Hak Jung Kim

Activation of CXCR2 by Extracellular Matrix Degradation Product Acetylated Pro-Gly-Pro Has Therapeutic Effects against Sepsis

RATIONALE Acetylated Pro-Gly-Pro (Ac-PGP) is an endogenous degradation product of extracellular collagen that binds to leukocyte-expressed chemoattractant receptor CXCR2. Although certain agents that block CXCR2-mediated signaling protect against experimental sepsis, the roles of Ac-PGP and CXCR2 in sepsis are unclear. OBJECTIVES To investigate the role of Ac-PGP and its receptor, CXCR2, in murine models of cecal ligation and puncture (CLP)-induced polymicrobial sepsis and organ injury. METHODS The impact of in vivo Ac-PGP treatment on animal survival after induction of experimental sepsis was assessed. Vital organ inflammation and immune cell apoptosis were evaluated by histology, and the modulation of proinflammatory cytokine production and bactericidal activity by Ac-PGP in mouse and human blood leukocytes was measured. MEASUREMENTS AND MAIN RESULTS The activation of CXCR2 by tripeptide agonist Ac-PGP dramatically improved survival in three experimental sepsis models. Ac-PGP elicited bactericidal activity via the generation of hydrogen peroxide, inhibited lung inflammation, and reduced immune cell apoptosis. Fluorescein isothiocyanate-labeled PGP bound directly to CXCR2, and the protective effect of Ac-PGP in sepsis was abolished in CXCR2-deficient mice. Ac-PGP treatment enhanced the production of type 1 cytokines (IFN-γ and IL-12) but inhibited the production of proinflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1β, and IL-6) in vivo. In vitro, Ac-PGP directly increased IFN-γ production and decreased the LPS-stimulated production of TNF-α by mouse splenocytes and human leukocytes. Furthermore, direct treatment of LPS-stimulated splenocytes with IFN-γ resulted in diminished secretion of TNF-α and IL-6. CONCLUSIONS CXCR2 and Ac-PGP are thus novel target and starting molecules, respectively, for the development of therapeutic agents against sepsis.

Activation of human monocytes by a formyl peptide receptor 2-derived pepducin.

We synthesized and investigated the effect of formyl peptide receptor 2 (FPR2)‐derived pepducins in human monocytes. The FPR2‐based cell‐penetrating lipopeptide, “pepducin” (F2pal‐16), stimulated intracellular calcium increase in human monocytes via pertussis toxin (PTX)‐sensitive G‐protein and phospholipase C (PLC) activity. From a functional aspect, we showed that F2pal‐16 stimulated monocyte chemotaxis. F2pal‐16 also stimulated the generation of superoxide anion in human monocytes. Moreover, F2pal‐16 dramatically increased the production of several kinds of pro‐inflammatory cytokines (CXCL8, CCL2, IL‐1β and TNF‐α) in human monocytes via NF‐κB activation. Since FPR2 plays an important role in immune responses, F2pal‐16 can serve as a useful reagent for the study of FPR2‐mediated immune modulation.

Sphingosylphosphorylcholine stimulates CCL2 production from human umbilical vein endothelial cells.

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein–dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3–, NF-κB–, and AP-1–mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.

A pertussis toxin sensitive G-protein-independent pathway is involved in serum amyloid A-induced formyl peptide receptor 2-mediated CCL2 production

Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of Gi protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.

Phospholipase C activator m-3M3FBS protects against morbidity and mortality associated with sepsis.

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS–dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-γ and IL-12 while inhibiting proseptic TNF-α and IL-1β production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-α and IL-1β following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.

A WKYMVm-Containing Combination Elicits Potent Anti-Tumor Activity in Heterotopic Cancer Animal Model

The development of efficient anti-cancer therapy has been a topic of intense interest for several decades. Combined administration of certain molecules and immune cells has been shown to be an effective form of anti-cancer therapy. Here, we examined the effects of administering an immune stimulating peptide (WKYMVm), 5-fluoro-uracil (5-FU), and mature dendritic cells (mDCs) against heterotopic cancer animal model. Administration of the triple combination strongly reduced tumor volume in CT-26-inoculated heterotopic cancer animal model. The induced anti-tumor activity was well correlated with FAS expression, caspase-3 activation, and cancer cell apoptosis. The triple combination treatment caused recruitment of CD8 T lymphocytes and natural killer (NK) cells into the tumor. The production of two cytokines, IFN-γ and IL-12, were strongly stimulated by administration of the triple combination. Depletion of CD8 T lymphocytes or NK cells by administration of anti-CD8 or anti-asialoGM1 antibody inhibited the anti-tumor activity and cytokine production of the triple combination. The triple combination strongly inhibited metastasis of colon cancer cells in a heterotopic cancer animal model as well as in a metastatic cancer animal model, and enhanced the survival rate of the mice model. Adoptive transfer of CD8 T lymphocytes and NK cells further increased the survival rate. Taken together, we suggest that the use of triple combination therapy of WKYMVm, 5-FU, and mDCs may have implications in solid tumor and metastasis treatment.

Serum amyloid A inhibits RANKL-induced osteoclast formation

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.

Chloroform Extract of Garlic (Allium sativum) Stimulates Production of Superoxide Anion and CXCL8 in Human Neutrophils

Garlic has garnered attention as a useful material which contains many bioactive molecules for the prevention and/or treatment of various human ailments, including infectious diseases. In this study we tested several extracts of garlic on cellular activity in human neutrophils. Stimulation of human neutrophils with chloroform extract of garlic (GCE) elicited intracellular calcium ([Ca2+]i) increases in a phospholipase C independent manner. GCE also stimulated superoxide anion production, which was completely blocked by a calcium (Ca2+) chelator. We also observed that stimulation of human neutrophils with GCE caused production of the chemokine CXCL8 in a concentration-dependent manner. GCE also stimulated activity of two important mitogen activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38 MAPK. Moreover, GCE-induced CXCL8 production was inhibited by a p38 MAPK inhibitor (SB203580) but not by an ERK inhibitor (PD98059). Taken together, our results demonstrate that GCE contains bioactive natural compounds that stimulate [Ca2+]i signaling, superoxide anion production, and CXCL8 production. Thus, GCE may be useful for the development of immune-modulating agents against infectious diseases.