Hyaluronan promotes intracellular ROS production and apoptosis in TNFα-stimulated neutrophils.

Background: Hyaluronan (HA) is an important structural component of the extracellular matrix and has well-described roles in maintaining tissue integrity and homeostasis. With inflammation, HA metabolism (synthesis and degradation) increases and results in higher concentrations of soluble HA. Previously, we demonstrated that (soluble) HA primed resting neutrophils for the oxidative burst in response to a secondary stimulus. Notably, HA-mediated priming was not dependent on degranulation, which is a hallmark of priming by classical agents such as TNFα. In this study, we queried the ability of HA to prime neutrophils to different stimuli and its capacity to modulate neutrophil function in the presence of TNFα. Methods: Blood neutrophils from healthy donors were stimulated ex vivo with HA in the absence and presence of classic neutrophil agonists, inclusive of TNFα. Western blotting was used to assess the activation (phosphorylation) of p38 MAPK, and key neutrophil functions associated with priming and activation, such as intracellular and extracellular ROS production, degranulation, and apoptosis, were evaluated by standard chemiluminescence assays (ROS) and flow cytometry. Results: Hyaluronan is capable of atypical priming and, with TNFα, co-priming neutrophils for an enhanced (rate and/or magnitude) oxidative burst to various secondary stimuli. In addition, HA can augment intracellular ROS production that is directly induced by TNFα in resting neutrophils, which coincided with the activation of p38 MAPK and apoptosis. Conclusions: These data demonstrate that the extracellular matrix component HA is a key modulator of neutrophil function(s) in the presence of inflammatory agents such as TNFα. Moreover, it provides additional evidence for the diversity and complexity of neutrophil priming and activation during inflammation.

Elevated ADA2 Enzyme Activity at the Onset of Chronic Graft-versus-Host Disease in Children.

Adenosinergic signaling has potent, context-specific effects on immune cells, particularly on the dysregulation of lymphocytes. This in turn may have a role in immune activation and loss of tolerance in such diseases as chronic graft-versus-host disease (chronic GVHD). We assessed whether changes in the enzymatic activity of adenosine deaminase 2 (ADA2), an enzyme that depletes adenosine in the extracellular space via conversion to inosine, may be associated with the onset of chronic GVHD. ADA2-specific enzyme activity was measured in plasma samples from 230 pediatric hematopoietic stem cell transplantation (HSCT) recipients enrolled on the Applied Biomarkers of Late Effects of Childhood Cancer (ABLE)/Pediatric Blood and Marrow Transplant Consortium (PBMTC) 1202 study and compared between patients developing chronic GVHD and those not developing chronic GVHD within 12 months of transplantation. ADA2 and its relationships with 219 previously measured plasma-soluble proteins, metabolites, and immune cell populations were evaluated as well. Plasma ADA2 enzyme activity was significantly elevated in pediatric HSCT recipients at the onset of chronic GVHD compared to patients without chronic GVHD and was not associated with prior history of acute GVHD or generalized inflammation as measured by C-reactive protein concentration. ADA2-specific enzyme activity met our criteria as a potential diagnostic biomarker of chronic GVHD (effect ratio ≥1.30 or ≤.75; area under the receiver operating characteristic curve ≥.60; P < .05) and was positively associated with markers of immune activation previously identified in pediatric chronic GVHD patients. These results support the potential of ADA2 enzyme activity, in combination with other biomarkers and subject to future validation, to aid the diagnosis of chronic GVHD in children post-HSCT.

Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression.

