Modern radiation therapy: the achievements and outlook

The ability of lipid A and the antitumour compound, ONO-4007 (sodium2-deoxy-2-[3S-(9-phenylnonanoyloxy)tetradecanoyl] amino-3-O-(9phenylnonanoyl)-D-glucopyranose 4-sulphate) to induce nitric oxide (NO) synthase was investigated in vitro and in vivo, in comparison to the effects of lipopolysaccharide and di- and monophosphoryl lipid A. In J744.2 macrophages, lipopolysaccharide, di-and monophosphoryl lipid A and ONO-4007 (10(-9) - 10(-5) g/ml) alone, or in combination with interferon-gamma, induced NO synthase (order of potency: lipopolysaccharide > diphosphoryl lipid A > monophosphoryl lipid A > ONO-4007). ONO-4007 increased the activity of the inducible NO synthase in the lung of anesthetised rats (20% of the increased caused by bacterial lipopolysaccharide). Thus, ONO-4007 is a weak inducer of the inducible isoform of NO synthase in vitro and in vivo. The finding that di- and monophosphoryl lipid A also induce NO synthase indicates that the lipid A moiety of lipopolysaccharide contributes to the induction of NO synthase by lipopolysaccharide. The induction of NO synthase by ONO-4007, resulting in the formation of cytotoxic NO may contribute to the antitumour activity of the compound.     

An apoptosis-inducing gene therapy for pancreatic cancer with a combination of 55-kDa tumor necrosis factor (TNF) receptor gene transfection and mutein TNF administration

Intratumoral injection of recombinant human tumor necrosis factor (TNF) for inoperable pancreatic cancer has shown some efficacy in suppressing tumor growth or decreasing tumor markers. However, complete regression has not yet been achieved, possibly due to a lack of TNF receptors on tumor cells or an abundance of intracellular resistance factors. Recently, two distinct types of TNF receptors, R55 and R75, were identified, which are responsible for signaling of cytotoxicity and of proinflammation, respectively. In this study, a novel type of suicide gene therapy is proposed that is based on transfection of the R55 gene into human pancreatic cancer cells (AsPC-1 and PANC-1) and subsequent administration of TNF. The transfectants from both cell lines showed higher TNF susceptibility than their parental cells. In vivo tumor formation of an AsPC-1 clone (clone 10) inoculated in nude mice was substantially suppressed by administration of TNF. For practical use of this strategy, however, the adverse effects of TNF may become an obstacle. We previously produced mutein TNF 471, which had a higher affinity for R55, superior antitumor activity, and fewer adverse effects. This mutein TNF 471 manifested greater antitumor activity against clone 10. Because the R55 receptor is known to be involved in augmentation of cellular immunity by TNF, mutein TNF 471 is also expected to be highly potent in this function. In fact, the mutein TNF 471 induced higher splenic natural killer cell activity in nude mice inoculated with clone 10 than did native TNF. This property of augumenting cellular responses may be advantageous in the eradication of viable tumor cells left untransfected in practical gene therapy regimens in which 100% transfection of the R55 gene into tumors is not feasible. Thus, gene therapy combining transfection of the TNF-R55 gene with administration of mutein TNF 471 may provide a new modality for the treatment of pancreatic cancer.     

Correlation between the capacity to activate macrophages in vitro and the antitumor activity in vivo of lipopolysaccharides from different bacterial species

