Abstract

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity. Antioxid. Redox Signal. 15, 2209–2219.

High-mobility group box 1 protein (HMGB1) is a prototypic alarmin that is released from activated and dying cells. Because of its proinflammatory activities, HMGB1 could mediate key events in the pathogenesis of systemic lupus erythematosus, a possibility supported by elevations of HMGB1 in patient blood and increased expression in renal biopsies. The biology of HMGB1 is complicated, however, and its activity is dependent on redox state as well as binding to other molecules such as cytokines. Defining more precisely the role of HMGB1 in lupus will require treatment studies to block the activity of this alarmin in animal models and ultimately patients.

Toll-like receptors (TLRs) are key receptors in innate immunity and trigger responses following interaction with pathogen-associated molecular patterns (PAMPs). TLR3, TLR4 and TLR9 recognize double stranded RNA, lipopolysaccharide (LPS) and CpG DNA, respectively. These receptors differ importantly in downstream adaptor molecules. TLR4 signals through MyD88 and TRIF; in contrast, the TLR3 pathway involves only TRIF while TLR9 signals solely through MyD88. To determine how differences in downstream signaling could influence gene expression in innate immunity, gene expression patterns were determined for the RAW264.7 macrophage cell line stimulated with LPS, poly (I:C), or CpG DNA. Gene expression profiles 6 and 24 hrs post-stimulation were analyzed to determine genes, pathways and transcriptional networks induced. As these experiments showed, the number and extent of genes expressed varied with stimulus. LPS and poly (I:C) induced an abundant array of genes in RAW264.7 cells at 6 hrs and 24 hrs following treatment while CpG DNA induced many fewer. By analyzing data for networks and pathways, we prioritized differentially expressed genes with respect to those common to the three TLR ligands as well as those shared by LPS and poly (I:C) but not CpG DNA. The importance of changes in gene expression was demonstrated by experiments indicating that RNA interference-mediated inhibition of two genes identified in this analysis, PLEC1 and TPST1, reduced IL-6 production by J774A.1 and RAW264.7 macrophages stimulated with LPS. Together, these findings delineate macrophage gene response patterns induced by different PAMPs and identify new genes that have not previously been implicated in innate immunity.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

Microparticles (MPs) are small membrane-bound vesicles that display proinflammatory and prothrombotic properties. These particles can be released by macrophages stimulated by ligands of the Toll-like receptors (TLRs) in a process that depends on nitric oxide (NO) production. Since sex hormones can modulate macrophage responses, we investigated the effects of progesterone and estradiol on macrophage particle release in vitro, comparing the responses with those induced by the glucocorticoid dexamethasone. As a model system for particle release, RAW 264.7 cells were stimulated in vitro with poly(I:C), a ligand of TLR3. Microparticles were measured by flow cytometry, while NO was measured by the Griess reaction. As the results of these studies showed, progesterone but not estradiol can block particle release by RAW264.7 cells treated with poly(I:C); dexamethasone was also active. Furthermore, while progesterone and dexamethasone inhibited NO production under the same culture conditions, neither agent blocked the production of particles stimulated by the NO donors dipropylenetriamine NONOate {(z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate} and (z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate. Studies using RU486 to assess the role of hormone receptors indicated that while this agent blocked the inhibition of particle and NO production by dexamethasone, it did not affect the inhibition by progesterone. Together, these results indicate that progesterone but not estradiol can inhibit particle release by stimulated macrophages and suggest a mechanism that may contribute to the immunomodulatory effects of this sex hormone.

Systemic lupus erythematosus (SLE) is prototypic autoimmune disease characterized by the production of autoantibodies to DNA among other nuclear molecules. These antibodies can form immune complexes that promote pathogenesis by stimulating cytokine production and depositing in the kidney to instigate nephritis. The antigens that form these complexes arise from the blood nucleome, a pool of circulating macromolecules comprised of DNA, RNA and nuclear proteins released from cells. Cell death is a major source of these molecules, releasing DNA in a process that can be modeled in mice by the administration of cells killed ex vivo. In the mouse model, the appearance of blood DNA requires macrophages and differs between males and females. This finding raises the possibility that augmented levels of extracellular DNA and other nuclear antigens can contribute to the increased frequency of SLE in females. Extracellular DNA can occur in both a soluble and particulate form, with microparticles generated in vitro displaying antigenically active DNA. Together, these findings suggest that cell death is an important event in lupus pathogenesis and can provide a supply of blood DNA essential for immune complex formation.

