Gamma/delta intraepithelial lymphocytes in the mouse small intestine.

Although many studies of intraepithelial lymphocytes (IELs) have been reported, most of them have focused on αß-IELs; little attention has been paid to γδ-IELs. The function of γδ-IELs remains largely unclear. In this article, we briefly review a number of reports on γδ-IELs, especially those in the small intestine, along with our recent studies. We found that γδ-IELs are the most abundant (comprising >70 % of the) IELs in the duodenum and the jejunum, implying that it is absolutely necessary to investigate the function(s) of γδ-IELs when attempting to delineate the in vivo defense system of the small intestine. Intraperitoneal injection of anti-CD3 mAb stimulated the γδ-IELs and caused rapid degranulation of them. Granzyme B released from their granules induced DNA fragmentation of duodenal and jejunal epithelial cells (paracrine) and of the IELs themselves (autocrine). However, perforin (Pfn) was not detected, and DNA fragmentation was induced even in Pfn-knockout mice; our system was therefore found to present a novel type of in vivo Pfn-independent DNA fragmentation. We can therefore consider γδ-IELs to be a novel type of large granular lymphocyte without Pfn. Fragmented DNA was repaired in the cells, indicating that DNA fragmentation alone cannot be regarded as an unambiguous marker of cell death or apoptosis. Finally, since the response was so rapid and achieved without the need for accessory cells, it seems that γδ-IELs respond readily to various stimuli, are activated only once, and die 2-3 days after activation in situ without leaving their site. Taken together, these results suggest that γδ-IELs are not involved in the recognition of specific antigen(s) and are not involved in the resulting specific killing or exclusion of the relevant antigen(s).

Autocrine DNA fragmentation of intra-epithelial lymphocytes (IELs) in mouse small intestine.

Intraepithelial lymphocytes (IELs) are present in the intestinal epithelium. Mechanisms of IELs for the protection of villi from foreign antigens and from infections by micro-organisms have not been sufficiently explained. Although more than 70% of mouse duodenal and jejunal IELs bear γδTCR (γδIELs), the functions of γδIELs are little investigated. We stimulate γδIELs by anti-CD3 monoclonal antibody (mAb) injection. The mAb activates γδIELs to release Granzyme B (GrB) into the spaces surrounding the γδIELs and intestinal villous epithelial cells (IECs). Released GrB induces DNA fragmentation in IECs independently of Perforin (Pfn). IECs immediately repair their fragmented DNA. Activated IELs reduce their cell size, remain for some time in the epithelium after the activation and are ultimately eliminated without leaving the site. We focus our attention on the response of IELs to the released GrB present in the gap surrounding IELs, after activation, in order to examine whether the released GrB has a similar effect on IELs to that observed on IECs in our previous studies. DNA fragmentation is also induced in IELs together with the repair of fragmented DNA thereafter. The time-kinetics of both events were found to be identical to those observed in IECs. DNA fragmentation in IELs is Pfn-independent. Here, we present Pfn-independent "autocrine DNA fragmentation" in IELs and the repair of fragmented DNA in IELs and discuss their biological significance. Autocrine DNA fragmentation has never been reported to date in vivo.

Activation of intra-epithelial lymphocytes; their morphology, marker expression and ultimate fate.

Intraepithelial lymphocytes (IELs) have been considered to play a key role in the defense system of the small intestine. Its mechanism has not been made sufficiently clear. Studies on IELs have been extremely limited to functions of αß T-cell receptor (αßTCR) IELs (αß-IELs). Since, in the mouse duodenum and jejunum, γδ-IELs consist 75 % of IELs, it thus would be inappropriate to argue the mechanism without extensive discussions over the functions of γδ-IELs. In previous studies, we found that the anti-CD3 monoclonal antibody (mAb) injection induced DNA fragmentation in intestinal epithelial cells (IECs) and DNA repair immediately after, that these responses were reproduced by anti-γδTCR mAb not by anti-αßTCR mAb and that the DNA fragmentation was induced by Granzyme B secreted by IELs, totally independent of Perforin. To further explore the functions of IELs in situ, we undertook experiments exclusively focused on IELs, on their changes and ultimate fate after the stimulation in mouse in vivo system. The current study demonstrated that the injected anti-CD3 mAb bound to CD3 on IELs, that the mAb activated γδ-IELs, leading to their degranulation, that changes occurred irreversibly in IELs and finally that activated IELs died in situ. γδ-IELs could be considered to respond to various stimulations most likely without the need of accessory cells ("always ready for rapid response"), to die in situ ("disposable") and thus to respond to the stimulation only once ("a one-shot responder"). These characteristics of γδ-IELs are important to further elucidate the functions of γδ-IELs in the intestinal defense system.

