Haruo Ohtani

CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3−B220low cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids.

Localization of transforming growth factor β1 and its latent binding protein in human chronic pancreatitis

BACKGROUND/AIMS Transforming growth factor beta 1 (TGF-beta 1) is thought to be the mediator of fibrosis in liver, glomerular, and pulmonary fibrosis. This study investigated the expression of TGF-beta 1 precursor (beta 1 latency-associated peptide), latent TGF-beta 1-binding protein (LTBP), and TGF-beta 1 messenger RNA (mRNA) in chronic pancreatitis. METHODS Beta 1 latency-associated peptide and LTBP expression were studied by immunohistochemistry, and TGF-beta 1 mRNA expression was studied by reverse-transcription polymerase chain reaction analysis in normal pancreatic parenchyma and in tissues from patients with chronic pancreatitis of different etiologies. RESULTS In normal specimens, TGF-beta 1 precursor was present in islet cells and in a few ductal and acinar cells but not in periductal connective tissue. No immunoreactivity for LTBP was detected. In chronic pancreatitis, TGF-beta 1 precursor was detected mainly in mononuclear cells located in the fibrotic areas and also in ducts damaged by fibrosis, more frequently in calcifying chronic pancreatitis. LTBP was present predominantly in mononuclear cells and in the extracellular matrix around them. TGF-beta 1 mRNA was either not expressed or was faintly expressed in normal tissue, whereas intense signals were detected in chronic pancreatitis. CONCLUSIONS The findings suggest the involvement of TGF-beta 1 in the development of fibrosis in chronic pancreatitis and the important role of inflammatory cells.

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model.

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.

Infiltrating CD8+ T Cells in Oral Lichen Planus Predominantly Express CCR5 and CXCR3 and Carry Respective Chemokine Ligands RANTES/CCL5 and IP-10/CXCL10 in Their Cytolytic Granules: A Potential Self-Recruiting Mechanism

Lichen planus is a chronic inflammatory disease of the skin and oral mucosa in which the cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. To understand its pathophysiology further, the present study examined the in situ expression of chemokines and chemokine receptors in oral lichen planus. Immunohistochemical analysis of 15 cases has consistently revealed that infiltrating CD4(+) and CD8(+) T cells in the submucosa predominantly expressed CCR5 and CXCR3. Furthermore, infiltrating T cells, particularly CD8(+) T cells, were positive for RANTES/CCL5 and IP-10/CXCL10, the ligands of CCR5 and CXCR3, respectively. By immunoelectron microscopy, these chemokines were localized in the cytolytic granules of CD8(+) T cells. Lesional keratinocytes also overexpressed the ligands of CXCR3, namely, MIG/CXCL9, CXCL10, and I-TAC/CXCL11. Our data suggest that the chemokines signaling via CCR5 and CXCR3, which are known to be selectively expressed by type 1 T cells, are predominantly involved in T-cell infiltration of oral lichen planus. Furthermore, the presence of CCL5 and CXCL10 in the cytolytic granules of tissue-infiltrating CD8(+) T cells expressing CCR5 and CXCR3 reveals a potential self-recruiting mechanism involving activated effector cytotoxic T cells.

Stromal reaction in cancer tissue: Pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions

Cancers are characterized by invasive growth and distant metastasis. Cancer cells not only destroy the pre‐existing extracellular matrix, but cancer invasion per se usually induces new matrix formation by activation of stromal cells; that is, desmoplastic reaction. This process includes both matrix production and degradation; that Is, the remodeling process. The similarity between desmoplastic reactions in cancer stroma and the wound healing process has already been pointed out, and it has been well documented that matrix‐degrading processes are actively involved In the wound healing process. A recent study revealed that most matrix‐degrading enzymes, generally considered to be one of the main mechanisms of cancer invasion and metastasis, are originated from stromal cells. Based on these preconditions, the present review postulates that the abundant expression of matrix‐degrading enzymes by fibroblasts, coupled with the abundant expression of type I procollagen, is involved in the matrix remodeling processes occurring in cancer stroma; that is, the mechanism similar to the wound healing process. Next, macrophages distributed along the invasive margin are known to express matrix‐degrading enzymes/factors. Data from past studies of colon carcinoma indicate that the tissue expression of matrix metalloproteinase‐9 and urokinase‐type plas‐mlnogen activator receptor Is inversely associated with simultaneous liver metastasis and infiltrating growth pattern. Previous clinicopathologic data have indicated that immune/Inflammatory cells are one of the factors for a favorable prognosis. This suggests that the expression of matrix‐degrading enzymes/factors by these host cells may be involved in host immune/inflammatory reactions, and that the net function of these cells can be defensive towards the host. Data from past studies of colon carcinoma on the expression of the intercellular adhesion molecule‐1 suggest that the interaction between macrophages, lymphocytes, and the phenotypes of venules distributed along the Invasive margin, further support the pro‐inflammatory milieu there. Therefore, the matrix degradation process in cancer tissue is multifunctional: besides the Involvement in cancer invasion and metastasis, the matrix degradation process is also involved in the tissue remodeling process and in the immune/inflammatory reaction occurring in the stroma.