The correlation between the activation of macrophages by lipopolysaccharides (LPS) from four different bacterial species and their antitumor effect in a rat model of colon cancer was investigated. The efficacy of LPS from Neisseria meningitidis (Nm), Salmonella minnesota (Sm), Escherichia coli (Ec) and Bordetella pertussis (Bp) was evaluated as the smallest concentration inducing rat peritoneal macrophages (pm psi) to produce tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and nitric oxide (NO). The cytokine production was measured in bioassays and NO production quantitatively with Griess reactant. Nm was the most effective LPS with concentrations of 1 ng/10(6) pm psi for the induction of TNF, IL-1 and IL-6 activities and 0.01 ng/10(6) pm psi for the induction of NO production. The range between efficacy of different LPS was broad from 1 to 10(4)-10(5) for TNF activity, 1 to 10(2)-10(3) for NO production and IL-6 activity and 1 to 10-10(2) for IL-1 activity. In vivo antitumor effect was evaluated on the growth of peritoneal carcinomatosis. Complete tumor regressions were observed, the LPS rating with respect to decreasing efficacy was Nm, Sm, Ec then Bp; Nm, Sm and Ec were very closed while Bp was not effective. These results show the correlation between the antitumor effect in vivo of LPS and their capacity to induce in vitro IL-1 activity, but not between their ability to induce NO production, TNF and IL-6 activities.     

Substance P augments tumor necrosis factor release in human monocyte-derived macrophages

Substance P (SP) is an undecapeptide that has the amino sequence Arg-Pro-Lys-Pro-Gin-Gln-Phe-Phe-Gly-Leu-Met-NH2 and that belongs to a family of structurally related peptides known as tachykinins, the latter are widely distributed in the central nervous system. SP is involved in the biological activities of cells in the immune system, including the induction of cytokines in immune cells. We have investigated the effects of SP on constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF) in cultured blood monocyte-derived macrophages (MDM). Cells cultured in vitro for 14 days were treated with SP at various concentrations (10(-10) to 10(-6M) in the presence of LPS before culture supernatants were harvested. TNF bioactivity in culture supernatants was measured with L929 cell line MDM from 10 of 12 donors treated with a SP alone showed increased TNF production. SP and LPS also interacted in a synergistic fashion in upregulating TNF production in MDM from responders. The stimulatory effect of SP was inhibited by two SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-7-D-Trp-9-leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). In addition, an anti SP polyclonal antibody blocked the SP effect on TNF production in cultured MDM, further indicating the specificity of these effects. These results demonstrate that SP is an important regulator of monokine production by human monocytes/macrophages.     

Regression of Ehrlich ascites carcinoma in BCG-sensitized mice

Intraperitoneal inoculation of CF1 mice with Bacillus Calmette-Guérin (BCG) protected many of them against the ascites form of Ehrlich carcinoma; and, for those that developed cancer, complete regression occurred in up to 50% of the cases at an advanced state of the neoplastic disease. In contrast, when a booster dose of BCG was administered in admixture with tumor cells, the incidence of the tumor was lower and tumor regressions were very rarely observed in mice that developed cancer. Trypan blue, an inhibitor of lysosomal enzymes of macrophages, was found to markedly suppress the natural (innate) antitumor resistance of control mice as well as the acquired resistance and tumor regressions of BCG-sensitized mice. Moreover, a comparison of the cytotoxic activity of the adherent (macrophages) and nonadherent (predominantly lymphocytes) cells isolated from the peritoneal cavity of BCG-sensitized mice, as measured by the inhibition of DNA synthesis, revealed that the effector cells were amongst the macrophages. In contrast, spleen macrophages were devoid of cytotoxicity. The spleen lymphocytes from both BCG-sensitized and control mice possessed about the same significant cytotoxic activity. These results indicate that the activated peritoneal macrophages, induced by a local injection of BCG, could play an important role in the antitumor immunity against Ehrlich carcinoma.     

Cytotoxic activity of BCG-activated macrophages against L929 tumor cells is nitric oxide-dependent