Antinuclear antibodies (ANAs) are venerable biomarkers for assessing the diagnosis and prognosis of patients with autoimmunity. While closely associated with diseases such as systemic lupus erythematosus, ANA expression occurs commonly in healthy people. The basis for this expression is unknown, although it may reflect features of the assays for antibody detection or intrinsic immunological disturbances in otherwise normal individuals. Like autoimmunity itself, ANA expression is more common among women than men, pointing to an important determinant of these responses. Future research will clarify the mechanisms of ANA expression and the utility of current assays as antecedent and screening biomarkers.

Microparticles (MPs) are small membrane-bound vesicles released from cells undergoing activation or cell death. These particles display potent biological activities that can impact on physiologic and pathologic processes. Previous studies with the Jurkat T leukemia cell line demonstrated that staurosporine (STS) induces the release of MPs as cells undergo apoptosis. To investigate further this process, we tested the effects of STS, its analogue, 7-hydroxystaurosporine (UCN-01), and other protein kinase C (PKC) and cyclin-dependent kinase (CDK) inhibitors. FACS analysis was used to assess MP release. Results of these studies indicate that STS and UCN-01 induce MP release by Jurkat cells; in contrast, other PKC and CDK inhibitors failed to induce comparable release, suggesting that release does not result from simple inhibition of either kinase alone. Time course experiments indicated that STS-induced particle release occurred as early as 2 h after treatment, with the early release MPs displaying low levels of binding of annexin V and propidium iodide (PI). Early-release MPs, however, matured in culture to an annexin V- and PI-positive phenotype. Together, these results indicate that STS and UCN-01 induce MPs that are phenotypically distinct and reflect specific patterns of kinase inhibition during apoptosis.

Microparticles (MPs) are small membrane-bound vesicles that are released from activated or dying cells by a blebbing process. These particles contain nuclear and cytoplasmic components and represent unique biomarkers for disease. The small size of particles, however, limits detection using flow cytometry with either light scatter or staining for surface markers. Because MPs contain DNA and RNA, we have explored the use of SYTO 13, a member of the class of SYTO dyes, for particle detection. SYTO 13 is cell permeable and has a high fluorescent yield when bound to DNA or RNA. In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of “noise” with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5 to 2.9 times as many particles as did light scatter. The increased sensitivity was observed with 3 different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content of MPs provides the basis for their detection in in vitro systems and suggests the utility of fluorescent dyes like SYTO 13 for more sensitive quantitative assays.

In preparation for randomised controlled trials (RCTs) of disease‐modifying antirheumatic drugs in patients with early inflammatory arthritis (EIA), formative research was conducted to enhance the design of such trials. The objectives of this research were to (1) determine patients' educational needs as they relate to the necessary elements of informed consent; and (2) assess patients' interest in enrolling in a hypothetical prevention trial. In‐depth interviews were conducted with nine patients. Seven patients were women and all but one white. The mean age was 48 years. During the 4‐month enrolment period, only three patients with EIA were identified; six patients with longer duration of symptoms were also interviewed. Most patients were able to express the primary aim of a hypothetical prevention trial presented. Factors cited by patients favouring enrolment were potential for direct medical benefit and knowledge that they would be withdrawn from the trial if they developed symptoms. Factors cited by patients against enrolment were the inclusion of a placebo and general uncertainty regarding treatment required by the RCT design. Pending larger‐scale empirical projects to explore patients' attitudes about prevention trials, small‐scale formative research in advance of such trials ought to be conducted.

Microparticles are small membrane-bound vesicles released from activated and dying cells. As shown in a study of primary Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis, levels of microparticles in the blood, as measured by a solid-phase prothrombinase assay or flow cytometry, are increased with autoimmunity. Among patients with these conditions, however, particle numbers were inversely related to disease activity and levels of the enzyme secretory phospholipase A2 that can digest membrane lipids and perhaps cause particle loss. These findings suggest microparticles as novel biomarkers for autoimmunity, with levels reflecting events leading to their loss as well as production.

Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.

The death of CD4+ CCR5+ T cells is a hallmark of human immunodeficiency virus (HIV) infection. We studied the plasma levels of cell death mediators and products—tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and plasma microparticles—during the earliest stages of infection following HIV type 1 (HIV-1) transmission in plasma samples from U.S. plasma donors. Significant plasma TRAIL level elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR-2, Fas ligand, and microparticle level elevations occurred concurrently with maximum VL. Microparticles had been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T-cell apoptotic microparticles also potently suppressed in vitro immunoglobulin G (IgG) and IgA antibody production by memory B cells. Thus, release of TRAIL during the onset of plasma viremia (i.e., the eclipse phase) in HIV-1 transmission may initiate or amplify early HIV-1-induced cell death. The window of opportunity for a HIV-1 vaccine is from the time of HIV-1 transmission until establishment of the latently infected CD4+ T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity during which a vaccine is able to extinguish HIV-1 infection and could place severe constraints on the amount of time available for the immune system to respond to the transmitted virus.

