Thy28 protects against anti-CD3-mediated thymic cell death in vivo.

Apoptotic cell death plays a pivotal role in the development and/or maintenance of several tissues including thymus. Deregulated thymic cell death is associated with autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), a prototype murine model for analysis of human multiple sclerosis. Because Thy28 expression is modulated during thymocyte development, we tested whether Thy28 affects induction of EAE as effectively as antigen-induced thymocyte deletion using Thy28 transgenic (TG) mice. Thy28 TG mice showed partial resistance to anti-CD3 monoclonal antibody (mAb)-induced thymic cell death in vivo, as assessed by annexin V-expression and loss of mitochondrial membrane potential. The resistance to anti-CD3 mAb-induced cell death in Thy28 TG mice appeared to correlate with a decreased c-Jun N-terminal kinase phosphorylation and reduced down-regulation of Bcl-xL. Moreover, thymic hyperplasia was detected in Thy28 TG mice, although thymocyte development was unaltered. Development of peripheral lymphoid tissues including spleen and lymph nodes was also unaltered. Thy28 TG spleen T cells showed an increased production of IFN-γ, but not IL-17, in response to both anti-CD3 and anti-CD28 mAbs. Finally, Thy28 TG mice displayed accelerated induction of EAE as assessed by disease incidence, clinical score, and pathology following immunization with myelin oligodendrocyte glycoprotein compared with control WT mice. These findings suggest that modulation of Thy28 expression plays a crucial role in the determination of thymic cell fate, which may contribute to the development of EAE through proinflammatory cytokine production.

B cell stimulatory factor 1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes.

Resting T cells proliferate in response to B cell stimulatory factor 1 (BSF-1; interleukin 4) plus phorbol myristate acetate (PMA). This response is obtained with highly purified T cells and is density independent, suggesting that accessory cells are not required. Both L3T4+ and Lyt-2+ T cells respond to BSF-1 plus PMA. Although BSF-1 alone does not cause T cell proliferation, it maintains the viability of small, dense T cells, indicating that it acts on resting T cells. Furthermore, BSF-1 is required early in the proliferative response of resting T cells to BSF-1 plus PMA, further supporting the concept that it acts on G0 or early G1 cells. However, BSF-1 is also needed late in the first round of division of T cells stimulated with BSF-1 plus PMA. Removing BSF-1 at 24 h of stimulation prevents entry into S phase. These results indicate that BSF-1 is involved in both the induction of competence and in the progression phases of T cell division.

Association of B cell antigen receptor with protein tyrosine kinase Lyn.

Antigen is thought to cross-link membrane-bound immunoglobulins (Igs) of B cells, causing proliferation and differentiation or the inhibition of growth. Protein tyrosine kinases are probably involved in signal transduction for cell proliferation and differentiation. The Src-like protein tyrosine kinase Lyn is expressed preferentially in B cells. The Lyn protein and its kinase activity could be coimmunoprecipitated with IgM from detergent lysates. Cross-linking of membrane-bound IgM induced a rapid increase in tyrosine phosphorylation of at least ten distinct proteins of B cells. Thus, Lyn is physically associated with membrane-bound IgM, and is suggested to participate in antigen-mediated signal transduction.

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy.

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.

Single cell observation of ligand-induced desensitization of B-cell membrane immunoglobulin-mediated calcium signals.

Using a digital imaging fluorescence microscope we have observed the membrane immunoglobulin (mIg)-induced desensitization of calcium signals in individual BAL17 B lymphoma cells which express two kinds of antigen receptors, mIgM and mIgD. The mIgD-mediated desensitization was partly abrogated by pretreating the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h, however, the mIgM-mediated one was not affected by the pretreatment. This supports the idea that at least two mechanisms are operative for mIg-induced desensitization in B cells.

Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoglobulin in BAL17 B lymphoma cells.

BAL17 cells pulsed with goat anti-IgM or anti-IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen-presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand-induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor-mediated antigen presentation by B cells.

Cytochrome c release, mitochondrial membrane depolarization, caspase-3 activation, and Bax-alpha cleavage during IFN-alpha-induced apoptosis in Daudi B lymphoma cells.

