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Interleukin (IL)-6 is a pleiotropic cytokine that has been shown to inhibit the growth of early stage and to promote the proliferation of advanced stage melanoma cells in vitro. In patients with metastasizing melanomas, highly increased IL-6 blood levels correlate with a poor response to chemotherapy and a worse overall prognosis, suggesting that IL-6 promotes melanoma progression in vivo. Here, we analyzed the role of IL-6 in melanoma development and progression in a transgenic mouse model. We bred IL-6-deficient mice with MT-ret transgenic animals predisposed for melanomas. While MT-ret transgenic animals develop severe melanosis of the skin and subcutis and subsequent melanomas at an incidence of 80% during their first year of life, MT-ret mice devoid of IL-6 developed preneoplastic melanosis and consecutive melanomas significantly less frequently (47%; P< 0.05). Moreover, the tumors were significantly smaller in the groups of MT-ret mice lacking one (P< 0.05) or both (P< 0.01) copies of the IL-6 gene. Immunoblot analysis revealed that ret transgene expression was not reduced in the skin of mice lacking IL-6, indicating that the observed decrease of melanoma incidence and of tumor sizes was not because of a down-regulation of transgene expression. Taken together, these results indicate that IL-6 enhances both the development of melanoma precursor lesions and the subsequent growth of the resulting tumors in the MT-ret model of melanoma development.

Regulation of immune system is of paramount importance to prevent immune attacks against self-components. Mice deficient in the interleukin (IL)-2/IL-15 receptor β chain, CD122, are model animals of such immune attacks and characteristically have a high number of abnormally activated T cells. Here, we show that the transfer of CD8+CD122+ cells into CD122-deficient neonates totally prevented the development of abnormal T cells. Furthermore, recombination activating gene–2−/− mice that received wild-type mice–derived CD8+CD122− cells died within 10 wk after cell transfer, indicating that normal CD8+CD122− cells become dangerously activated T cells in the absence of CD8+CD122+ T cells. CD8+CD122+ cells could control activated CD8+ or CD4+ T cells both in vivo and in vitro. Our results indicate that the CD8+CD122+ population includes naturally occurring CD8+ regulatory T cells that control potentially dangerous T cells.

Background:Human serum immunoglobulin A1 (IgA1) has a unique mucine-like structure in its hinge region that contains O-glycans and proline-rich peptides. We previously reported the under-O-glycosylation of the hinge in serum IgA1 and deposited IgA1 in glomeruli (glomerular IgA1) in IgA nephropathy. The clinical development and exacerbation of IgA nephropathy frequently are preceded by episodes of upper respiratory tract infections. Therefore, tonsils, which represent the predominant immunocompetent tissue of the upper respiratory tract, may be related to the pathogenesis of IgA nephropathy. In this study, we investigated the O-glycan structure of IgA1 produced by tonsillar lymphocytes (tonsillar IgA1), suspecting that tonsillar IgA1 is one of the origins of glomerular IgA1 in patients with IgA nephropathy. Methods:Extracted tonsils were obtained from 7 patients with IgA nephropathy and 5 patients with chronic tonsillitis as controls. Tonsillar lymphocytes separated from extracted tonsils were cultured for 7 days, and IgA1 in the culture medium was purified. The varieties of O-glycans in tonsillar IgA1 were determined from the molecular weights measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results:A significant increase in the percentage of asialo-agalacto type O-glycans was found in tonsillar IgA1 in 4 of 7 patients with IgA nephropathy (57.1%) compared with controls. Between the IgA nephropathy and control groups, the difference was statistically significant (P= 0.047). Conclusion:This study provides precise information about the structure of O-glycans in tonsillar IgA1 in patients with IgA nephropathy. Our results suggest that tonsils produced the underglycosylated IgA1 molecules in patients with IgA nephropathy.

Ageing-dependent dysfunctions develop for each of various cell types in living organisms. Amongst them, immunosenescence, an ageing-dependent deterioration of the immune system, seriously affects the health condition of an aged individual. The central problem in immunosenescence is a decrease in the ability of T-cells to respond to antigens for proliferation and cytokine production, accompanied by accumulation of T-cells with a memory cell phenotype (CD44-high CD45RB-low) replacing those with a naive cell phenotype (CD44-low CD45RB-high). Recently, a transgenic mouse model and a genetically modified mouse model have been reported to display promoted immunosenescence. One is a mouse line transgenic to human CD2 promoter / enhancer-guided rabbit protein kinase C(PKC)α, and the other is a mouse line in which the p53 gene is deficient (p53- / -). Both of these mouse lines display accelerated accumulation of memory Tcell replacing naive T-cells during ageing, accompanying progressively diminishing responsiveness to sheep red blood cell antigens for cytokine production. T-cells activate PKCα when they receive either an antigenic or stress stimulus. Repetitions of antigenic and stress stimuli that recurrently activate PKCα are probably mimicked by continuously elevated PKCα activity in the PKCα transgenic mice. Activated PKC-α probably counteracts the apoptosis-inducing signal, which prevents activation-induced cell death of T-cells and causes accumulation of memory T-cells as the descendants of activated T-cells that have survived. On the other hand, p53 is known to mediate the signaling for apoptosis induction that follows DNA damage due to oxidative stress. The apoptotic signal pathway fails to work well in p53- / - mice. During ageing of these mice, T-cells must encounter a number of antigenic and stress stimuli for activation, and activation of Tcells that is not followed by cell-death causes accumulation of memory T-cells. Results of experiments using the two transgenic mouse models for immunosenescence introduced here support the view that immunosenescence develops by chronic exposure to antigenic and stress stimuli, which is promoted by a defect in the mechanism for efficient elimination of activated T-cells.

