Nobunao Ikewaki

Immunological Actions of Sophy β‐Glucan (β‐1,3‐1,6 Glucan), Currently Available Commercially as a Health Food Supplement

We examined the immunological actions of Sophy β‐glucan (Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004‐329077), a type of β‐1,3–1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO‐202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy β‐glucan significantly (P<0.01) stimulated the 3H‐thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme‐linked immunoassays (EIAs) revealed that Sophy β‐glucan stimulated the production of interleukin‐8 (IL‐8) or soluble Fas (sFas), but not that of IL‐1β, IL‐2, IL‐6, IL‐12 (p70+40), interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte‐like cell line, U937. The induction by Sophy β‐glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen‐class II (HLA‐class II), Toll‐like receptor‐2 (TLR‐2), and Toll‐like receptor‐4 (TLR‐4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR‐2 and TLR‐4 did not block the Sophy β‐glucan ‐induced production of IL‐8 in the U937 cells. Furthermore, a mAb to the β‐glucan receptor, Dectin‐1, significantly (P<0.05) blocked the Sophy β‐glucan‐induced DNA synthesis in the PBMCs, and Sophy β‐glucan ‐induced production of IL‐8 in the U937 cells. The Sophy β‐glucan‐induced production of IL‐8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H‐89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase‐polymerase chain reaction (RT‐PCR) showed that Sophy β‐glucan stimulated the expression of IL‐8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy β‐glucan also blocked the stimulator cell induction of DNA synthesis and IFN‐γ production in the responder cells in a one‐way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy β‐glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy β‐glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T‐Cell Line Established from the Mouse Intestinal Epithelium

Growth factor‐dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long‐term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A‐stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy‐1+ CD5–TCRαβ+ CD4+CD8 α+β– (double‐positive; DP) IEL and Thy‐1+ CD5– TCRαβ+ CD4–CD8α+β– (CD8 single‐positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T‐cell receptor (TCR)‐directed stimuli. The DP IEL cell line expressed high levels of the CD3‐TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ‐directed stimuli, which indicated that TCRαβ‐mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.

A unique monoclonal antibody mNI-11 rapidly enhances spread formation in human umbilical vein endothelial cells.

We previously reported a novel monoclonal antibody (MAb), designated mNI-11, recognizing an adhesion-associated antigen distinct from any previously reported ones. In this article, this adhesion-associated antigen with a molecular weight of about 97 kDa was found to be strongly expressed on human umbilical vein endothelial cells (HUVECs) by fluorescence-activated cell sorter (FACS) analysis. Expression of this antigen on HUVECs was slightly increased in response to the exposure to tumor necrosis factor-α (TNF-α) or phorbol myristate acetate (PMA). As a biological function exerted by this antigen, it was of great interest that immobilized mNI-11 directly and rapidly enhanced the spread formation of HUVECs, whereas MAbs binding other adhesion-associated antigens such as mNI-58A (anti-CD11a), L130 (anti-CD18), L133.1 (anti-CD31), L178 (anti-CD44), L25.3 (anti-CD49d), or LB-2 (anti-CD54) did not carry such activity under the same conditions. The HUVECs spread formation enhanced by mNI-11 was completely blocked in the presence of a microfilament formation inhibitor, cytochalasin D (CyD), a Ca2+-calmodulin inhibitor, W-7, EDTA, and was partially blocked by a microtubule formation inhibitor, nocodazole, a protein kinase C (PKC) inhibitor, H-7, and a protein synthesis inhibitor, cycloheximide (CHX). However, a protein tyrosine kinase (PTK) inhibitor, genistein, did not affect the spread formation under the same conditions. Taken together, it was suggested that the spread formation of HUVECs enhanced by mNI-11 was mainly associated with the influx of Ca2+ and microfilament reorganization. In addition, the novel property associated with mNI-11 to enhance the spread formation of HUVECs was possibly mediated through its reaction against a unique epitope on HUVECs.

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances.

