ability of granulysin to attract both iDC and mDC. Only mDC (high expression of CD1a, CD83, CD86, and HLA-DR), and not iDC (expressing low CD83, low CD1a and CD86 and moderate HLA-DR), were attracted by granulysin (Fig. 4 and Table III).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Granulysin chemoattracts mature but not iDC. iDC were prepared by culturing adherent cells from human PBMC with GM-CSF and IL-4 for 7 days. A portion of these cells was then used to generate mDC by adding TNF- for 2 more days of culture. The phenotype of these cells is shown in Table III. Results are representative of three similar experiments. The p value is <0.05 for a chemotaxis index >2.

To characterize the receptor(s) for granulysin-mediated chemotaxis, cells were pretreated with pertussis toxin to disrupt signaling through G_i-protein-coupled receptors (17). T cells, monocytes, and DC pretreated with pertussis toxin no longer responded to granulysin, indicating that granulysin-mediated chemotaxis involves G_i-protein-coupled receptor(s) (Fig. 5).

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Pertussis toxin blocks granulysin-induced chemotaxis. Monocytes, CD4+ T cells, or CD8+ T cells were purified from PBMC; mDC were prepared as described in Materials and Methods. Cells were preincubated with medium () or with 200 ng/ml pertussis toxin () for 30 min before addition to the chemotaxis assay. Results are representative of three similar experiments.

U937 cells secrete TNF- when cultured with LPS or LTA (18). We asked whether granulysin could affect TNF- secretion (Fig. 6). U937 cells were incubated with LPS or LTA in the presence or absence of granulysin and release of TNF- was quantitated by ELISA. Granulysin increases TNF- production stimulated by LPS/LTA (p < 0.05 at 100 nM and 1 µM granulysin). TNF- mRNA increased in parallel with TNF- protein (data not shown), suggesting that the effect of granulysin on TNF- was due to increased transcription. Although granulysin-induced chemotaxis for U937 cells was maximal at 10 nM, granulysin-induced increase in TNF- expression increased from 1 nM to 1 µM, indicating that these effects are most likely mediated by different pathways.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Granulysin synergizes with LPS and LTA to stimulate TNF- production. U937 cells were incubated with 100 ng/ml PMA for 2 days, washed, and then incubated with 100 ng/ml S. typhimurium LPS (•), 100 ng/ml E. coli 0111:B4 LPS (), or 1 µg/ml S. aureus LTA () in the absence or presence of granulysin for 4 h. Granulysin alone (). TNF- production was then measured by ELISA. Results are representative of three similar experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Granulysin activates U937 cells to express mRNA for some proinflammatory cytokines and chemokines. U937 cells were stimulated with medium alone () or 10 nM granulysin () for 4 h. mRNA expression was measured by real-time PCR and fold increase was calculated as described in Materials and Methods.

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Granulysin increases expression of MCP-1 and RANTES in monocytes and U937 cells. Monocytes () and U937 cells () were stimulated with the indicated concentrations of granulysin for 4 h. The supernatant was collected and MCP-1 and RANTES were measured by ELISA. Results are presented as the mean of three experiments ± SD.

	Discussion

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Other cationic peptides, including human defensins and cathelicidin, have been reported to be chemotactic for selected leukocytes (25, 26). The defensins are chemoattractants for naive resting CD4⁺CD45RA T cells, some CD8⁺ T cells, and iDC while defensins attract cells expressing CCR6, including some iDC, resting memory CD4⁺CD45RO T cells, and some CD8⁺ T cells (26, 27). Human defensin 3 also is chemotactic for monocytes, cells that do not express CCR6, suggesting the presence of additional receptors. Cathelicidin (LL37) is broadly chemoattractant for monocytes, neutrophils, and T cells but not iDC (8, 10, 25). Its receptor is the formyl peptide receptor-like 1, another G-protein-coupled receptor (28). The subsets of leukocytes attracted by granulysin are distinct from those attracted by the defensins or LL37, indicating that granulysin binds to a different, and as yet unidentified, receptor.

Granulysin induces chemotaxis in only some NK cell populations. We found that both CD16^–CD56⁺ and CD16⁺CD56⁺ cells are attracted by granulysin while NKT cells (CD3⁺CD56⁺) do not respond to granulysin. Peripheral blood CD56⁺ lymphocytes derive from multiple lineages and can be divided into distinct subset populations based on cell surface phenotype (29, 30). NKT cells express CD3, comprise 5% of PBMC in normal human adults and respond well to MIP-1. CD3^– NK cells can be subdivided based upon CD16 expression. CD16⁺ cells comprise 5% of PBMC and respond to IL-8 while CD16^– cells, the population most responsive to granulysin, comprise 1% of PBMC and have higher levels of CD94 and CD56. Thus granulysin contributes to heterogeneity of NK cell population trafficking and activity.

Cells treated with pertussis toxin did not migrate in response to granulysin, indicating involvement of a G_i-protein-coupled receptor. To date we have not been able to identify this receptor using either a panel of cells transfected with orphan G_i-protein-coupled receptors or Abs against some of the known chemokine receptors. In addition, mouse cells apparently do not express this receptor: neither primary murine cells nor any the murine cell lines tested up-regulated cytokines or migrated in response to granulysin.

Granulysin activates immune cells to express and secrete proinflammatory cytokines. Granulysin enhances TNF- production by monocytes stimulated with LPS (18). Gram-negative bacteria contain LPS, inducing a strong cellular response and many toxic effects. LPS interacts directly with endothelial cells, smooth muscle, granulocytes, platelets, and macrophages/monocytes. The binding of LPS to CD14 on macrophages/monocytes induces the expression of adhesion molecules, cytokines, and bioactive substances including TNF- (31). This prompted us to examine the effect of granulysin on expression of proinflammatory cytokines. We found that several cytokines contributing to effector cell function were induced by granulysin. Collectively, these studies indicate that granulysin contributes to inflammation at several stages, including recruitment of inflammatory cells, activation of the secretion of proinflammatory mediators, and in higher concentrations, direct cell death. However, these effects occur at different concentrations. At nanomolar concentrations, granulysin causes chemotaxis and activates monocytes to produce a variety of proinlammatory molecules. At micromolar concentrations, granulysin is lytic against both microbes and mammalian cells. Thus, it is likely that at the site of granule exocytosis, the high concentration of granulysin and other lytic molecules, e.g., perforin and granzyme, function primarily in cytolysis. Further from the site of granule release, the concentration of granulysin is much lower and most likely functions to attract and to activate additional immune cells.

	Disclosures

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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 a grant to A.M.K. from the National Institutes of Health (AI43348). A.M.K. is the Shelagh Galligan Professor of Pediatrics.

² A.D. and S.C. contributed equally to the study.

³ Current address: Changzheng Hospital, 415 Fengyang Road, Shanghai 200003, China.

⁴ Current address: Department of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan.

⁵ Address correspondence and reprint requests to Dr. Alan M. Krensky, Department of Pediatrics, Stanford University School of Medicine, Center for Clinical Sciences Research 2105, 300 Pasteur Drive, Stanford, CA 94305-5164. E-mail address: Krensky{at}Stanford.edu

⁶ Abbreviations used in this paper: LTA, lipoteichoic acid; DC, dendritic cell; iDC, immature DC; mDC, mature DC; SDF-1, stromal cell-derived factor 1.

Received for publication October 15, 2004. Accepted for publication February 9, 2005.

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