T-cell death, which occurs either for ontogenic T-cell selection or for activated T-cell elimination, is normally induced through binding of a specific ligand to cell-surface T-cell receptor for crosslinkage. Heavy metals and carbonyl compounds that bind to protein-reactive groups such as cysteine sulfhydryl groups and lysine ε-amino groups may also induce crosslinkage of cell-surface proteins, in part replacing or modifying the ligand-mediated action. This chemical event has been found to accompany clustering of membrane rafts, to which signal-transducing elements such as glycosylphosphatidylinositol-anchored proteins and Src family protein tyrosine kinases (PTKs) are attached, and to trigger the signal transduction for apoptotic T-cell death, inducing mitochondrial membrane potential reduction, caspase activation and DNA fragmentation. As signals potentially upstream of this signaling, activations of PTKs and mitogen-activated protein (MAP) family kinases and production of reactive oxygen species (ROS) were induced following the cell-surface event, and crucial roles of activation of c-Jun amino-terminal kinase and apoptosis signal-regulating kinase 1 by a redox-linked mechanism in the cell-death signaling were demonstrated. Intriguingly, ROS production as well as PTK/MAP family kinase activation occurred in a membrane raft integrity-dependent manner. The redox-linked and cell surface-oriented signal delivery pathway demonstrated here may play an important role in induction of immune disorders by protein reactive group-binding chemicals.

Signals for cell-death induction by menadione were studied in Jurkat T cells. Low concentrations of menadione (10-20 μM) and H2O2 (10-50 μM) induced cell death accompanying low (menadione: <5%) or moderate (H2O2: 10-15%) levels of DNA fragmentation in Jurkat cells. These concentrations of menadione (10 μM) and H2O2 also caused membrane (necrotic) cell death at unproportionally high (80%) and proportional (10-30%) levels, respectively. Higher concentrations (100-5,000 μM) of H2O2 exclusively induced membrane cell death. Unexpectedly, 30-300 μM menadione induced ever-decreasing levels of necrotic cell death in a concentration-dependent manner. An in vitro kinase assay showed that 20-50 μM, but not >100 μM, menadione induced activation of c-Jun NH2-terminal kinase (JNK), whereas a striking activation of JNK was induced by 500-5,000 μM H2O2. Induction of cell death by a low concentration of menadione was partially inhibited in dominant negative JNK gene-transfected Jurkat/VPF cells. A high concentration (300 μM) of menadione was found to inhibit cell-death induction by high concentrations (200-5,000 μM) of H2O2. The JNK inhibitory activity of menadione was also demonstrated in a cell-free system. However, menadione did not activate JNK in vitro. These results suggest that JNK is required for induction of not only apoptotic cell death, but also necrotic cell death in Jurkat T cells and that menadione biphasically controls this JNK-linked signal for inducing cell death.

The signaling for activation of protein tyrosine kinases (PTKs) is usually started by binding of ligands to cell-surface receptors. However, recent evidence suggests the presence of ligand binding-independent signaling pathways that are mediated by oxidative stress. Oxidation and reduction of protein cysteine sulfhydryl (SH) groups may work as a molecular switch to start or to stop the signaling. It is known that oxidation of cysteine SH groups on protein tyrosine phosphatases switches off the action of protein tyrosine phosphatases. This event may not, however, signal for initial autophosphorylation of previously unphosphorylated PTKs, whereas it certainly prevents dephosphorylation of once-phosphorylated PTKs. We have suggested new mechanisms for oxidative stress-mediated PTK activation. First, cell-surface glycosylphosphatidylinositol-anchoring proteins and a phosphoglycolipid/cholesterol-enriched membrane microdomain termed a "raft" can be the direct targets of oxidative stress for inducing their clustering through an S-S-bonded or S-X-S-bonded crosslinking of cell-surface proteins and subsequent activation of raft-associating Src family PTKs. Second, intracellular specific cysteine SH groups on PTK proteins can be another target of oxidative stress for inducing a conformational change necessary for initial activation of PTKs. A possible relationship between cell-surface and intracellular events is that the former frequently induces superoxide production as the second messenger for the latter.

Galactose-α1,3-galactose (αGal) epitopes, the synthesis of which requires the enzyme product of α1,3-galactosyltransferase (α1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The αGal-negative species as well as mice with deletion of the α1,3GT gene produce abundant anti-αGal antibodies. The evolutionary loss of αGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the α1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of αGal expression, may have implications in medical research.

