IL-3 dependence of a Thy-1lo, B220+, IL-3 receptor-positive antigen-specific DTH-initiating clone.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different antigen-specific Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This clone WP-3.27 produces an antigen-specific factor, OX-F, that acts in an Ag-specific manner to initiate DTH. The clone was phenotyped as a Thy-1+, B220+, CD3-, CD4-, CD8- cell. In this report, we further detail the characteristics of this unusual Ag-specific DTH-initiating cell clone. By flow cytometry analysis, WP-3.27 is Thy-1lo, Lyt-1+ (CD5+), but CD3-, TCR-alpha beta-, and TCR-gamma delta-. Moreover, WP-3.27 does not express surface immunoglobulins but expresses B220 (CD45RA), and also some macrophage markers such as Mac-1, F4-80, and MHC class II after gamma-IFN treatment. Interestingly, this clone also expresses IL-3 receptors (IL-3R) and not IL-2R. In addition to the Ag-specific DTH-initiating factor, WP-3.27 constitutively produces IL-3. Inhibition of proliferation of WP-3.27 with an anti-mouse IL-3 monoclonal antibody suggests that the clone WP-3.27 is IL-3-dependent, at least partially. WP-3.27 also constitutively produces IL-1 and IL-6, but not TNF-alpha. LPS activation of the clone resulted in a net increase of IL-1, IL-6, and TNF-alpha production. Thus, this Ag-specific DTH-initiating cell clone makes a unique set of cytokines. Northern blot analysis demonstrated that clone WP-3.27 transcribes mRNA encoding IL-1, IL-3, IL-6, and TNF-alpha, but not for TNF-beta (lymphotoxin). The nature of this unusual cell, which displays characteristics of more than one cell lineage, is discussed.     

Suppression and contrasuppression in athymic nude mice: nude mice produce the antigen-specific component of a T suppressor factor that inhibits the late 24-hr phase of DTH but do not generate suppression nor contrasuppression of the early initiating phase of DTH.

Previous studies demonstrated that the initiation of murine delayed-type hypersensitivity (DTH), as exemplified by contact sensitivity induced by picryl chloride (PCI) or oxazolone (OX), is due to antigen-specific, T cell-derived, DTH-initiating factors called, respectively, PCl-F and OX-F. These factors participate in the extravascular recruitment of CD4+, Th-1, DTH effector T cells in the elicitation of DTH. Related factors also participate, together with nonantigen binding factors derived from CD8+ T cells, to constitute an antigen-specific T cell-derived suppressor factor (TsF) that can down regulate the ability of Th-1 effector T cells to mediate DTH. Since it was shown recently that athymic nude mice can make antigen-specific, DTH-initiating T cell factors, the current study tested whether nude mice also could produce the antigen-specific component of the TsF that suppresses DTH effector T cells. We found that antigen-specific factors from nu/nu mice could complement the nonantigen-binding subfactor produced in normal mice to constitute the whole antigen-specific TsF. Additional studies showed that the successful adoptive cell transfer of DTH-initiating T cell activity from nude mice into normal mice required cyclophosphamide treatment of the recipient. In contrast, transfer of DTH-initiating cell activity from nu/+ mice did not require cyclophosphamide treatment of the recipients. We hypothesized that nude mice lacked contrasuppressor cells. Although nude mice were able to manifest the early, initiating phase of DTH, we found that there was no suppression of early DTH-initiating T cells in nude mice, compared to nu/+. Therefore the production of DTH-initiating T cell factor could be boosted in nude mice. The ability to boost DTH-initiating cells in nude mice should facilitate the development of cell lines and clones with the ability to initiate DTH.     

An antigen-specific DTH-initiating cell clone. Functional, phenotypical, and partial molecular characterization

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different Ag-specific Thy-1+ cells. An early-acting DTH-initiating cell in the lymphoid organs produces a circulating, Ag-specific factor that is functionally analogous to IgE antibody and initiates DTH by sensitizing the local tissue for release of the vasoactive amine serotonin. In picryl chloride (PC1) or oxazolone (OX) contact sensitivity, this DTH-initiating factor is called PC1-F and OX-F respectively, and is Ag-specific, but MHC-unrestricted. The phenotype of polyclonal DTH-initiating cells was recently shown to be unusual for an Ag-specific cell. The phenotype was: Thy-1+, Lyt-1+ (CD5), triple negative (CD4-, CD8-, and CD3-), B220+ (Ly-5, CD45RA), positive for IL-3 receptors, but not IL-2 receptors, and positive for antibodies that react with a putative constant or framework portion of DTH-initiating factors such as anti-PC1-F antibodies and 14-30 mAb. We report here the generation of an Ag-specific DTH-initiating cell clone from nude mice that were immunized and boosted by contact sensitization with OX. By flow microfluorometry analysis, this clone has a similar unique surface phenotype, and by in vivo assay has the same functional abilities, as polyclonal DTH-initiating cells. The clone produces Ag-specific OX-F that acts in an Ag-specific manner to initiate DTH. Moreover, specific cDNA probes and Northern blot analysis of the clone demonstrated that the Ag-specific DTH-initiating cells are Thy-1+, CD3-, and IL-3R+. Thus, DTH initiation is due to an Ag-specific lymphoid cell, that produces an Ag-specific factor, and that has a unique surface phenotype for Ag-specific cells; namely, Thy-1+, CD5+, sIg-, CD4-, CD8-, CD3-, CD45RA+, IL-2R-, and IL-3R+.     