Granzyme B-dependent and perforin-independent DNA fragmentation in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes.

We previously found that an i.p. injection of anti-CD3 monoclonal antibody (mAb) into mice caused DNA fragmentation in the intestinal villous epithelial cells (IVECs) of the duodenum and the jejunum. In this study, in order to elucidate the mechanism of DNA fragmentation in IVECs, we searched for the inducer(s) of DNA fragmentation by using immunohistochemistry. The release of cytoplasmic granules from intraepithelial lymphocytes (IELs) and the formation of large gaps between IELs and IVECs were observed electron microscopically after antibody administration. The presence and distribution pattern of Granzyme B (GrB), a serine protease in cytolytic granules present in cytotoxic T lymphocytes and natural killer cells and considered to be the responsible molecule for DNA fragmentation in target cells, was examined in detail in intestinal villi by immunohistology. GrB was detected in cytoplasmic granules in nearly all IELs. The time-kinetics of granule release from IELs after mAb injection coincided not only with that of the extracellular diffusion of GrB, but also with that of DNA fragmentation in IVECs. On the other hand, perforin (Pfn), assumed to cooperate with GrB in DNA fragmentation, could not be detected in IELs, and its release was not confirmed after the anti-CD3 mAb injection. Anti-CD3 mAb injection also induced DNA fragmentation in IVECs in Pfn-knockout mice. These results support the notion that DNA fragmentation in IVECs by the stimulated IELs in the present study is induced by a mechanism involving GrB, but independent of Pfn.

The interaction of monocytes with rheumatoid synovial cells is a key step in LIGHT-mediated inflammatory bone destruction.

Formation of osteoclasts and consequent joint destruction are hallmarks of rheumatoid arthritis (RA). Here we show that LIGHT, a member of the tumour necrosis factor (TNF) superfamily, induced the differentiation into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) of CD14(+) monocytes cocultured with nurse-like cells isolated from RA synovium, but not of freshly isolated CD14(+) monocytes. Receptor activator of nuclear factor-kappaB ligand (RANKL) enhanced this LIGHT-induced generation of TRAP-positive MNCs. The MNCs showed the phenotypical and functional characteristics of osteoclasts; they showed the expression of osteoclast markers such as cathepsin K, actin-ring formation, and the ability to resorb bone. Moreover, the MNCs expressed both matrix metalloproteinase 9 (MMP-9) and MMP-12, but the latter was not expressed in osteoclasts induced from CD14(+) monocytes by RANKL. Immunohistochemical analysis showed that the MMP-12-producing MNCs were present in the erosive areas of joints in RA, but not in the affected joints of osteoarthritic patients. These findings suggested that LIGHT might be involved in the progression of inflammatory bone destruction in RA, and that osteoclast progenitors might become competent for LIGHT-mediated osteoclastogenesis via interactions with synoviocyte-like nurse-like cells.

Inhibition of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 reduces the severity of collagen-induced arthritis.

OBJECTIVE: To investigate whether the blockade of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) has any therapeutic effects on rheumatoid arthritis. METHODS: A functional blocking monoclonal antibody for SHPS-1 (anti-SHPS-1 mAb) was administered at various doses to collagen-induced arthritis (CIA) mice, and severity of the arthritis was evaluated by clinical and histological scores of the limbs. To clarify the mechanisms of action of the antibody, the serum concentration of anti-type II collagen antibody was measured in those mice, and in vitro experiments were conducted to determine the effects of the antibody on the induction of osteoclasts and the release of cytokines from mouse spleen cells. RESULTS: Compared with mice given control IgG, the administration of anti-SHPS-1 mAb significantly reduced the severity of inflammation and destruction of bone and cartilage in CIA mice. This therapeutic effect was observed even when the antibody treatment was started after the onset of arthritis. The appearance of anti-type II collagen antibody in CIA mice was not altered by the antibody treatment. In in vitro experiments, the anti-SHPS-1 mAb significantly inhibited osteoclastogenesis of bone marrow cells, and significantly reduced the release of interleukin 1beta (IL-1beta), IL-2, IL-12, interferon-gamma, and tumor necrosis factor-alpha, but not that of IL-4 or IL-10, from the spleen cells after stimulation with concanavalin A. CONCLUSION: Administration of a monoclonal antibody for SHPS-1 reduced the severity of arthritis in CIA mice. Regulation of biological functions of SHPS-1 may be a novel and potent strategy to treat patients with rheumatoid arthritis.

