Anti-adhesive functions of CD43 expressed on colon carcinoma cells through the modulation of integrins.

CD43 has conflicting roles in both pro- and anti-adhesive function in cell-to-cell adhesion in hematopoietic cells. We examined the role of CD43 glycoprotein in a colorectal carcinoma cell line. We expressed human CD43 antigen on HT-29 cells, a colon adenocarcinoma cell line, and compared the adhesion to the extracellular matrix with that of mock-transduced cells in vitro. CD43 expression inhibited the adhesion to extracellular matrix, such as collagen type IV and laminin. As the expression of ß1 integrin was downregulated in CD43-expressing HT-29 cells, the anti-adhesive effect of CD43 might be implicated in its expression. Our findings suggest that the anti-adhesive function of CD43 in colon carcinoma cells plays a role in the tumorigenesis and metastasis of colorectal carcinoma cells.

In situ induction of dendritic cell-based T cell tolerance in humanized mice and nonhuman primates.

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

Targeting of a developmentally regulated epitope of CD43 for the treatment of acute leukemia.

Previously, we developed a JL1 mouse monoclonal antibody that specifically recognizes the leukemic cells of T, B, and myeloid lineages, but not the peripheral blood cells and pluripotent hematopoietic stem cells. Here, we identified that JL1 mAb recognized a specific epitope of human CD43 and validated its potential as an anti-leukemic targeting agent. After the comprehensive screening of JL1 Ag in the human thymocyte cDNA library, multiple fusion gene constructs encoding human CD43 were generated to identify its specific epitope to JL1 antibody. JL1 antibody interacted with a developmentally regulated and non-glycosylated epitope of the human CD43 extracellular domain (AA 73-81, EGSPLWTSI). In an in vivo leukemia model using NOD/SCID mice injected with CCRF-CEM7 cells, JL1 antibody induced effective cytotoxicity in tumor cells and prolonged survival (p < 0.05). Saporin conjugation to JL1 antibody effectively depleted tumor cells in in vitro cytotoxic assays and also prolonged survival in a leukemic mouse model (p < 0.001). These preclinical results further support the therapeutic potential of the JL1 antibody in the management of acute leukemia.

CD99 is essential for leukocyte diapedesis in vivo.

Recruitment of leukocytes into inflamed tissue requires migration of leukocytes from the blood stream across the endothelial lining and the basement membrane of the local blood vessels. CD99 in humans is a 32-kDa highly O-glycosylated cell surface protein expressed on most leukocytes. The authors recently found CD99 to be expressed in leukocytes and at human endothelial cell contacts. Human CD99 is involved in homophilic interaction between the two cell types and participates in the transendothelial migration of monocytes and polymorphonuclear neutrophils (PMNs) in vitro. To test the role of CD99 in vivo, the authors cloned murine CD99 (muCD99), expressed it in vitro, and generated a blocking monoclonal antibody against it. We first showed that muCD99 is expressed on mouse leukocytes as well as enriched at the endothelial cell borders. Transfection of cells with muCD99 imparts on them the ability to aggregate in a CD99-dependent homophilic manner. Cells expressing muCD99 did not bind to cells expressing murine or human platelet endothelial call adhesion molecule (PECAM) or human CD99. In the thioglycollate peritonitis model of inflammation, anti-CD99 monoclonal antibody blocked the recruitment of neutrophils and monocytes by over 40% and 80%, respectively, at 18 h. Microscopy showed that this blocking occurred at the luminal surface of venules. The authors conclude that CD99 plays a major role in the emigration of leukocytes in vivo.

PKB/Akt inhibits ceramide-induced apoptosis in neuroblastoma cells by blocking apoptosis-inducing factor (AIF) translocation.

Ceramide is a sphingolipid that is abundant in the plasma membrane of neuronal cells and is thought to have regulatory roles in cell differentiation and cell death. Ceramide is known to induce apoptosis in a variety of different cell types, whereas the physiological significance of gangliosides, another class of sphingolipids, in these processes is still unclear. We examined the mechanisms of ceramide-induced cell death using a human neuroblastoma cell line. Treatment of the human neuroblastoma cell line SH-SY5Y with ceramide induced dephosphorylation of the PKB/Akt kinase and subsequent mitochondrial dysfunction. In addition, ceramide-induced neuronal cell death was not completely blocked by inhibition of caspase activity. This incomplete inhibition appeared to be attributable to the translocation of apoptosis-inducing factor to the nucleus. Furthermore, overexpression of active PKB/Akt or Bcl-2 successfully blocked ceramide-induced neuronal cell death through inhibition of the translocation of apoptosis-inducing factor.

Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells.

AIM: To investigate the combined chemotherapeutic effects of celecoxib when used with 5-FU in vitro. METHODS: Two human colon cancer cell lines (HCT-15 and HT-29) were treated with 5-FU and celecoxib, alone and in combination. The effects of each drug were evaluated using the MTT [3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, flow cytometry, and western blotting. RESULTS: 5-FU and celecoxib showed a dose-dependent cytotoxic effect. When treated with 10(-3) mol/L 5-FU (IC(50)) and celecoxib with its concentration ranging from 10(-8) mol/L to 10(-4) mol/L of celecoxib, cells showed reduced cytotoxic effect than 5-FU (10(-3) mol/L) alone. Flow cytometry showed that celecoxib attenuated 5-FU induced accumulation of cells at subG1 phase. Western blot analyses for caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage showed that celecoxib attenuated 5-FU induced apoptosis. Western blot analyses for cell cycle molecules showed that G2/M arrest might be possible cause of 5-FU induced apoptosis and celecoxib attenuated 5-FU induced apoptosis via blocking of cell cycle progression to the G2/M phase, causing an accumulation of cells at the G1/S phase. CONCLUSION: We found that celecoxib attenuated cytotoxic effect of 5-FU. Celecoxib might act via inhibition of cell cycle progression, thus preventing apoptosis induced by 5-FU.

CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells.

CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.

T cell expression of CIITA represses Th1 immunity.

Despite the fact that major histocompatibility complex class II transactivator (CIITA) has been known to be involved in Th1/Th2 balance in addition to its major role as a master regulator for the expression of MHC class II genes, the exact role of CIITA in Th1/Th2 balance is still controversial. To investigate whether the Th1/Th2 balance could be modulated by T cell specific expression of CIITA, we generated CIITA-transgenic mice, in which the CIITA expression is controlled by the distal promoter of p56lck, resulting in constitutive expression of CIITA predominantly in peripheral T cells. Naive CD4+ T cells from CIITA-transgenic mice exhibited a low level of IFN-gamma secretion as well as impaired Th1 polarization in vitro, while IL-4 secretion was enhanced under Th2 condition. In addition, the development of experimental autoimmune encephalomyelitis (EAE), a prototype of Th1-mediated disease, was repressed in CIITA-transgenic mice. Resistance to EAE was correlated with reduced production of IFN-gamma in response to MOG35-55, while the proliferation of MOG35-55 -specific T cells was not affected in CIITA-transgenic mice. Together, these data demonstrate that overexpression of CIITA in T cells inhibits Th1 differentiation and function, suggesting that the expression of CIITA in T cells might play a role in the regulation of the Th1/Th2 balance during the T cell lineage commitment.

TCR-independent and caspase-independent apoptosis of murine thymocytes by CD24 cross-linking.

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat.

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.

Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis.

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.

Identification of antigenic peptide recognized by the anti-JL1 leukemia-specific monoclonal antibody from combinatorial peptide phage display libraries.

PURPOSE: In the present study an antigen-mimetic peptide of the anti-JL1 leukemia-specific monoclonal antibody (mAb) was identified and characterized. METHODS: From combinatorial peptide phage display libraries displaying the random linear heptapeptides and dodecapeptides, we selected clones with affinity to anti-JL1 mAb through repeated rounds of panning on a mAb-coated ELISA plate. The antigenicity and immunogenicity of the peptide epitopes were then studied using chemically synthesized peptides. RESULTS: The selected clones had the LXPSIP consensus sequence. Two synthetic peptides LPPSIPFGLTVGGGGS and LLPSIPNQAYLGGGGS specifically reacted with anti-JL1 mAb in ELISA. These two peptides were found to inhibit the interaction between anti-JL1 mAb and JL1 antigen-positive Molt-4 cells. Although the immune sera raised against the keyhole limpet hemocyanin-conjugated peptides failed to react with Molt-4 cells, it showed strong reactivity to the peptide epitope. However, one mAb raised by peptide immunization successfully bound to Molt-4 cells. CONCLUSION: An epitope-mimetic peptide of anti-JL1 mAb was found using combinatorial peptide phage display libraries. It induced strong humoral response against itself, but only a limited fraction of this humoral response was cross-reactive with the original JL1 antigen.