Interferon-alpha (IFN-alpha) displays antitumor action by inducing direct cytotoxicity against tumor cells in addition to generation of cytotoxic cells. The IFN-alpha-induced direct cytotoxicity is at least partly due to induction of apoptosis. In the present study, we examined signaling pathways implicated in IFN-alpha-induced apoptosis in Daudi cells. Release of cytochrome c from mitochondria to cytosol was found after 12 h incubation with IFN-alpha, followed by a decline in mitochondrial membrane potential (Delta psi(m)) and procaspase-3 activation at 24 and 36 h, respectively. Cleavage of endogenous Bax-alpha (21 kDa), generating an 18-kDa fragment (p18 Bax-alpha), was found at 36 h. Although the endogenous p21 Bax-alpha was located in both cytosol and mitochondrial membranes, the p18 Bax-alpha resided only on mitochondrial membranes. IFN-alpha-induced apoptosis occurred 48 h after stimulation, with a further increase in proportion up to 72 h. Pretreatment with pancaspase inhibitor Z-VAD-fmk substantially inhibited the IFN-alpha-mediated Bax-alpha cleavage and apoptosis, but not the decline in Delta psi(m), suggesting the possibility that caspase-3 activation is implicated in the Bax-alpha cleavage, probably leading to amplification of the apoptotic processes. Our results suggest that modulation of endogenous p21 Bax-alpha is implicated in IFN-alpha-induced apoptosis.

Participation of Bax-alpha in IFN-alpha-mediated apoptosis in Daudi B lymphoma cells.

Although interferon-alpha (IFN-alpha) has proved beneficial in the treatment of some tumors, the basis for this is still uncertain. In this study, we examined the effects of IFN-alpha on the growth of tumor cells in vitro, using the Daudi line of B lymphoma cells as a model. There was a dose-dependent accumulation of these cells in the G1 phase of the cell cycle 24-48 h from the time of exposure to IFN-alpha. This was followed between 48 h and 96 h by an increasing degree of apoptosis, as assessed by cell survival, propidium iodine staining, and transmission electron microscopy. Concomitantly with the apoptosis, there was the appearance of pl8 Bax-alpha, an apparently novel variant low molecular weight form of the p21 Bax-alpha found in normal cells. There was also a slight diminution in Bcl-xL, with a resultant drop in the Bcl-xL:Bax-alpha ratio. Treatment of cells with CD40-L partially inhibited the development of apoptosis in response to IFN-alpha. At the same time, generation of p18 Bax-alpha was reduced, which suggests that this plays a part in the apoptotic process. These findings may throw light on the development of lymphomas and perhaps point to future ways of improving therapy with IFN-alpha.

Differential sensitivity to antigenic competition in antigen-specific and -nonspecific antigen presentation by B cells.

We have previously shown that a specific Ag presentation by B cells is different from a nonspecific one in the sensitivity to protein synthesis inhibition. In the present study we have compared the sensitivity of these Ag presentations to antigenic competition. A20-HL cells expressing TNP-specific IgM were pulsed with anti-mouse IgM goat IgG (aMGG) or trinitrophenylated goat IgG (TNP-NGG) as an Ag internalized through Ag receptor or NGG as an Ag internalized by fluid-phase pinocytosis. The pulsed cells induced IL-2 production by NGG-specific cloned T cells. The presence of dog IgG during pulsing A20-HL cells severely inhibited the presentation of NGG but not of aMGG or TNP-NGG. The presence did not decrease the internalization of 125I-NGG into A20-HL cells, suggesting that the inhibition was localized into the complex formation of antigenic peptides with MHC class II molecules. Thus, a specific Ag presentation by A20-HL cells is different from a nonspecific one in its sensitivity to antigenic competition.

Proteolytic cleavage of vitamin K-dependent bovine plasma protein S by alpha-thrombin and plasma serine protease.

It is known that protein S, a vitamin K-dependent plasma protein, isolated from a human source, gives a closely spaced doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and that this heterogeneity in molecular size results from a limited proteolysis of protein S mediated by alpha-thrombin in human species. We found here that alpha-thrombin also rapidly converted single-chain bovine protein S to a nicked form, which consisted of the NH2-terminal segment containing gamma-carboxyglutamic acid and the COOH-terminal large segment bridged by a disulfide linkage(s). These two segments were isolated and chemically characterized after S-alkylation of the nicked protein S. The results suggest that the alpha-thrombin-catalyzed hydrolysis of protein S probably occurs at a peptide linkage (Arg-Ser) located in the NH2-terminal portion. The conversion of single-chain protein S to the nicked form was also mediated by plasma kallikrein and plasmin, in addition to alpha-chymotrypsin and trypsin. However, the alpha-thrombin-catalyzed conversion did not occur when calcium ions were added to the reaction mixture.