Increased production of glyoxal (GO) and methylglyoxal (MGO) under oxidative stress is harmful to the cells. In this study, we examined the early signaling effect of GO/MGO on cultured human umbilical vein endothelial cells. Both GO and MGO induced tyrosine phosphorylation and aggregation of a number of cellular proteins. Aggregation occurred mainly for cell surface proteins such as Flk-1 and VE-cadherin, but barely for the majority of intracellular proteins. Interestingly, however, GO/MGO caused both aggregation and dephosphorylation of intracellular phospho-ERK for inactivation. This phospho-ERK dephosphorylation was mediated by orthovanadate-sensitive phosphatase activity accompanying chemical recruitment of MKP-1 to the aggregated phospho-ERK. Evidence was provided that GO/MGO upregulated MKP-1 activity that in turn dephosphorylated possibly co-aggregated phospho-ERK efficiently for inactivation. These results together suggest that GO and MGO trigger a novel pathway for chemical reaction-mediated downregulation of ERK.

We showed that osmotic stress induces activation of c-RET and second-set activation of constitutively activated RET-MEN2B. A few percentage of RET proteins normally formed disulfide-bonded dimers in the cell, and osmotic stress promoted formation of these dimers. The disulfide-bonded dimers displayed higher levels of autophosphorylation and catalytic activity per molecule than did monomers. Osmotic stress also promoted activation and disulfide-bonded dimerization of the extracellular domain-depleted mutant RET (RET-PTC-1), suggesting that the target amino acid(s) for dimerization is located intracellularly rather than in the cysteine-rich region of the extracellular domain. In the mutant c-RET and RET-PTC-1 in which Cys987 of c-RET or Cys376 of RET-PTC-1 was replaced with Ala, the levels of intrinsic kinase activity were greatly reduced and barely increased in response to osmotic stress. Correspondingly, the Cys376-defective RET-PTC-1 did not form any demonstrable levels of dimers even after exposure to osmotic stress. In contrast, another RET-PTC-1 mutant that had a replacement of Cys365 with Ala mostly behaved like parental RET-PTC-1. These results suggest that Cys987 of c-RET or Cys376 of RETPTC-1 plays a crucial role in maintenance and promotion of dimerization and activation of the RET kinases.

ABSTRACTInterleukin-2 (IL-2)/IL-15 receptor β (IL-15Rβ)−/−mice were susceptible to infection with avirulent Salmonella entericasubsp. entericaserovar Choleraesuis, whereas IL-2−/−mice were resistant. A natural killer cell response was not evident for both types of deficient mice. A Th1 response was detected in IL-2−/−but not in IL-2/IL-15Rβ−/−mice infected with Salmonella, suggesting that IL-2/IL-15Rβ signaling is important for the generation of protective Th1 cells.

ABSTRACTThe catalytic activities of Ret tyrosine kinases as the products of oncogene RETwith multiple endocrine neoplasia type 2A (Ret-MEN2A) or 2B (Ret-MEN2B) mutations and the hybrid gene from c-RETand RFP(Rfp-Ret) were higher than those of c-Ret. We demonstrated that ultraviolet light (UV) irradiation induced activation of c-Ret and superactivation of genetically mutated, and thereby constitutively activated, Ret-MEN2A, Ret-MEN2B, and Rfp-Ret. We found that small proportions of c-Ret and Ret-MEN2B and a large proportion of MEN2A were dimerized due to disulfide bonds and that high kinase activity resided in these fractions. The UV-induced activation of c-Ret and superactivation of Ret-MEN2A and Ret-MEN2B were then shown to be closely associated with promotion of the disulfide bond-mediated dimerization of the Ret proteins. Furthermore, we showed that a large proportion of Rfp-Ret was dimerized or polymerized and that almost all kinase activities resided in the highly polymerized but not dimerized fraction. The UV-induced superactivation of Rfp-Ret was also found to be closely associated with promotion of polymerization but not with dimerization of Rfp-Ret. Further experiments revealed that UV induced intracellular dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Most importantly, the levels of basal kinase activity and dimerization of Ret-TPC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-TPC-1 was replaced with alanine, were low and were not increased by UV irradiation. These results suggest that the cysteine at this position works as the primary target of dimerization of Ret proteins inside the cell for both the maintenance of the basal kinase activity and its promotion by UV, possibly in co-operation with the cysteine(s) in the extracellular domain of Ret-MEN2A and Rfp-Ret, which is the target of dimerization and polymerization outside the cell. The potential biological significance of the UV-mediated superactivation of mutant Ret through the newly proposed mechanism in oncogenesis is discussed.

Transgenic mice carrying the REToncogene under the control of the metallothionein promoter exhibit severe pigmentation of the whole skin and melanocytic tumors. The genetic background influences melanoma development in RETmice; founder mice crossed with BALB/c mice show decreased incidence and increased latency of melanocytic tumors, whereas progeny of C57BL/6 mice show the opposite effect. Using partially congenic RETmice on a C57BL/6 genetic background (N3/RETmice), we studied genetic linkage in (N3/RETxBALB/c)xN3/RETbackcross mice. We mapped three melanoma modifier loci, on chromosome 1 (Melm1and Melm2) and chromosome 11 (Melm3), that are linked with early melanoma incidence and latency. Mapping of Melmloci and of five additional regions on chromosomes 6, 8, 9, 12, and 13 indicated allelic imbalance in N3/RETmice, with a significant excess of BALB/c alleles, suggesting the presence of additional putative melanoma modifier loci on these chromosomes.

