C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.

OBJECTIVE: To achieve disease-inducible expression of recombinant antiinflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. METHODS: We compared the inducible 2-component expression system (C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV]) with the constitutive cytomegalovirus (CMV) promoter in the polyarticular collagen-induced arthritis (CIA) model in mice. DBA/1 mice were immunized with bovine type II collagen and were given boosters on day 21. On day 22, mice were injected intraarticularly with the adenoviral vectors AdCMVLuc, AdCMVhIL-1Ra, AdC3-Tat/HIV-Luc, or AdC3-Tat/HIV-hIL-1Ra. The injected knee joints and hind paws were then scored for signs of arthritis, and knee joint histology was compared. RESULTS: The CMV-driven interleukin-1 receptor antagonist (IL-1Ra) expression resulted in a high constitutive expression and amelioration of CIA. C3-Tat/HIV-driven IL-1Ra expression could be detected only on days 24, 29, and 35. Fourteen days after injection of the vectors, CIA was significantly better inhibited by the C3-Tat/HIV-driven IL-1Ra expression compared with the CMV-driven IL-1Ra expression. Moreover, prevention of CIA in the knee joints also prevented CIA in the untreated hind paws. CONCLUSION: Our data demonstrate for the first time the feasibility of an inducible expression system for local production of IL-1Ra for treatment of arthritis in the CIA model.

Endogenous regulation of a therapeutic transgene restores homeostasis in arthritic joints.

The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-alpha, IL-1 beta, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall-induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.

The crucial role of systemic responses in the innate (non-adaptive) host defense.

We suggest that successful defense against microbial invasion requires both local inflammation and systemic anti-inflammation. The key systemic responses involve the hypothalamic-pituitary-adrenocortical axis, the sympathetic-adrenomedullary axis, acute phase protein production, thermoregulation and alterations in leukocyte responsiveness to agonists such as bacterial endotoxin. These integrated responses raise blood and tissue concentrations of several anti-infective molecules, mobilize leukocytes into the circulation, and increase blood flow to injured or infected sites. They also neutralize cytokines, proteases and oxidants that enter the bloodstream from inflamed local sites and forestall endothelial activation in distant vessels. Together, these forces help concentrate activated phagocytes at injured or infected local sites while preventing potentially damaging inflammation in uninvolved tissues.

Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins.

CD14, a myeloid cell-surface receptor and soluble plasma protein, binds LPS and other microbial molecules and initiates the innate immune response to bacterial invasion. The blood concentration of soluble CD14 (sCD14) increases during the systemic response to infection. Although high sCD14 blood levels have correlated with increased risk of dying from severe sepsis, sCD14 can diminish cell responses to LPS. We show here that in human serum, sCD14 increases the rate at which cell-bound LPS is released from the monocyte surface and binds to plasma lipoproteins. This enhanced rate of LPS efflux is associated with a significant reduction in the ability of monocytes to produce cytokines in response to LPS. Serum from septic patients reduced the LPS-monocyte interaction by as much as tenfold, and depletion of sCD14 from the serum restored LPS-monocyte binding and release kinetics to near normal levels. In serum from septic patients, monocyte-bound LPS also moved more rapidly into lipoproteins, which completely neutralized the biologic activity of the LPS that bound to them. In human plasma, sCD14 thus diminishes monocyte responses to LPS by transferring cell-bound LPS to lipoproteins. Stress-related increases in plasma sCD14 levels may help prevent inflammatory responses within the blood.

In vitro evaluation of an enhanced human serum amyloid A (SAA2) promoter-regulated soluble TNF receptor fusion protein for anti-inflammatory gene therapy.