Background: Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with Deficiency of ADA2 (DADA2), an autosomal recessive disease associated with an unusually broad range of inflammatory manifestations including vasculitis, hematological defects and cytopenia. Previous work by our group led to the identification of ADA2 variants of novel association with DADA2, among which was a unique c.1052T>A (p.Leu351Gln; herein referred to as L351Q) variant located in the catalytic domain of the protein. Methods: Mammalian (Flp-IN CHO) cells were engineered to stably express wild-type ADA2 and ADA2 protein variants, including the pathogenic L351Q variant identified in DADA2 patients. An enzyme assay and immunoblotting were used to assess ADA2 catalytic activity and secretion, respectively, and the outcome of experimentally induced inhibition of protein processing (Golgi transport and N-linked glycosylation) was assessed. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to determine the relative expression of Type I Interferon stimulated genes (ISGs), IFIT3 and IRF7. Results: In addition to abrogating catalytic activity, the L351Q variant impaired secretion of L351Q ADA2 resulting in an intracellular accumulation of L351Q ADA2 protein that was not observed in cells expressing wild-type ADA2 or other ADA2 protein variants. Retention of L351Q ADA2 was not attributable to impaired glycosylation on neighboring asparagine residues and did not impact cell growth or integrity. Constitutive expression of Type I ISGs IFIT3 and IRF7 was observed in cells expressing L351Q ADA2. Conclusions: The impaired secretion of L351Q ADA2 may be an important factor leading to the severe phenotype observed in patients with this variant further emphasizing the importance of assessing impacts beyond catalytic activity when evaluating genotype-phenotype relationships in DADA2.

Latent gammaherpesvirus exacerbates arthritis through modification of age-associated B cells.

Epstein-Barr virus (EBV) infection is associated with rheumatoid arthritis (RA) in adults, though the nature of the relationship remains unknown. Herein, we have examined the contribution of viral infection to the severity of arthritis in mice. We have provided the first evidence that latent gammaherpesvirus infection enhances clinical arthritis, modeling EBV's role in RA. Mice latently infected with a murine analog of EBV, gammaherpesvirus 68 (γHV68), develop more severe collagen-induced arthritis and a Th1-skewed immune profile reminiscent of human disease. We demonstrate that disease enhancement requires viral latency and is not due to active virus stimulation of the immune response. Age-associated B cells (ABCs) are associated with several human autoimmune diseases, including arthritis, though their contribution to disease is not well understood. Using ABC knockout mice, we have provided the first evidence that ABCs are mechanistically required for viral enhancement of disease, thereby establishing that ABCs are impacted by latent gammaherpesvirus infection and provoke arthritis.

Rheumatoid arthritis is one of the most common autoimmune diseases, leaving patients in pain as their immune system mistakenly attacks the lining of their joints. The precise cause is unknown, but research suggests a link to the Epstein-Barr virus, the agent responsible for mononucleosis (also known as glandular fever). After infection and recovery, the virus remains in the body, lying dormant inside immune 'B cells' which are often responsible for autoimmune diseases. Of particular interest are a sub-group known as 'age-associated B-cells', which are mostly cells left over from fighting past infections such as mononucleosis. Yet, the link between Epstein-Barr virus and rheumatoid arthritis remains hard to investigate because of the long gap between the two diseases: the virus mostly affects children and young people, while rheumatoid arthritis tends to develop in middle age. To investigate how exactly the two conditions are connected, Mouat et al. created a new animal model: they infected young mice with the murine equivalent of the Epstein-Barr virus, and then used a collagen injection to trigger rheumatoid arthritis-like disease once the animals were older. Next, Mouat et al. monitored the paws of the mice, revealing that viral infection early in life worsened arthritis later on. These animals also had more age-associated B cells than normal, and the cells showed signs of participating in inflammation. On the other hand, early viral infection did not make arthritis worse in mice unable to produce age-associated B cells. Taken together, these results suggest that the immune cells are required to enhance the effect of the viral infection on rheumatoid arthritis. This new insight may help to refine current treatments that work by reducing the overall number of B cells. Ultimately, the animal model developed by Mouat et al. could be useful to identify better ways to diagnose, monitor and treat this debilitating disease.

Adenosine deaminase 2 activity negatively correlates with age during childhood.