1,3-galactosyltransferase (1,3GT) is an enzyme that produces carbohydrate chains termed Gal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost 1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated.

To define the role of cytokine binding to the IL-2/IL-15Rβ chain in protective immunity against systemic infection with herpes simplex virus type 2 (HSV-2), IL-2/IL-15 receptor(R)β knock-out mice were inoculated intraperitoneally with HSV-2 strain 186. IL-2/IL-15Rβ-deficient mice were susceptible to systemic HSV-2 infection compared with their heterozygous littermates. The emergence of natural killer (NK) cells was impaired in IL-2/IL-15Rβ knock-out mice, but CD4+T cell receptor (TCR) αβ+T cells were normally detected in the peritoneal cavity after infection with HSV-2. However, the generation of HSV-2-specific CD4+T helper (Th) 1 cells producing interferon-γ was impaired in IL-2/IL-15Rβ knock-out mice following HSV-2 infection. The serum IL-15 level in control mice was increased in the early stage after HSV-2 infection but was not detectable in IL-2/IL-15Rβ knock-out mice. In vivoadministration of recombinant IL-15 protected normal mice from HSV-2-induced lethality, accompanied by increases in numbers of NK cells and HSV-2-specific Th1 cells. Taken together, these results suggest that IL-15, using the IL-2/IL15Rβ chain, plays an important role in mounting protective immunity during the course of systemic HSV-2 infection.

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 μM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3–4%) and Ret-MEN2B (1–2%) and large percentages of Ret-MEN2A (30–40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond–mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond–mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain–deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1–C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.

We recently established a metallothionein‐I(MT)/RETtransgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma develop stepwise. Malignant melanoma cells but not benign melanocytic tumor cells had metastatic ability in transgenic mice. In the present study, we investigated the expression of several matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MT1– MMP, TIMP‐1 and TIMP‐2, in these tumors. Western and northern blot analyses revealed that malignant transformation of melanocytic tumors developed in MT/RETtransgenic mice accompanied with upregulation of MMP‐9and downregulation of TIMP‐2.Expression of other MMP and TIMPgenes examined was very low or undetectable in both benign and malignant tumors. Since activation of MMP‐9in malignant tumors was detected by gelatin zymography, these results suggest that imbalance of expression of the MMP‐9and TIMP‐2genes might be associated with metastatic ability of melanoma cells developed in MT/RETtransgenic mice.

c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N2O3scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitrolabeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivolabeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na3VO4did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N2O3-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N2O3-provoked S-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.

Although interleukin 2 (IL-2) has been thought to be the most important cytokine for T cell growth, animals lacking IL-2 or a component of its receptor molecules have more expanded T cells with activated memory phenotype, indicating an indispensable role for the IL-2/IL-2 receptor system in regulating the size and activity of the T cell population. In this study, we investigated the possible mechanism of abnormal expansion of activated T cells in IL-2 receptor β chain (IL-2Rβ)−/− mice using the systems of bone marrow transplantation and T cell transfer. Here, we show that IL-2Rβ2/− T cells in mice reconstituted with a mixture of IL-2Rβ2/− and IL-2Rβ1/+ bone marrow cells did not develop into an abnormally activated stage, and that already activated IL-2Rβ2/− T cells were effectively eliminated by IL-2Rβ1/+ T cells when both cells were cotransferred to T cell–deficient host mice. This regulation and/or elimination was dependent on T cells bearing α/β type T cell receptor, especially on CD8+ T cells and independent of the Fas–Fas ligand (FasL) system. IL-2Rβ1/+ T cells that eliminated activated IL-2Rβ2/− T cells expressed FasL, perforin, granzyme B, and tumor necrosis factor α/β. These results indicate a novel function of IL-2Rβ that is necessary for the induction of regulatory T cells acting to eliminate activated T cells.

The immunoregulatory action of saikosaponin-d (SSd), which was isolated from the root of Bupleurum falcatumL. and has a steroid-like structure, was examined on splenic T lymphocytes of C57BL/6 mice. SSd displayed a definite action in vitroto bidirectionally control the growth response of T lymphocytes stimulated by concanavalin A, anti-CD3 monoclonal antibody, and calcium ionophore A23187 plus phorbol 12-myristate 13-acetate. Low concentrations (I-3 μg/ml) of SSd upregulated the responses to suboptimum stimuli of agonists, particularly during the relatively late stage of the responses, whereas it downregulated the responses to supraoptimal stimuli. Under appropriate experimental conditions, SSd promoted interleukin-2 (IL-2) production and IL-2 receptor expression. It also accelerated c-fosgene transcription, but it did not modulate the level of tyrosine phosphorylation of cellular proteins. We concluded from these results that SSd uniquely modulates T lymphocyte function and that at least one target of the action of SSd is located at or before the step of c-fosgene transcription and after T-cell receptor/CD3-mediated protein tyrosine kinase activation.

