Effect of antisera against recombinant tumor necrosis factor and the monocyte-derived cytotoxin(s) on monocyte-mediated killing of various tumor cells

Antisera raised against recombinant tumor necrosis factor (TNF) and against the monocyte-derived cytotoxic/cytostatic protein factor (CF), which is related to recombinant TNF, have been compared with respect to their ability to inhibit monocyte-mediated killing of various types of cells which differ in their sensitivity to recombinant TNF. During 6 hr of coculturing monocytes and target cells, the recombinant TNF antiserum inhibited killing of the extremely TNF-sensitive WEHI 164 clone 13 cells and actinomycin D-treated WEHI 164 cells from which the clone 13 cells were derived (parental WEHI 164 cells (P-WEHI 164 cells]. The CF antiserum also inhibited monocyte-mediated killing of these cells during 6 hr of coculturing with monocytes, but on a per volume basis it was less potent than the recombinant TNF antiserum, consistent with the fact that the CF antiserum also was much less potent in inhibiting the cytotoxic activity of recombinant TNF. However, during 72 hr of coculturing with monocytes and target cells, the CF antiserum inhibited monocyte-mediated killing of P-WEHI 164 cells more efficiently than the recombinant TNF antiserum. Moreover, during 72 hr of coculturing with monocytes, only the CF antiserum was able to significantly inhibit monocyte-mediated killing of the relatively recombinant TNF-resistant K562 cells. This suggests that a factor immunologically different from recombinant TNF, perhaps a form of natural TNF differing somewhat immunologically from recombinant TNF, was involved in the killing of K562 cells, and possibly in the killing of P-WEHI 164 cells, during 72 hr of coculturing with monocytes. Although this factor was present extracellularly, it appears that it may act as a monocyte-associated factor in monocyte-mediated killing of K562 cells, since exposure to recombinant interferon-gamma (rIFN-gamma) in the absence of Escherichia coli endotoxin (lipopolysaccharide, LPS) activated the monocytes to mediate killing of K562 cells more efficiently than exposure to LPS alone, despite the fact that only little cytotoxic/cytostatic activity was released from the monocytes without the addition of LPS. The ability of rIFN-gamma and LPS to activate monocytes to produce and release CF has also been studied.     

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.     

Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors

The effect of protease inhibitors on invasion of rhesus erythrocytes by Plasmodium knowlesi merozoites was evaluated. Chymostatin, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) inhibited invasion. Leupeptin, antipain, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) had no effect. TLCK and TPCK inhibited attachment of merozoites to host erythrocytes. Chymostatin had no adverse effect on attachment, and in its presence junction formation between the merozoite and host erythrocyte occurred. Both chymostatin and leupeptin inhibited normal rupture of schizont-infected erythrocytes. It is suggested that proteolytic activity may be important both in the rupture of schizont-infected erythrocytes and in the invasion of erythrocytes by malaria parasites.     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

Human monocytes cultured in a specially prepared medium free of lipopolysaccharide (LPS) constitutively produced a small, though significant, amount of tumor necrosis factor (TNF). Upon addition of LPS, the amount produced remained constant until the LPS concentration reached 1-10 ng/ml, whereupon the production of TNF dramatically increased, eventually becoming 100-fold greater than when the LPS concentration was below 1 ng/ml. Priming the monocytes with recombinant interferon-gamma (rIFN-gamma) before LPS exposure resulted in a 2- to 10-fold increase in TNF production, the highest relative increase being obtained at lower LPS concentrations and in the absence of LPS. Monocyte-produced TNF appears to be the effector molecule in monocyte-mediated killing of some target cell types, since antiserum against recombinant TNF inhibited killing of both actinomycin D-treated and untreated WEHI 164 cells by human monocytes. However, it also appears that TNF may not in all cases be an effector molecule in monocyte-mediated killing, since cytolysis of K562 cells mediated by IFN-gamma/LPS-activated monocytes was not inhibited by antiserum against recombinant TNF. Antiserum which was raised against a monocyte-derived cytotoxic factor and which neutralized recombinant TNF did, however, inhibit monocyte-mediated cytolysis of K562 cells, suggesting that an extracellular factor, perhaps related to TNF, was also involved in monocyte-mediated killing of K562 cells. A TNF-like activity was associated with the monocyte surface membrane, since paraformaldehyde-fixed monocytes expressed cytotoxic activity which was neutralized by antiserum against recombinant TNF. Fixed monocytes activated with rIFN-gamma in addition to LPS before fixation were generally more cytotoxic than those exposed to LPS alone, and those exposed to LPS were much more cytotoxic than those not exposed to LPS. Thus it is possible that high local TNF concentrations may be generated near the target cell upon direct contact between effector and target cells, and that also monocyte-associated TNF may in this way be involved in monocyte-mediated cytotoxicity.     