BACKGROUND: Human adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered ADA2 enzyme activity has been associated with some viral infections and rheumatic diseases. The potential utility of ADA2 as a biomarker is, however, limited by the absence of established ranges of ADA2 concentration and enzyme activity in the healthy population. It is known that ADA2 enzyme activity is lower in adults, but when (and why) this decline happens is not known. The purpose of this study was to establish normative ranges of ADA2 enzyme activity and protein concentration in the healthy pediatric population. METHODS: We modified a commercially available ADA2 enzyme activity assay to enable higher throughput analysis of fresh, frozen and hemolyzed blood samples. With this assay and ADA2 protein immunoblotting, we analyzed ADA2 enzyme activity and protein concentration in blood plasma from a cohort of children and adolescents (n = 94) aged 5 months to 18 years. One-way ANOVA with subsequent Tukey multiple comparison test was used to analyze group differences. Reference intervals were generated using the central 95% of the population (2-97.5 percentiles). RESULTS: ADA2 enzyme activity was consistent in fresh, frozen, and hemolyzed sera and plasma as measured by our modified assay. Analysis of plasma samples from the healthy pediatric cohort revealed that ADA2 enzyme activity is significantly lower in older children than in younger children (p < 0.0001). In contrast, there was no significant correlation between ADA2 protein concentration and either age or ADA2 enzyme activity. CONCLUSION: We observed that ADA2 enzyme activity, but not ADA2 protein concentration, negatively correlates with age in a cohort of children and adolescents. Our findings stress the importance of appropriate age-matched controls for assessing ADA2 enzyme activity in the clinical setting.

Identification of Novel Adenosine Deaminase 2 Gene Variants and Varied Clinical Phenotype in Pediatric Vasculitis.

OBJECTIVE: Individuals with deficiency of adenosine deaminase 2 (DADA2), a recently recognized autosomal recessive disease, present with various systemic vascular and inflammatory manifestations, often with young age at disease onset or with early onset of recurrent strokes. Their clinical features and histologic findings overlap with those of childhood-onset polyarteritis nodosa (PAN), a primary "idiopathic" systemic vasculitis. Despite similar clinical presentation, individuals with DADA2 may respond better to biologic therapy than to traditional immunosuppression. The aim of this study was to screen an international registry of children with systemic primary vasculitis for variants in ADA2. METHODS: The coding exons of ADA2 were sequenced in 60 children and adolescents with a diagnosis of PAN, cutaneous PAN, or unclassifiable vasculitis (UCV), any chronic vasculitis with onset at age 5 years or younger, or history of stroke. The functional consequences of the identified variants were assessed by ADA2 enzyme assay and immunoblotting. RESULTS: Nine children with DADA2 (5 with PAN, 3 with UCV, and 1 with antineutrophil cytoplasmic antibody-associated vasculitis) were identified. Among them, 1 patient had no rare variants in the coding region of ADA2 and 8 had biallelic, rare variants (minor allele frequency <0.01) with a known association with DADA2 (p.Gly47Arg and p.Gly47Ala) or a novel association (p.Arg9Trp, p.Leu351Gln, and p.Ala357Thr). The clinical phenotype varied widely. CONCLUSION: These findings support previous observations indicating that DADA2 has extensive genotypic and phenotypic variability. Thus, screening ADA2 among children with vasculitic rash, UCV, PAN, or unexplained, early-onset central nervous system disease with systemic inflammation may enable an earlier diagnosis of DADA2.

Clinical practice variation and need for pediatric-specific treatment guidelines among rheumatologists caring for children with ANCA-associated vasculitis: an international clinician survey.

