Periodate oxidized ATP (oATP) reduces hyperalgesia in mice: involvement of P2X7 receptors and implications for therapy.

Some inflammatory mediators play an important role not only in the pathogenesis of the inflammatory pain, but also in that of neuropathic and visceral pain. We previously showed the antihyperalgesic effect of oATP, the inhibitor of the P2X7 receptors for the pro-nociceptive ATP, in experimental inflammation. Here we show the antihyperalgesic effect of oATP in mouse models of neuropathic and visceral pain, other than in a model of arthritic pain mimicking rheumatoid arthritis in humans. We also show that mice lacking P2X7 receptors (KO) are resistant to hyperalgesic thermal stimuli following the induction of arthritic, neuropathic and visceral pain. Local (injection into the right hind paw) pre-treatment with oATP is able to prevent the successive induction of ATP-dependent hyperalgesia in wild type mice. In addition, KO mice are not insensitive to intraplantar treatment with ATP. Our data suggest that, even if oATP is able to inhibit purinoceptors different from P2X7, the latter are the more important involved in pain transmission.

Identification and characterization of a novel class of interleukin-1 post-translational processing inhibitors.

Lipopolysaccharide (LPS)-activated monocytes and macrophages produce large quantities of pro-interleukin (IL)-1beta but externalize little mature cytokine. Efficient post-translational processing of the procytokine occurs in vitro when these cells encounter a secretion stimulus such as ATP, cytolytic T cells, or hypotonic stress. Each of these stimuli promotes rapid conversion of 31-kDa pro-IL-1beta to its mature 17-kDa species and release of the 17-kDa cytokine. In this study, two novel pharmacological agents, CP-424,174 and CP-412,245, are identified as potent inhibitors of stimulus-coupled IL-1beta post-translational processing. These agents, both diarylsulfonylureas, block formation of mature IL-1beta without increasing the amount of procytokine that is released extracellularly, and they inhibit independently of the secretion stimulus used. Conditioned medium derived from LPS-activated/ATP-treated human monocytes maintained in the absence and presence of CP-424,174 contained comparable quantities of IL-6, tumor necrosis factor-alpha (TNFalpha), and IL-1RA, but 30-fold less IL-1beta was generated in the test agent's presence. As a result of this decrease, monocyte conditioned medium prepared in the presence of CP-424,174 demonstrated a greatly diminished capacity to promote an IL-1-dependent response (induction of serum amyloid A synthesis by Hep3B cells). Oral administration of CP-424,174 to mice resulted in inhibition of IL-1 in the absence of an effect on IL-6 and TNFalpha. These novel agents, therefore, act as selective cytokine release inhibitors and define a new therapeutic approach for controlling IL-1 production in inflammatory diseases.

LL-Z1271alpha: an interleukin-1beta production inhibitor.

LL-Z1271alpha, a fungal metabolite, dose-dependently inhibited interleukin-1beta (IL-1beta) production in lipopolysaccharide (LPS)-stimulated human whole blood. Oral administration of LL-Z1271alpha to LPS-challenged mice caused significant lowering in the IL-1beta levels in peritoneal cavity. Data presented suggest that LL-Z1271alpha inhibits IL-1beta production by a novel mechanism as the inhibitory activity was not due to effects on caspase-1 (IL-1beta converting enzyme), the ATP-induced release mechanism or a lysosomotrophic effect.

Altered cytokine production in mice lacking P2X(7) receptors.

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 beta 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 beta comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1 beta 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 beta 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 ATP-treated, 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 beta and initiation of a cytokine cascade.

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, 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-1beta, 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 P2X(7) receptor involvement. Quantities of IL-1beta 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-alpha 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-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

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-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). 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.

Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice null for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

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 stimulus-coupled IL-1beta posttranslational processing: modulation via monovalent cations.