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q‐mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up‐regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta‐isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte‐like cell line (U937) treated with various apoptosis‐inducing chemical substances: an RNA‐synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein‐synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA‐synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two‐color flow cytometry with Annexin V and 7‐amino actinomycin D (7AAD). The above‐mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) cells, was then investigated using a CD93 mAb (mNI‐11), originally established in our laboratories, and flow cytometry using a fluorescence‐activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI‐11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA‐class I (HLA‐A, B, C), HLA‐class II (HLA‐DR), CD18 (lymphocyte function‐associated antigen‐1 β; LFA‐1β) and CD54 (intercellular adhesion molecule‐1; ICAM‐1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (—) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above‐mentioned chemical substances closely resembled those of HLA‐class I (HLA‐A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI‐11) on the U937 cells treated with various apoptosis‐inducing chemical substances had clearly decreased. On the other hand, an enzyme‐linked immunoassay (EIA) showed that although PMA‐treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above‐mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however, the U937 cells with apoptotic properties induced by various apoptosis‐inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI‐11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis‐inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.

High serum glyceraldehyde-3-phosphate dehydrogenase levels in patients with liver cirrhosis

We have developed a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and established a form of semi-quantitative latex particle aggregation as a first-screening technique for determining serum levels of GAPDH. Focusing on chronic liver diseases, we measured serum GAPDH in 213 patients to clarify its clinical significance. Serum GAPDH levels were similar in healthy control subjects, patients who were hepatitis B or C virus carriers, or with chronic hepatitis. However, the levels were significantly increased in patients with cirrhosis (P<0.001). Logistic regression analysis indicated that the presence of hepatocellular carcinoma and a high gamma-globulin level were closely associated with high GAPDH levels (P=0.006 and 0.011, respectively). Elevated serum GAPDH was also confirmed in a patient with cirrhosis associated with hepatocellular carcinoma using Western blotting analysis. The mechanism(s) of serum GAPDH elevation remains unclear, but one possible source is increased leakage of the GAPDH molecule into the serum from damaged cirrhotic hepatocytes underlying hepatocarcinogenesis or chronic inflammation.

Regulation of CD93 Cell Surface Expression by Protein Kinase C Isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)‐mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte‐like cell line (U937), a human NK‐like cell line (KHYG‐1), and a human umbilical vein endothelial cell line (HUV‐EC‐C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H‐89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde‐fixed cellular enzyme‐linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI‐11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down‐regulated CD93 expression on the U937 cells in a dose‐dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down‐regulated by Go6976, but not by Rottlerin, in the KHYG‐1 cells and by both Rottlerin and Go6976 in the HUV‐EC‐C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up‐regulated CD93 expression on the cell surface of all three cell‐lines and induced interleukin‐8 (IL‐8) production by the U937 cells and interferon‐γ (IFN‐γ) production by the KHYG‐1 cells. In addition, both Go6976 and Rottlerin inhibited the up‐regulation of CD93 expression induced by PMA and IL‐8 or IFN‐γ production in the respective cell‐lines. Whereas recombinant tumor necrosis factor‐α (rTNF‐α) slightly up‐regulated CD93 expression on the U937 cells, recombinant interleukin‐1β (rIL‐1β), recombinant interleukin‐2 (rIL‐2), recombinant interferon‐γ (rIFN‐γ) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Depolymerization of actin filament by cytochalasin E induces interleukin-8 production and up-regulates CD54 in the HeLa epithelial cell line.

We previously reported that the depolymerization of actin filament by cytochalasin E enhances low affinity Fcε receptor II (CD23) expression on the human monocyte‐like cell line, U937 (J. Clin. Immunol. 20: 235, 2000). In this study, we found that cytochalasin E strongly induces interleukin‐8 through an epithelial cell line, HeLa, in dose‐ and time‐dependent manners as assessed by enzyme‐linked immunoassay and reverse transcription‐polymerase chain reaction techniques. In addition, interleukin‐8 production in the HeLa cells cultured with cytochalasin E was blocked in the presence of protein kinase C inhibitors, Go6976 and H‐7. On the other hand, it was found that CD54 (intercellular adhesion molecule‐1; ICAM‐1) expression on the HeLa cells and the secretion of soluble CD54 were significantly up‐regulated after culturing with cytochalasin E, and that these up‐regulations of CD54 were also suppressed by Go6976. Taken together, these findings indicate that cytochalasin E activates protein kinase C under the depolymerization of actin filament, leading to the induction of interleukin‐8 production and the up‐regulation of CD54 in HeLa cells.

