Seong-A Ju

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

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‐α, 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‐α), 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.

Trp-Lys-Tyr-Met-Val-D-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes.

Among the phagocytic leukocytes, monocytes have the important role of clearing out parasitic microorganisms. They accomplish this through production of toxic metabolites of oxygen. Trp‐Lys‐Tyr‐Met‐Val‐D‐Met (WKYMVm), a peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes, including monocytes, binds to a unique cell surface receptor and stimulates superoxide generation, killing of Staphylococcus aureus, and activation of phospholipase D (PLD) in human monocytes. Preincubation of the cells with a PI‐specific phospholipase C (PLC) inhibitor (U‐73122), protein kinase C inhibitor (GF109203X), or intracellular Ca2+ chelator (BAPTA/AM) before the peptide stimulus totally inhibits the peptide‐induced PLD activation and superoxide generation. On the other hand, tyrosine kinase inhibitor genistein only partially inhibits the peptide‐induced processes. The peptide‐induced bacteria killing activity shares regulatory mechanisms for PLD activation with the superoxide generation, which is inhibited in the presence of 1‐butanol. We suggest that the peptide stimulates PLD downstream of PLC activation and PLD activation in turn is essential for the peptide‐induced immunological functions such as the superoxide generation and killing of bacteria by human monocytes. J. Leukoc. Biol. 65:241–248; 1999.

4-1BB (CD137) Is Required for Rapid Clearance of Listeria monocytogenes Infection

ABSTRACT 4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is a T-cell-costimulatory receptor that is expressed on activated T cells, dendritic cells, and NK cells. Little has been reported about its role in early host defense against bacterial infection. In this study, we report that 4-1BB-deficient (4-1BB−/−) mice are much more susceptible to Listeria monocytogenes (intracellular bacteria) infections than wild-type mice. Upon L. monocytogenes infection, 4-1BB−/− mice showed a lower survival rate, a higher bacterial burden in organs, and larger hepatic microabscesses than 4-1BB+/+ mice. 4-1BB−/− mice also had impairment in clearance of bacteria from the bloodstream. Neutrophils from 4-1BB+/+ mice constitutively expressed 4-1BB, which could be activated to induce intracellular Ca2+ influx by ligation with anti-4-1BB antibody. On the other hand, neutrophils from 4-1BB−/− mice were defective in reactive oxygen species generation, phagocytic activities, and intracellular Ca2+ mobilization. In addition, mice pretreated with anti-4-1BB monoclonal antibody were much more resistant to L. monocytogenes infection than control antibody-treated mice. Our results support the notion that 4-1BB may play a major role in host defense against intracellular pathogens through neutrophil activation.

Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumour-infiltrating lymphocytes

4‐1BB costimulates T cells to carry out effector functions such as eradication of established tumours. 4‐1BB (CD137) is a member of the TNF receptor family, and its triggering by either 4‐1BB ligand or antibody ligation induces T‐cell activation and growth. We analysed tumour‐infiltrating lymphocytes (TIL) in the experimental B16F10 melanoma model to determine the mechanisms involved in 4‐1BB‐mediated tumour suppression. 4‐1BB+/+ mice survived longer than 4‐1BB–/– mice, and survival was further prolonged by triggering 4‐1BB with an agonistic mAb. The number of metastatic B16F10 colonies in the lung was much greater in 4‐1BB–/– mice than in their 4‐1BB+/+ littermates. Administration of agonistic anti‐4‐1BB mAb increased the number of TIL in the tumour masses in the lungs of 4‐1BB+/+ mice. The numbers of CD4+ T, CD8+ T and CD11b+ TIL increased in these mice. Anti‐4‐1BB mAb induced not only CD8+ 4‐1BB+ T cells but also a CD8+ IFN‐γ+ T‐cell population. B16F10 cells from the lungs of anti‐4‐1BB‐treated mice showed enhanced expression of MHC class Ι and II antigens compared with the same cells from control IgG‐treated mice. Thus, the increase in number of CD8+ T cells and enhanced MHC Ι and II expression in B16F10 cells that result from augmented IFN‐γ production in response to anti‐4‐1BB mAb may lead to suppression of tumour growth and metastasis.