The tumoricidal activity of activated macrophages has been attributed largely to the release of tumor necrosis factor (TNF), or to the production of reactive oxygen or nitrogen intermediates. The L929 tumor cell line (a murine fibroblast-like cell) when treated with actinomycin D (ActD) has been used to measure TNF alpha cytotoxicity. In the present study, we determined the cytotoxic activity of BCG-activated peritoneal macrophages against ActD-untreated L929 tumor cells. Furthermore, we measured the production of hydrogen peroxide (H2O2), nitric oxide (NO) and TNF by macrophages cultured in the presence or absence of L929 cells. As expected, BCG-activated macrophages produced significant amounts of H2O2 (16.0 +/- 3.0 microM), TNF (512 U/ml) and NO (71.5 +/- 3.2 microM). TNF (256 U/ml) and NO (78.9 +/- 9.7 microM) production was unchanged in co-cultures of L929 cells with BCG-activated macrophages but H2O2 production was totally inhibited. The cytotoxic activity was dependent on NO release since L-NAME (2.5, 5.0 and 10 mM), which blocks NO synthase, inhibited the killing of L929 cells. Addition of anti-TNF (20 micrograms/ml) antibodies to the cultures did not affect the tumoricidal activity of macrophages. Our results indicate that macrophage-mediated killing of L929 cells is largely dependent on NO production but independent of H2O2 or TNF release.     

Divergent effects of TNF alpha in the adoptive immunotherapy of a murine sarcoma

We have previously described an in vitro sensitization (IVS) procedure which enabled the generation of therapeutic T cells from tumor-bearing mice for adoptive immunotherapy. The procedure involved culture of tumor-draining lymph node (TDLN) cells with irradiated tumor in the presence of interleukin-2 (IL-2). The availability of many recombinant cytokines affords an opportunity to examine their effects on the immune response to tumor. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF alpha) on the generation and function of IVS cells utilized in adoptive immunotherapy of the murine MCA 106 sarcoma. TNF alpha administered iv at nontherapeutic doses was found to enhance the antitumor efficacy mediated by IVS cells plus IL-2 in the treatment of pulmonary metastases. In contrast, TNF alpha administration to mice bearing progressive footpad tumors had inhibitory effects on the sensitization of tumor-reactive cells in TDLN since IVS cells generated from these animals displayed a diminished antitumor effect. This effect appeared to be due to a reduced number of tumor-reactive lymphoid cells in the TDLN since TNF alpha added to IVS cultures did not alter the antitumor efficacy of the resultant IVS effector cells. These findings indicate the divergent effects of TNF alpha on the immune response to tumor and adoptive immunotherapy with IVS cells.     

Cytotoxicity induced by recombinant human tumor necrosis factor-alpha dependent on the types of its receptors on canine cells

Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-alpha has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-alpha on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-alpha receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-alpha induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-alpha. Combined with ACT-D, rh-TNF-alpha showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-alpha depends on the presence of TNF RI receptors on canine tumor cells.     

Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-kappaB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls

This study was undertaken to evaluate the role of CD14 and complement receptors type 3 (CR3) and 4 (CR4) in mediating TNF release and NF-kappaB activation induced by LPS and cell wall preparations from group B streptococci type III (GBS). LPS and GBS caused TNF secretion from human monocytes in a CD14-dependent manner, and soluble CD14, LPS binding protein, or their combination potentiated both LPS- and GBS-induced activities. Blocking of either CD14 or CD18, the common beta-subunit of CR3 and CR4, decreased GBS-induced TNF release, while LPS-mediated TNF production was inhibited by anti-CD14 mAb only. Chinese hamster ovary cell transfectants (CHO) that express human CD14 (CHO/CD14) responded to both LPS and GBS with NF-kappaB translocation, which was inhibited by anti-CD14 mAb and enhanced by LPS binding protein. While LPS showed fast kinetics of NF-kappaB activation in CHO/CD14 cells, a slower NF-kappaB response was induced by GBS. LPS also activated NF-kappaB in CHO cells transfected with either human CR3 or CR4 cDNA, although responses were delayed and weaker than those of CHO/CD14 cells. In contrast to LPS, GBS failed to induce NF-kappaB in CHO/CR3 or CHO/CR4 cells. Both C3H/OuJ (Lps[n]) and C3H/HeJ (Lps[d]) mouse peritoneal macrophages responded to GBS with TNF production and NF-kappaB translocation, whereas LPS was active only in C3H/OuJ macrophages. Thus, LPS and GBS differentially involve CD14 and CR3 or CR4 for signaling NF-kappaB activation in CHO cells and TNF release in human monocytes, and engage a different set of receptors and/or intracellular signaling pathways in mouse macrophages.     