Selective infiltration of CCR5(+)CXCR3(+) T lymphocytes in human colorectal carcinoma.

T cell infiltration in colorectal cancer is associated with a favorable prognosis, suggesting an occurrence of a certain degree of anti‐tumor immunity. T helper type 1 (Th1) and Th2 cells are now known to selectively express CC‐chemokine receptor 5 (CCR5)/CXC‐chemokine receptor 3 (CXCR3) and CCR4, respectively. To clarify the mechanism of T cell infiltration, we examined in situ expression of these chemokine receptors and their respective chemokine ligands in 40 cases of human colorectal cancer. Immunohistochemistry showed a predominant accumulation of T cells expressing CCR5 and CXCR3 mainly along the invasive margin, whereas those expressing CCR4 were rare. Flow cytometric analysis showed that more than half of CD8+ T cells and a fraction of CD4+ cells isolated from fresh tumor tissues co‐expressed CCR5 and CXCR3, and CD8+ T cells and CD4+ cells predominantly produced interferon‐γ (IFN‐γ) over interleukin‐4 (IL‐4) after in vitro stimulation. RANTES/CCL5, a ligand of CCR5, was localized within infiltrating CD8+ T cells in a granular pattern, whereas IP‐10/CXCL10, a ligand of CXCR3, was localized in cancer cells and macrophages along the invasive margin. These data were consistent with an active recruitment of T cells expressing CCR5 or CXCR3 into the invasive margin of colorectal cancer. With the previous clinicopathological studies showing a favorable prognostic impact of T cell infiltration in colorectal cancer, our study supports the occurrence of a certain level of Th1‐shifted cellular immune responses in human colorectal cancer.

Stromal expression of fibroblast activation protein/seprase, a cell membrane serine proteinase and gelatinase, is associated with longer survival in patients with invasive ductal carcinoma of breast.

Fibroblast activation protein (FAP)/seprase is a serine integral membrane proteinase with gelatinase activity, which is expressed by activated fibroblasts in the stroma of various epithelial cancers, mesenchymal tumors and breast‐cancer cells, as well as during wound repair. However, the pathophysiologic significance of its expression remains poorly understood. The present study was designed to reveal the impact of stromal expression of FAP/seprase on survival in human breast cancer. Immunohistochemical expression of FAP/seprase was restricted to stromal fibroblasts adjacent to tumor‐cell nests but not cancer cells, which was confirmed by double‐labeling immunohistochemistry. Clinicopathologic analysis revealed that more abundant FAP/seprase expression in 112 cases of invasive ductal carcinoma is associated with longer overall and disease‐free survival. Multivariate analysis with other clinicopathologic factors demonstrated that FAP/seprase expression is an independent prognostic factor. The effect on the survival rate of FAP/seprase was also apparent in cases with lymph node metastasis. FAP/seprase expression is one of the manifestations of the stromal reaction (i.e., matrix turnover); thus, invasive ductal carcinomas with fewer stromal reactions expressing FAP/seprase may be more aggressive.

