Splenic natural cytotoxic activity is enhanced during growth of a murine fibrosarcoma.

We have shown previously that the natural killer (NK) cell activity of DBA/2J mice bearing M-1 fibrosarcomas is consistently depressed at the later stages of tumor growth. The apparent mechanisms of inhibition are suppressor cell activation and prostaglandin E (PGE) production by tumor and lymphoid cells. In contrast, we show here that the natural cytotoxic (NC) activity of cells from the spleen, blood, and lymph nodes of mice bearing M-1 tumors is enhanced when compared to that of age- and sex-matched control mice. This enhanced NC activity does not appear to be due to increased cytolytic activity of macrophages but, rather, to enhanced cytolytic activity of multiple populations of non-adherent cells including B and T cells. Correlated with this is the finding that the NC activity of normal spleen cells is not inhibited in vitro by either PGE1 or PGE2 at levels which are inhibitory to NK cells. NC activity, although independent of PGE, is in fact enhanced by PGE1 in a dose-related fashion. These data indicate that NK and NC cells are regulated differently by PGE and during tumor growth. Utilizing a Winn assay, we also demonstrate that a cloned cell line with NC activity is capable of slowing tumor growth in vivo and that this action is improved if mice are treated with indomethacin concomitantly.

Novel intracellular signaling function of prostaglandin H synthase-1 in NF-kappa B activation.

Many potent nonsteroidal antiinflammatory drugs (NSAIDs) exert their effects by inhibiting the cyclooxygenase activity of prostaglandin H synthase-1 (PGHS1, thus disrupting prostaglandin biosynthesis. However, these drugs do not block the activation of NF-kappa B, an inducible transcription factor which regulates numerous inflammation-related genes. Here we demonstrate that PGHS1 peroxidase, a NSAID-insensitive activity of PGHS1, mediates NF-kappa B activation through an intracellular reactive oxygen signaling pathway. Overexpression of PGHS1 strongly potentiated NF-kappa B activation by phorbol esters and dramatically elevated the generation of intracellular reactive oxygen species (ROS) in response to low concentrations of t-butyl peroxide. Both functions were dependent on PGHS1 peroxidase activity and could be suppressed by the potent antioxidant pyrrolidine dithiocarbamate. In contrast, elimination of PGHS1 cyclooxygenase activity by NSAIDs or site-directed mutagenesis failed to block ROS production or NF-kappa B activation. Thus, PGHS1 peroxidase serves an intracellular signaling function leading to NF-kappa B activation, separable from its role in prostaglandin synthesis.

The involvement of protein kinase C, calcium, and 5-lipoxygenase in the production of tumor necrosis factor by a cloned interleukin-3 dependent cell line with natural cytotoxic activity.

The cloned interleukin-3 dependent cell line, M1-A5 was studied to determine whether protein kinase C, calcium mobilization, and 5-lipoxygenase activity were involved in the signal transduction pathways required for the production of TNF. TNF release was stimulated by 10 ng/ml phorbol myristate acetate (PMA), 2 microM calcium ionophore A23187, and 1 microgram/ml lipopolysaccharide (LPS) with synergism seen between PMA and A23187. All signals were blocked by phloretin and the PMA signal was blocked by H-7, both drugs acting as protein kinase C inhibitors. Desensitization of protein kinase C by PMA (1 microgram/ml for 24 h) provided evidence that both PMA- and LPS-stimulated TNF production were protein kinase C-dependent while A23187-stimulated TNF production was not. Both the calcium chelator, EGTA, and the intracellular calcium antagonist, TMB-8, inhibited TNF production stimulated by all agents, indicating that TNF stimulation by all agents was calcium dependent. Finally, the 5-lipoxygenase inhibitors, ketoconazole and L-656,224, but not the cyclo-oxygenase inhibitor ASA, inhibited TNF stimulated by all agents. These findings indicate that, although TNF production by M1-A5 cells can be stimulated either by a calcium/protein kinase C- or by a calcium-dependent signal, there is a convergence of signals at the level of 5-lipoxygenase activation.

Murine strain variation in the natural killer cell and proliferative responses to the immunostimulatory compound 7-allyl-8-oxoguanosine: role of cytokines.

