Search

The innate immune system utilizes many approaches for defense against invading microorganisms, including complement-mediated lysis, engulfment, formation of neutrophil extracellular traps, and release of antimicrobial peptides. Although classically thought to be driven by adaptive immunity, the development of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus is increasingly associated with dysregulated innate immune pathways. An emerging theme within this literature is the contribution of antimicrobial peptides to the development of autoimmune disorders. This is best exemplified in atopic dermatitis and psoriasis where the defensins and the single human cathelicidin, LL-37, may contribute to disease. Furthermore, in the past few years, a role for LL-37 has emerged in the pathogenesis of systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis, and possibly other diseases. In this review, we discuss the role of LL-37 and its murine ortholog, mCRAMP, in the modulation of immune and inflammatory pathways and their effects on autoimmune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Spectacular images of neutrophils ejecting nuclear chromatin and bactericidal proteins, in response to microbes, were first reported in 2004. As externalized chromatin could entangle bacteria, these structures were named neutrophil extracellular traps (NETs). Subsequent studies identified microorganisms and sterile conditions that stimulate NETs, as well as additional cell types that release extracellular chromatin. The release of NETs is the most dramatic stage in a cell death process called NETosis. Experimental evidence suggests that NETs participate in pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. Exaggerated NETosis or diminished NET clearance likely increases risk of auto-reactivity to NET components. The biological significance of NETs is just beginning to be explored. A more complete integration of NETosis within immunology and pathophysiology will require better understanding of NET properties associated with specific disease states and microbial infections. This may lead to the identification of important therapeutic targets. [ABSTRACT FROM AUTHOR]

A response by Marian J. Kaplan and J. Michelle Kahlenberg to a letter to the editor about an article "Inflammasome Activation of IL-18 Results in Endothelial Progenitor Cell Dysfunction in Systemic Lupus Erythematosus" published in this issue of the journal is presented.

Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes. Neutrophils undergoing NETosis induced by various physiological stimuli showed distinct changes, with a loss of multilobulated nuclei, as well as nuclear decondensation followed by membrane compromise, and were accurately counted by applying filters based on fluorescence intensity and nuclear size. Findings were confirmed and validated with the established method of immunofluorescence microscopy. The platform was also validated to rapidly assess and quantify the dose-dependent effect of inhibitors of NETosis. In addition, this method was able to distinguish among neutrophils undergoing NETosis, apoptosis, or necrosis based on distinct changes in nuclear morphology and membrane integrity. The IncuCyte ZOOM platform is a novel real-time assay that quantifies NETosis in a rapid, automated, and reproducible way, significantly optimizing the study of neutrophils. This platform is a powerful tool to assess neutrophil physiology and NETosis, as well as to swiftly develop and test novel neutrophil targets. [ABSTRACT FROM AUTHOR]

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women. [ABSTRACT FROM AUTHOR]

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA. [ABSTRACT FROM AUTHOR]

Inappropriate activation of type I IFN plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). In this study, we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM-resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre-B cells, suggesting an inhibition in early B cell development and an expansion of B cells at the transitional stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN-high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice, similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type-I IFNR. BM neutrophils were abundant, responsive to, and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil-mediated IFN activation and alterations in B cell ontogeny and selection. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-ip and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous manifestations including severe organ damage and vascular dysfunction leading to premature atherosclerosis. IFN-α has been proposed to have an important role in the development of lupus and lupus-related cardiovascular disease, partly by repression of IL-1 pathways leading to impairments in vascular repair induced by endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs). Counterintuitively, SLE patients also display transcriptional upregulation of the IL-1β/IL-18 processing machinery, the inflammasome. To understand this dichotomy and its impact on SLE-related cardiovascular disease, we examined cultures of human and murine control or lupus EPC/CACs to determine the role of the inflammasome in endothelial differentiation. We show that caspase-1 inhibition improves dysfunctional SLE EPC/CAC differentiation into mature endothelial cells and blocks IFN-α-mediated repression of this differentiation, implicating inflammasome activation as a crucial downstream pathway leading to aberrant vasculogenesis. Furthermore, serum IL-18 levels are elevated in SLE and correlate with EPC/CAC dysfunction. Exogenous IL-18 inhibits endothelial differentiation in control EPC/CACs and neutralization of IL-18 in SLE EPC/CAC cultures restores their capacity to differentiate into mature endothelial cells, supporting a deleterious effect of IL-18 on vascular repair in vivo. Upregulation of the inflammasome machinery was operational in vivo, as evidenced by gene array analysis of lupus nephritis biopsies. Thus, the effects of IFN-a are complex and contribute to an elevated risk of cardiovascular disease by suppression of IL-1β pathways and by upregulation of the inflammasome machinery and potentiation of IL-18 activation. [ABSTRACT FROM AUTHOR]