BACKGROUND: Because pediatric antineutrophil cytoplasmic antibody-associated vasculitis is rare, management generally relies on adult data. We assessed treatment practices, uptake of existing clinical assessment tools, and interest in pediatric treatment protocols among rheumatologists caring for children with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS: A needs-assessment survey developed by an international working group of pediatric rheumatologists and two nephrologists was circulated internationally. Data were summarized with descriptive statistics. Pearson's chi-square tests were used in inferential univariate analyses. RESULTS: The 209 respondents from 36 countries had collectively seen ~1600 children with GPA/MPA; 144 had seen more than two in the preceding 5 years. Standardized and validated clinical assessment tools to score disease severity, activity, and damage were used by 59, 63, and 36%, respectively; barriers to use included lack of knowledge and limited perceived utility. Therapy varied significantly: use of rituximab rather than cyclophosphamide was more common among respondents from the USA (OR = 2.7 [1.3-5.5], p = 0.0190, n = 139), those with >5 years of independent practice experience (OR = 3.8 [1.3-12.5], p = 0.0279, n = 137), and those who had seen >10 children with GPA/MPA in their careers (OR = 4.39 [2.1-9.1], p = 0.0011, n = 133). Respondents who had treated >10 patients were also more likely to continue maintenance therapy for at least 24 months (OR = 3.0 [1.4-6.4], p = 0.0161, n = 127). Ninety six percent of respondents believed in a need for pediatric-specific treatment guidelines; 46% supported adaptation of adult guidelines while 69% favoured guidelines providing a limited range of treatment options to allow comparison of effectiveness through a registry. CONCLUSIONS: These data provide a rationale for developing pediatric-specific consensus treatment guidelines for GPA/MPA. While pediatric rheumatologist uptake of existing clinical tools has been limited, guideline uptake may be enhanced if outcomes of consensus-derived treatment options are evaluated within the framework of an international registry.

The importance of considering monogenic causes of autoimmunity: A somatic mutation in KRAS causing pediatric Rosai-Dorfman syndrome and systemic lupus erythematosus.

OBJECTIVES: Clinicians need to be aware of the growing list of defined monogenic etiologies of autoimmune diseases. This is particularly relevant when evaluating children, as these rare monogenic forms of autoimmunity tend to present very early in life. METHODS AND RESULTS: By harnessing the transformative power of next generation sequencing, we made the unifying diagnosis of RAS-associated autoimmune leukoproliferative disease (RALD), caused by the somatic gain-of-function p.G13C KRAS mutation, in a boy with the seemingly unrelated immune dysregulatory conditions of Rosai-Dorfman and systemic lupus erythematosus (SLE). CONCLUSIONS: This case expands our understanding of the clinical phenotypes associated with the extremely rare condition of RALD, and emphasizes the importance of always considering the possibility of a monogenic cause for autoimmunity, particularly when the disease manifestations begin early in life and do not follow a typical clinical course.

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes.

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.

Endotoxin free hyaluronan and hyaluronan fragments do not stimulate TNF-α, interleukin-12 or upregulate co-stimulatory molecules in dendritic cells or macrophages.

The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free.

Innate defense regulator peptide 1018 protects against perinatal brain injury.

OBJECTIVE: There is currently no pharmacological treatment that provides protection against brain injury in neonates. It is known that activation of an innate immune response is a key, contributing factor in perinatal brain injury; therefore, the neuroprotective therapeutic potential of innate defense regulator peptides (IDRs) was investigated. METHODS: The anti-inflammatory effects of 3 IDRs was measured in lipopolysaccharide (LPS)-activated murine microglia. IDRs were then assessed for their ability to confer neuroprotection in vivo when given 3 hours after neonatal brain injury in a clinically relevant model that combines an inflammatory challenge (LPS) with hypoxia-ischemia (HI). To gain insight into peptide-mediated effects on LPS-induced inflammation and neuroprotective mechanisms, global cerebral gene expression patterns were analyzed in pups that were treated with IDR-1018 either 4 hours before LPS or 3 hours after LPS+HI. RESULTS: IDR-1018 reduced inflammatory mediators produced by LPS-stimulated microglia cells in vitro and modulated LPS-induced neuroinflammation in vivo. When administered 3 hours after LPS+HI, IDR-1018 exerted effects on regulatory molecules of apoptotic (for, eg, Fadd and Tnfsf9) and inflammatory (for, eg, interleukin 1, tumor necrosis factor α, chemokines, and cell adhesion molecules) pathways and showed marked protection of both white and gray brain matter. INTERPRETATION: IDR-1018 suppresses proinflammatory mediators and cell injurious mechanisms in the developing brain, and postinsult treatment is efficacious in reducing LPS-induced hypoxic-ischemic brain damage. IDR-1018 is effective in the brain when given systemically, confers neuroprotection of both gray and white matter, and lacks significant effects on the brain under normal conditions. Thus, this peptide provides the features of a promising neuroprotective agent in newborns with brain injury.

Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages.

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with ß-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.

Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses.

The human cathelicidin peptide, LL-37, is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study, we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF-α and NO levels produced by LPS and IFN-γ-polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4-polarized M2-BMDM to upregulate arginase activity, although it did inhibit LPS-induced TNF-α secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However, LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF-α secretion in ex vivo alveolar macrophages, whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF-α secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM, M2-BMDM, and tissue macrophage production of the proinflammatory cytokine TNF-α in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.

Galectin 3 aggravates joint inflammation and destruction in antigen-induced arthritis.

OBJECTIVE: Galectin 3, an endogenous ß-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis. METHODS: Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay. RESULTS: The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice. CONCLUSION: Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis.

Intracellular generation of superoxide by the phagocyte NADPH oxidase: how, where, and what for?

Professional phagocytes increase their consumption of molecular oxygen during the phagocytosis of microbes or when encountering a variety of nonparticulate stimuli. In these circumstances, oxygen is reduced by the phagocyte NADPH oxidase, and reactive oxygen species (ROS), which are important for the microbicidal activity of the cells, are generated. The structure and function of the NADPH oxidase have been resolved in part by studying cells from patients with chronic granulomatous disease (CGD), a condition characterized by the inability of phagocytes to assemble a functional NADPH oxidase and thus to produce ROS. As a result, patients with CGD have a predisposition to infections as well as a variety of inflammatory symptoms. A long-standing paradigm has been that NADPH oxidase assembly occurs exclusively in the plasma membrane or invaginations thereof (phagosomes). A growing body of evidence points to the possibility that phagocytes are capable of NADPH oxidase assembly in nonphagosomal intracellular membranes, resulting in ROS generation within intracellular organelles also in the absence of phagocytosis. The exact nature of these ROS-producing organelles is yet to be determined, but granules are prime suspects. Recent clinical findings indicate that the generation of intracellular ROS by NADPH oxidase activation is important for limiting inflammatory reactions and that intracellular and extracellular ROS production are regulated differently. Here we discuss the accumulating knowledge of intracellular ROS production in phagocytes and speculate on the precise role of these oxidants in regulating the inflammatory process.

Treating neonatal brain injury - promise and inherent research challenges.

In this review we discuss current challenges faced by researchers and clinician-scientists in the pursuit of therapeutics to treat hypoxic-ischemic (HI) brain injury in term infants. At present, there is an absence of neuroprotective drugs that are safe and effective for the protection of neonates from neurological sequels after HI. We discuss secondary neurotoxic processes elicited by HI that may be targets for therapeutic interventions with a specific focus on inflammatory mechanisms. Advances in research to unravel these cellular processes and molecular mechanisms that drive injurious processes after HI have traditionally been plagued by conflicting results when assessing different times for intervention, different models for brain injury, and the adult versus neonate brain. We attribute impeded drug development in part to such disparate results and general difficulties to conduct a stringent, comprehensive analysis of candidate drugs prior to clinical trials. It will be imperative to implement changes in the clinic and laboratory in order for future drug initiatives to achieve success. We also provide a brief discussion on the pursuit of anti-inflammatory molecules and monitoring methods that are the focus of current patents and that, in our opinion, may lead to important new developments in the treatment of HI brain injury in newborn infants.

Divergent effects on phagocytosis by macrophage-derived oxygen radicals.