Monocyte-mediated drug-dependent cellular cytotoxicity: effects on different WEHI 164 Target cell lines

Summary The contribution of monocyte cytotoxic protein factor (CF) to monocyte-mediated drug-dependent cellular cytotoxicity (DDCC) has been investigated. Cell lines which have been derived from murine WEHI 164 cells (termed WEHI 164 parental) by selecting for high (WEHI 164 clone 3) and low (R-WEHI 164) sensitivity to CF-mediated cytotoxicity were used as target cells in DDCC. By comparing the CF doses which produced 50% dead cells (LD 50) we found that WEHI 164 clone 3 was approximately 30 times more sensitive than WEHI 164 parental which in turn was 70 times more sensitive than R-WEHI 164. Actinomycin D (Act D) treatment of WEHI 164 parental and R-WEHI 164 greatly increase susceptibility to CF-mediated cytotoxicity. The susceptibility of WEHI 164 clone 3 was apparently somewhat increased at low dilutions of CF, whereas no significant increase was observed at high dilutions. The susceptibility to DDCC of the three target cell lines (WEHI 164 parental, WEHI 164 clone 3, and R-WEHI 164) correlated with the sensitivity pattern obtained in CF-mediated cytotoxicity of Act D-treated target cells. Monocyte- and CF-mediated cytotoxicity against Act D-treated WEHI 164 clone 3 and R-WEHI 164 was inhibited by neutralizing CF antiserum. These data indicate that CF is an effector molecule in monocyte-mediated DDCC.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Human monocyte cytotoxic factor mediates cytolysis of WEHI 164 cells

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.     

Antiproteinases protect cultured lung endothelial cells from endotoxin injury

To determine whether the effects of endotoxin on cultured lung endothelium involve proteolytic mechanisms, we incubated bovine pulmonary arterial endothelial cells with endotoxin in medium 199 + 10% fetal bovine serum (FBS) in the presence and absence of several proteinase inhibitors. Three chloromethyl ketone (CK) derivatives [N-tosyl-L-lysine (CK)-(TLCK), N-tosyl-L-phenylalanine CK(TPCK), methoxysuccinyl-Ala-Ala-Pro-Val CK(SPCK)] and a single synthetic proteinase substrate [N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME)] attenuated endotoxin-induced cytotoxicity (lactate dehydrogenase release) and prostacyclin production in a dose-related fashion. The most effective inhibitors of endotoxin-induced cytotoxicity were TLCK and TPCK. TLCK and TAME most effectively attenuated endotoxin-stimulated prostacyclin production. Two chemically unrelated substances, soybean trypsin inhibitor and alpha 1 proteinase inhibitor also attenuated the endotoxin response. In the absence of FBS or in the presence of 10% heat-inactivated FBS, antiproteases attenuated endotoxin-induced prostacyclin production but had less effect on cytotoxicity than with 10% FBS. We also measured the capacity of the CK inhibitors to scavenge superoxide radicals generated in a cell-free xanthine/xanthine oxidase system by measuring inhibition of cytochrome c reduction. Percent scavenging of superoxide by these inhibitors was as follows: TLCK, 62.7 +/- 5.8 (SE); TPCK, 83.9 +/- 7.7; TAME, 24.5 +/- 6.4; SPCK, 0. We conclude that certain proteinase inhibitors attenuate endotoxin-induced endothelial cytotoxicity and prostacyclin production and that direct scavenging of superoxide radicals fails to explain the protective effects of proteinase inhibition. We speculate that the effects of endotoxin on lung endothelium may involve proteolytic mechanisms even in the absence of neutrophils.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. 5. Role of proteases, inhibitors, and substrates