Lipopolysaccharide-activated human monocytes produce prointerleukin (pro-IL)-1beta but release little of this inflammatory cytokine as the biologically active species. Efficient externalization of mature 17-kDa cytokine requires that the activated monocytes encounter a secondary stimulus such as ATP. To identify cation requirements of the ATP-induced process, lipopolysaccharide-activated monocytes were treated with ATP in media containing different Cl- salts or sucrose. Media devoid of Na+ did not support IL-1beta processing. Titration of NaCl into choline chloride- or sucrose-based media restored 17-kDa IL-1beta production. Na+ replacement, however, was not sufficient to support ATP-induced production of 17-kDa IL-1beta in the presence of >/=37 mM extracellular K+ or Li+. Inhibition by K+ suggests that efflux of this cation is a necessary component of the stimulus-coupled response. The inhibitory effect achieved by Na+ depletion is not due to inactivation of the ATP receptor and is distinct from a caspase-1 inhibitor. Stimulus-coupled IL-1beta posttranslational processing, therefore, requires extracellular Na+ for a step downstream of the initiating stimulus but preceding caspase-1 activation.

Post-translational processing of murine IL-1: evidence that ATP-induced release of IL-1 alpha and IL-1 beta occurs via a similar mechanism.

In response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1 beta can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1 beta initiates within 7.5 min of ATP addition; 17-kDa mature IL-1 beta is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1 alpha. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1 alpha, IL-1 beta, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1 beta exceeds that of IL-1 alpha in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) block ATP-induced proteolysis of pro-IL-1 beta and prevent release of pro-IL-1 alpha/beta and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1 beta-converting enzyme inhibitor blocks proteolysis of IL-1 beta, but it does not prevent release of pro-IL-1 alpha, pro-IL-1 beta, or LDH. These results indicate that ATP stimulates externalization of both IL-1 alpha and IL-1 beta. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.

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.

Tenidap and other anion transport inhibitors disrupt cytolytic T lymphocyte-mediated IL-1 beta post-translational processing.

LPS-activated murine peritoneal macrophages produce IL-1 beta but externalize little mature cytokine in the absence of a secondary stimulus, and CTLs previously were reported to serve this capacity. The release of 17-kDa IL-1 beta from LPS-activated BALB/c macrophages occurred rapidly after the addition of C57/B1-derived allogeneic CTLs; within 30 min of coculture, mature IL-1 beta was observed in the medium, and maximum release was achieved within 4 h. CTL-induced post-translational processing was efficient, and >80% of newly synthesized pro-IL-1 beta was released into the medium as the 17-kDa species. Externalization of IL-1 beta required active recognition of the macrophage target by the CTL preparation; C57/B1 CTLs promoted the release of mature IL-1 beta from allogeneic BALB/c macrophages, but not from syngeneic C57/B1 macrophages. In contrast, extracellular ATP promoted mature IL-1 beta release from both macrophage populations. CTL-induced cytokine post-translational processing was blocked by anion transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, UK5099, and the anti-inflammatory agent tenidap. An analogue of tenidap, CP-100,829, was more effective as an inhibitor of both IL-1 beta post-translational processing and anion transport. In contrast, the close structural analogue CP-236,492 inhibited neither process. Tenidap's activity was reversible and was not mimicked by cyclooxygenase inhibitors or by cycloheximide. Therefore, tenidap disrupted CTL-induced IL-1 beta post-translational processing by a mechanism dependent on anion transport inhibition. Multiple stimuli are likely to operate in vivo to promote IL-1 beta post-translational processing, and anion transport inhibitors such as tenidap that suppress cytokine processing independently of the initiating stimulus thus represent attractive candidates as therapeutic regulators of IL-1 production.

Envelopment of varicella-zoster virus: targeting of viral glycoproteins to the trans-Golgi network.