A characteristic property of vitamin K-dependent plasma protein Z.

Evidence is presented for rapid, limited proteolysis of protein Z by alpha-thrombin. This alpha-thrombin-catalyzed proteolysis of protein Z occurred at a single peptide linkage, between Arg-365 and Gly-366, located in the COOH-terminal portion. The resulting NH2-terminal large fragment (PZt) and the COOH-terminal peptide (C-peptide) were isolated and chemically characterized. The C-peptide consisted of 31 amino acid residues including one galactosamine-type Thr residue and was assigned to the position from Gly-366 to the COOH-terminal residue of Val-396 in protein Z. The NH2-terminal large fragment, PZt, constituted the remainder of protein Z. The abilities to bind calcium of intact protein Z, PZt, and the derivative of protein Z devoid of the NH2-terminal gamma-carboxyglutamic acid (Gla) domain (Gla-domainless), prepared with the known chymotrypsin treatment, were examined by equilibrium dialysis. The results indicated that intact protein Z and PZt contain four calcium binding sites with dissociation constants of 0.1 mM. Moreover, the Scatchard plot analysis showed positive cooperativity, suggesting the presence of at least two initial sites for calcium binding. In contrast, the Gla-domainless protein Z had no calcium binding site, indicating that the domain of protein Z functional for calcium binding occurs within the NH2-terminal Gla domain. This differed from factor X, factor IX, protein S, and protein C, all of which contain one or two calcium binding site(s) independent on their Gla-domains.

Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain.

Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).

Anti-human factor IX monoclonal antibodies specific for calcium ion-induced conformations.

Four monoclonal antibodies have been produced, which are specific for the Ca2+ or Sr2(+)-induced conformation of human factor IX. Certain, but not all, gamma-carboxy-glutamic acid residues in factor IX are involved in the epitope expression together with the conformation stabilized by the adjacent region of Gla-domain and a disulfide bridge. All the antibodies interfered with the binding of factor IX to phospholipids and inhibited the procoagulant activity of factors IX and IXa beta.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

'Clear line' formation by immunodiffusion of tetanus toxoid and mixtures of distinct monoclonal antibodies.

Single monoclonal antibodies could not form any precipitin line with tetanus toxoid in Ouchterlony analysis. However, some mixtures of two distinct monoclonal antibodies formed a visible precipitin line (conventional line), while others formed a transparent, just barely visible line, which we call a 'clear line'.

Etoposide-mediated sensitization of squamous cell carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced loss in mitochondrial membrane potential.

Squamous cell carcinoma (SCC) cell lines (MIT7-x(L), MIT8, and MIT16) that overexpress Bcl-x(L) have been demonstrated to show resistance to multiple chemotherapeutic drugs. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which belongs to the TNF family of proteins, induces apoptosis in tumor, but not in normal, cells. In the present study, we examined whether etoposide sensitizes tumor cells with multiple-drug-resistance to TRAIL-induced apoptosis. Sequential treatment with etoposide and TRAIL resulted in a synergistically induced cell death in the two resistant lines (MIT7-x(L) and MIT16) but not MIT8, as assessed by WST-8 assay. As expected, MIT7 cells (a drug-sensitive line) were sensitive to the combined treatment. The cell death caused by both etoposide and TRAIL appears to involve apoptosis, since the combined treatment caused a loss in mitochondrial membrane potential (DeltaPsim), which is closely associated with apoptosis induction. The density of the TRAIL-receptors (TRAIL-Rs) was not appreciably modulated by the etoposide treatment, suggesting that etoposide targets molecule(s) downstream of the TRAIL-Rs. Regardless of the molecular mechanisms underlying the cell death, sequential treatment with etoposide and TRAIL could be useful in the design of treatment modalities for patients with SCC, especially those with elevated levels of Bcl-x(L).

BcI-xL antisense treatment sensitizes Bcl-xL-overexpressing squamous cell carcinoma cells to carboplatin.