T-cell antigen receptor is a heterodimer of disulfide-linked α- and β-chains. Although the essential features of T-cell receptor seem to be rather similar to those of immunoglobulin, the amount of T-cell receptor expressed on the surface of a T-cell is not large enough to be analyzed physico-chemically. In this study, the DNA fragment encoding 120 amino acids from the 116th to the 235th of the murine, β-chain which corresponds to the presumed constant domain was inserted into an expression vector in E. coli. A large amount of this 18 kDa protein was observed to be synthesized in E. coli, and might be a good source for the three dimensional analysis of the T-cell receptor molecule.

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae(CPS‐K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS‐K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS‐K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coliO55 or Salmonella enteritidisexhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coliO111 or that by Boivin's method from E. coliO55 was similar to that of CPS‐K. It is concluded therefore that CPS‐K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS‐K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS‐K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS‐K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.

The serological specificity of the neutral polysaccharide possessing extraordinarily strong adjuvanticity originally isolated from the culture supernatant of KlebsiellaK1 strain Kasuya has been investigated. Among all of the reference strains (K1–K82) of Klebsiellaobtained from the International Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, only 13 strains have been shown to produce the adjuvant polysaccharide by the passive hemagglutination inhibition test. All of these 13 strains belong to the 03 group, and the strains which belong to other O groups or O groups of which were not identifiable did not produce it. The gel precipitation test has demonstrated that the adjuvant polysaccharide is antigenically identical to O3 antigen isolated from the cells of the decapsulated mutant (strain LEN 1) of KlebsiellaK1 strain Kasuya and to O9 antigen of Escherichia coliisolated from either the culture supernatant or the cells, which has already been shown to be antigenically and structurally identical to the O3 antigen of Klebsiella.

In culture fluid, Klebsiella pneumoniaetype 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type‐specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type‐specific capsular polysaccharide was isolated from ultraviolet‐irradiated cells of K. pneumoniaetype 1 Kasuya strain which had been labeled with leucine‐requiring marker by selecting unagglutinable cells with the antiserum to the type‐specific capsular polysaccharide. Serological tests showed that the type‐specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniaewas discussed.

The effect of the capsular polysaccharide of Klebsiella pneumoniaetype 1 Kasuya strain (CPS‐K) on the formation of macrophage colonies in cultures of mouse spleen cells was investigated by the liquid culture technique during an incubation period of 7–8 days. CPS‐K markedly inhibited further generation of macrophage colonies when added at any time after the beginning of culture, whereas it showed no destructive effect on macrophage colonies which were already formed before its addition. When CPS‐K was present throughout the incubation period, such a low concentration as 0.05 μg/ml significantly inhibited colony formation, and the intensity of its inhibitory effect depended on its dose in the range of 0.005–50 μg/ml. The inhibitory effect persisted even if CPS‐K was washed out after spleen cells were kept in contact with 20 μg of CPS‐K per ml at 37 C for 6 hr. It was found that the inhibitory effect of CPS‐K on colony formation was not mediated through its action on T cells, B cells or macrophages, and that it was not due to the generation of suppressor cells capable of inhibiting colony formation. It is concluded therefore that CPS‐K directly inhibits the proliferation of macrophage colony‐forming cells. The active substance responsible for the inhibitory effect of CPS‐K on colony formation is the neutral polysaccharide fraction of CPS‐K.

The adjuvant action of the O3 antigen of Klebsiella(KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti‐SRBC plaque‐forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia colilipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 107‐2 × 108sonicated SRBC at 6 × 104gfor 30 min [Sup‐SRBC]), whose ability to inhibit anti‐SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC‐primed mice. However, Sup‐SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup‐SRBC involved helper memory for anti‐trinitrophenyl group (TNP) response to challenge with TNP‐conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup‐SRBC‐primed mice was also demonstrated by an in vitroculture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup‐SRBC. Repeated stimulation with Sup‐SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (106) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.

Previously we found that KlebsiellaO3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant of strain Kasuya (O3:K1) or its decapsulated mutant strain LEN‐1 (O3: K1–) exhibited very strong adjuvant activity in augmenting antibody response and delayed‐type hypersensitivity to protein antigens in mice. The preparation of KO3 LPS after deproteinization by four cycles of treatment with chloroform–butanol (5: 1) usually contained a small percentage of proteins and a definite amount of another antigen which was destroyed by heating at 100 C for 1 hr. This antigen proved to be derived from type 1 fimbriae which are responsible for mannose‐sensitive hemagglutination of guinea pig erythrocytes. The preparation of KO3 LPS isolated from culture supernatant of the strains which did not produce type 1 fimbriae exhibited strong adjuvant activity similar to that of the preparation from those which produced them. The preparation of KO3 LPS treated with hot phenol water which is known to remove lipid A‐associated proteins exhibited a similar strong adjuvant activity. The preparation of KO3 LPS after extensive deproteinizing, two cycles of pronase treatment followed by ten cycles of treatment with chloroform–butanol, no longer contained detectable amounts of proteins and the fimbrial antigen, but this preparation also exhibited similar strong adjuvant activity. Moreover, there was no difference in strength of the adjuvant activity between the preparation of KO3 LPS isolated from culture supernatant and that isolated by the phenol method from bacterial cells. The present study demonstrates that the strong adjuvant activity of the preparation of KO3 LPS does not depend in any way on proteins contaminating the preparation.

