Yasuo Hitsumoto

Two mechanisms for platelet-mediated killing of tumour cells: one cyclo-oxygenase dependent and the other nitric oxide dependent.

We have tried to identify the cytotoxic effectors in platelet‐mediated tumour cell killing, using two tumour cell lines K562 (a chronic myelogenic leukaemic cell line) and LU99A (a lung cancer cell line), which are both sensitive to platelet cytotoxicity. Cyclo‐oxygenase inhibitors, acetylsalicylic acid (ASA) and indomethacin, effectively inhibited the platelet‐mediated killing of K562 cells, but not that of LU99A cells. In contrast, inhibitors of the nitric oxide (NO) pathway, NG‐nitro‐L‐arginine (L‐NA), haemoglobin and methylene blue, reduced the cytotoxic activity of platelets against LU99A, but not against K562. Synthetic analogues of platelet–cyclo‐oxygenase products thromboxane A2/prostaglandin H2 (TXA2/PGH2) exerted cytotoxicity against K562 cells but not against LU99A cells. Electron microscopic study showed that TXA2/PGH2 analogues induced bleb formation and disruption of the plasma membrane of K562 cells. K562 cells enhanced the production of TXA2 by platelets, as inferred from the accumulation of thromboxane B2 (TXB2), a spontaneous hydrolysis product of TXA2. LU99A cells had no such effects. These results indicate that platelets kill these two tumour cell lines through different mechanisms. In K562, the cyclo‐oxygenase products TXA2/PGH2 possibly play a significant role, but in LU99A the NO pathway seems to be involved.

In vitro degradation of very low density lipoprotein from diabetic patients by lipoprotein lipase

Fatty acid release by incubation with lipoprotein lipase (LPL) in vitro from very low density lipoproteins (VLDL) obtained from diabetic patients was low compared with that from healthy subjects, though the compositions were similar in both VLDL. Percentages of the large size VLDL decreased and those of the small size VLDL increased after the incubation with LPL. At the same time, on polyacrylamide gel disk electrophoresis, the smaller catabolic products from these VLDL appeared at a similar position to that of low density lipoproteins (LDL) and at the running front where high density lipoproteins (HDL) had migrated. The amount of the small size VLDL and the LDL-like lipoproteins produced from diabetic VLDL were less than those from normal VLDL and inversely correlated with the percent decrease of the large original size VLDL. This fact suggests that VLDL from diabetic patients are poor substrates for LPL compared with normal VLDL.

Role of membrane-associated lymphotoxin (mLT) in the killing activity of lymphokine-activated killer (LAK) cells towards various tumour cell lines.

Human lymphokine‐activated killer (LAK) cells developed by an incubation of peripheral mononuclear cells with IL‐2 express the membrane‐associated lymphotoxin (LT)‐related molecule (mLT). By a further cultivation of mLT expressing (mLT‐positive) LAK ceils for 24 h without IL‐2. mLT disappears (mLT‐negative LAK cells). Cytotoxicities of various tumour cell lines by either mLT‐positive or ‐negative LAK cells were compared. Eight out of 12 tumour cell lines, less susceptible to mLT‐negative LAK cells than mLT‐positive LAK cells, were categorized as group A, Two tumour ceils (K562 and Moit‐4) had the same susceptibility to both kinds of LAK cells. The others (Daudi and Jurkat) had less susceptibilities only when they were assessed at E:T ratios of less than 5. The four tumour cell lines in the latter two cases, containing K562. Moit‐4. Daudi and Jurkat cells, were categorized as group B. The cytotoxicities of group A tumour cells, but not group B tumourceils, by LAK cells were significantly suppressed by the presence of anti‐LT antibody. Group A tumour ceils had higher LT‐binding ability (2‐82‐16‐44 fmol/106 cells) than group B tumour cells (less than 1 46 fmol/106 cells). Both mLT‐positive and ‐negative LAK cells had similar perform activities and tumour cell‐binding capacities. These results suggest that the mLT‐mediated killing mechanism is involved in tumour ceil killing by LAK cells. Further, various tumour cell lines can be classified into two large groups according to their susceptibilities to the mLT‐mediated killing by LAK cells.

Enhancement of CD3‐mediated thymocyte apoptosis by the cross‐linkage of heat‐stable antigen

Heat‐stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4−CD8− double‐negative and CD4+CD8+ double‐positive thymocytes but not CD4+ or CD8+ single‐positive thymocytes. Effects of anti‐HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single‐cell suspension culture system. Immobilized R13 enhanced the CD3‐mediated DNA fragmentation and killing of thymocytes but not the dexamethasone‐induced or phorbol myristate acetate‐induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3‐mediated apoptosis when HSA and T‐cell receptor (TCR)/CD3 were co‐cross‐linked by a cross‐reactive secondary antibody. Even without the cross‐reactive secondary antibody, soluble R13 enhanced CD3‐mediated apoptosis, although a greater than 100‐fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3‐mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation‐induced apoptosis of thymocytes and the involvement of HSA in negative selection.

Mechanisms of Neutralization of Endotoxin by Monoclonal Antibodies to O and R Determinants of Lipopolysaccharide

The protective potentials of antibodies to O and R core regions of lipopolysaccharide (LPS) have been amply substantiated (2, 7). However, the efficacy of antibodies to the toxic lipid A moiety itself is still ambiguous, and how antibodies to regions distal from lipid A can neutralize the toxicity remains to be clarified. We have compared the effects of mouse monoclonal antibodies (mAbs) of IgG class to these three regions of LPS on the biological activities as well as micellic structure of LPS, in order to shed light on the mechanism of neutralization of endotoxin by these antibodies.