Tumour necrosis factor (TNF)-alpha contributes to the pathogenesis of many inflammatory diseases. Recombinant soluble TNF receptor fusion proteins (sTNFR:Ig) are potent TNF antagonists, both in vitro and in vivo. The concentration of serum amyloid A (SAA) increases by up to 1000-fold during inflammation, largely owing to cytokine-driven transcriptional upregulation. A reporter plasmid, comprising the proximal 0.7 kb of the human SAA2 promoter fused to a luciferase gene, was used in transient transfection experiments in human HepG2 hepatoma cells to assess the quantitative and qualitative TNF antagonist properties of a construct in which sTNFR:Ig synthesis is under the control of a chimera of the SAA2 promoter and a tat/HIV element. The SAA2-tat/HIV-sTNFR:Ig construct retained the fine-tuned cytokine responsiveness of the SAA2 promoter, while exhibiting the quantitatively enhanced level of protein expression conferred by the tat/HIV element. It produced a biologically significant TNF inhibition that was at least as strong as that achieved using a CMV promoter-driven sTNFR:Ig construct. There was a dose- and time-dependent relationship between the pro-inflammatory cytokine used, and the generation of TNF antagonist activity by SAA2-tat/HIV-sTNFR:Ig. Although sTNFR:Ig protein can be induced by either TNF-alpha or interleukin (IL)-1beta, its antagonist activity is limited to the former cytokine. The SAA2-tat/HIV-sTNFR:Ig construct, and derivatives thereof, may therefore be ideally suited to gene therapy applications that require the local production of potent and specific immune modifiers only when there is active pathology. It may consequently be of particular use in the future treatment of diseases such as rheumatoid arthritis.

Gene therapy to prevent organophosphate intoxication.

The specific hydrolytic activity of PON1 paraoxonase/arylesterase enzymes in liver and blood provides a natural barrier against the entry of organophosphate toxins into the central and peripheral nervous systems. Inherited differences in PON1 enzyme concentrations may determine levels of susceptibility to organophosphate injury in humans. To test whether boosting serum levels of PON1 enzymes by gene therapy might provide increased protection, we compared the degree of inactivation of whole brain acetylcholinesterase of mice exposed to chlorpyrifos 4 days after intravenous injection of recombinant adenoviruses containing PON1-LQ or PON1-LR genes or no PON1 gene. Both recombinant viruses containing PON1 genes boosted serum arylesterase concentrations by approximately 60% and significantly prevented the inactivation of brain acetylcholinesterase. Some mice were completely protected. These findings indicate that boosting serum levels of PON1 enzymes by a gene delivery vector raises the threshold for organophosphate toxicity by hydrolytic destruction before the chemical can enter the brain.

Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes.

Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using native Salmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained (3)H-labeled lipopolysaccharide (LPS) and (35)S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [(3)H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.

Plasma constituents regulate LPS binding to, and release from, the monocyte cell surface.

Innate immunity to Gram-negative bacteria involves regulated mechanisms that allow sensitive but limited responses to LPS. Two important pathways that lead to host cell activation and LPS deactivation involve: (i) LPS interactions with CD14 and Toll-like receptor 4 on cells (activation); and (ii) LPS sequestration by plasma lipoproteins (deactivation). Whereas these pathways were previously thought to be independent and essentially irreversible, we found that they are connected by a third pathway: (iii) the movement of LPS from host cells to plasma lipoproteins. Our data show that, in the presence of human plasma, LPS binds transiently to monocyte surfaces and then moves from the cell surface to plasma lipoproteins. Soluble CD14 enhances LPS release from cells in the presence of lipoproteins, whereas LPS binding protein and phospholipid transfer protein do not. The transfer of cell-bound LPS to lipoproteins is accompanied by reduced cell responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo. Preliminary data suggest that changes that occur in the plasma after trauma or during sepsis decrease LPS binding to leukocytes while greatly increasing the rate of LPS release from cells.

Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate.

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.

Plasma lipoproteins promote the release of bacterial lipopolysaccharide from the monocyte cell surface.

When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.

CD14-dependent internalization and metabolism of extracellular phosphatidylinositol by monocytes.

