Macrophage Tumoricidal Activity Research Articles

Tumoricidal response of liver macrophages isolated from rats bearing liver metastases of colon adenocarcinoma

Intraportal inoculation of CC531 adenocarcinoma cells into syngeneic rats causes an increase of liver macrophage cell number but not of major histocompatibility complex class II antigen expression. On day 1 after inoculation of 10(5) CC531 cells, a fixed number of isolated liver macrophages lysed significantly more target cells in vitro than did control cells. This effect was still present after 4 weeks. A 10-fold higher initial tumor dose significantly suppressed the macrophage response during the first 2 weeks. In contrast to tumoricidal activity induced by lipopolysaccharide in vitro, the tumoricidal response following in vivo challenge with tumor cells appeared not closely related to the production of reactive nitrogen intermediates, as in the latter case it was not abrogated in the presence of nitric oxide synthase inhibitor. Furthermore, the liver macrophage population appeared not fully activated after tumor inoculation as lipopolysaccharide further increased tumoricidal activity in vitro. The observed numerical and functional response of liver macrophages to intraportally inoculated tumor cells points at an important role of these cells in aspecific immune reactivity aimed at the reduction of local tumor growth. Results suggest that mechanistic differences exist between macrophage tumoricidal activity induced by tumor cells as compared with lipopolysaccharide.

Inhalation exposure to isobutyl nitrite inhibits macrophage tumoricidal activity and modulates inducible nitric oxide

Abuse of nitrite inhalants is common among male homosexuals and a history of abuse has been correlated with seropositivity to HIV and with the incidence of Kaposi's sarcoma among AIDS patients. The present study shows that inhalation exposure of mice to 900 ppm isobutyl nitrite for 45 min/day for 14 days compromised macrophage tumoricidal activity by up to 40% and it remained compromised for at least 7 days after terminating exposures. The inhalation exposures did not affect tumor cell binding but did inhibit inducible nitric oxide (NO zero). The NO zero synthase inhibitor NG-methyl-L-arginine totally inhibited both NO zero production and cytotoxicity, suggesting that reductions in NO zero due to inhalant exposure may be responsible for the reduced cytotoxic activity. Exposure to the inhalant increased constitutive production of tumor necrosis factor-alpha (TNF-alpha). TNF-alpha has been reported to stimulate the replication of HIV and the proliferation of Kaposi's sarcoma cells in vitro.

Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages

To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 (-/-) mice by gene targeting. NF-IL6 (-/-) mice were highly susceptible to infection by Listeria monocytogenes. Electron microscopic observation revealed the escape of a large number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 (-/-) mice. Furthermore, the tumor cytotoxicity of macrophages from NF-IL6 (-/-) mice was severely impaired. However, cytokines involved in macrophage activation, such as TNF and IFNγ, were induced normally in NF-IL6 (-/-) mice. Nitric oxide (NO) formation was induced to a similar extent in macrophages from both wild-type and NF-IL6 (-/-) mice. These results demonstrate the crucial role of NF-IL6 in macrophage bactericidal and tumoricidal activities as well as the existence of a NO-independent mechanism of these activities. We also demonstrate that NF-IL6 is essential for the induction of G-CSF in macrophages and fibroblasts.

Potential requirement of a functional double-stranded RNA-dependent protein kinase (PKR) for the tumoricidal activation of macrophages by lipopolysaccharide or IFN-alpha beta, but not IFN-gamma.

We analyzed the expression of the dsRNA-dependent protein kinase (PKR) during the activation of murine macrophages to the tumoricidal state by LPS and/or IFNs. LPS induced PKR expression in a dose-dependent manner at levels that were comparable with those observed in response to IFNs. By using the PKR inhibitor 2-aminopurine (2-AP), we have shown that the pathways of macrophage tumoricidal activation elicited by LPS and IFN-alpha beta, but not by IFN-gamma, included a 2-AP-sensitive step. In fact, LPS- and IFN-alpha beta-induced activation was inhibited by 2-AP, whereas the activation by IFN-gamma was not affected by the presence of the inhibitor. 2-AP did not affect the activation of protein kinase C or protein kinase A in intact cells. In the presence of 2-AP the up-regulation of IFN-beta mRNA by LPS was specifically inhibited, whereas the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA or the induction of PKR remained unchanged, thereby demonstrating that 2-AP inhibited selective macrophage genes. The differential sensitivity to 2-AP suggested that the expression of a functional PKR may be required for the macrophage tumoricidal response triggered by LPS and IFN-alpha beta but not IFN-gamma.