In a series of our earlier studies, the O3 antigen isolated from culture supernatant of Klebsiella pneumoniaestrain Kasuya (O3:K1) (KO3) was shown to exhibit very strong adjuvant activity in mice. KO3 obtained was homogeneous in analyses by either gel filtration or ultracentrifugation. Its molecular weight determined by ultracentrifugal analysis was greater than 2 × 106. It contained 37.9% C, 6.20% H, 0.24% N, and less than 0.1% P. KO3 was degraded into the polysaccharide moiety and lipid moiety (about 20%) by hydrolysis with 1% acetic acid at 100 C for 1 hr. The molecular weight of the polysaccharide moiety obtained by the hydrolysis was 16,200 as determined by the Somogyi‐Nelson method. Chemical analyses using methylation analysis and Smith degradation as the principal methods indicated that the polysaccharide moiety consisted of a mannan which has a pentasaccharide repeating unit of α‐mannosyl‐1,3‐α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐α‐mannose joined through α‐1,3‐mannosyl linkages. The number of repetitions was less than 20. The fact that minor components such as 2‐keto‐3‐deoxyoctonate and glucose were detected suggests the presence of a core oligosaccharide, but its precise structure is unknown.

Previously we showed that KlebsiellaO3 lipopolysaccharide (KO3 LPS) is much more potent than other kinds of LPS including Escherichia coliO127 LPS (EO127 LPS) in adjuvant activity in augmenting antibody response and delayed‐type hypersensitivity to protein antigens and in the ability to enlarge the regional lymph node. Various denned uniform salt forms, the triethylamine, sodium, potassium, ammonium, tris(hydroxymethyl)ammomethane, and calcium salt forms, of KO3 LPS and EO127 LPS were prepared by removing basic materials present in LPS preparations by electrodialysis and neutralizing the electrodialyzed LPS preparations with various kinds of alkali. The triethylamine salt form showed the best solubility and consisted of the smallest granules and, on the other hand, the calcium salt form showed the lowest solubility, compared with the natural form and the other uniform salt forms. Even if the natural forms of KO3 LPS and EO127 LPS were converted to the defined uniform salt forms, adjuvanticity of KO3 LPS and EO127 LPS in augmenting delayed‐type hypersensitivity to ovalbumin and the ability to enlarge the regional lymph node did not significantly differ from those of the respective natural forms. From these results it is concluded that the difference in strength of the adjuvanticity between KO3 LPS and EO127 LPS is not due to the difference in their salt forms, solubility or physical state. Moreover, there were no significant differences in lethal toxicity for mice by the intraperitoneal route among the natural form and all the uniform salt forms of KO3 LPS tested.

The lymphocyte signal transduction, as determined by intracellular free Ca2+mobilization of concanavalin A‐stimulated T lymphocytes and of antiimmunoglobulin μ chain antibody‐stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine Pl.HTR mastocytoma‐induced ascites and in spleen cells treated with the ascites in vitro.The suppression was observed both at the peak level and in the reactive pattern of Ca2+influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic Pl.HTR tumor‐derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor‐derived factor‐mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

We investigated the proliferation‐promoting effects of murine amniotic fluid (MAF) on in vitrocultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation‐promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells from 2‐week‐old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1‐year‐old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti‐mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti‐mouse SCF antibody revealed a band at 30–32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type‐dependently antagonized by TNF‐α and TGF‐β. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.

Alloreactive CTL clone D2‐23 proliferated in response to antigenic cells without IL‐2 production. Among subclones of D2‐23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fc receptors. Cyclosporin A, which totally blocks IL‐2 production of Th1 cells, barely or only partially inhibited PMA‐ or aCD3‐induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL‐2 plus antigenic cells, or even IL‐2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR‐linked mechanism for CTL proliferation that is independent of costimulatory cell‐ or cytokine‐mediated signaling, but is originally prepared by prior stimulation with IL‐2.

A high concentration (30 μg/ml or more) of Con A caused the death of not only thymocytes but also splenic cells of BALB/c mice, whereas a moderate concentration (3 μg/ml) of Con A induced proliferation of these cells. A high concentration of Con A also induced the death of splenic cells of athymic BALB/c‐nu/nu mice and the bone marrow cells of BALB/c mice which mainly consist of non‐T cells. However, any concentration (1‐30 μg/ml) of Con A failed to induce the proliferation of these cells. Specific binding of tetrameric Con A to mannose‐containing receptors was required for the induction of cell death. DNA fragmentation was observed by both laser flow cytometry and electrophoresis in Con A‐stimulated T cells and non‐T cells. This indicated that the mechanism of induction of apoptosis with Con A is not necessarily TCR‐dependent. Con A induced tyrosine phosphorylation of a number of proteins in various types of cells. Interestingly, phosphorylation of the 40 kDa protein developed only in the thymocytes and spleen cells that contain T cells, whereas phosphorylation of the 80 and 120 kDa proteins appeared in both T cells and non‐T cells. These results suggested that the Con A‐induced apoptosis of T cells and non‐T cells involves different but possibly mutually related protein tyrosine phosphorylation‐linked signals.