The DTH-initiating Thy-1+ cell is double-negative (CD4-, CD8-) and CD3-, and expresses IL-3 receptors, but no IL-2 receptors

The elicitation of delayed-type hypersensitivity (DTH) requires an early-acting Thy-1+ cell that produces an Ag-specific, non-MHC-restricted factor that initiates DTH by sensitizing the local tissue for release of the vasoactive amine serotonin. We characterized the phenotype of this DTH-initiating cell by treating cells from sensitized mice with different antibodies and then either with rabbit C or anti-Ig panning or bead separation to deplete various subpopulations. We then transferred these cells i.v. into naive recipients that were challenged to elicit DTH. Our findings indicate that the early DTH-initiating cell is Thy-1+, Lyt-1+, CD4-, CD8- and CD3-, whereas the classical, late DTH effector T cell is Thy-1+, Lyt-1+, CD4+, CD8-, and CD3+. We hypothesize that DTH-initiating cells are primitive T cells with Ag receptors that can bind Ag without MHC-restriction. This hypothesis was supported by the finding that two different antibodies, that both bind T cell-derived Ag-binding molecules, eliminated the DTH-initiating, cell but did not affect the late component, MHC-restricted CD4+, CD3+ T cell. Additional experiments with antibodies against restricted determinants of the T-200 glycoprotein family (CD45R) showed that the early but not the late cell is positive for B220, which is usually present on B cells, and on some activated T cells. Also, the DTH-initiating cell is Il-2R-, but Il-3R+; whereas the late component DTH T cell is IL-2R+ and IL-3-. Our findings suggest that DTH-initiating cells may be Ag-specific lymphoid precursor cells that arise before final differentiation along the pathway leading to mature T or B cells. Our results indicate that antigen-specific Thy-1+, CD3-, CD4-, CD8- cells function in vivo to initiate DTH reactions.     

Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia

In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance. However, IFN-gamma-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.     

Lps induces IL-6 in the brain and in serum largely through TNF production

We investigated the relative contribution of IL-6 and PGE2 directly induced by LPS and indirectly induced via TNF, using in vivo and in vitro models in the mouse. In these models we have used as tools an anti-TNF antibody and a cyclooxygenase inhibitor, the S enantiomer of ketoprofen (S-KPF). Anti-TNF antibodies inhibited LPS-induced IL-6 production in three different models: IL-6 production by mouse peritoneal macrophages in vitro; serum IL-6 levels induced by intraperitoneal LPS; and brain IL-6 levels induced by an intracerebroventricular injection of LPS. However, in vitro anti-TNF antibodies, did not inhibit LPS-induced PGE2, indicating that this effect is not mediated by TNF. Since PGE2 has an opposite effect on TNF and IL-6 production, inhibiting that of TNF but inducing that of IL-6, we investigated the effect of S-KPF on TNF and IL-6 production in vivo following LPS injection. Both TNF and IL-6 induction was augmented by S-KPF, but anti-TNF antibodies abolished the augmentation of IL-6 production. Thus, the effect of anti-inflammatory drugs on IL-6 production in some models can be secondary to their effect on TNF production.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-alpha, endothelin (ET)-1, interleukin (IL)-1alpha, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.     

The DTH effector response and IL-2 are unaffected by cyclosporine A in autoimmune B6D2F1 mice

Delayed-type hypersensitivity (DTH) is classically defined as inflammation involving activated Th1 cells and cytokine production. DTH paw swelling, along with the cytokines IL-2, IFNγ, MCP-1 and TNFα, were inhibited in Balb/c mice by cyclosporine A (CsA). Surprisingly, the DTH response in the B6D2F1 mice was unaffected by CsA, despite a decrease in TNFα and IFNγ levels. IL-2 levels, however, were not decreased. To determine if the IL-2 production in the B6D2F1 strain is occurring through CD28-mediated costimulation, both CsA and CTLA-4Ig were administered. Paw swelling and IL-2 levels were decreased, indicating a role for costimulation. Co-administration of temsirolimus and CsA also reduced DTH and IL-2 levels in B6D2F1 mice, demonstrating involvement of the mTORC1 pathway. These results indicate that the cell activation pathways responsible for DTH differ with mouse strain. It is important to understand these differences in order to accurately interpret the results using potential therapeutic agents.     