Taxol provides a second signal for murine macrophage tumoricidal activity.

The anticancer drug, taxol, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and bacterial LPS induce strikingly similar responses in murine macrophages. Here we report that taxol, like LPS, provides a "second" signal for murine macrophage activation to tumoricidal activity. Tumoricidal activity was determined by the release of 51Cr from prelabeled P815 mastocytoma target cells. Taxol or LPS alone weakly induced C3H/OuJ (Lpsn) murine macrophages to kill P815 mastocytoma cells, and tumoricidal activity was not induced by the classic "priming" signal, IFN-gamma. However, combinations of taxol or LPS with IFN-gamma synergized to activate macrophages to lyse tumor cells. Taxol activation of macrophages required an intact LPS signaling pathway, as taxol did not induce IFN-gamma-treated C3H/HeJ (Lpsd) macrophages to lyse target cells. In normal (Lpsn) murine macrophages, IFN-gamma, LPS, or taxol alone induced low or moderate levels of nitric oxide synthase gene expression and nitric oxide secretion. However, this gene and cytostatic metabolite were induced synergistically by combinations of taxol or LPS with IFN-gamma. Secretion of nitric oxide correlated with tumor cell killing, and taxol-activated macrophages failed to kill tumor targets in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase. The data illustrate the potential for taxol to activate macrophage mediated-antitumor mechanisms in addition to its better characterized role as an anti-mitotic agent.

Dietary n-3 polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice. Modulation of macrophage secretory activities.

We examined the effects of dietary n-3 polyunsaturated and saturated fatty acids on the development of the atherogenic process in mice and on the macrophage ability to secrete several effector molecules that may be involved in the atherogenic process. The secretion of inflammatory proteins such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) and the production of lipoprotein lipase (LPL), nitrogen oxide (NO2), and prostaglandin E2 (PGE2) were evaluated in peritoneal macrophages isolated from atherosclerosis-susceptible C57BL/6J mice. The mice were assigned at random to three experimental groups: the first group was fed a semi-defined control diet (control diet); the second group was maintained on the control diet supplemented with 10% menhaden oil (menhaden diet); and the third group received the control diet supplemented with 10% palm oil plus 2% cholesterol (saturated fat diet). Macrophages derived from mice fed the menhaden diet showed a suppression of their basal TNF-alpha mRNA expression and production. They also presented a dramatically decreased ability to express TNF-alpha and IL-1 beta mRNAs in response to exposure to lipopolysaccharide (LPS) compared with the macrophages from the control group. LPL mRNA and protein expression were downregulated after 6 and 15 weeks of menhaden-diet feeding. Significantly higher NO2 production in response to interferon gamma was found, both after 6 and 15 weeks of diet feeding, in the menhaden group compared with the control group. In addition, prostaglandin production and macrophage tumoricidal activity in response to LPS were decreased in this group compared with the control group. Macrophages derived from the saturated fat group did not show any significant alterations in TNF-alpha, LPL, NO2, or PGE2 secretion compared with controls. Interestingly, we observed a progressive increase of the LPS-induced IL-1 beta gene expression and secretion among macrophages harvested from mice receiving the dietary supplement of saturated fatty acids. At 6 and 15 weeks histologic examination of the atherosclerotic lesions did not reveal any important lesions in the control and menhaden groups, whereas a gradual development of fatty streaks was observed in the menhaden experimental diets for 10 additional weeks resulted in a major development of lesions in the control group, whereas only slight lesions were observed in the menhaden group.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein tyrosine kinase inhibitors decrease induction of nitric oxide synthase activity in lipopolysaccharide-responsive and lipopolysaccharide-nonresponsive murine macrophages.