Carboplatin (CBDCA) has been widely used for the treatment of oral squamous cell carcinoma (SCC). The Bcl-2 family member Bcl-xL has been demonstrated to provide resistance to chemotherapeutic agents including CBDCA. Morpholino Bcl-xL antisense oligonucleotides (oligos) were employed to down-regulate Bcl-xL in CBDCA-resistant (MIT8, MIT16) as well as CBDCA-sensitive (MIT7) SCC cell lines. The oligos were delivered to adherent cells using a scrape-load procedure. The Bcl-xL antisense reduced Bcl-xL levels without altering the level of control actin, suggesting the specificity of this agent. The addition of Bcl-xL antisense oligos substantially prevented the cell growth of both CBDCA-sensitive and-resistant cells. The CBDCA-induced partial prevention of cell growth was further augmented by the addition of the Bcl-xL, but not the control, antisense oligos. The morpholino type Bcl-xL antisense oligos may be useful for the treatment of SCC, especially multidrug-resistant tumors with enhanced Bcl-xL levels.

Cleavage of Bax-alpha and Bcl-x(L) during carboplatin-mediated apoptosis in squamous cell carcinoma cell line.

Although carboplatin (CBDCA) has been used for the treatment of several types of tumors, the complete response rate has been limited, probably because of inherent or CBDCA-induced resistance. As a first step to overcome these problems, we tried to elucidate the mechanisms of CBDCA-mediated cytotoxicity in the squamous cell carcinoma cell line MIT7. The treatment of cells with CBDCA resulted in apoptosis in a dose-dependent manner, as assessed by the propidium iodide staining method and DNA degradation in a nucleosomal pattern. The induction of apoptosis was accompanied by the decline of mitochondrial membrane potential (Deltapsi(m) ) at 12 h following CBDCA stimulation. Variant forms of p18 Bax-alpha and p16 Bcl-x(L) were generated with the down-regulation of both Bax-alpha (p21) and Bcl-x(L) (p31) at 36 and 48 h following CBDCA stimulation, suggesting that the modulation of Bcl-2 family proteins Bax-alpha and Bcl-x(L) play some role in CBDCA-mediated apoptosis. The activation of caspase-3 and -8 occurred at 12 and 24 h following the stimulation, respectively. The pretreatment of cells with pan-caspase inhibitor Z-VAD-fmk markedly prevented CBDCA-mediated cytotoxicity/apoptosis and the modulation of Bcl-2 family proteins (generation of p18 Bax-alpha and p16 Bcl-x(L) ) with only slight prevention of decline of Deltapsi(m). Taken together, these results may suggest that activation of several caspases, including caspase-3 and -8, plays some role in CBDCA-mediated apoptosis, probably through the modification of Bcl-2 family proteins, Bax-alpha and Bcl-x(L). Moreover, caspase activation may occur downstream of membrane depolarization.

Bcl-x(L) confers multi-drug resistance in several squamous cell carcinoma cell lines.

Carboplatin (CBDCA) alone or in combination with irradiation and other chemotherapeutic agents has been used for the treatment of oral squamous carcinoma. However, there are some limitations for such therapy because of inherent or acquired resistance to CBDCA. To gain some insights into the association of CBDCA resistance with Bcl-2 family level or p53 status, we established eight carcinoma cell lines, consisting of two resistant (MIT8, MIT16), two sensitive (MIT6, MIT7), and four intermediate lines. All of the five cell lines with p53 mutation belonged to the resistant approximately intermediate group, whereas two of three other lines with wild-type p53 were in the sensitive group. Interestingly, both of the two resistant cell lines showed elevated levels of Bcl-x(L), almost double that of sensitive line (MIL5), whereas either Bcl-2 or Bax-alpha level did not correlate with the CBDCA-resistance. To further verify the association between the Bcl-x(L) level and the drug resistance, two transformants (x(L)-3, x(L)-6) overexpressing Bcl-x(L) in the CBDCA-sensitive cell line MIT7 were established using the gene transfer method. Both clones showed resistance to multiple chemotherapeutic agents, including CBDCA, actinomycin D, etoposide, and mitomycin C. Moreover, MIT8 and MT16 also displayed cross-resistance to these agents. These findings suggest that Bcl-x(L) may function as one of the key components conferring multiple drug-resistance in squamous cell carcinomas.