Predictors of functional disability in patients with rheumatoid arthritis

The ability of nonactivated peritoneal macrophages to induce nitric oxide (NO) secretion in transformed and tumor cells of the same origin differing in tumorigenic (TGA) and spontaneous metastatic activities (SMA) was examined. Low tumorigenic and nonmetastatic spontaneously transformed in vitro hamster embryo cells of the STHE strain in contact with macrophages, or their non-purified soluble product were the highest producers of NO. In vivo selected STHE cell variants characterized by middle or high TGA demonstrated low, or no NO production, respectively (independently of SMA- or SMA+). Two highly tumorigenic RSV-SR (v-src) transformed cell strains (SMA- and SMA+) were both negative in NO production. Thus, NO production by tumor cells appeared to be inversely correlated with TGA level and less dependent on SMA.     

GE3, a novel hexadepsipeptide antitumor antibiotic, produced by Streptomyces sp. I. Taxonomy, production, isolation, physico-chemical properties, and biological activities

GE3, a novel cyclic hexadepsipeptide antibiotic, was isolated from the culture broth of Streptomyces sp. GE3. GE3 was weakly active against some Gram-positive and Gram-negative bacteria and showed potent cytotoxicity against human tumor cell lines. GE3 also exhibited antitumor activity against human pancreatic carcinoma, PSN-1, in vivo. GE3B, a linear peptide form of GE3, which was isolated from the same culture broth with GE3, showed no antibiotic and cytotoxic activities, suggesting the necessity of the cyclic structure of GE3 for its biological activities.     

Stimulation of nitric oxide production in macrophages by Babesia bovis

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.     

Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis

Dendritic cells (DCs), which are antigen presenting cells of potential use in human antitumor vaccination trials, are presently the subject of intense investigation. Many recent studies have reported the possibility of generating ex vivo large numbers of DCs with high antigen presenting capacity by the culture of bone marrow or blood progenitors. In this study, we examined the differentiation into DCs of CD34+ progenitors isolated from the G-CSF mobilized blood of 3 healthy donors and 5 patients with breast cancer and cultured in the presence of GM-CSF + IL-13. The characteristics of the cells were compared to those of cells obtained in the presence of GM-CSF + TNF alpha. By day 15, one third of the bulk cells cultured with IL-13 were CD1a+/CD14- and strongly expressed CD1c, CD40, CD80 and HLA-DR. In contrast, cells obtained with TNF alpha expressed CD1a on one in three cells but with a considerably lower fluorescence intensity than on IL-13-cultured cells and strongly expressed CD14 on more than 50% of cells. CD1a+/CD14- cells emerged in IL-13 cultures at day 5, while in TNF alpha cultures CD14+ cells appeared before CD1a+ cells. Cells grown in the presence of IL-13 had an increased capacity to present antigens to autologous lymphocytes and to stimulate allogeneic T-lymphocytes. This effect was greater than that of cells grown in the presence of TNF alpha. These cells should therefore have greater effector potential in any therapeutic applications in humans.     

Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.     

Proliferation and survival of mammary carcinoma cells are influenced by culture conditions used for ex vivo expansion of CD34(+) blood progenitor cells

Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34(+) blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1beta (IL-1beta), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34(+) BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-beta1 (TGF-beta1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1beta + IL-3 + IL-6 + EPO, but was suppressed by TGF-beta1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 x 10(5)/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34(+) BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34(+) BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.     

