Monocytes are resistant to apoptosis in systemic juvenile idiopathic arthritis
Introduction
Systemic juvenile idiopathic arthritis (SJIA) is a chronic inflammatory disease of childhood characterized by a combination of systemic features (fever, rash, adenopathy, serositis) and arthritis. Although SJIA represents only 10–20% of all JIA, it accounts for more than 2/3 of JIA mortality [1]. Approximately 10% of SJIA patients develop a potentially fatal complication known as “macrophage activation syndrome” (MAS). MAS is characterized by uncontrolled activation of macrophages and T lymphocytes, resulting in fever, hepatic dysfunction, severe cytopenia, disseminated intravascular coagulation, and neurological involvement [2].
Monocytes show a tendency for expansion and activation in SJIA even in the absence of clinically diagnosed MAS [3], [4]. Compared to normal monocytes, SJIA monocytes produce more pro-inflammatory cytokines, including IL-1β and IL-6 [5], [6]. They also show enhanced proteolytic activity, degrading more bone in vitro than normal monocytes [7]. Additionally, SJIA serum contains significantly elevated levels of S100A8, S100A9, and S100A12, proteins that are secreted during activation of neutrophils and monocytes [8]. Levels of macrophage migration inhibitory factor (MIF), which up-regulates phagocytic function and secretion of pro-inflammatory cytokines by macrophages, are also significantly elevated in SJIA serum and synovial fluid [9] and may drive monocyte activation in SJIA, at least in part. Activated monocytes are found in inflamed joints of SJIA patients [10], and circulating levels of chemotactic factors for activated monocytes are found during periods of active disease (6).
Uncontrolled monocyte activation may result from a killing defect in NK cells. NK cytolytic activity and perforin expression are reported to be lower in at least a subgroup of SJIA patients (even in the absence of MAS) compared to other JIA subtypes and controls [11], [12], [13]. As NK cells have been shown to regulate macrophage activity by directly killing activated macrophages [14], dysfunctional NK cytolytic activity could contribute to uncontrolled activation of monocytes in SJIA.
There is some evidence that activation of normal monocytes is associated with resistance to apoptosis [15], [16]. We hypothesized that monocyte activation in SJIA may be associated with dysregulation of apoptosis, either as a cause or effect. Apoptosis is the primary form of programmed cell death and is crucial to balancing cell activation/proliferation with cell death [17]. Apoptosis results in a chromatin condensation and characteristic cell morphology, involving cell shrinkage, blebbing, and breakage into smaller apoptotic bodies. Under normal conditions, monocytes develop in the bone marrow daily and survive for 24–48 h in the circulation before undergoing spontaneous apoptosis [18].
Apoptosis is mediated by the extrinsic (death receptor) or the intrinsic (mitochondrial) pathways [19]. In the extrinsic pathway, a death-inducing ligand (e.g. Fas ligand) binds to its receptor (e.g. Fas) on target cells, initiating signal transduction and the formation of the death-inducing signaling complex (DISC). The adapter molecule Fas-associated death domain protein (FADD) is recruited to DISC and then recruits pro-caspase 8, which undergoes autocatalytic cleavage/activation. Active caspase 8 is released from DISC and initiates the caspase cascade by cleaving caspase 3. Caspase 3 activation results in DNA fragmentation, degradation of key cellular proteins, and cell death. FLIP proteins act as dominant negative regulators of this pathway by interfering with Fas-induced DISC formation.
