Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Susceptibility of cells with different stage of malignancy to cytostatic action of resident peritoneal macrophages of Syrian hamsters

Syrian hamster non-activated resident peritoneal cells (PC) and peritoneal macrophages (Mph) was demonstrated. The in vivo selection of highly tumourigenic and highly metastatic variants of this strain correlated with their resistance to CSA PC and Mph in four cell variants out of five examined. The highly tumourigenic Syrian hamster embryo cells in vitro transformed by Rous sarcoma virus were highly resistant to CSA PC without selection in vivo. The resistance of highly malignant cells to CSA PC appeared to be unrelated to their ability to produce immunosuppressing prostaglandins of E type.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

The resistance of in vivo selected tumor cells of malignant phenotype to the cytolytic action of activated peritoneal macrophages in vitro]

Using radioisotope cytolytic 3H-thymidine release assay the sensitivity of low-malignant spontaneously in vitro transformed hamster embryo cells (STHE strain) and its in vivo selected malignant variants (STHE-LM-4, STHE-LM-8, STHE-75/18) as well as Rous sarcoma virus (RSV) (Schmidt-Ruppin strain) tumorigenic transformants (HET-SR-1, TU-SR) were tested to cytolysis by peritoneal Syrian hamster macrophages (MP) activated in vitro with levan, LPS, MDP and PMA. It has been shown that the only parental cells STHE strain (non-selected in vivo) were sensitive to cytolysis by activated MP. All in vivo selected malignant variants of STHE cell sublines as well as tumorigenic RSV-SR-transformants were resistant to cytolysis by activated MP. Thioglycollate-elicited but not activated MP did not destroy any tumour target cells.     

The circadian system of the Turkish hamster, Mesocricetus brandti: responses to light

The circadian system of the Turkish hamster controlling wheel-running activity responded to single 1-hr light pulses and to repeated 1-hr pulses in a similar way as that of Syrian hamsters studied previously. At constant light of 100 lx, the period length (tau) of the freerunning activity rhythm of Turkish hamsters was longer and the activity time (alpha) was shorter than that of Syrian hamsters. Among individuals, the ability of the system to be entrained by one 1-hr light pulse per cycle was related to the range (advance plus delay amplitude) of the phase response curve (PRC) derived from single light pulses and to the compression of alpha caused by the pulse Zeitgeber. The data support the hypothesis derived from experiments on Syrian hamsters that the range of the PRC is functionally related with alpha, possibly reflecting the phase relations (coupling) between two oscillators.     

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.     

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.     

Transient and Persistent Depolarization-Induced Changes of Protein Phosphorylation in a Molluscan Nervous System

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea-water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ [( Ca2+]o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10-fold by high K+, irrespective of the level of [Ca2+]o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF- and EDTA-sensitive protein phosphatase capable of dephosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on [Ca2+]o even in normal low K+ (10 mM) medium. Removal of [Ca2+]o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long-lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of thrombin-induced myosin phosphorylation and the assembly of cytoskeletal structures in platelets by the adenylate cyclase stimulants prostaglandin D2 and forskolin

Stimulation of platelets by thrombin causes an increase in the amount of cytoskeleton proteins insoluble in 1% Triton X-100, i.e. myosin, actin, actin-binding protein, an alpha-actinin-like protein of Mr = 105,000, unidentified polypeptides of Mr = 150,000, 31,00, and under some conditions, 56,000. Concurrently the Mr = 20,000 light chains of myosin and a cytoplasmic Mr = 42,000 polypeptide are phosphorylated, presumably by calmodulin-Ca2+-dependent myosin light chain kinase and a phospholipid-Ca2+-dependent kinase, respectively. The adenylate cyclase stimulators prostaglandin D2 (PGD2) and forskolin increased platelet cyclic AMP and prevented the phosphorylation of these polypeptides and the increase in Triton-insoluble cytoskeleton proteins. When added to platelets after stimulation by thrombin they caused rapid complete reversal of myosin light chain and Mr = 42,000 polypeptide phosphorylation; simultaneously the association of myosin with the cytoskeleton proteins and the increase in the content of each of the Triton-insoluble cytoskeleton proteins (except the Mr = 56,000 polypeptide) was reversed. The amount of Triton-insoluble myosin was affected more readily by PGD2 or forskolin than were the other proteins. Increasing thrombin from 0.1 to 1.0 unit/ml inhibited all the responses to PGD2 and forskolin possibly due to concentration-dependent effects of thrombin that inhibit adenylate cyclase. These results suggest that cytoskeleton assembly and activation of the contractile apparatus in intact platelets are readily reversible by cyclic AMP-dependent reactions.     

Secretion of prostaglandin type E (PGE) by tumor cells of Syrian hamsters in in vitro contact with natural killers

The ability of Syrian hamster tumor cells of the same origin but with different degrees of malignancy to secrete prostaglandin E (PGE) was studied following their in vitro contact with Syrian hamster natural killer cells (NK cells). Syrian hamster NK cells were shown to lose significantly cytotoxic activity after their contact with malignant tumor cells. Short-term in vitro contact of malignant tumor cells with human and Syrian hamster NK cells resulted in a rapid PGE secretion into the culturing medium. PGE was determined in the culturing medium, using the biological test, described in the paper, or direct radioimmunoassay. No PGE secretion was observed after the treatment of tumor cells with indomethacin. It is assumed that PGE secretion by malignant tumor cells is one of the mechanisms of their protection against natural killer cells.     

Rat peritoneal macrophage procoagulant and fibrinolytic activities. An expression of the local inflammatory response.