The effect of protease inhibitors and substrates on the killing of tumor cells by in vitro activated macrophages was examined. Phenylmethylsulfonyl fluoride, a serine esterase inhibitor, and the active site titrants TPCK and TLCK (chloromethyl ketone derivatives of tosyl-l-phenylalanine and tosyl-l-lysine) inhibited macrophage-mediated cytotoxicity in a dose-dependent and irreversible fashion. The synthetic protease substrates, tosyl-lysine methyl ester and tosyl-arginine methyl ester and the natural esterase inhibitors soybean trypsin inhibitor and antithrombin III had no effect. These results suggest that an activated macrophage-associated esterase may play a modulating role in the cytolytic interaction between activated macrophages and tumor cells.     

Interferon-gamma enhances target cell sensitivity to monocyte killing

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.     

The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.

Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.     

Inhibition of the transformation-specific kinase in ASV-transformed cells by N-alpha-tosyl-L-lysyl chloromethyl ketone.

We find that the protease inhibitor N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits the transformation-specific kinase activity (Collett and Erikson, 1978) associated with p60^src, the avian sarcoma virus (ASV) gene product responsible for the transformation of fibroblasts. TLCK has been shown to induce the phenotypic reversion of ASV-transformed cells to normal (Weber, 1975). Kinase activity was measured in extracts of chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of ASV (SR-ASV) with antiserum from rabbits bearing ASV-induced tumors. The immunoprecipitates were incubated with γ-³²P-ATP under conditions in which the phosphorylation of the IgG heavy chain in the immunoprecipitate was directly proportional to the concentration of cell extract. When ASV-transformed CEF were treated with 0.1 mM TLCK, the kinase activity was reduced by 60% after 2 hr and by 80% after 6 hr, and continued to remain low for up to 40 hr when TLCK was present. When TLCK was removed, the kinase activity rose slowly over a period of many hours, suggesting that the enzyme is irreversibly inactivated by TLCK and new enzyme must be synthesized. The effect of TLCK in vivo is concentration-dependent and specific. Other serine protease inhibitors had no effect on kinase activity. At low concentrations (0.03 mM), TPCK produced partial inhibition (≤20%), but at higher concentrations TPCK was extremely toxic to the cells and therefore could not be tested. The inhibition by TLCK was not due to its ability to inhibit protein synthesis since cycloheximide treatment (1 μg/ml) did not significantly reduce kinase activity. TLCK also inhibited kinase activity when added directly to cell extracts, but about 5 times higher concentrations of TLCK were required to produce 50% inhibition. Under these conditions both TLCK and TPCK were comparable inhibitors, whereas PMSF had no effect. Our finding that the inhibition of the kinase by TLCK in vivo parallels the reversion of cell morphology to normal suggests that the kinase has an important role in transformation and offers a biochemical rationale for treatment of tumors with this agent.     

绿僵菌分解昆虫外壳蛋白酶MAP-21的纯化与特性

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶 。发酵液经超滤、Ultrogel AcA 54凝胶层析、制备IEF电泳,纯化了一种蛋白酶MAP-21,SDS-PAGE电泳后经银染色呈单带。该酶的Mr为27kD左右,pI为76。它的特异识别氨基酸为Arg,其活性可被PMSF和TLCK抑制,表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leupeptin、Antipain及STI等所抑制,而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP-21是类胰蛋白酶。此外,该酶还可被EDTA所抑制,表明金属离子为其活性所必需。另外还研究了MAP-21的最适作用温度和pH,以及温度耐受性等特性。     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations In Vitro

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.     

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

The role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.