Purification of the murine heat-stable antigen from erythrocytes

The rat anti-mouse erythrocyte (MRBC) monoclonal antibody (mAb), R13, has been developed. The MRBC membrane protein recognized by R13 (R13-Ag) can be purified by loading the butanol-extracted MRBC membrane solution on a R13-conjugated Cellulofine column in the presence of 0.1% CHAPS followed by elution with 1% CHAPS. The amino acid sequence of the affinity-purified R13-Ag corresponded to that predicted from the cDNA for the murine heat-stable antigen. It was revealed that the actual heat-stable antigen was composed of 27 amino acids.

IgG isotype and isotype specificity of murine monoclonal IgG rheumatoid factors.

Immune complexes of lipopoly saccharide (LPS) with homologous IgG antibody induces rheumatoid factor (RF) predominantly of the IgG class in normal mice, while LPS alone induces mostly IgM RF directed to homologous IgG1. In this study, IgG monoclonal RFs (mRF) were prepared from hybridomas derived from spleen cells of BALB/c mice which were immunized with complexes of TNP‐LPS with anti‐TNP mouse IgG and their specificity to mouse IgG subclasses was assessed by analysing dissociation kinetics of the ligands due to RF‐specific and non‐specific interactions. Of the 19 IgG mRFs (11 IgG1, five IgG2a, one IgG2b and two IgG3 types) tested, 14 were directed to either IgG3 or IgG2b or both, while only one exhibited a significant binding capacity to IgG1. Other mRFs, although reactive to rabbit IgG, exhibited little homophilic activity. None of these mRFs reacted strongly with their own isotypes. The results suggest that the IgG RF producing cells are not direct progenies of the IgG1‐directed IgM RF‐producing cells but may have developed via a rigorous selection process to eliminate clones that produce self‐reactive RF.

Inhibition of human and mouse complement-dependent hemolytic activity by mouse fibronectin.

Not only does mouse complement (C) have low hemolytic activity, but mouse serum has an inhibiting effect on hemolysis by human C. To purify and identify the putative mouse serum factor inhibiting human C activity, a sequential procedure of fractionated precipitation by PEG, followed by chromatographies with a heparin-Sepharose column, a phenyl-Sepharose column, a Protein G column, and a gel-filtration column was performed. The amino acid sequence analyses of two polypeptides obtained by digestion of the purified serum factor with TPCK-trypsin revealed that it was mouse fibronectin (FN). Highly purified mouse FN, but not human FN, has an inhibiting effect on human C-dependent hemolysis. Moreover, the hemolysis of sensitized rabbit erythrocytes by mouse C was also inhibited by the addition of mouse FN in a dose-dependent fashion, but not by the addition of human FN. These results suggest that FN is the putative internal C inhibitor in the mouse system.

Preparation of membrane fraction from herpes simplex virus-infected cells which induce cytotoxic T lymphocytes.

The immunogenic capacity of herpes simplex virs (HSV)‐infected cells and their subcellular membrane fractions was investigated by assessing the anti‐HSV cytotoxic T lymphocyte (CTL) response in cultures of spleen lymphocytes from HSV‐primed BALB/c mice. Methylchloranthrane‐induced fibrosarcoma (Meth A) cells infected with HSV (HSV‐Meth A) were fixed either with glutaraldehyde or by heating at 56 C to preserve their immunogenic competence and then used as a stimulator. Microsomes and plasma membranes were prepared from HSV‐Meth A and their immunogenic activities were determined. Though the recovery of stimulatory activity in the plasma membrane fraction was half of that in the microsome fraction, the activity in the former was much more stable than in the latter and the plasma membrane fraction proved to be well qualified as an immunogen for anti‐HSV CTL induction. Upon purification, the specific activity of the membrane fraction, on the basis of protein concentration, increased 43‐fold.

Compositions of very low density lipoprotein subfractions from patients with polydisperse low density lipoproteins

In some hyperlipidemic patients, low density lipoprotein (LDL) shows several peaks (polydisperse) on polyacrylamide gel disc electrophoreses, though LDL usually shows a single peak (monodisperse). In order to clarify the relationship between the LDL polydispersion and VLDL heterogeneity, LDL and VLDL were prepared from hyperlipidemic patients sera with mono- and polydisperse LDL by sequential ultracentrifugation and fractionated by gradient ultracentrifugation and their compositions were analyzed. Polydisperse LDL was rich in triacylglycerol (TG) and poor in esterified cholesterol (CE) as compared with monodisperse LDL and consisted of the lowest and the medium density subfractions when the LDL was separated into six subfractions. The monodisperse LDL was composed of a single major subfraction of a medium density. VLDL from the patients with polydisperse LDL was relatively rich in the dense and poor in the buoyant subfractions as compared with that from the patients with monodisperse LDL. The subfractions in the former contained more CE and less TG than the corresponding subfractions in the latter. There were no significant differences in the apolipoprotein compositions between those VLDLs. The results suggest that polydisperse LDL might be originated from VLDL that differs in particle sizes, densities and compositions from ordinary VLDL.