Discovery of novel non-peptide thrombopoietin mimetic compounds that induce megakaryocytopoiesis.

We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.

Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients.

BACKGROUND: The epidermal growth factor (EGF) and EGF receptor (EGFR) families play important roles in the hyperplastic growth of several tissues as well as tumor growth. Since synovial hyperplasia in rheumatoid arthritis (RA) resembles a tumor, involvement of the EGF/EGFR families in RA pathology has been implied. Although several reports have suggested that ErbB2 is the most important member of the EGFR family for the synovitis in RA, it remains unclear which members of the EGF family are involved. To clarify the EGF-like growth factors involved in the pathology of RA, we investigated the expression levels of seven major EGF-like growth factors in RA patients compared with those in osteoarthritis (OA) patients and healthy control subjects. METHODS: The expression levels of seven EGF-like growth factors and four EGFR-like receptors were measured in mononuclear cells isolated from bone marrow and venous blood, as well as in synovial tissues, using quantitative RT-PCR. Further evidence of gene expression was obtained by ELISAs. The proinflammatory roles were assessed by the growth-promoting and cytokine-inducing effects of the corresponding recombinant proteins on cultured fibroblast-like synoviocytes (FLS). RESULTS: Among the seven EGF-like ligands examined, only amphiregulin (AREG) was expressed at higher levels in all three RA tissues tested compared with the levels in OA tissues. The AREG protein concentration in RA synovial fluid was also higher than that in OA synovial fluid. Furthermore, recombinant human AREG stimulated FLS to proliferate and produce several proinflammatory cytokines, including angiogenic cytokines such as interleukin-8 and vascular endothelial growth factor (VEGF), in a dose-dependent manner. The VEGF mRNA levels in RA synovia and VEGF protein concentrations in RA synovial fluid were significantly higher than those in the corresponding OA samples and highly correlated with the levels of AREG. CONCLUSION: The present findings suggest that AREG functions to stimulate synovial cells and that elevated levels of AREG may be involved in the pathogenesis of RA.

LIGHT induces cell proliferation and inflammatory responses of rheumatoid arthritis synovial fibroblasts via lymphotoxin beta receptor.

OBJECTIVE: To investigate the effects of LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) on the proliferation and gene expression of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: We measured LIGHT levels in RA synovial fluids (SF) by ELISA, and compared them with those in osteoarthritis (OA) SF. Levels of LIGHT and its receptors in RA-FLS and synovium were assessed using real-time quantitative polymerase chain reaction (PCR). RA-FLS proliferation was examined by a bromodeoxyuridine assay. Expression of intercellular adhesion molecule-1 (ICAM-1) and several chemokines, such as interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha), was examined by real-time quantitative PCR, ELISA, and flow cytometry. The effects of LIGHT on nuclear factor-kappaB (NF-kappaB) activation were investigated using immunofluorescence and Western blotting. RESULTS: LIGHT was upregulated in both SF and synovium of RA patients compared with OA patients. Herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTbetaR), but not LIGHT, were detected in RA-FLS. LIGHT significantly promoted RA-FLS proliferation and induced expression of MCP-1, IL-8, MIP-1alpha, and ICAM-1 by RA-FLS. As well, LTbetaR small interfering RNA (siRNA), but not HVEM siRNA, inhibited these effects of LIGHT. LIGHT induced IkappaBa degradation and NF-kappaB translocation, and a NF-kappaB inhibitor suppressed the effects of LIGHT on RA-FLS. CONCLUSION: Our findings suggest that LIGHT signaling via LTbetaR plays an important role in the pathogenesis of RA by affecting key processes such as the proliferation and activation of RA-FLS. Regulation of LIGHT-LTbetaR signaling may represent a new therapeutic target for RA treatment.

Characterization of novel non-peptide thrombopoietin mimetics, their species specificity and the activation mechanism of the thrombopoietin receptor.

A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells. To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain. Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should be helpful for structure-activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.

Immunosuppression by morphine-induced lymphocyte apoptosis: is it a real issue?

