Interleukin-1 (IL-1) interacts with cells through two types of binding molecules, IL-1 type I receptor (IL-1R I) and IL-1R II. The function of IL-1R II is unknown. In studies using monoclonal antibodies, IL-1 prolonged the in vitro survival of polymorphonuclear cells (PMN) through IL-1R I, and IL-4 antagonized the action of IL-1 by inducing expression and release of IL-1R II. Dexamethasone also induced expression and release of the IL-1R II in PMN. These results, together with the effect of antibodies to IL-1R on IL-1-induced production of cytokines in monocytes, indicate that IL-1 acts on myelomonocytic cells through IL-1R I and that IL-1R II inhibits IL-1 activity by acting as a decoy target for IL-1. The existence of multiple pathways of regulation emphasizes the need for tight control of IL-1 action.

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1β and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1β, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.

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Mature circulating polymorphonuclear cells (PMN) have the shortest half- life among leukocytes and undergo rapid programmed cell death in vitro. In this study, we have examined the possibility that inflammatory signals (cytokines and bacterial products) can regulate PMN survival. PMN in culture were found to rapidly die, with percentages of survival at 24, 48, 72, and 96 hours of 97.3% +/- 1.9%, 36.8% +/- 5.3%, 14.5% +/- 3.1%, and 4.2% +/- 2.9%, respectively (mean +/- SE of 20 different donors). PMN incubated with interleukin-1 beta (IL-1 beta), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and interferon-gamma (IFN-gamma), but not with prototypic chemoattractants (fMLP, recombinant C5a, and IL-8), showed a marked increase in survival, with values ranging at 72 hours of incubation from 89.5% +/- 5.8% for IL-1 beta to 47.6% +/- 6.4% for IFN- gamma. The calculated half-life was 35 hours for untreated and 115 hours for IL-1-treated PMN. PMN activated with lipopolysaccharide (LPS) or inactivated streptococci also showed a longer survival compared with untreated cells (94.4% +/- 3.2% and 95.5% +/- 2.4%, respectively, at 72 hours). PMN surviving in response to LPS or IL-1 beta retained the capacity to produce superoxide anion when treated with phorbol esters or fMLP. All inducers of PMN survival protect these cells from programmed cell death because they reduced cells with morphologic features of apoptosis and the fragmentation of DNA in multiples of 180 bp. Thus, certain cytokines and bacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial permissive step for certain cytokines and microbial products that activate gene expression and function in PMN.

Abstract. When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a halflife of ,x,10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-ArgGly-Asp-Ser-coated microbeads (~10 microbeads/ cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.T HE formation and regression of new vascular structures is a regulated process that governs organ development during embryogenesis (for review see reference 11). This suggests that not only capillary proliferation, but also capillary involution, depends on physiological control mechanisms. The composition and organization of the extraceUular matrix are known to markedly influence remodeling of blood vessels. Capillary basement membrane dissolution correlates with microvessel retraction, endothelial cell (EC) 1 rounding, and associated capillary regression (21,24). This series of events are likely caused by the fact that EC must be adherent to matrix proteins to survive and proliferate. When EC are cultured under conditions that prevent adhesion and spreading, they stop growing and lose viability (20,24,31).

Mature circulating polymorphonuclear cells (PMN) have the shortest half- life among leukocytes and undergo rapid programmed cell death in vitro. In this study, we have examined the possibility that inflammatory signals (cytokines and bacterial products) can regulate PMN survival. PMN in culture were found to rapidly die, with percentages of survival at 24, 48, 72, and 96 hours of 97.3% +/- 1.9%, 36.8% +/- 5.3%, 14.5% +/- 3.1%, and 4.2% +/- 2.9%, respectively (mean +/- SE of 20 different donors). PMN incubated with interleukin-1 beta (IL-1 beta), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and interferon-gamma (IFN-gamma), but not with prototypic chemoattractants (fMLP, recombinant C5a, and IL-8), showed a marked increase in survival, with values ranging at 72 hours of incubation from 89.5% +/- 5.8% for IL-1 beta to 47.6% +/- 6.4% for IFN- gamma. The calculated half-life was 35 hours for untreated and 115 hours for IL-1-treated PMN. PMN activated with lipopolysaccharide (LPS) or inactivated streptococci also showed a longer survival compared with untreated cells (94.4% +/- 3.2% and 95.5% +/- 2.4%, respectively, at 72 hours). PMN surviving in response to LPS or IL-1 beta retained the capacity to produce superoxide anion when treated with phorbol esters or fMLP. All inducers of PMN survival protect these cells from programmed cell death because they reduced cells with morphologic features of apoptosis and the fragmentation of DNA in multiples of 180 bp. Thus, certain cytokines and bacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial permissive step for certain cytokines and microbial products that activate gene expression and function in PMN.

Two receptors for the proinflammatory cytokine interleukin 1 (IL-1) have been cloned and characterized biochemically. While it has been well established that the type I (80-kDa) IL There are two known receptors for IL-1, which differ in both size and tissue distribution (2-4). The ligand-binding portions of the two receptors are similar, but whereas the cytoplasmic region of the type I (-80-kDa) receptor contains -215 amino acids, that of the type II (=60-kDa) receptor is only 29 amino acids long. It is well established that the type I receptor is capable of mediating biological responses (5, 6). In studies reported elsewhere, it has also been clearly shown that the type I and type II receptors are not subunits of a multimeric receptor complex but instead bind IL-1 independently of one another (7). This raises the possibility that each receptor might couple to different signal transduction pathways. In the studies reported here, however, we have been unable to find any evidence that the type II receptor signals at all.

Aluminum hydroxide (Alum) is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. In this study, we show that Alum activates caspase-1 and induce secretion of mature IL-1β and IL-18. Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists released only traces of IL-1β or IL-18, despite the fact that the IL-1β mRNA was readily induced by both TLR agonists. In contrast, cells costimulated with TLR agonists plus Alum released large amount of IL-1β and IL-18. Alum-induced IL-1β and IL-18 production was not due to enhancement of TLR signaling but rather reflected caspase-1 activation and in mouse dendritic cells occurred in a MyD88-independent fashion. Secretion of other proinflammatory cytokines such as IL-8 was not affected by Alum treatments. However, TLR-induced production of IL-10 was increased and that of IFN-γ-inducible protein decreased by Alum cotreatment. Considering the immunostimulatory activities of these cytokines and the ability of IL-1β to act as adjuvant, our results suggest a mechanism for the adjuvanticity of Alum.