7-Allyl-8-oxoguanosine (loxoribine) is a di-substituted guanine ribonucleoside which has been shown previously to enhance murine NK activity, B lymphocyte proliferation, and antibody synthesis. In this study we examined the relationship among enhancement of NK activity, proliferation, and cytokine synthesis in the responses of different strains of mice to loxoribine to provide insight into the role of cytokines in these biological activities. The NK response of mice was enhanced both in vitro and in vivo in all strains tested with the exception of the NK-deficient beige (BgBg) mouse. However, there was a marked difference in the degree of NK enhancement noted in other inbred strains, with C3H and CBA mice producing the highest responses, C57BL/6, BALB/c, and DBA/2 strains giving intermediate responses, and SJL mice manifesting low responses. Striking enhancement of NK cell activity was seen in SCID mice. A somewhat different effect was seen in humans. Loxoribine treatment enhanced both the NK and LAK activity of cells from individuals with low and high spontaneous NK activity. The degree of enhancement was similar for both groups, and thus the general hierarchy of NK activity among different donors was maintained. There was less interstrain variation in the murine proliferative response to loxoribine although nude (NuNu) mice showed the highest activity and SJL mice produced substantially lower responses than other strains. All strains produced IL-6, TNF alpha, IFN-alpha/beta, and IFN-gamma when spleen cells were cultured for 48 hr with loxoribine. Interstrain variability of cytokine synthesis displayed no consistent pattern from one cytokine to another, and all failed to correlate with interstrain variability of NK cell activity or B lymphocyte proliferation. When anti-cytokine antibodies were tested for the ability to block the immunostimulatory effects of loxoribine, only anti-IFN-alpha/beta and, to a lesser degree, anti-IFN-beta, partially inhibited NK activation. Similarly, only anti-IFN alpha/beta antibodies partially blocked the proliferative response to loxoribine. In both cases, reconstitution of the responses was achieved by adding back IFN-alpha/beta to cultures containing antibodies against IFN-alpha/beta. These data suggest that, although several cytokines are produced in response to loxoribine, only IFN-alpha and IFN-beta are directly involved in the NK activation and proliferative responses. The pattern of strain variation appears to be reflective of variation in NK cell responsiveness to IFN-alpha/beta.

Loxoribine (7-allyl-8-oxoguanosine) activates natural killer cells and primes cytolytic precursor cells for activation by IL-2.

Guanine ribonucleosides that have been substituted at the C8 position with bromine or thiol groups have been shown previously to activate NK cells and to act as sparing agents for IL-2 in the generation of LAK cells. Herein, we examined a disubstituted guanosine, 7-allyl-8-oxoguanosine (loxoribine), for the ability to activate NK cells and to interact with IL-2 in the generation of LAK cells. Loxoribine enhanced the NK activity of murine spleen cells with optimal activity occurring after 10 h of culture at concentrations ranging from 50 to 150 microM. The response was, however, short lived, approaching baseline levels by 24 h of culture. In contrast, if spleen cells were cultured with a suboptimal concentration of IL-2 (10 U/ml) in combination with loxoribine, a prolonged and enhanced cytolytic activity was seen. The enhancement was greatest if the loxoribine and IL-2 were both added to the cultures at the beginning of the incubation period. Analysis of the expression of the alpha-chain of the IL-2 receptor after loxoribine stimulation indicated that gene transcription was enhanced within 4 h, and cell surface expression was observed on NK1.1+ Thy1+ and NK1.1+ Thy1- cell populations within 24 h of loxoribine treatment. The priming of LAK cell precursors by loxoribine did not appear to be mediated by IFN-alpha/beta, because anti-IFN antibodies did not block either the activation of cytolytic cells by IL-2 or the expression of IL-2 receptors after culture with loxoribine. These data suggest that one mechanism by which cytolytic precursor cells are primed by loxoribine to respond to IL-2 faster and with enhanced cytolytic activity may be through the expression of high affinity IL-2 receptors due to the up-regulation of the alpha-chain.

The immunostimulatory compound 7-allyl-8-oxoguanosine (loxoribine) induces a distinct subset of murine cytokines.

C8- and N7, C8-substituted guanine ribonucleosides comprise a class of molecules with potent immunostimulatory activity for a variety of humoral and cellular immune responses. Although it has been suggested that the immunostimulatory activity may be partially mediated by cytokine production, to date there has been no systematic evaluation of the spectrum of cytokines elicited by these nucleosides. In this study, we examine the cytokines produced by murine spleen cells in response to the di-substituted guanosine analog loxoribine (7-allyl-8-oxoguanosine). First, the levels of cytokine mRNA in spleens from vehicle- or loxoribine-treated mice were compared using PCR analysis with a panel of cytokine-specific primers. Enhancement of IL-1 alpha, TNF-alpha, TNF-beta, IL-6, IFN-alpha, and IFN-gamma mRNA was seen in the spleens of loxoribine-treated mice. IL-12 mRNA responses were more complex, with an increase in the p40 chain and a decrease in the p35 chain. In contrast, no increase was seen for mRNA levels of IL-2, IL-3, IL-4, IL-5, IL-7, or GM-CSF. ELISA assays on the supernatants of loxoribine-treated spleen cells demonstrated that IL-1 alpha, IL-6, TNF-alpha, and IFN-gamma were all produced in a dose-dependent fashion with TNF-alpha produced first, followed by IL-6 and IFN-gamma, and last by IL-1 alpha. IFN-alpha beta activity rose as quickly as TNF-alpha, leveling off at 8 to 12 hr, and was supplanted by a later-occurring surge of IFN-gamma production. IL-1 alpha, IL-6, TNF-alpha, and IFN-gamma were also detected in the sera of mice injected with loxoribine. When antibodies against the relevant cytokines were tested, only anti-IFN-alpha beta inhibited NK activity or lymphocyte proliferation and, in both cases, activity was partially restored by the addition of exogenous IFN-alpha beta. Taken together, these data indicate that loxoribine induces the production of a selective cohort of cytokines all of which have been shown to have immunostimulatory activity. However, only IFN-alpha beta appears to play a role in the enhancement of NK activity and lymphocyte proliferation.

