The P2X 7 receptor (P2X 7 R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1␤ post-translational processing, we have generated a P2X 7 R-deficient mouse line. P2X 7 R ؊/؊ macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1␤ comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1␤ produced by the P2X 7 R ؊/؊ cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1␤ from P2X 7 R ؊/؊ macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X 7 R ؊/؊ animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATPtreated, LPS-primed P2X 7 R ؊/؊ animals. Absence of the P2X 7 R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency, in vivo cytokine signaling cascades are impaired in P2X 7 R-deficient animals. Together these results demonstrate that P2X 7 R activation can provide a signal that leads to maturation and release of IL-1␤ and initiation of a cytokine cascade.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-Å resolution and has a characteristic GST fold (Protein Data Bank entry code 1eem). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1β, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1β, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X7 receptor involvement. Quantities of IL-1β generated by an individual donor’s blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-α levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1β and IL-18, and suggest that circulating blood monocytes constitutively express a P2X7-like receptor.

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Interleukin (IL)-1␤ produced by monocytes and macrophages is not released via the normal secretory apparatus, and prior to its release, this cytokine must be proteolytically processed to generate a mature biologically active species. Biochemical mechanisms that regulate these posttranslational steps are not well understood. Lipopolysaccharide (LPS) is a poor activator of IL-1 posttranslational processing despite serving as a potent inducer of IL-1 synthesis. For example, freshly isolated human monocytes treated with LPS released <30% of their newly synthesized IL-1␤ as the mature 17-kDa cytokine species, and monocytes that were aged overnight in culture prior to LPS treatment released no 17-kDa cytokine. In contrast, addition of extracellular ATP promoted IL-1␤ posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P 2Z -type receptors, promoted major intracellular ionic changes. To explore whether these ionic changes were required for cytokine posttranslational processing, LPS-stimulated human monocytes were maintained in ionically altered media. Hypotonic conditions promoted an efficient and selective release of mature 17-kDa IL-1␤ from LPS-activated monocytes in the absence of ATP. In contrast, hypertonic conditions blocked the ATP-induced posttranslational processing reactions. Both hypotonic stress-and ATP-induced processing were blocked when NaI was substituted for NaCl within the medium; substitution with NaSCN or NaNO 3 also blocked the ATP response, but these salts were less inhibitory against the hypotonic stimulus. Sodium glucuronate substitution did not inhibit cytokine processing induced by either stimulus. Removal of divalent cations from the medium did not affect the ATP response, but pretreatment of monocytes with the phosphatase inhibitor okadaic acid dose-dependently suppressed ATP-induced IL-1␤ posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1␤ posttranslational processing is initiated. The strong dependence of this cytokine release mechanism on chloride anions suggests that selective anion transporters function as important components of this response.