A Very Late Activating Antigen-α4 (CD49d) Monoclonal Antibody, BU49 Induces Phosphorylation of a cAMP Response Element-Binding Protein (CREB), Resulting in Induction of Homotypic Cell Aggregation and Enhancement of Interleukin-8 (IL-8) Production

A very late activating antigen‐α4 (CD49d) monoclonal antibody (mAb), BU49 was found to induce phosphorylation of a cAMP response element‐binding protein (CREB) in the human monocyte‐like cell line, U937. This phosphorylation of CREB was completely inhibited by a protein kinase A (PKA) inhibitor H‐89 with the optimum concentration (completely inhibits PKA). Furthermore, BU49 strongly and rapidly (within 5 hr) induced homotypic cell aggregation in the U937 cells accompanied by CREB phosphorylation. This cell aggregation was also completely inhibited by the addition of H‐89. Interestingly, both of two mAbs (mAb13 and 4B4) recognizing different epitopes on the CD29 (β1 integrin) completely inhibited this aggregation at the late phase (18 to 24 hr) but not at the early phase (5 hr) after cultured with BU49. On the other hand, BU49 significantly enhanced interleukin‐8 (IL‐8) production from the U937 cells into the culture supernatant. In addition, this IL‐8 production was significantly blocked in the presence of H‐89 with the optimum concentration. However, a CD29 mAb which inhibits homotypic cell aggregation could not block this IL‐8 production. Taken together, these findings indicate that BU49 induces CREB phosphorylation mainly mediated by PKA, which finally results in the induction of homotypic cell aggregation and the enhancement of IL‐8 production. Furthermore, these findings also indicate that the enhancement of IL‐8 production from the U937 cells induced by BU49 partially depends on CREB phosphorylation mainly mediated by PKA.

A Microfilament Formation Inhibitor, Cytochalasin Strongly Enhances the Low-Affinity Fc ε Receptor II (CD23) Expression on the Human Monocyte-Like Cell Line, U937

Enhancement of the low-affinity Fc ε receptor (CD23) expression by cytochalasin was analyzed on the human monocytelike cell line, U937. The CD23 expression on the U937 cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.

Unique properties of cluster of differentiation 93 in the umbilical cord blood of neonates.

It has previously been reported by these authors that cluster of differentiation (CD) 93 is co‐expressed on naive T‐lymphocytes (CD4+CD45RA+ cells) in neonatal umbilical cord blood cells (UCBCs) but not on normal adult peripheral blood cells (PBCs). In this study, expression of CD93 on other lymphocyte subsets and the concentration of soluble formed CD93 (sCD93) in serum or culture supernatants from neonatal umbilical cord blood (UCB) was examined. It was found that CD93 is also co‐expressed on CD2+, CD16+, CD56+ or CD25+ cells in the lymphocyte population of neonatal UCBCs, but not on normal adult PBCs. The concentrations of sCD93 in serum and culture supernatants from neonatal UCB were significantly greater than those from normal adult peripheral blood. The concentrations of sCD93 in culture supernatants from neonatal UCBCs and normal adult PBCs treated with phorbol 12‐myristate 13‐acetate (PMA) were significantly enhanced compared with those without PMA treatment. The degree of enhancement of sCD93 by PMA in culture supernatants from neonatal UCBCs was significantly greater than that of normal adult PBCs and enhancement of sCD93 by PMA in the culture supernatants from neonatal UCBCs and normal adult PBCs was significantly suppressed by PKC inhibitor. Interestingly, the high concentration of serum sCD93 in neonates was significantly decreased in sera from infants at 1 month after birth. Expression of CD93 on the lymphocyte population of PBCs from infants at 1 month after birth was also significantly decreased, compared with that for neonatal UCBCs. These findings indicate that CD93 in neonatal UCB has unique properties as an immunological biomarker.