The intrinsic pathway of apoptosis is initiated by DNA damage, hypoxia, or other severe cell stress. These stimuli influence the Bcl-2 protein family, which may be induced, repressed, or post-translationally modified to regulate activity. Cell stress can result in p53 stabilization and accumulation in the nucleus, where it enhances the expression of a number of pro-apoptotic genes involved in the intrinsic pathway, including BAX, NOXA, and PUMA. In addition, p53 can exert pro-apoptotic effects by direct binding of anti-apoptotic Bcl-2 proteins or by activating pro-apoptotic Bcl-2 proteins [20]. The balance between pro-apoptotic Bcl-2 family members (e.g., Bad, PUMA) and anti-apoptotic members (e.g., Bcl-2, Bcl-w) controls the intrinsic pathway. Pro-apoptotic members induce loss of integrity of the outer mitochondrial membrane but can be inhibited by anti-apoptotic members. Upon mitochondrial membrane permeabilization, cytochrome c and the SMAC/DIABLO protein translocate into the cytosol. SMAC/DIABLO promotes apoptosis by blocking inhibitors of apoptosis proteins (IAPs), which inactivate caspases. Cytochrome c binds Apaf-1 (apoptotic protease activating factor-1), forming the apoptosome. Procaspase-9 is recruited into the apoptosome, where it is allosterically activated. Activated caspase 9 in turn activates the downstream effector caspases 3, 6, and/or 7.
Fas pathway signaling through DISC assembly and caspase-8 activation is insufficient to induce apoptosis in certain cell types, such as hepatocytes and pancreatic β-cells [21], [22], [23], [24]. These cells, termed “type II” cells, depend upon the mitochondrial apoptosis pathway to amplify the initial death receptor signal [25]. Specifically, activated caspase-8 cleaves the pro-apoptotic Bcl-2 family member Bid, which translocates to the mitochondrial membrane, induces loss of membrane potential and release of cytochrome c, activating the caspase cascade. Bcl2a1 and Bcl-w proteins directly inhibit Bid cleavage.
Apoptotic death of activated monocytes could be a mechanism for controlling inflammation. Dysregulated apoptosis has been postulated to play a role in several autoimmune diseases [26], [27], [28]. The few studies investigating apoptosis in SJIA focus on peripheral blood lymphocytes and describe increased susceptibility to apoptosis [29], [30]. To date, no study has investigated monocyte apoptosis in SJIA. We hypothesized that SJIA monocytes are resistant to apoptosis, enabling these cells to persist in and enhance pro-inflammatory microenvironments.
Section snippets
Study population and blood collection
SJIA patients were recruited through the Stanford Pediatric Rheumatology Clinic. All SJIA study subjects met American College of Rheumatology criteria for the diagnosis of SJIA [31] and were enrolled after informed consent. The study was approved by the Stanford Institutional Review Board. Clinical data were collected at each visit when blood samples were drawn; clinical data included history, physical exam, and clinical laboratory values (including CBC, ESR). A disease activity scoring system
In active SJIA, PBMC express altered levels of genes regulating apoptosis
In an initial screen for gene expression changes associated with active SJIA disease, we classified samples into two disease categories: active (systemic score ≥ 2 and/or arthritis score ≥ B) and inactive (systemic score ≤ 1 and arthritis score = A) (see Supplementary Tables 1 and 2). We compared PBMC transcript profiles in paired samples (active/inactive disease) from 14 children with SJIA. Unsupervised clustering of gene expression data revealed distinct patterns of gene expression associated with
Discussion
In this study, we find that SJIA monocytes are resistant to apoptosis induced by several stimuli. Activation of normal monocytes is characterized in part by resistance to apoptosis [43], [44]. Activation confers survival signals, enabling monocytes to persist and function in sites of inflammation containing cytotoxic inflammatory mediators [16]. As such, the anti-apoptotic phenotype of SJIA monocytes may be a consequence of activation, arising from similar mechanisms that operate in normal
Acknowledgments
We would like to thank the patients, their families, and the medical staff of Pediatric Rheumatology and Pediatric Endocrinology Clinics at Lucile Packard Hospital for Children. This work was supported by The Wasie Foundation, the Dana Foundation, the Child Health Research Program of Stanford University, and the National Institutes of Health. J.L. Park is supported by the American College of Rheumatology Research and Education Foundation Physician Scientist Development Award and the Ernest and
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Signals and mechanisms regulating monocyte and macrophage activation in the pathogenesis of juvenile idiopathic arthritis
2021, International Journal of Molecular Sciences
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Current address: Department of Immunology, University of Washington, Seattle, WA, USA.