Epidemiology of trichinellosis in lynx in Finland

To examine the possibility of cytokine gene therapy in relation to pancreatic cancer, we evaluated the antitumor effect of human pancreatic carcinoma cells (AsPC-1) which were retrovirally-transduced with several kinds of cytokine genes. These cells were inoculated into BALB/c nude mice and their tumor volumes were assessed. The in vitro growth rate of the transduced cells was not different from that of a parental cell line. Among the transduced cells, human interleukin (IL)-6-transduced AsPC-1 and mouse granulocyte macrophage colony-stimulating factor-transduced AsPC-1 cells showed a significant retardation of tumor growth compared with a parental cell line. In the cases of AsPC-1 cells transduced with the human IL-2 or mouse IL-4 gene, small tumors were generated but thereafter they regressed completely. Histological examinations showed monocytic cell infiltration around the tumors of IL-2- or IL-4-producing cells. These data suggest that secretion of IL-2 or IL-4 from tumor cells can induce an antitumor effect even in the defective condition of mature T cells.     

Establishment of tumor cell culture (ILA) derived from hamster pancreatic islets treated with BOP

ILA cells were established from tumors induced by the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in hamster islets. The proliferation, morphology, karyotype, immunoreactivity with certain antibodies and growth factor secretion of these tumor cells were compared with the same parameters in tumor cells induced by BOP in hamster ductal cells (TAKA-1-BOP) established in a previous study. Minor differences were found in the morphology and ultrastructure of the 2 cell lines. Contrary to TAKA-1-BOP cells, ILA cells did not express cytokeratins 8.13, 13 or 18 but did express DU-PAN-2 and TAG-72, 2 known human pancreatic cancer-associated antigens. No endocrine cell markers were expressed. A significant difference also was found in the chromosomal pattern in that there were more abnormalities and marker chromosomes in ILA cells than in TAKA-1-BOP cells and the Y or X chromosomes were missing in ILA cells. ILA cells produced TGF-alpha, IGF-I, bombesin and gastrin and expressed specific binding sites for hEGF. TGF-alpha secretion from ILA cells was much greater than that from TAKA-1-BOP cells. Our results indicate that pancreatic cancer cells grown in vitro are not a single clone. We conclude that there are some genetic and biological differences between tumors arising from pancreatic duct and islets and that pancreatic ductal adenocarcinomas originating from islets have a profound malignant potential.     

Involvement of reactive oxygen intermediates in tumor necrosis factor alpha-dependent bacteriostasis of Mycobacterium avium

We studied the involvement of reactive oxygen intermediates and reactive nitrogen intermediates in the bacteriostasis of two Mycobacterium avium strains differing in virulence by resident peritoneal macrophages. We found that both the highly virulent strain (25291) and the low-virulence strain (1983) of M. avium induced superoxide production but inhibited nitrite production in vitro. This inhibition was due to the production of superoxide, a nitric oxide scavenger. The stimulation of superoxide production was two- to fivefold higher in strain 1983-infected than in strain 25291-infected resident peritoneal macrophages and was independent of contaminating T cells or NK cells. Superoxide secretion was dependent on the tumor necrosis factor (TNF) produced endogenously by the macrophages. This was also true when macrophages were isolated from infected mice. Addition of TNF to the infected resident peritoneal macrophages caused only a slight, albeit significant, increase in superoxide production by strain 25291-infected macrophages. Incubation of resident peritoneal macrophages with different scavengers of reactive oxygen intermediates showed that strain 1983 was susceptible to hydrogen peroxide produced by resident peritoneal macrophages. Strain 25291 was shown to decrease superoxide secretion inside heavily infected bone marrow-derived macrophages. This strain was also shown to be a better trigger for production of reactive oxygen intermediates than strain 1983. In summary, strain 1983 induced high levels of TNF synthesis that acted in an autocrine fashion to stimulate production of reactive oxygen intermediates by macrophages leading to growth restriction mediated by hydrogen peroxide. The highly virulent strain 25291 induced low levels of TNF synthesis, and therefore little reactive oxygen intermediate production, and could also inhibit superoxide production by the infected macrophages.     

Protein kinase C regulates macrophage tumor necrosis factor secretion: direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance

BACKGROUND: Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. METHODS: Murine macrophages were cultured +/- LPSp for 24 hours. Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator. Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay. RESULTS: LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA. PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells. Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages. PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells. CONCLUSIONS: Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages. Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.