Previous studies suggested that varicella-zoster virus derives its final envelope from the trans-Golgi network (TGN) and that envelope glycoproteins (gps) are transported to the TGN independently of nucleocapsids. We tested the hypothesis that gpI is targeted to the TGN as a result of a signal sequence or patch encoded in its cytosolic domain. cDNAs encoding gpI wild type (wt) and a truncated mutant gpI(trc) lacking transmembrane and cytosolic domains were cloned by using the PCR. Cells transfected with cDNA encoding gpI(wt) or gpI(trc) synthesized and N glycosylated the proteins. gpI(wt) accumulated in the TGN, some reached the plasmalemma, but none was secreted. In contrast, gpI(trc) was retained and probably degraded in the endoplasmic reticulum; none was found on cell surfaces, but some was secreted. The distribution of gpI(trc) was not affected by deletion of potential glycosylation sites. To locate a potential gpI-targeting sequence, cells were transfected with cDNA encoding chimeric proteins in which the ectodomain of a plasmalemmal marker, the interleukin-2 receptor (tac), was fused to different domains of gpI. A chimeric protein in which tac was fused with the transmembrane and cytoplasmic domains of gpI was targeted to the TGN. In contrast, a chimeric protein in which tac was fused only with the gpI transmembrane domain passed through the TGN and concentrated in endosomes. We conclude that gpI is targeted to the TGN as a result of a targeting sequence or patch in its cytosolic domain.

Reduction of intracellular pH by tenidap. Involvement of cellular anion transporters in the pH change.

Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H- indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na+/H+ antiporter. Fibroblasts incubated with [14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-N-[2-(3,4-dimethoxyphe nyl) ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including alpha-cyano-beta-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [14C]tenidap. In contrast, ethacrynic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not impair the efflux process. Likewise, tenidap analogs that produced a sustained intracellular acidification blocked the efflux of [14C]tenidap, but non-acidifying species did not. These data suggest that movements of tenidap into and/or out of cells is a facilitated process subject to pharmacological intervention. Together, the structural selectivity of the acidification response and the evidence of facilitated transport suggest that the pHi modulating activity of tenidap is dependent on its unique physicochemical properties. Due to the dependence of these physicochemical properties on environmental and cellular conditions, in vivo expression of the acidification activity is likely to occur only within restricted environments that favor this tenidap-induced process.

Inhibition of interleukin-1 beta production by SKF86002: evidence of two sites of in vitro activity and of a time and system dependence.

Cytokine-suppressing anti-inflammatory drugs (CSAIDs) are reported to inhibit production of proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) by affecting a stress-induced kinase. To gain a better understanding of the selectivity and cellular dynamics of this type of inhibitor, we studied in vitro the prototype member of this class of agents, SKF86002. Lipopolysaccharide (LPS)-activated human monocytes treated with SKF86002 produced less proIL-1 beta but normal amounts of the noncytokine lysozyme. Two-dimensional gel analysis indicated that only eight polypeptides produced by monocytes were decreased by SKF86002. Inhibition of IL-1 beta production was achieved by affecting two separate steps in this cytokine's biogenesis. First, SKF86002 lowered proIL-1 beta synthesis. By pulse-chase analysis, this effect was localized to a posttranscriptional site of action; maximal inhibition was observed when SKF86002 was added at the time of cytokine translation. Exposure of monocytes to SKF86002 for > 2 hr led to a loss of IL-1 beta inhibitory activity, suggesting that these cells adapted to this agent. Moreover, LPS-activated monocytes that were pretreated with granulocyte-macrophage colony-stimulating factor were less sensitive to the proIL-1 beta inhibitory effect of SKF86002, and production of proIL-1 beta by cytokine-stimulated human fibroblasts was impaired only modestly by the CSAID. A second effect of SKF86002 was to inhibit release of IL-1 beta into the medium in response to high concentrations of LPS; this effect is observed only with freshly isolated human monocytes as other IL-1 beta-producing cells do not release significant cytokine in response to LPS. The ability of SKF86002 to inhibit this posttranslational mechanism was mimicked by lysosomotrophic agents such as chloroquine, quinacrine, and methylamine. In contrast, chloroquine, and quinacrine were not effective inhibitors of monocyte proIL-1 beta translation. Thus, SKF86002 inhibits IL-1 beta production by affecting at least two distinct steps in the biosynthesis of this cytokine. Manifestation of these two effects, however, is dependent on the length of time for which cells are exposed to this agent and the nature of the cytokine-producing cellular system.

