ATP treatment of human monocytes promotes caspase-1 maturation and externalization.

Mechanisms that regulate conversion of prointerleukin-1beta (pro-IL-1beta) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1beta, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1beta secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1beta. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1beta and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. On the other hand, anion transport inhibitors and alkylating agents arrest ATP-treated monocytes in a state where membrane latency is maintained. The data provided support the hypothesis that stimulus-coupled IL-1 post-translational processing involves a commitment to cell death.

Human monocyte ATP-induced IL-1 beta posttranslational processing is a dynamic process dependent on in vitro growth conditions.

Despite a large production capacity, freshly isolated lipopolysaccharide (LPS)-activated human monocytes release only a small percentage of their newly synthesized interleukin (IL)-1 beta into the medium. Extracellular ATP, acting via surface P2z-type purino-receptors, increases cytokine posttranslational processing. To explore whether this ATP response was affected by culture conditions, monocytes were maintained for different time periods in the absence and presence of various media components including fetal bovine and human sera and recombinant human cytokines. The ability of monocytes to produce radiolabeled pro-IL-1 beta in response to LPS and to posttranslationally process the procytokine after ATP stimulation was affected both by time in culture and by the presence of specific media components. These observations indicate that ATP's ability to promote human monocyte IL-1 beta posttranslational processing is a dynamic process that is subject to regulation by cytokines and/or growth factors. Changes in monocyte/macrophage ATP responsiveness may provide an important regulatory mechanism for the control of IL-1 biological activity in vivo.

Human monocyte interleukin-1beta posttranslational processing. Evidence of a volume-regulated response.

Interleukin (IL)-1beta 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-1beta 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-1beta posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P2Z-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-1beta 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 NaNO3 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-1beta posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1beta 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.

Inhibition of cytokine activation processes in vitro by tenidap, a novel anti-inflammatory agent.

Tenidap is a novel anti-inflammatory and anti-arthritic agent that lowers intracellular pH and suppresses anion transport when applied to cells in vitro. Both of these parameters are known to influence pro-inflammatory cell function. To investigate whether tenidap can modulate cellular responses to cytokine stimulation, several in vitro cytokine-driven assays were characterized with respect to their tenidap sensitivity. Human monocytes treated with granulocyte-macrophage colony stimulating factor (GM-CSF) demonstrated an increased production of IL-6 as well as an increased total translational activity. Tenidap dose-dependently inhibited both cytokine-induced responses; the effect on IL-6, however, occurred at lower tenidap concentrations than those required to prevent the increase in total translational activity. In contrast, the known translational inhibitor cycloheximide did not demonstrate selectivity for IL-6; this agent decreased the GM-CSF-induced increase in total translational activity in parallel with its effects on IL-6. GM-CSF-treated monocytes also produced greater amounts of IL-1 beta in response to LPS stimulation than did non-GM-CSF-treated cells, and tenidap again suppressed this cytokine-induced activation. Human Hep3B cells treated with a combination of interleukin (IL)-1 beta and IL-6 demonstrated an acute phase-type of response. These hepatoma cells increased production of the positive acute phase protein serum amyloid A (SAA) while they decreased production of a negative acute phase protein human serum albumin (HSA). Tenidap dose-dependently inhibited the cytokine-induced increase in SAA production without effecting synthesis of HSA or total TCA-precipitable macromolecules. Importantly, the ability of tenidap to alter these various cytokine responses was not shared with piroxicam, a potent cyclooxygenase inhibitor. Finally, human neutrophils treated with either GM-CSF or tumor necrosis factor (TNF)-alpha demonstrated an increased chloride conductance as measured by the loss of radioactive chloride from 36Cl-loaded cells. When tenidap was included within the medium during cytokine stimulation, loss of radioactive chloride was prevented. Thus, tenidap inhibited the cytokine-induced increase in anion transport. Together, these results indicate that tenidap can suppress cellular activation processes induced by a variety of cytokines. This functional antagonism is not dependent on cyclooxygenase inhibition but, rather, appears to link to tenidap's unique ability to alter ionic homeostasis. These in vitro observations, therefore, may help to explain how this novel anti-inflammatory agent acts to lower acute phase proteins and IL-6 levels in man.