Systemic lupus erythematosus is a systemic autoimmune disease characterized by the production of antinuclear antibodies (ANAs). Recent research into human and murine lupus suggests that disease susceptibility results from genetic polymorphisms regulating immune responses as well as impairing the clearance of apoptotic cells. Because the products of dead cells, including nucleic acids, have immunologic activity, this situation can promote antigen-driven ANA responses. Furthermore, immune complexes of ANAs can drive the production of proinflammatory cytokines, inducing the 'interferon signature', and intensifying disease. Together, these findings point to new genetic and immunologic markers of disease as well as targets for new therapies.

Cell death is critical to normal homeostasis, although this process, when increased aberrantly, can lead to the production of pro-inflammatory mediators promoting autoimmunity. Two novel intercellular mediators of inflammation generated during cell death are high mobility group box 1 (HMGB1) protein and microparticles (MPs). HMGB1 is a nuclear protein that functions in transcription when inside the nucleus but takes on pro-inflammatory properties when released during cell death. Microparticles are small, membrane-bound structures that extrude from cells when they die and contain cell surface proteins and nuclear material from their parent cells. MPs circulate widely throughout the vasculature and mediate long-distance communication between cells. Both MPs and HMGB1 have been implicated in the pathogenesis of a broad spectrum of inflammatory diseases, including the prototypic autoimmune conditions systemic lupus erythematosus and rheumatoid arthritis. Given their range of activity and association with active disease, both structures may prove to be targets for effective therapy in these and other disorders.

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. To function as an alarmin, HMGB1 translocates from the nucleus of the cell to the extra-cellular milieu, a process that can take place with cell activation as well as cell death. HMGB1 can interact with receptors that include RAGE (receptor for advanced glycation endproducts) as well as Toll-like receptor-2 (TLR-2) and TLR-4 and function in a synergistic fashion with other proinflammatory mediators to induce responses. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. New approaches to therapy for these diseases may involve strategies to inhibit HMGB1 release from cells, its interaction with receptors, and downstream signaling.

Nitric oxide (NO) may play important roles in rheumatoid arthritis (RA). RA is an inflammatory disease involving joints and other systems including salivary glands. To assess NO production in RA patients, we compared levels of serum, urine, and salivary nitrite and nitrate (NOx) in patients with RA and normal subjects, and we examined the relationships of these measures to disease activity. Serum, urine, and NOx levels as well as renal creatinine, NOx clearance and fractional excretion rates were compared in 25 RA patients and 20 age- and gender-matched healthy controls. Subjects were hospitalized for 3 days and placed on a NOxrestricted diet. NOx was assayed using nitrate reductase and the Griess reagent. RA activity was assessed using standard clinical and laboratory measures. While consuming a restricted diet for 3 days to eliminate the effects of oral intake of NOx, 24 hour urinary NOx excretion decreased in both RA patients and healthy controls. Urine NOx levels at all time points were not significantly different between RA patients and normal subjects. Serum NOx levels also decreased during the 3 days of NOx restriction, but RA patients had higher serum NOx levels at all time points compared with the control group. Likewise, serum NOx/creatinine ratios were higher in RA patients than in controls. Although basal salivary flow rate and tear flow were lower in RA patients, salivary NOx levels did not differ between normal and RA subjects. While renal creatinine clearance was not different between the two groups, we found that RA patients had lower renal NOx clearance and lower renal NOx fractional excretion. After correction of p values for multiple comparisons, there were no significant relationships for the RA group between measures of disease activity and the urinary NOx, serum NOx, or urinary NOx clearance. Despite interest in the use of NO as a marker of disease activity, alterations in renal NOx clearance and fractional excretion in RA make it difficult to assess in vivo NO production even with strict dietary restriction of NOx intake.

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4+ CD8+ thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4+ CD8+ thymocytes and in vitro evidence that thromboxane A2 (TXA2) causes apoptosis of these cells, we tested whether enhanced generation of TXA2 plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 μg of LPS intraperitoneally displayed a marked increase in generation of TXA2 and prostaglandin E2 in the thymus as well as apoptotic deletion of CD4+ CD8+ thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA2 mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.

Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E2 (PGE2) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 μg of CpG DNA/ml increased NO and PGE2 production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE2 production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE2 production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells converted l-arginine to l-citrulline, but the NOS inhibitor NG-monomethyl-l-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE2 production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, and N-iminoethyl-l-lysine effectively blocked NO production and increased the production of PGE2 in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE2 occurring in an interdependent manner.