We investigated tumoricidal activation and induction of nitric oxide synthase (NOS) activity in macrophages from LPS-responsive (C3H/HeN) and -unresponsive mice (C3H/HeJ). Macrophages were incubated in vitro with a synthetic lipopeptide or with LPS and IFN-gamma. LPS and IFN-gamma activated C3H/HeN but not C3H/HeJ macrophages to lyse B16 melanoma cells. In contrast, lipopeptide and IFN-gamma activated macrophages from both strains of mice. Genistein, a specific inhibitor of protein tyrosine kinase, significantly blocked tumoricidal activation of macrophages from both strains of mice. Genistein did not affect tumor cell binding but significantly inhibited the production of nitric oxide. Genistein, herbimycin A, and tyrphostin inhibited the induction of NOS activity in macrophages from both strains of mice. These data suggest that protein tyrosine kinase activity is involved in the signal transduction pathway of LPS and other synthetic bacterial-related immunomodulators at a point preceding triggering of macrophage tumoricidal activation and expression of inducible NOS activity.

Functional characteristics of the rat liver macrophage population after a single intravenous injection of liposome-encapsulated muramyl peptides.

In this study, we investigated different functional characteristics of the rat liver macrophage population after a single i.v. injection of liposome-encapsulated muramyl dipeptide (MDP) or its lipophilic derivative muramyl tripeptide-phosphatidylethanolamine (MTP-PE). The in situ induced tumoricidal activity of the liver macrophage population was determined in vitro against C26 colon adenocarcinoma cells. For investigating which cells are responsible for the observed cytotoxic effects, subfractions of the liver macrophage population, differing in cell size, were isolated at different intervals after injection of the liposomal muramyl peptides. From these subfractions, the number of cells, degree of cytotoxicity, and the response to an additional activation with free MDP in vitro were determined. Maximal induction of tumoricidal activity of the liver macrophage population was reached between 12 and 24 hours after injection of liposomal MDP, while no significant differences between the subfractions were observed. Heterogeneity of tumor cytolytic capacity was observed in subfractions of macrophages isolated at 2 and 48 hours after injection. At these time points, highest cytolytic activity was observed for the small to intermediate-size macrophages. No significant cytotoxicity was detectable in any subfraction 72 hours after injection of liposomal MDP. An identical pattern of macrophage tumoricidal activity was observed after injection of liposomal MTP-PE, although slightly lower cytotoxicity levels were found. When isolated during the first 12 hours after injection of liposomal MDP, the macrophage population was unable to respond to a subsequent in vitro exposure to MDP, with respect to tumor cytotoxicity. Twenty-four and 48 hours after injection, the smallest cells could be slightly reactivated, whereas the larger cells still remained unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of tumoricidal properties in macrophages by lipopolysaccharide requires protein-tyrosine kinase activity.

The purpose of these studies was to determine whether triggering murine peritoneal macrophages to a tumoricidal state by lipopolysaccharide (LPS) requires protein-tyrosine phosphorylation. The LPS-triggered activation of mouse macrophages to lyse syngeneic B16 melanoma cells was significantly inhibited in a dose-dependent manner by the protein-tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin. Genistein was effective only when added to macrophages prior to or simultaneously with LPS. Genistein potently inhibited the productive interaction of macrophages with LPS but had only a minor effect on the action of interferon-gamma. The effects of genistein on LPS-triggered macrophage activation were not due to nonspecific changes in macrophage metabolism or toxicity because genistein did not prevent lysis of tumor cells by activated macrophages, nor did it reduce the capacity of macrophages to phagocytose antibody-opsonized sheep erythrocytes. Western blot analysis with antiphosphotyrosine monoclonal antibody revealed that incubation of macrophages with LPS produced a rapid increase in tyrosine phosphorylation of several proteins and that the induced phosphorylation could be inhibited by effective concentrations of genistein, herbimycin A, or tyrphostin. Taken together, these data indicate that protein-tyrosine phosphorylation plays an important role in LPS-induced tumoricidal activation of macrophages.