Eradication of established renal cell carcinoma by a combination of 5-fluorouracil and anti-4-1BB monoclonal antibody in mice.

Renal cell carcinoma (RCC), one of the most incurable malignancies, is highly resistant to chemotherapy and radiotherapy. Cytokine immunotherapy has been the standard approach, but the overall response rate is still very low. Administration of agonistic anti‐4‐1BB monoclonal antibody (mAb) has been shown to induce regression of several animal tumors but its effect on RCC is unknown. We show here that monotherapy with either anti‐4‐1BB mAb or the cytotoxic drug, 5‐fluorouracil (5‐FU), has little effect on established RCC, Renca tumors, but combination therapy with anti‐4‐1BB mAb and 5‐FU eradicates the tumors in more than 70 % of mice. The regressing tumor tissues from mice receiving the combination therapy contained more apoptotic tumor cells and tumor infiltrating lymphocytes than tumor tissues from mice receiving 5‐FU or anti‐4‐1BB mAb monotherapy. The number of lymphocytes in the spleens and tumor‐ draining lymph nodes (TDLNs) of the combination therapy mice was greatly increased compared to that of control or 5‐FU monotherapy mice. Mice that had recovered due to the combination therapy rapidly rejected rechallenge with the tumor, pointing to the establishment of long‐lasting tumor‐specific memory. Our results indicate that targeting tumors with 5‐FU, and immune cells with 4‐1BB stimulation, could be a useful strategy for treating incurable RCC.

Stimulation of the Molecule 4-1BB Enhances Host Defense against Listeria monocytogenes Infection in Mice by Inducing Rapid Infiltration and Activation of Neutrophils and Monocytes

ABSTRACT The tumor necrosis factor receptor family molecule 4-1BB (CD137) has diverse roles in adaptive and innate immune responses. However, little is known of its role in bacterial infections. Previously, we showed that 4-1BB-deficient mice have enhanced susceptibility to Listeria monocytogenes infection, and mice pretreated with agonistic anti-4-1BB antibody (3E1) were much more resistant to L. monocytogenes infection than mice treated with control antibody. In this study, we report that stimulating 4-1BB by administering 3E1 in the early phase of L. monocytogenes infection is critical for promoting the survival of mice by inducing rapid infiltration of neutrophils and monocytes into L. monocytogenes-infected livers. The levels of tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1 in the livers of 3E1-treated mice increased as early as 30 min postinfection and peaked by 1 to 2 h, while those in mice treated with control antibody started to increase only at 16 h postinfection. Monocytes and neutrophils from the 3E1-treated mice had higher levels of activation markers, phagocytic activity, and reactive oxygen species than those from control mice. In vitro stimulation of 4-1BB induced the production of the inflammatory cytokines/chemokines of neutrophils, but not those of monocytes. These results suggest that 4-1BB stimulation of neutrophils in the early phase of L. monocytogenes infection causes rapid production of inflammatory cytokines/chemokines and that the subsequent infiltration of neutrophils and monocytes is crucial for eliminating the infecting L. monocytogenes.

Administration of 6‐gingerol greatly enhances the number of tumor‐infiltrating lymphocytes in murine tumors