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-a synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role. [ABSTRACT FROM AUTHOR]

IL-17 and IL-23 are known to be absolutely central to psoriasis pathogenesis because drugs targeting either cytokine are highly effective treatments for this disease. The efficacy of these drugs has been attributed to blocking the function of IL-17-producing T cells and their IL-23-induced expansion. However, we demonstrate that mast cells and neutrophils, not T cells, are the predominant cell types that contain IL-17 in human skin. IL-17+ mast cells and neutrophils are found at higher densities than IL-17+ T cells in psoriasis lesions and frequently release IL-17 in the process of forming specialized structures called extracellular traps. Furthermore, we find that IL-23 and IL-1β can induce mast cell extracellular trap formation and degranulation of human mast cells. Release of IL-17 from innate immune cells may be central to the pathogenesis of psoriasis, representing a fundamental mechanism by which the IL-23-IL-17 axis mediates host defense and autoimmunity. [ABSTRACT FROM AUTHOR]

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4(+) and CD8(+) T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8(+) TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8(+) TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8(+) TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8(+) and CD4(+) T cell subsets in AA patients, including CD8(+) and CD4(+) TSCMs. CD8(+) TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4(+) and CD8(+) TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8(+) TSCM subset is a novel biomarker and a potential therapeutic target for AA.

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study, we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and β, IL-1R1, and vascular endothelial growth factor A, and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, by decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs, and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not anti-neutrophil cytoplasmic Ab-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1R antagonist, downregulation of vascular endothelial growth factor A, and glomerular and blood vessel decreased capillary density, compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients because of the presence of low-density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function, and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils, and healthy control neutrophils were compared with regard to their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile, and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-alpha, and IFN-gamma, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in systemic lupus erythematosus by simultaneously mediating enhanced vascular damage and inhibiting vascular repair.

T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-gamma release by lupus T cells, and production was proportional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease.

Individuals with systemic lupus erythematosus (SLE) have a striking increase in the risk of premature atherosclerosis, a complication preceded by significant subclinical vascular damage. A proposed mechanism leading to accelerated vascular disease in SLE is an imbalance between vascular damage and repair, as patients with this disease display significant abnormalities in phenotype and function of endothelial progenitor cells. In addition, individuals with SLE have a higher incidence of insulin resistance which may further contribute to the increased cardiovascular risk. This study examined the role of the peroxisome proliferator activated receptor gamma agonist pioglitazone in improving endothelial function, endothelial progenitor cell numbers and functional capacity, metabolic parameters, and disease activity in the lupus-prone murine model New Zealand Black/New Zealand White (NZB x NZW)F(1). Ten-week-old prenephritic female NZB/NZW F(1) mice were exposed to 10 or 25 mg/kg/day of oral pioglitazone or vehicle for 15 or 24 wk. Mice exposed to pioglitazone exhibited pronounced enhancement in endothelial-dependent vasorelaxation of thoracic aortas and in endothelial progenitor cell function, as assessed by the capacity of bone marrow-derived endothelial progenitor cells to differentiate into mature endothelial cells. Pioglitazone-treated mice showed improvement in insulin resistance, adipokine, and lipid profile. Kidneys from pioglitazone-treated mice showed significant decreases in immune complex deposition, renal inflammation, T cell glomerular infiltration, and intrarenal synthesis of TNF-alpha, IL-1beta, and VCAM-1. These results indicate that peroxisome proliferator-activated receptor gamma agonists could serve as important tools in the prevention of premature cardiovascular disease and organ damage in SLE.