Activation of Human Monocytes and Alveolar Macrophages by a Synthetic Peptide of C-Reactive Protein

We have previously shown that native human C-reactive protein (CRP) produces antitumor effects in experimental animals, and that these effects are mediated primarily through macrophages. More recently, we have observed that RS-83277, a synthetic peptide derived from CRP, appears to mimic the antitumor effects of native CRP. The purpose of this study was to determine the effects of RS-83277 on normal human monocyte and alveolar macrophage tumoricidal activity, and cytokine secretion. At optimal doses of 250-500 micrograms/ml, RS-83277 significantly enhanced tumoricidal activity of both monocytes and macrophages. RS-83287, a CRP peptide derived from a different site, had no effect at these doses. Specificity of RS-83277 for monocyte/macrophage-mediated cytotoxic activity was demonstrated by the failure of RS-83277 to enhance either natural killer (NK) or lymphokine-activated killer (LAK) cell-mediated activity. RS-83277 also augmented secretion of interleukin-1-beta (IL-1 beta) and interleukin-6 (IL-6) by monocytes. These data suggest a role for synthetic CRP peptide, RS-83277, as a novel biological response modifier in cancer therapy.

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.

Effect of tumor burden and route of administration on the immunotherapeutic properties of polyinosinic-polycytidylic acid stabilized with poly- l-lysine in carboxymethyl cellulose [Poly(I,C)-LC

We examined the immunomodulatory and therapeutic activities of poly(I,C-LC. Mice received a subcutaneous (s.c.) injection of sufficient numbers of MBL-2 lymphoma cells to produce in 1 week either a high or low tumor burden. A week after tumor cell injection, poly(I,C)-LC treatment was initieated; the agent was administered intraperitoneally (i.p.) at 5 mg/kg twice a week or at 2.5 or 0.5 mg/kg every day or as an intravenous (i.v.) injection at 0.5, 0.05, or 0.005 mg/kg three times a week. Poly(I,C)-LC treatment significantly increased antitumor effector cell functions in a variey of organs (including spleen, lungs, and peritoneum), as shown by increased killing of MBL-2 cells in vitro and increased tumor cell killing by natural killer cells and macrophages. Furthermore, prolongation of survival correlated with peritoneal macrophage tumoricidal activity when poly(I,C)-LC was given i.p. and with pulmonary effector cell function (including natural killer, cytolytic T-lymphocyte and macrophage tumoricidal activity) when the agent was administered i.v.

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.

TNF-α differentially regulates Ia antigen expression and macrophage tumoricidal activity in two murine macrophage cell lines

We have assessed tumor necrosis factor-α (TNF-α) production and its autocrine effects on activation in two murine macrophage cell lines which have distinct responses to the activation stimuli interferon-γ (IFN-γ) and bacterial lipopolysaccharide (LPS), and compared these responses to those observed in thioglycollate-elicited peritoneal macrophages. IFN-γ induced TNF-α production in RAW 264.7 cells and this induction was regulated at the transcriptional level. IFN-γ did not stimulate TNF-α production in either WEHI-3 cells or peritoneal macrophages, although MHC class II antigen expression was induced. LPS stimulated TNF-α production in the RAW 264.7 cell line and peritoneal macrophages; however, no TNF-α was detected in WEHI-3 cells activated with LPS. We also assessed the ability of endogenous TNF-α to serve as an autocrine regulator of two aspects of IFN-γ-mediated macrophage activation, namely, induction of antibody-independent tumoricidal activity and induction of MHC class II antigen expression. These studies revealed that TNF-α could act synergistically or antagonistically with IFN-γ in the regulation of these two functions, depending on both the macrophage population used and the function assessed. The results of our experiments suggest that the mechanism of induction of TNF-α production by IFN-γ or LPS, and the ultimate autocrine contribution of such TNF-α to a given activation response, is dependent on the activated macrophage target population under analysis. The WEHI-3 and RAW 264.7 cell lines provide a model system for comparative exploration of the mechanistic basis of this differential regulation.

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages.

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.

