Hidero Kitasato

Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3−/−) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3−/−, in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3−/−, compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3−/−. These results demonstrate significance of host stromal PGE2-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.

Cyclooxygenase-1 and cyclooxygenase-2 selectivity of non-steroidal anti-inflammatory drugs: investigation using human peripheral monocytes.

Since the pharmacological profiles of various non‐steroidal anti‐inflammatory drugs (NSAIDs) might depend on their differing selectivity for cyclooxygenase 1 (COX‐1) and 2 (COX‐2), we developed a new screening method using human peripheral monocytes. Monocytes from healthy volunteers were separated, and the cells were incubated with or without lipopoly‐saccharide (LPS). Monocytes without LPS stimulation exclusively expressed COX‐1 on Western blotting analysis, whereas LPS stimulation induced COX‐2 expression. Unstimulated monocytes (COX‐1) and LPS‐stimulated monocytes (COX‐2) were then used to determine the COX selectivity of various NSAIDs. The respective mean IC50 values for COX‐1 and COX‐2 IC50 (μm), and the COX‐1/COX‐2 ratio of each NSAID were as follows: celecoxib, 82, 6.8, 12; diclofenac, 0.076, 0.026, 2.9; etodolac, > 100, 53, > 1.9; ibuprofen, 12, 80, 0.15; indometacin, 0.0090, 0.31, 0.029; meloxicam, 37, 6.1, 6.1; 6‐MNA (the active metabolite of nabumetone), 149, 230, 0.65; NS‐398, 125, 5.6, 22; piroxicam, 47,25, 1.9; rofecoxib, > 100,25, > 4.0; S‐2474, > 100,8.9, > 11; SC‐560, 0.0048, 1.4, 0.0034. The percentage inhibition of COX‐1 activity at the IC50 of COX‐2 also showed a wide variation among these NSAIDs. The bioassay system using human monocytes to assess the inhibitory effects of various NSAIDs on COX‐1 and COX‐2 may become a clinically useful screening method.

Selective recruitment of CCR6-expressing cells by increased production of MIP-3α in rheumatoid arthritis

Infiltration of various types of leucocytes has been shown to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Macrophage inflammatory protein‐3α (MIP‐3α) is a recently identified chemokine which is a selective chemoattractant for leucocytes such as memory T cells, naïve B cells and immature dendritic cells. In this study, we investigated the expression of MIP‐3α and its specific receptor CCR6 in the inflamed joints of patients with RA. Increased amounts of MIP‐3α were found by ELISA in synovial fluids (SF) of patients with RA. MIP‐3α was apparently detected in all synovial tissue specimens of RA patients (n = 6), but it could not be detected in that of osteoarthritis (OA) patients (n = 4). Expression of MIP‐3α was detected especially in the sublining layer, and infiltrating mononuclear cells in RA synovial tissue. Gene expression of MIP‐3α was also found in six out of 11 RA‐synovial fluid cells by RT‐PCR. Cultured synovial fibroblasts derived from either RA or OA patients were capable of producing MIP‐3α in response to IL‐1β and TNFα in vitro. Furthermore, expression of CCR6 was found in infiltrating mononuclear cells in the cellular clusters and around the vessels of RA synovial tissue. These findings indicate that increased production of MIP‐3α may contribute to the selective recruitment of CCR6‐expressing cells in RA.

Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries blaIMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The kcat/Kmvalue of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

COX-2/VEGF-dependent facilitation of tumor-associated angiogenesis and tumor growth in vivo.

Nonsteroidal anti-inflammatory drugs are known to suppress the occurrence and progression of malignancies such as colorectal cancers. However, the precise mechanism of these actions remains unknown. We have evaluated the role of an inducible cyclo-oxygenase (COX-2) in tumor-associated angiogenesis and tumor growth, and identified the downstream molecules involved using a ddy mouse model of sponge angiogenesis, which mimics tumor angiogenesis and is COX-2 and vascular endothelial growth factor (VEGF) dependent. In this model, VEGF expression was down-regulated by selective COX-2 inhibition with NS-398. To find out the involvement of COX-2/VEGF pathway in tumor-associated angiogenesis, we estimated angiogenesis occurring around implanted Millipore chambers containing sarcoma-180 (S-180) cells or Lewis lung carcinoma cells. Daily oral administration of NS-398 or of aspirin, a nonselective COX inhibitor, suppressed angiogenesis seen around the Millipore chambers. S-180 cells implanted in ddy mice formed substantial tumors with extensive angiogenesis markedly suppressed by aspirin and COX-2 inhibitors NS-398 and JTE522, but not by mofezolac, an inhibitor of constitutive COX-1. Tumor-associated angiogenesis was also significantly suppressed by a neutralizing antibody against VEGF. S-180 tumor growth in the subcutaneous tissues was also suppressed by aspirin, COX-2 selective inhibitors, and the VEGF antibody, but not by the COX-1 inhibitor. These results demonstrate that the inhibition of the COX-2/VEGF-dependent pathway was effective in tumor-associated angiogenesis, tumor growth, and tumor metastasis.