UNLABELLED: Morphine has been an optimal choice for cancer pain management. However, several recent studies suggested that morphine induces apoptosis in human peripheral blood lymphocytes (PBLs), raising a serious concern about the use of opioid-based analgesic strategies. In this study, therefore, we aimed to evaluate whether morphine induced apoptosis in cultured human PBLs. Apoptotic events were assessed by flow-cytometrical detection of surface phosphatidylserine and nuclear fragmentation, as well as Fas, Bcl-2, and Caspase-3 activity in PBLs gated on a light-scatter basis. Peripheral blood mononuclear cells isolated from healthy subjects were cultured with etoposide, morphine, or vehicle (medium) for 48 h. During co-culture with etoposide, apo-ptosis was significantly induced in PBLs, and the cells did not survive for 48 h. In comparison, morphine had no effect on the expression rate of any of the detected molecules, suggesting that no apparent apoptotic processes were induced during the incubation. Furthermore, co-incubation with a Fas-specific antibody did not increase apoptotic cell rates in the morphine cultures. These results do not support the hypothesis that morphine directly modulates PBL apoptosis resulting in immunosuppression. We believe that the choice of opioids for optimal pain relief should not be discouraged until further studies clarify this issue. IMPLICATIONS: Recent reports that morphine potentially induces apoptosis in human lymphocytes in vitro have raised a concern about the use of opioid-based analgesic strategies. Regarding this issue, we present rather contradictory findings that morphine has no effects on the cell expression of various apoptosis-related molecules in cultured human lymphocytes.

Evidence for existence of oligoclonal tumor-infiltrating lymphocytes and predominant production of T helper 1/T cytotoxic 1 type cytokines in gastric and colorectal tumors.

Tumor-infiltrating lymphocytes (TIL) play a central role in cellular immunity against tumor. We have revealed the characteristics of TILs in terms of T-cell receptor (TCR) repertoire, T-cell clonality, and cytokine production. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of gastric or colorectal cancers surgically resected from 11 patients. The cytokine expression was measured by real-time quantitative polymerase chain reaction. TCR repertoires were similar among multiple tissue specimens from different sites of the same tumor. Similar peak patterns of CDR3 size spectratyping were observed among these tumor specimens, but not in normal tissues or PBMCs. In addition, identical peaks were detected in multiple specimens of the same tumor. The ratio of the levels of IFN-gamma to that of IL-4 is significantly higher for tumor lesions compared with PBMCs. These results suggested that a limited number of TILs locally expand in response to tumor antigens exiting within gastric or colorectal cancers and local predominant production of the T helper 1/T cytotoxic 1 type cytokine may affect the anti-tumor immune response of TILs.

DNA fragmentation and detachment of enterocytes induced by anti-CD3 mAb-activated intraepithelial lymphocytes.

To elucidate the role of intraepithelial lymphocytes (IEL) and enterocytes in the defense mechanism of the small intestine, we designed experiments to stimulate the IEL by anti-CD3epsilon, anti-TCRalphabeta, or anti-TCRgammadelta monoclonal antibodies (mAbs), and to examine the subsequent changes to the enterocytes. The enterocytes of the duodenum and jejunum, but not of the ileum, showed massive DNA fragmentation 30 min after intraperitoneal injection of anti-CD3 mAb. These responses were also induced by anti-TCRgammadelta mAb, but not by anti-TCRalphabeta mAb, and were completely inhibited by cyclosporin A. Nearly half of the enterocytes of the villi in the duodenum and jejunum were exfoliated into the lumen 4 h after the injection of the mAb. Administration of anti-CD3 mAb also induced DNA fragmentation in Fas-deficient MRL/lpr mice, indicating that the Fas-Fas ligand system was not involved in these events. The anti-CD3 mAb treatment also induced massive DNA fragmentation in the intestinal epithelium of the duodenum and jejunum in TNF-receptor-1-deficient mice, whereas TNF-alpha induced only the detachment of intestinal epithelium of wild-type mice, implying the dissociation of two independent factors and/or mechanisms for DNA fragmentation and the subsequent epithelial cell detachment in the murine duodenum and jejunum. The mAb failed to exfoliate the epithelium in TNF-R1-deficient mice. Thus, TCRgammadelta(+) IEL, when treated with anti-CD3 or anti-TCRgammadelta mAbs, induced rapid DNA fragmentation and subsequent detachment of the duodenal and jejunal epithelia, but not in the ileum ("the silent ileum"), partly because of the paucity of TCRgammadelta(+) IELs in the ileum.