Staphylococcal exotoxins stimulate nitric oxide-dependent murine macrophage tumoricidal activity

The staphylococcal exotoxins toxic shock syndrome toxin 1 (TSST-1) and enterotoxin B were tested for their ability to stimulate murine peritoneal macrophages (PM) for tumoricidal activity. Both toxins were found to stimulate oil-elicited, gamma interferon-primed PM monolayers to kill nonadherent P815 tumor targets. The mechanism of killing of toxin-stimulated tumoricidal activity involved the production of nitric oxide, as nitrite could be demonstrated in culture fluids, and NG-monomethyl-L-arginine, an inhibitor of nitric oxide production, abrogated toxin-stimulated tumoricidal activity. TSST-1 stimulated the secretion of tumor necrosis factor by PM monolayers in the presence and absence of gamma interferon. The mechanism of toxin-stimulated tumoricidal activity was also determined to be independent of the production of reactive oxygen intermediates in that TSST-1 failed to stimulate H2O2 production by PM. These results demonstrate that the staphylococcal exotoxins are capable of stimulating macrophage production of nitric oxide for tumor cytotoxicity and suggest that the nitric oxide thus produced may subsequently play a role in the pathogenesis of the diseases caused by these toxins.

Protein kinase C in tumoricidal activation of mouse macrophage cell lines

A potential role of protein kinase C (PKC) in lipopolysaccharide- (LPS-) induced tumoricidal activation of macrophages was investigated by using two mouse macrophage cell lines (P388D1 and J774). J774 cells are stimulated by LPS to kill target P815 mastocytoma cells, whereas P388D1 cells fail to develop such an ability. Pretreatment of J774 cells with H-7 or phorbol myristate acetate resulted in a significant inhibition of LPS-induced cytotoxicity, whereas pretreatment with H-8, ML-7, HA1004, or W-7 did not. Since these results suggested a critical role of PKC in the activation process, the properties of PKC in the two cell lines were compared. Western blotting with rabbit antiserum specific for the PKC beta regulatory domain allowed detection of a protein of 79 kilodaltons (kDa) in the detergent lysates of both cell lines that were not stimulated by LPS. However, LPS treatment resulted in the appearance of a second protein of 40 kDa only in J774 cells and not in P388D1 cells. Furthermore, two forms of protein kinase (one basic and the other acidic) were identified in the cytosol of J774 cells by HPLC on DEAE-5PW, whereas only the basic form was found in P388D1 cells. On the basis of the response of the basic and acidic form protein kinases to phosphatidylserine (PS), diolein, and Ca2+, the basic form was found to contain both regulatory and catalytic domains of PKC, whereas the acidic form was suggested to represent the PKC catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of L-arginine on the retention of macrophage tumoricidal activity.

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.

Liposomal muramyl dipeptide therapy of experimental M5076 liver metastases in mice.

The effectiveness of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) or of liposomes containing a lipophilic MDP derivative, MDP-glyceroyldipalmitate MDP-GDP in inhibiting the growth of M5076 reticulum cell sarcoma liver metastases in C57BL/6 mice has been determined. MDP (100 micrograms) or liposomal MDP-GDP (2.5 mumol containing 1 microgram) were equally effective in inhibiting liver metastatic growth when given as a single treatment 3 days before tumor cell injection. Therapeutic treatment, initiated 3 days after tumor cell injection and continued for a period of 2 weeks, failed to inhibit metastatic growth. Activation of thioglycollate-elicited peritoneal macrophages or Kupffer cells in vitro with MDP or liposomal MDP-GDP resulted in the expression of tumoricidal activity against M5076 tumor cells. Adoptive cellular therapy with four injections of 2 x 10(6) macrophages was ineffective: activation of the macrophages with either MDP or liposomal MDP-GDP prior to injection was effective in inhibiting liver metastatic growth. Incorporation of the macrophage toxin dichlorodimethylene diphosphonate within liposomes containing MDP-GDP abolished the ability of such liposomes to induce macrophage or Kupffer cell tumoricidal activity in vitro as well as the antitumor activity when administered 3 days before tumor cell challenge.