Tumor‐infiltrating lymphocytes (TILs) play critical roles in host antitumor immune responses. It is known that cancer patients with tumor‐reactive lymphocyte infiltration in their tumors have better prognoses, while patients with tumors infiltrated by immunosuppressive cells have worse prognoses. We found that administration of 6‐gingerol, which is a component of ginger, inhibited tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, which were established by inoculating tumor cells on the flanks of mice. However, administration of 6‐gingerol did not lead to complete eradication of the tumors. 6‐Gingerol treatment of tumor‐bearing mice caused massive infiltration of CD4 and CD8 T‐cells and B220+ B‐cells, but reduced the number of CD4+Foxp3+ regulatory T‐cells. The CD8 tumor‐infiltrating T lymphocytes in 6‐gingerol‐treated mice strongly expressed IFN‐γ, a marker of activation of cytotoxic T lymphocytes (CTL) CD107a and chemokine receptors that are expressed on TH1 cells, such as CXCR3 and CCR5. To test whether 6‐gingerol could promote infiltration of tumor antigen‐specific CD8 T‐cells into tumors, we adoptively transferred CFSE‐labeled OT‐1 CD8 T‐cells into EG7 tumor‐bearing mice. We found that CD8 T cells isolated from 6‐gingerol pretreated OT‐1 mice, but not from control OT‐1 mice, massively infiltrated tumors and tumor draining lymph nodes and divided several times. Our results strongly suggest that 6‐gingerol can be used in tumor immunotherapy to increase the number of TILs.

HVEM signaling in monocytes is mediated by intracellular calcium mobilization

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 NFκB activation as a result of IκBα 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-α production, and enhances the bactericidal activities of monocytes. Immunoprecipitation and Western blotting analyses revealed phosphorylation of phospholipase Cγ1 (PLCγ1) but not PLCγ2. rhLIGHT-induced Ca2+response was completely abolished by silencing PLCγ1, 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 IκBα degradation, generation of reactive oxygen species, TNF-α production and the bactericidal activities of monocytes. Our results indicate that Ca2+is a downstream mediator of the LIGHT/HVEM interaction in monocytes.

Tristetraprolin suppresses the EMT through the down-regulation of Twist1 and Snail1 in cancer cells

Inhibition of epithelial-mesenchymal transition (EMT)-inducing transcription factors Twist and Snail prevents tumor metastasis but enhances metastatic growth. Here, we report an unexpected role of a tumor suppressor tristetraprolin (TTP) in inhibiting Twist and Snail without enhancing cellular proliferation. TTP bound to the AU-rich element (ARE) within the mRNA 3′UTRs of Twist1 and Snail1, enhanced the decay of their mRNAs and inhibited the EMT of cancer cells. The ectopic expression of Twist1 or Snail1 without their 3′UTRs blocked the inhibitory effects of TTP on the EMT. We also observed that TTP overexpression suppressed the growth of cancer cells. Our data propose a new model whereby TTP down-regulates Twist1 and Snail1 and inhibits both the EMT and the proliferation of cancer cells.

Blockade of CD137 Signaling Counteracts Polymicrobial Sepsis Induced by Cecal Ligation and Puncture

ABSTRACT Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Previously, we have shown that CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, plays critical roles in eradicating infective Listeria monocytogenes, a gram-positive bacterium, and that stimulation of CD137 protects mice from sepsis-induced death. In this study, we unexpectedly found that CD137 activation aggravated polymicrobial sepsis due to mixed gram-positive and gram-negative bacterial infection induced by cecal ligation and puncture (CLP). CD137-deficient (CD137−/−) mice showed significantly lower mortality than CD137-sufficient (CD137+/+) mice in the CLP model. Administration of an agonistic anti-CD137 monoclonal antibody (MAb) to CD137+/+ mice decreased their survival in this infection model, while administration of a blocking anti-CD137 ligand MAb (TKS-1) to such mice increased their survival. CD137−/− mice and TKS-1-treated CD137+/+ mice had lower levels of chemokines/proinflammatory cytokines (monocyte chemoattractant protein 1, interleukin-6 [IL-6], tumor necrosis factor alpha, IL-12) and an anti-inflammatory cytokine (IL-10), exhibited improved bacterial clearance in the peritoneum, liver, and blood, and had greater numbers of infiltrated peritoneal neutrophils and macrophages in the CLP model than control mice. Our data suggest that CD137 activation aggravates polymicrobial sepsis induced by CLP.