Differential expression of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) in ulcerative colitis and Crohn's disease

Mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) is selectively expressed in the endothelial cells of intestinal mucosa and gut‐associated lymphoid tissue (GALT). Engagement of MAdCAM‐1 to its ligand, integrin α4β7, on lymphocytes is associated with the homing of gut‐associated lymphocytes to normal gastrointestinal tract and inflammation sites. The present study was designed to elucidate differences between Crohns disease (CrD) and ulcerative colitis (UC) from the expression patterns of MAdCAM‐1. Samples were taken from 40 patients with CrD and 24 patients with UC at surgical resection. Using frozen sections, immunohistochemistry was performed for MAdCAM‐1, E‐selectin and CD34. MAdCAM‐1+ venules were abundant in inflamed mucosa in both UC and CrD. In contrast, clear differences were noted between UC and CrD in the inflammatory area in the ulcer base, that is, MAdCAM‐1+ venules were more abundant in CrD than in UC (P < 0.001), while E‐selectin was expressed equally in these venules in both diseases. Furthermore, CrD was characterized by the occurrence of MAdCAM‐1+ venules in deeper layers of the intestinal tissue, mainly in lymphoid aggregates. Our data indicated more extensive expression of MAdCAM‐1 in CrD, which could contribute not only to mucosal inflammation, but also to transmural inflammation in CrD.

Macrophage-Derived Chemokine (MDC/CCL22) and CCR4 Are Involved in the Formation of T Lymphocyte-Dendritic Cell Clusters in Human Inflamed Skin and Secondary Lymphoid Tissue

Our previous study demonstrated formation of T cell-dendritic cell (DC) clusters in inflamed dermis of intraorally autotransplanted skin flaps. Such T cell-DC clusters are supposed to be important for close interactions between T cells and DCs including the specific antigen presentation. Here we show the involvement of the macrophage-derived chemokine (MDC/CCL22) and its specific receptor CC chemokine receptor 4 (CCR4) in the formation of T cell-DC clusters. Reverse transcriptase-polymerase chain reaction analysis revealed high levels of mRNA expression for MDC and CCR4 in inflamed skin and neck lymph nodes (LNs), but not in normal skin. Immunohistochemically, MDC(+) cells and CCR4(+) cells were mainly located within the T cell-DC clusters both in the dermis of inflamed skin and the T cell area of LNs. MDC(+) cells were identified to be DCs both in inflamed skin and LNs. The majority of CCR4(+) cells were CD4(+) T cells, accounting for approximately one-third of total CD4(+) T cells in the inflamed skin. Our data suggest that the MDC-CCR4 system plays an important role in the formation of T cell-DC clusters both in inflamed skin and LNs.

DUAL OVER-EXPRESSION PATTERN OF MEMBRANE-TYPE METALLOPROTEINASE-1 IN CANCER AND STROMAL CELLS IN HUMAN GASTROINTESTINAL CARCINOMA REVEALED BY IN SITU HYBRIDIZATION AND IMMUNOELECTRON MICROSCOPY

Membrane‐type metalloproteinase‐l (MTI‐MMP) is a transmembrane metalloproteinase, which activates pro‐gelatinase A. There has been disagreement as to whether the cell types expressing MTI‐MMP are cancer cells or stromal fibroblasts. Using human gastrointestinal carcinomas, the present study disclosed the tissue localization of MTI‐MMP mRNA by in situ hybridization and ultrastructural localization of its protein by immunoelectron microscopy. In normal colon and stomach tissues, MTI‐MMP mRNA and protein were negative or faintly positive both in epithelial cells and in stromal fibroblasts, except in the fundic gland of the stomach, which showed the positivity for MTI‐MMP. In contrast, gastrointestinal cancer tissue showed over‐expression of MTI‐MMP mRNA and protein both in cancer cells and in stromal cells (fibroblasts). Stromal fibroblasts also expressed mRNA for gelatinase A and type‐l procollagen. Double immunohistochemistry revealed that macrophages were also positive for MTI‐MMP. Immunoelectron microscopy showed that MTI‐MMP was localized along the plasma membrane of cancer cells and macrophages and in rough endoplasmic reticulum of fibroblasts. The present study reveals a dual over‐expression pattern of MTI‐MMP both in cancer cells and in stromal fibroblasts; the expression in cancer cells may be related to the invasive growth, whereas that in fibroblasts may be related to the tissue remodeling process caused by invasive growth of cancer cells.