Abstract: The chronic donor organ shortage has led to the production of transgenic animals. We assume that cells or organs derived from possible animal donors carrying a large amount of α‐galactosyl epitopes should not be transplanted into humans, because a corresponding amount of immunosuppressants would be needed to prolong the survival of such xenografts in the recipients. This may not only make the recipients compromised hosts but also introduce some unknown or uncontrollable pathogens into society at large. We also assume that gene manipulation itself should not be a detriment to possible transgenic animals. To explore possibilities that not only can minimize the possible detrimental factors to humans, such as α‐galactosyl epitopes, but also can minimize the possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory sequences, we have produced a DNA construct that replaces the mouse oc(1,3)‐galactosyltransferase gene (GT) with the human a(1,2)‐fucosyltransferase (FT) minigene (promoterless for the expression of FT) at the GT locus. The mouse fibrosarcoma cell line, L929, was transfected with the construct. Colonies were obtained after incubation with non‐heat‐inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at the GT locus without integration of any selectable marker genes. The immunostaining analysis with lectins showed that the transfectants expressed H antigens, which suggested that H antigens were expressed by the intrinsic GT promoter. Thus gene replacement, knock‐in, of the mouse GT with the human FT without integration of any selectable marker genes in the GT locus was shown to be possible. This is especially important in producing transgenic animals for the clinical application of xenografts into humans.

A close correlation was observed between body weight and length of the survival time of mice inoculated intravenously (i.v.) with Cryptococcus neoformans (p<0.001). An endotoxic substance of C. neoformans (Cr-ET) increased the susceptibility of mice to i.v. infection of C. neoformans only when more than 50 µg of Cr-ET was injected i.v. 24 hours before infection. Intraperitoneal (i.p.) administration of dimethyl sulfoxide which is found to enhance bacterial infection did not enhance death rate of mice infected i.p. with C. neoformans.

FACS analysis together with PIPLC treatment was applied to Pi-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylchol-inesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.

Interleukin-4 (IL-4) and intercellular adhesion molecule 1 (ICAM-1) are assumed to be involved in the pathogenesis of allergic disease. In this study, we examined the potential link between IL-4 and soluble ICAM-1 (sICAM-1) levels in patients with allergic rhinitis. The levels of sICAM-1 and IL-4 in sera and in nasal epithelial lining fluids (ELF) from 12 patients with Japanese cedar pollinosis were measured preseason through postseason, and the results were compared with those from 7 healthy subjects. In sera from the allergic subjects, the levels of sICAM-1 were upregulated during the early part of the season and downregulated during the middle of the season, with upregulation of the IL-4 levels. Moreover, a negative correlation was found between the serum sICAM-1 levels and serum IL-4 levels during the middle (r = –.80) and late (r = –.73) parts of the season. In ELF from allergic subjects, the levels of sICAM-1 were significantly upregulated during the early and middle parts of the season, and began to be downregulated during the late part of the season, with upregulation of the levels of IL-4. In conclusion, IL-4 possibly acts as a potential suppressor of sICAM-1 in the pathogenesis of seasonal allergic rhinitis, at least under provocation by a small amount of natural allergen.

Glutathione-S-transferase (GST) in one of several factors that are proposed to affect tumor sensitivity to anticancer drugs, including cisplatin (CDDP). Attempts are made herein to evaluate the significance of the enzymes in resistance to CDDP in clinical samples of gastric cancer. A total of 22 gastric cancer specimens, 16 of which were obtained with matching normal mucosae, underwent immunoblotting with polyclonal antibodies against GST-a and GST-p. At the same time, the chemosensitivity of 15 gastric cancer specimens to CDDP was evaluated by the succinic dehydrogenase inhibition (SDI) test. The expression of GST-p was detected in all the specimens, and its content in the neoplasms exhibited a significant positive correlation with that in the matched normal mucosae. The expression of GST-a was detected in 18 of 22 cancer specimens (82%), but its content in the neoplasms did not correlate with that in the matched mucosae. A comparison of the drug-sensitivity findings with the results of immunoblotting revealed a weak but interesting correlation between the protein levels of GST-a and CDDP resistance. The cellular content of GST-a correlated weakly with CDDP resistance in gastric cancer, and its quantification could contribute to prediction of the clinical effects of CDDP in patients with gastric cancer.