We report that membrane CD14 (mCD14), a cell surface receptor found principally on leukocytes, can mediate the uptake and metabolism of extracellular phosphatidylinositol (PtdIns). mCD14 facilitates PtdIns internalization, targeting it to intracellular sites where, following stimulation with a calcium ionophore, it can be acted upon by cytosolic phospholipase A(2). The [(14)C]arachidonate released from mCD14-acquired [(14)C]arachidonyl-PtdIns is either esterified to triacylglycerol and retained in the cell or secreted as free arachidonate or leukotrienes. Although less than 10% of the arachidonate-derived lipids secreted from endogenous cellular stores are 5-lipoxygenase metabolites, over one-half of the secreted (14)C-lipids derived from mCD14-acquired PtdIns are hydroxyeicosatetraenoic acids or leukotriene B(4). mCD14 may allow these highly active blood cells to acquire and use extracellular PtdIns as a source of arachidonate for leukotriene synthesis.

Deacylation of purified lipopolysaccharides by cellular and extracellular components of a sterile rabbit peritoneal inflammatory exudate.

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14(+) MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites.

Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways.

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

Physiologically responsive gene therapy.

The goal of physiologically responsive gene therapy is to allow a host's endogenous regulatory mechanisms to control the production of therapeutic proteins (effectors). Ideally, effector production would be switched on in response to specific signals, stay within therapeutic limits and be switched off when no longer needed. In this way, the unwanted consequences of constitutive, high-level effector expression could be avoided. While recent studies have shown that transgenes can be regulated within animal hosts, they have also highlighted significant problems that require much further research.

Phosphatidylinositides bind to plasma membrane CD14 and can prevent monocyte activation by bacterial lipopolysaccharide.

Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo.

CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS.

We analyzed the impact of ligand aggregation and LPS-induced signaling on CD14-dependent LPS internalization kinetics in human monocytic THP-1 cells and murine macrophages. Using two independent methods, we found that the initial rate and extent of LPS internalization increased with LPS aggregate size. In the presence of LPS binding protein (LBP), large LPS aggregates were internalized extremely rapidly (70% of the cell-associated LPS was internalized in 1 min). Smaller LPS aggregates were internalized more slowly than the larger aggregates, and LPS monomers, complexed with soluble CD14 in the absence of LBP, were internalized very slowly after binding to membrane CD14 (5% of the cell-associated LPS was internalized in 1 min). In contrast, the initial aggregation state had little or no effect on the stimulatory potency of the LPS. Previous studies suggest that LPS-induced signal responses may influence the intracellular traffic and processing of LPS. We found that elicited peritoneal macrophages from LPS-responsive (C3H/HeN) and LPS-hyporesponsive (C3H/HeJ) mice internalized LPS with similar kinetics. In addition, pre-exposure of THP-1 cells to LPS had no effect on their ability to internalize subsequently added LPS, and pre-exposure of the cells to the LPS-specific inhibitor, LA-14-PP, inhibited stimulation of the cells without inhibiting LPS internalization. In these cells, LPS is thus internalized by a constitutive cellular mechanism(s) with kinetics that depend importantly upon the physical state in which the LPS is presented to the cell.

A two-component expression system that responds to inflammatory stimuli in vivo.

A therapeutic dilemma often complicates the management of inflammatory diseases; the benefits gained from reducing inflammation must be balanced against the potentially harmful consequences of chronic immunosuppression. Gene therapy might address this dilemma by producing anti-inflammatory proteins in response to a patient's endogenous signals, so that recombinant drug production is linked to the intensity and duration of the inflammatory condition. To test this, we have developed inflammation-inducible systems for regulating recombinant protein production in vivo. We describe a two-component expression construct in which (1) the murine complement factor 3 (C3) promoter regulates production of the human immunodeficiency virus (HIV) transactivator of transcription (Tat), and (2) the Tat protein then stimulates protein expression from genes inserted downstream of the the HIV promoter. When incorporated into a nonreplicating adenovirus (Ad.C3-tat/HIV-luc) and studied in a murine model, the construct produces large amounts of recombinant protein in vivo in response to two different inflammatory stimuli.