Bafilomycin A1 inhibits IL-1-stimulated proteoglycan degradation by chondrocytes without affecting stromelysin synthesis.

Interleukin-1 alpha (IL-1) stimulated the release of degraded proteoglycan from primary cultures of chondrocyte monolayers in a time- and dose-dependent fashion. Bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase, efficiently blocked acidification of the chondrocyte vacuolar system. Under these conditions IL-1-stimulated proteoglycan degradation was inhibited by bafilomycin A1 with an IC50 of < 10 nM in both chondrocyte monolayers and articular cartilage explants. This concentration was at least 100-fold less than that required to partially inhibit total protein synthesis. In chondrocyte monolayers, bafilomycin A1 could be added several hours after IL-1 and complete inhibition was still observed. Tumor necrosis factor-alpha and retinoic acid also stimulated proteoglycan degradation in chondrocyte monolayers, and in both cases the response was inhibited by bafilomycin A1. These results suggest that maintenance of vacuolar acidity is required for cytokine stimulated proteoglycan degradation and that this requirement is at a point distal to receptor binding and early signal transduction events. IL-1 also stimulated the synthesis and secretion of prostromelysin by chondrocyte monolayers, however, under conditions in which IL-1 stimulated proteoglycan release was totally blocked by bafilomycin A1, there was no effect on IL-1-stimulated stromelysin secretion or stromelysin enzyme activity. These results, in which stromelysin synthesis and proteoglycan degradation were dissociated, suggest that an additional enzyme is responsible for proteoglycan degradation in this chondrocyte monolayer system.

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.

Synovial fluid from rheumatoid arthritis patients contains sufficient levels of IL-1 beta and IL-6 to promote production of serum amyloid A by Hep3B cells.

The presence of positive acute phase proteins within the circulation of rheumatoid arthritis patients suggests that elevated cytokine production associated with this chronic inflammatory disorder initiates the hepatic acute phase response. Cytokines produced at inflammatory lesions are believed to travel via the circulation to the liver where they induce acute phase protein production by hepatocytes. To test whether serum from rheumatoid arthritis patients contained sufficient levels of cytokines to promote an acute phase response in vitro, a bioassay was developed that employed the human hepatoma cell line Hep3B. These cells produced the acute phase protein serum amyloid A (SAA) in response to a combination of recombinant IL-1 beta and IL-6 or to monocyte conditioned medium. Serum (or plasma) from normal individuals or from rheumatoid arthritis patients did not induce SAA production by Hep3B cells. Moreover, these serum samples did not prevent SAA production induced by monocyte conditioned medium, indicating that they did not contain inhibitors of cytokine activity. Despite the inactivity of serum samples, synovial fluid samples obtained from rheumatoid arthritis patients were active in the hepatocyte bioassay and promoted SAA synthesis. One synovial fluid sample was analysed in detail to identify cytokines responsible for the SAA-inducing activity. Neutralizing antisera against IL-6 and IL-1 beta blocked this activity by > 90% whereas anti-IL1 alpha and anti-TNF-alpha sera were without effect. Absolute cytokine levels within the synovial fluid sample were determined by ELISA; IL-6, IL-beta and TNF-alpha, but not IL-1 alpha, were confirmed to be present. Moreover, the synovial fluid sample contained a large amount of the IL-1 receptor antagonist. These data indicate, therefore, that synovial fluid recovered from an inflamed joint contains all the necessary cytokines in balance with inhibitors to promote SAA production by Hep3B cells. The steady state levels of these factors within the plasma compartment, however, were insufficient to induce the acute phase response by cultured Hep3B cells, suggesting that this system does not mimic the relationship between the circulation and the liver that likely exists in rheumatoid arthritis patients.

Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network.

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.