1. Lysates of rat (Rattus norvegicus, Wistar strain) peritoneal macrophages selected by adherence were analysed for procoagulant activity (PCA) and plasminogen activator activity (PAA). 2. PCA is expressed following in vitro stimulation by lipopolysaccharide and in vivo (respectively 118 +/- 19.1 and 147.2 +/- 45.2 mUnits/10(6) M phi) very early after the intraperitoneal injection of thioglycollate. 3. PAA present in 24 hr thioglycollate stimulated cells (79.5 +/- 26.1 mI.U./10(6) M phi), disappears in a time dependent fashion after in vitro lipopolysaccharide stimulation. 4. Our findings indicate that rat peritoneal macrophages regulate their coagulolytic activities in a specific manner. 5. Rat peritoneal cavity may be proposed as a model for the study of inflammatory reaction with PCA and PAA as its indexes.     

Recombinant human lymphotoxin effects on osteoblastic cells

Lymphotoxin, or tumor necrosis factor beta, has been shown to be a potent bone resorbing cytokine. In the present study, the effect of recombinant human lymphotoxin on osteoblastic cell proliferation and prostaglandin synthesis was investigated. Lymphotoxin (10(-10)-10(-7) M) caused a significant, dose-dependent decrease of rat osteoblastic cell proliferation. This appeared to be an indirect, prostaglandin-dependent action, since in the presence of indomethacin (1 microM) the lymphotoxin effect was reversed. Subsequently, prostaglandin E2 and prostacyclin (assayed as 6-keto-prostaglandin F1 alpha) levels produced by the osteoblastic cells in response to lymphotoxin were measured. The cytokine caused a dose-dependent increase of these arachidonic acid metabolites, with the maximum effect at 10(-8) M. These results suggest that lymphotoxin's mechanism of action on bone may involve increases in arachidonic acid metabolite synthesis and an indirect, prostanoid-mediated decrease in the proliferation rate of osteoblastic cells.     

Induction of penicillinase by staphylococci in vitro and in vivo

Eyckmans, Luc (Cornell University Medical College, New York, N.Y.), and Edward W. Hook. Induction of penicillinase by staphylococci in vitro and in vivo. J. Bacteriol. 91:997-1003. 1966.-Staphylococci in mice with peritoneal infection showed no significant increase in penicillinase activity 6 hr after administration of penicillin G. In contrast, induction of penicillinase was readily demonstrated in leukopenic animals under similar conditions. Induction of penicillinase by staphylococci in vitro was inhibited by including leukocytes and immune serum in the mixtures. The role of leukocytes in inhibiting induction of penicillinase by staphylococci in response to penicillin was investigated.     

Potentiation of cytotoxic effect of lymphotoxin by anti-cancer drugs and elevated temperatures

The cytotoxic lymphokine, lymphotoxin (LT), has been shown to possess antitumor effect in vitro and in vivo. We examined the effect of the combination of partially purified LT with anti-cancer drugs and elevated temperatures on mouse transformed fibroblast cell line, L-929, and two human carcinoma of the cervix cell lines, HeLa and ME180. The cells were treated for 7 hr with Adriamycin, cisplatin, or bleomycin. These cells were then incubated for 24 hr in the presence of LT. At the end of the incubation period, cytotoxicity was measured by the neutral red dye uptake assay. There was 10- to 47-fold potentiation of cytotoxicity of LT on L-929 cells. The potentiation of cytotoxicity on human carcinoma of cervix cell lines ranged from 3- to 23-fold. L-929 cells and ME180 cells were incubated for 7 hr at 40 or 42 degrees C followed by 24 hr of incubation in the presence of LT. The elevated temperature treatment also enhanced (5- to 9-fold) the cytotoxic effect of LT. DNA, RNA, and protein syntheses of the ME180 cells was measured following incubation at 42 degrees C. It was observed that all three parameters were suppressed by incubation at this temperature. It was, therefore, possible that the repair of LT damaged cells was hampered by the elevated temperature treatment. It is suggested that LT may have a potential as an anti-tumor agent in combination with selected therapeutic drugs and hyperthermia.     

Structural studies of T lymphocyte Fc receptors. Association of Gc protein with IgG binding to Fc gamma

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of Mr 56,000, pI 4.8-5.1 and Mr 60,000, pI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the T-cell membrane. While the Mr 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the Mr 60,000 protein remains unknown. Three other components were detected, although with less consistency; Mr 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that less than 5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of Mr 60,000, 42,000, and 20,000-25,000, complexed IgG binding to Fc gamma of human peripheral blood T-cells also becomes spatially associated with Gc protein.     

Increasing the sensitivity of in vitro transformed hamster embryo cells (STHE strain) to cytolysis by resident and activated macrophages

The sensitivity of low-malignant spontaneously in vitro transformed hamster embryo cells (STHE strain) to cytolysis of both resident and activated macrophages has been examined with cytolytic 3H-thymidine release assay. Activated macrophages were obtained from Syrian hamster peritoneal exudate cells 5 days following priming with 3% thioglycollate-broth and subsequent in vitro activation with proper-myl, levan, LPS, MDP, 1,4-dihydropyridine-derivate PP-256 and PMA. It has been shown that the STHE strain cells were sensitive to cytolysis by only fully activated macrophages. Both resident and non-activated (Thioglycollate-elicited) macrophages have not developed significant levels of the cytolytic activity against STHE cell targets. Short-term treatment of STHE cells with actinomycin D result in augmentation of their sensitivity to cytolysis by resident and activated macrophages.