Recent advances in crystal-induced acute inflammation.

Purpose of reviewThe aim of this article is to highlight recent advances suggesting essential involvement of the innate immune system in crystal-induced acute inflammation. Recent findingsGout is a disease caused by the deposition of monosodium urate monohydrate crystals. Precise mechanisms underlying the initiation of monosodium urate monohydrate crystal-induced acute inflammation, however, are not known. Recent investigations provided novel evidence in the pathology of acute gout. Immunological study indicated that monosodium urate monohydrate crystals can act as a ‘danger signal’ that resembles exogenous adjuvants. Two laboratories have documented interesting findings that Toll-like receptor-mediated pathways or MyD88-dependent pathways are involved in monosodium urate monohydrate crystal-induced acute inflammation. Upregulation of the triggering receptor expressed on myeloid cells 1 (TREM-1) in phagocytes by the stimulation with monosodium urate monohydrate crystals has been demonstrated. Furthermore, pathological significance of NALP 3 inflammasome in gout has been shown. These findings provide a new concept that the innate immune system may play a crucial role on the triggering of crystal-induced acute inflammation. Spontaneous resolution is a characteristic feature of acute gout. Involvement of nuclear hormone receptors, peroxisome proliferator-activated receptor γ and liver X receptor α, during the termination of acute gout has been also shown. SummaryThese studies provided a new insight into the mechanisms underlying the initiation and the termination of monosodium urate monohydrate crystal-induced acute inflammation.

Lipopolysaccharide-Induced Up-Regulation of Triggering Receptor Expressed on Myeloid Cells-1 Expression on Macrophages Is Regulated by Endogenous Prostaglandin E2

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface molecule that is expressed by neutrophils and monocytes. TREM-1 expression is modulated by various ligands for TLRs in vitro and in vivo. However, the influence of PGE2, a potential mediator of inflammation, on TREM-1 expression has not been elucidated. In this study, we examined the effects of PGE2 on LPS-induced TREM-1 expression by resident murine peritoneal macrophages (RPM) and human PBMC. PGE2 significantly induced murine TREM-1 (mTREM-1) expression by RPM. Up-regulation of TREM-1 expression was specific to PGE2 among arachidonic acid metabolites, while ligands for chemoattractant receptor-homologous molecule expressed on Th2 cells and the thomboxane-like prostanoid receptor failed to induce mTREM-1 expression. PGE2 also increased expression of the soluble form of TREM-1 by PBMC. LPS-induced TREM-1 expression was regulated by endogenous PGE2 especially in late phase (>2 h after stimulation), because cyclooxygenase-1 and -2 inhibitors abolished this effect at that points. A synthetic EP4 agonist and 8-Br-cAMP also enhanced mTREM-1 expression by RPM. Furthermore, protein kinase A, PI3K, and p38 MAPK inhibitors prevented PGE2-induced mTREM-1 expression by RPM. Activation of TREM-1 expressed on PGE2-pretreated PBMC by an agonistic TREM-1 mAb significantly enhanced the production of IL-8 and TNF-α. These findings indicate that LPS-induced TREM-1 expression on macrophages is mediated, at least partly, by endogenous PGE2 followed by EP4 and cAMP, protein kinase A, p38 MAPK, and PI3K-mediated signaling. Regulation of TREM-1 and the soluble form of TREM-1 expression by PGE2 may modulate the inflammatory response to microbial pathogens.

Human papillomavirus 57 identified in a plantar epidermoid cyst

We report a 23‐year‐old Japanese man who had plantar warts on the right sole, beneath one of which an epidermoid cyst developed. On microscopic examination, an acanthotic epidermis markedly invaginated into the underlying dermis, resulting in an open epidermoid cyst. Not only the polymerase chain reaction but also an in situ hybridization detected HPV 57 DNA in the cyst. HPV 60 is the only type of HPV that has been identified in epidermoid cysts. To our knowledge, this is the first case report of an epidermoid cyst, in which a different type of virus from HPV 60 was identified. Histological features of the cyst were also different from those of HPV 60‐associated epidermoid cysts.

Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis.

Cyclooxygenase (COX)‐2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E2 (PGE2)–prostaglandin E2 receptor (EP)3 signaling appears critical for tumor‐associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX‐2 inhibitor NS‐398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX‐1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)‐9 and vascular endothelial growth factor (VEGF)‐A was suppressed in NS‐398‐treated mice compared with PBS‐treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3−/−) mice compared with WT mice. The expression of MMP‐9 and VEGF‐A was downregulated in metastatic lungs of EP3−/− mice. An immunohistochemical study revealed that MMP‐9‐expressing endothelial cells were markedly reduced in EP3−/− mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP‐9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP‐9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis. (Cancer Sci 2009; 100: 2318–2324)

Production of Macrophage Inflammatory Protein 3α (MIP-3α) (CCL20) and MIP-3β (CCL19) by Human Peripheral Blood Neutrophils in Response to Microbial Pathogens

ABSTRACT Effects of bacterial pathogens on the production of macrophage inflammatory protein 3α (MIP-3α) and MIP-3β from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.