Glutathione-S-transferase (GST) in one of several factors that are proposed to affect tumor sensitivity to anticancer drugs, including cisplatin (CDDP). Attempts are made herein to evaluate the significance of the enzymes in resistance to CDDP in clinical samples of gastric cancer. A total of 22 gastric cancer specimens, 16 of which were obtained with matching normal mucosae, underwent immunoblotting with polyclonal antibodies against GST-a and GST-p. At the same time, the chemosensitivity of 15 gastric cancer specimens to CDDP was evaluated by the succinic dehydrogenase inhibition (SDI) test. The expression of GST-p was detected in all the specimens, and its content in the neoplasms exhibited a significant positive correlation with that in the matched normal mucosae. The expression of GST-a was detected in 18 of 22 cancer specimens (82%), but its content in the neoplasms did not correlate with that in the matched mucosae. A comparison of the drug-sensitivity findings with the results of immunoblotting revealed a weak but interesting correlation between the protein levels of GST-a and CDDP resistance. The cellular content of GST-a correlated weakly with CDDP resistance in gastric cancer, and its quantification could contribute to prediction of the clinical effects of CDDP in patients with gastric cancer.

In this study an endotoxic substance was extracted from the cells ofCryptococcus neoformans and the physicochemical and biological properties of this substance (Cr-ET) were investigated. In comparison with endotoxin of gram-negative bacteria, the lethality of Cr-ET for mice and chick embryos was low and such biological activities were weak as the pyrogenic effect on rabbits and effects on the leucocyte count and blood sugar level in rabbits. Skin reactions (both primary and Shwartzman reactions) were elicited in rabbits by relatively large dose of Cr-ET. Unlike bacterial endotoxin, hyperreactivity to Cr-ET was not induced in mice by prior infection with BCG.

The biological significance of the action of glycosylphosphatidylinositol (GPI)-anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI-anchored proteins play a crucial role in the recently defined heavy metal (HgCl 2)-triggered signal delivery to T lymphocytes. Thiol-reactive HgCl 2, a multi-potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy-1, a representative GPI-anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl 2is diminished by the pre-treatment of cells with phosphatidylinositol-specific phospholipase C, which partially cleaved GPI-anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI-anchored proteins in the HgCl 2-mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan-class A gene (PIG-A), and its restoration in a transfectant with PIG-A.

Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI-specific phospholipase C (PIPLC) from Bacillus thuringiensiswas shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex-induced signal delivery for cell activation. This was demonstrated for very early protein tyrosine phosphorylation, early c- fostranscription and late DNA synthesis. For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released form the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane-bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the T cell receptor function is discussed.

The biological significance of the action of glycosylphosphatidylinositol (GPI)‐anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI‐anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)‐triggered signal delivery to T lymphocytes. Thiol‐reactive HgCl2, a multi‐potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy‐1, a representative GPI‐anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2is diminished by the pre‐treatment of cells with phosphatidylinositol‐specific phospholipase C, which partially cleaved GPI‐anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI‐anchored proteins in the HgCl2‐mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan‐class A gene (PIG‐A), and its restoration in a transfectant with PIG‐A.

FACS analysis together with PIPLC treatment was applied to Pi‐anchoring antigens such as DAF (decay‐accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement‐regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI‐anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylchol‐inesterase and granulocyte alkaline phosphatase which were also known to be PI‐anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.

Exposure of lymphocytes to 0.2–2 mM HgCl2, a thiol‐reactive heavy metal, induced extensive tyrosine phosphorylation of multiple cellular proteins. The phosphorylation started as quickly as 5 s after exposure to HgCl2, and was irreversible. Another 3 thiol‐reactive chemicals also displayed similar, though less marked, actions, whereas dithiothreitol, a reducing agent, antagonized the HgCl2action. The demonstrated new action of HgCl2indispensably required membrane‐intact cells as a target. Whereas exposure of lymphocytes to > 0.2 mM HgCl2caused rapid cell death, 0.01–0.1 mM HgCl2affected the cells so as to accelerate their c‐fostranscription. These results suggest a novel redox‐linked mechanism of cell surface triggering of intracellular protein kinase activity, which is independent of receptor‐ligand interactions.

Abstract: Immunogenicity of allogeneic immunoglobulins in mice were studied, measuring the allotype-specific antibody activity by agglutination of allogeneic antibody-coated red blood cells. It was found that the serum from C.B-20 mice (Igh b , BALB/c-congenic) was uniquely immunogenic in BALB/c mice for allotype antibody response. Whereas the C57BL/6 (Igh b ) serum was immunogenic only when heat aggregated and/or combined with adjuvant, the ultracentrifugation-deaggregated C.B-20 serum was definitely immunogenic when administered in a moderate dose (100 μl/mouse). Even more surprising was the fast that very low doses (0.01–0.1 μl) of soluble C.B-20 serum, but not C57BL/6 serum, down regulated the allotype-specific response effectively. Genetic analysis on congenic mice suggested that the immunogenicity is controlled by donorIgh orIgh-V(Id-C.B) inasmuch as the serum from BALB/c-congenic C.B-20 (Igh-V b C b ), but not BALB/c-congenic BAB/14 (Igh-V a C b ), mice was active in BALB/c mice in soluble form. Further studies showed that the Id-C.B was dominantly expressed on the immunoglobulins of (BALB/cC.B-20)F1 and (C56BL/6C.B-20)F1 strains, and was originally derived from the C57BL/Ka strain. The major determinant for the antibody production was encoded inIgh-C, but not inIgh-V. It is suggested thatId-C.B controls the allotype-specific antibody response in an unusual manner, possibly acting as a unique determinant activating helper T cells.