Clearance of senescent cells from damaged tissue is a crucial step to successfully resolve an inflammatory response. Poor clearance has been attributed to defects in neutrophil apoptosis, a process that is regulated in part by reactive oxygen species (ROS) produced by activated neutrophils. Using ROS-deficient and wild-type macrophages treated with extracellular ROS scavengers we demonstrate that macrophage-derived ROS also influence phagocytosis and do so by direct and indirect mechanisms.

Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.

Galectin-3, a beta-galactoside binding, endogenous lectin, takes part in various inflammatory events and is produced in substantial amounts at inflammatory foci. We investigated whether extracellular galectin-3 could participate in the phagocytic clearance of apoptotic neutrophils by macrophages, a process of crucial importance for termination of acute inflammation. Using human leukocytes, we show that exogenously added galectin-3 increased the uptake of apoptotic neutrophils by monocyte-derived macrophages (MDM). Both the proportion of MDM that engulfed apoptotic prey and the number of apoptotic neutrophils that each MDM engulfed were enhanced in the presence of galectin-3. The effect was lactose-inhibitable and required galectin-3 affinity for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins, since a mutant lacking this activity was without effect. The enhanced uptake relied on the presence of galectin-3 during the cellular interaction and was paralleled by lectin binding to apoptotic cells as well as MDM in a lactose-dependent manner. These findings suggest that galectin-3 functions as a bridging molecule between phagocyte and apoptotic prey, acting as an opsonin. The process of clearance, whereby apoptotic neutrophils are removed by macrophages, is crucial for the resolution of acute inflammation and our data imply that the increased levels of galectin-3 often found at inflammatory sites could potently affect this process.

ROS-deficient monocytes have aberrant gene expression that correlates with inflammatory disorders of chronic granulomatous disease.

Chronic granulomatous disease is an immunodeficiency caused by an inability to produce reactive oxygen species. While the mechanism of hyper-sensitivity to infection is well understood in CGD, the basis for debilitating inflammatory disorders that arise in the absence of evident infection has not been fully explained. Herein it is demonstrated that resting and TLR-activated monocytes from individuals with CGD expressed significantly higher levels of inflammatory mediators than control cells; the expression in CGD cells resembled normal cells stimulated with lipopolysaccharide. The lack of acute illness, infection or circulating endotoxin in the blood of the CGD patients at the time of sampling was consistent with infection-free inflammation. The enhanced expression of inflammatory mediators correlated with elevated expression of NF-kappaB and was dependent on ERK1/2 signalling. The results are consistent with the hypothesis that ROS are anti-inflammatory mediators that control gene expression and potentially limit the development of sterile inflammatory disorders.

Enhanced inflammatory responses of chronic granulomatous disease leukocytes involve ROS-independent activation of NF-kappa B.

Reactive oxygen species (ROS) generated by the cellular NADPH-oxidase are crucial for phagocytic killing of ingested microbes and have been implicated as signaling molecules in various processes. For example, ROS are thought to be involved in activation of the transcription factor NF-kappaB, central for mediating production of proinflammatory cytokines in response to inflammatory stimuli. Several studies have demonstrated that inhibitors of the NADPH-oxidase interfere with NF-kappaB activation and production of proinflammatory cytokines. Curiously, patients with chronic granulomatous disease (CGD), an immunodeficiency characterized by an inability to produce ROS, are not only predisposed to severe infections, but also frequently develop various inflammatory complications indicative of exaggerated inflammatory responses. Here, we show that human CGD leukocytes display a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to stimulation with Toll-like receptor agonists. The hyperinflammatory phenotype was also evident in mononuclear cells from CGD mice (gp91(phox) -/-), but not in control cells in the presence of NADPH-oxidase inhibitor diphenyleneiodonium, probably reflecting NADPH-oxidase-independent effects of the inhibitor. Furthermore, we show that the major steps involved in NF-kappaB activation were intact in human CGD cells. These data indicate that ROS were nonessential for activation of NF-kappaB and their production may even attenuate inflammation.