Systemic lupus erythematosus (SLE) is characterized by a systemic autoimmune response with profound and diverse T cell changes. Dendritic cells (DCs) are important orchestrators of immune responses and have an important role in the regulation of T cell function. The objective of this study was to determine whether myeloid DCs from individuals with SLE display abnormalities in phenotype and promote abnormal T cell function. Monocyte-derived DCs and freshly isolated peripheral blood myeloid DCs from lupus patients displayed an abnormal phenotype characterized by accelerated differentiation, maturation, and secretion of proinflammatory cytokines. These abnormalities were characterized by higher expression of the DC differentiation marker CD1a, the maturation markers CD86, CD80, and HLA-DR, and the proinflammatory cytokine IL-8. In addition, SLE patients displayed selective down-regulation of the maturation marker CD83 and had abnormal responses to maturation stimuli. These abnormalities have functional relevance, as SLE DCs were able to significantly increase proliferation and activation of allogeneic T cells when compared with control DCs. We conclude that myeloid DCs from SLE patients display significant changes in phenotype which promote aberrant T cell function and could contribute to the pathogenesis of SLE and organ damage.

Increased monocyte/macrophage (Mphi) apoptosis occurs in patients with systemic lupus erythematosus (SLE) and is mediated, at least in part, by an autoreactive CD4(+) T cell subset. Furthermore, autoreactive murine CD4(+) T cells that kill syngeneic Mphi in vitro induce a lupus-like disease in vivo. However, it is unclear whether increased Mphi apoptosis in SLE per se is sufficient to accelerate/promote autoimmunity. We have investigated whether increased Mphi apoptosis in vivo, induced by the administration of clodronate liposomes, can exacerbate the autoimmune phenotype in NZB x SWR (SNF(1)) lupus-prone mice, and induce autoantibody production in haplotype-matched BALB/c x DBA1 (DBF(1)) non-lupus-prone mice. Lupus-prone mice SNF(1) mice that were treated with clodronate liposomes, but not mice treated with vehicle, developed significant increases in autoantibodies to dsDNA, nucleosomes, and the idiotypically related family of nephritic Abs Id(LN)F(1), when compared with untreated SNF(1) mice. Furthermore, clodronate treatment hastened the onset of proteinuria and worsened SNF(1) lupus nephritis. When compared with vehicle-treated controls, clodronate-treated non-lupus-prone DBF(1) mice developed significantly higher levels of anti-nucleosome and Id(LN)F(1) Abs but did not develop lupus nephritis. We propose that Mphi apoptosis contributes to the pathogenesis of autoantibody formation and organ damage through both an increase in the apoptotic load and impairment in the clearance of apoptotic material. This study suggests that mechanisms that induce scavenger cell apoptosis, such as death induced by autoreactive cytotoxic T cells observed in SLE, could play a pathogenic role and contribute to the severity of the disease.

Inhibiting DNA methylation in CD4+ T cells causes aberrant gene expression and autoreactive monocyte/macrophage killing in vitro, and the hypomethylated cells cause a lupus-like disease in animal models. Similar decreases in T cell DNA methylation occur in idiopathic lupus, potentially contributing to disease pathogenesis. The genes affected by DNA hypomethylation are largely unknown. Using DNA methylation inhibitors and oligonucleotide arrays we have identified perforin as a methylation-sensitive gene. Our group has also reported that DNA methylation inhibitors increase CD4+ T cell perforin by demethylating a conserved methylation-sensitive region that is hypomethylated in primary CD8+ cells, which express perforin, but is largely methylated in primary CD4+ cells, which do not. As lupus T cells also have hypomethylated DNA and promiscuously kill autologous monocytes/macrophages, we hypothesized that perforin may be similarly overexpressed in lupus T cells and contribute to the monocyte killing. We report that CD4+ T cells from patients with active, but not inactive, lupus overexpress perforin, and that overexpression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells and demethylated by DNA methylation inhibitors. Further, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4+ lupus T cells, suggesting that the perforin is functional. We conclude that demethylation of specific regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. Our results also suggest that aberrant perforin expression in CD4+ lupus T cells may contribute to monocyte killing.

Individuals with systemic lupus erythematosus show evidence of a significant increase in monocyte apoptosis. This process is mediated, at least in part, by an autoreactive T cell subset that kills autologous monocytes in the absence of nominal Ag. We have investigated the apoptotic pathways involved in this T cell-mediated process. Expression of the apoptotic ligands TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and Fas ligand on lupus T cells was determined, and the role of these molecules in the monocyte apoptotic response was examined. We report that these apoptotic ligands mediate the autologous monocyte death induced by lupus T cells and that this cytotoxicity is associated with increased expression of these molecules on activated T cells, rather than with an increased susceptibility of lupus monocytes to apoptosis induced by these ligands. These results define novel mechanisms that contribute to increased monocyte apoptosis characterizing patients with lupus. We propose that this mechanism could provide a source of potentially antigenic material for the autoimmune response and interfere with normal clearing mechanisms.