Interleukin 1- and tumor necrosis factor-stimulation of prostaglandin E2 synthesis in MDCK cells, and potentiation of this effect by cycloheximide

The effects of interleukin (IL)-1 alpha, IL-1 beta and TNF alpha on prostaglandin-E2 synthesis in Madin-Darby canine kidney (MDCK) cells were investigated. IL-1 beta time- and dose-dependently stimulated prostaglandin-E2 synthesis. While TNF alpha produced a comparatively small but significant stimulation of PGE2 release, coincubation of IL-1 beta with TNF alpha produced a marked synergistic stimulation of PGE2 release. The effect of IL-1 beta and of IL-1 beta and TNF alpha was apparent as early as after 2 h of incubation. The enhanced PGE2 synthesis was inhibited by indomethacin as well as actinomycin D, while cycloheximide surprisingly potentiated PGE2 synthesis in response to both IL-1 beta and TNF alpha. IL-1 alpha alone was ineffective in stimulating a significant release of PGE2 at concentrations as high as 10 nM. However, it also showed a marked synergistic interaction with TNF alpha in stimulating PGE2 release.     

Several functions of immune cells in mice changed by oxidative stress caused by endotoxin

We have studied natural killer (NK) activity, lymphoproliferative response, the release of several cytokines (IL-2, TNF alpha and IL-1 beta) and the ROS production in peritoneal leukocytes obtained 0, 2, 4, 12 and 24 h after lipopolysaccharide (LPS) injection. Lethal septic shock (100 % mortality occurred at 30 h after LPS administration) was caused in female BALB/c mice by intraperitoneal injection of 100 mg/kg of E. coli LPS. Cytotoxicity and lymphoproliferation assay were preformed together with the measurement of IL-1 beta, IL-2 and TNF alpha production, and quantification of ROS. Natural killer activity, spontaneous lymphoproliferative response, IL-2, TNF alpha, IL-beta release and ROS production were increased after LPS injection. In conclusions, ROS and proinflammatory mediators produced by immune cells in response to LPS are involved in the oxidative stress of endotoxic shock. This oxidative state alters some functional characteristics of leukocytes (proliferation and NK activity).     

Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.     

Regulation of murine mononuclear phagocyte inflammatory products by macrophage colony-stimulating factor. Lack of IL-1 and prostaglandin E2 production and generation of a specific IL-1 inhibitor

The influence of macrophage (M)-CSF on the production of inflammatory mediators has been examined in murine peritoneal macrophages. Cultures of macrophages treated with up to 30,000 U/ml of human rM-CSF or with 10,000 U/ml of L929-derived M-CSF did not reveal either PGE2, IL-1, or IL-6 secretion. In contrast, LPS, which served as a positive control, stimulated production of significant levels of PGE2, IL-1, and IL-6. Furthermore, Northern blot analysis of macrophage RNA revealed a strong induction of IL-1 alpha and IL-6 mRNA by LPS but not by M-CSF. Conditioned medium from macrophage cultures treated with purified L929 or human rM-CSF in combination with LPS exhibited a significant reduction of IL-1 bioactivity as compared with an LPS challenge alone. To investigate the mechanism involved in this M-CSF-dependent reduction of IL-1 bioactivity, we measured IL-1 alpha gene expression. The addition of M-CSF to LPS-treated macrophages did not affect IL-1 alpha mRNA levels suggesting that M-CSF may regulate production of an IL-1 inhibitor. This hypothesis was shown to be valid because removal of IL-1 alpha from conditioned medium of LPS plus M-CSF-treated cells allowed the detection of a nondialyzable factor that blocked IL-1-dependent thymocyte proliferation. The inhibitor appeared to be specific because it did not inhibit IL-2 and TNF bioactivities. Furthermore, this IL-1 inhibitor, which binds to cells and not to IL-1, competed with the binding of radioactive IL-1 alpha or beta to EL-4.6.1 cells. The results demonstrate that M-CSF alone does not induce proinflammatory mediators and PGE2 as was previously published. The data also suggest that M-CSF may play a role in the down-regulation of inflammatory responses.     

Stimulation of human chondrocyte prostaglandin E2 production by recombinant human interleukin-1 and tumour necrosis factor

In this study we have examined the effects of recombinant cytokine preparations on the production of prostaglandin E2 (PGE2) by human articular chondrocytes in both chondrocyte monolayer and cartilage organ cultures. The cytokines chosen for this study included only those reported to be present in rheumatoid synovial fluids and which therefore could conceivably play a role in chondrocyte activation in inflammatory arthritis. Of the cytokines tested, interleukin-1 (IL-1; alpha and beta forms) consistently induced the highest levels of PGE2 production followed, to a lesser extent, by tumour necrosis factor (TNF; alpha and beta forms). The IL-1s were effective at concentrations 2-3 orders of magnitude less than the TNFs, with each cytokine demonstrating a dose-dependent increase in PGE2 synthesis for the two culture procedures. The increased PGE2 production by the chondrocytes exhibited a lag phase of 4-8 h following the addition of the IL-1 or TNF and was inhibited by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. Our results suggest that IL-1 may be the key cytokine involved in modulating chondrocyte PGE2 production in inflammatory arthritis; they further extend the list of human chondrocyte responses which are affected by both IL-1 and TNF.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production.

ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages. Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1. The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta. The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP. Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted. Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages. We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release. We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.