In vivo activation of natural killer cells and priming of IL-2 responsive cytolytic cells by loxoribine (7-allyl-8-oxoguanosine).

Guanine ribonucleosides which have been substituted at the N7 and/or C8 positions have been shown previously to activate natural killer (NK) cells and to act as sparing agents for interleukin-2 (IL-2) in the in vitro generation of lymphokine activated killer (LAK) cells. In this paper we examined a disubstituted guanosine, 7-allyl-8-oxoguanosine (loxoribine), for the ability to activate NK cells and to interact with IL-2 in the generation of LAK cells in vivo. Following iv administration, loxoribine enhanced murine splenic NK activity in a dose-related fashion, with optimal responses occurring at 3 mg/mouse. Enhanced lysis of YAC-1 cells was seen within 6 hr of injection and NK activity remained elevated for over 96 hr. Mature B and T cells were not required for NK activation since SCID mice responded to loxoribine within the same dose range as did the normal, immunocompetent mice. Both effector and precursor cells were eliminated by the administration of anti-asialo GM1 antibodies and NK activation was totally blocked in mice injected with anti-NK 1.1 antibodies. To test whether loxoribine would act as a sparing agent for IL-2 stimulated LAK activation, mice were injected with 2 mg loxoribine followed by twice daily administration of 10,000 units IL-2. In assays performed 48, 72, and 96 hr after injection of loxoribine, the cytolytic activity with the combination therapy exceeded the activity expected from the algebraic sum of the responses to the individual agents. Single injections of 2 mg loxoribine and 25,000 units IL-2 also stimulated NK/LAK activity, but the greatest enhancement was seen when loxoribine was administered 24 hr before the IL-2. Analysis of mRNA transcripts for the alpha chain of the IL-2 receptor indicated that gene transcription was enhanced within hours of loxoribine administration.

A leukocyte lipid up-regulates the avidity of lymphocyte function-associated antigen-1.

An acidic lipid termed leukocyte adhesion lipid (LAL) was isolated from PMA stimulated lymphoid and myeloid cell lines HL60, Jurkat, K562 and U937 but not from unstimulated cells or PMA treated Cos7 cells. LAL treated peripheral blood leukocytes (PBL) adhered strongly to IL-1 beta activated human umbilical vein endothelial cells (HUVEC), and the interaction could be inhibited by antibodies to intercellular adhesion molecule (ICAM-1) or lymphocyte function-associated antigen-1 (LFA-1). Leukocytes treated with LAL maintained the high avidity state of LFA-1 for at least 1 hr whereas the avidity of LFA-1 in PMA treated cells declined after 30 min. LAL was stable to heat (100 degrees C, 10 min), alkaline phosphatase and proteinase K treatments. Chemical analysis suggested that LAL contained unsaturated lipids. Our findings provide evidence for the involvement of lipids in LFA-1 activation.

Tepoxalin enhances the activity of an antioxidant, pyrrolidine dithiocarbamate, in attenuating tumor necrosis factor alpha-induced apoptosis in WEHI 164 cells.

The nuclear transcription factor-kappaB (NF-kappaB) and free radicals are known to be involved in apoptosis. We studied the effects of a series of di-aryl-substituted pyrazole NF-kappaB inhibitors including tepoxalin on tumor necrosis factor alpha (TNFalpha)-induced apoptosis in murine fibrosarcoma WEHI 164 cells. We found that potent inhibitors of NF-kappaB were also effective in attenuating apoptosis. WEHI 164 cells that had been dually treated with tepoxalin and the antioxidant pyrrolidine dithiocarbamate (PDTC) were significantly protected from TNFalpha-induced killing. To study the role of free radicals in mediating TNFalpha-induced apoptosis, stable WEHI 164 cells overexpressing Bcl-2, an antioxidant protein, were generated. These cells were protected from TNFalpha-induced apoptosis and neither tepoxalin nor PDTC provided further significant protection. These results suggest that Bcl-2, PDTC, and tepoxalin may attenuate apoptosis in this system by affecting the same signaling pathway or converging pathways. Because tepoxalin suppresses the release of free radicals, PDTC scavenges free radicals and Bcl-2 is an antioxidant protein, free radicals are among the key mediators of this TNF-induced killing event. Tepoxalin and antioxidants may be useful in developing new therapeutics for treating neurodegenerative diseases, autoimmune deficiency syndrome, and ischemia-reperfusion injuries.