Kyohei Nakamura

Effect of silica particle size on macrophage inflammatory responses.

Amorphous silica particles, such as nanoparticles (<100 nm diameter particles), are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area) and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1β secretion, cell death and lung inflammation). Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1β secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.

Inhibitory Receptor Paired Ig-like Receptor B Is Exploited by Staphylococcus aureus for Virulence

The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host–bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1β secretion. Consequently, Pirb−/− mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory responses. In vivo, however, an LTA-deficient S. aureus mutant was highly virulent and poorly recognized by macrophages in both wild-type and Pirb−/− mice, demonstrating that LTA recognition by PRRs other than PIR-B mediates effective bacterial elimination. These results provide direct evidence that bacteria exploit the inhibitory receptor for virulence, and host immune system counterbalances the infection.

Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand

The natural killer group 2 membrane D (NKG2D) activating receptor plays crucial roles not only in host defense against tumors and viral infections, but also in autoimmune diseases. After NKG2D-mediated activation, Natural killer (NK) cells must be regulated to avoid potentially harmful reactivity. However, the negative regulation of these activated NK cells is poorly understood. Here, we reveal that the engagement of NKG2D by its ligand elicits not only target cell lysis, but also NK cell fratricide. Conventional mouse NK cells underwent cell death when cocultured with RMA cells expressing the NKG2D ligand retinoic acid early-inducible protein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells from mice deficient for DAP10 and DAP12 or perforin did not undergo death, highlighting the importance of the NKG2D pathway for NK cell death. However, NKG2D does not transmit direct death signals in NK cells. Rather, the interaction between NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae-1 by a membrane transfer process known as ”trogocytosis,” which was associated with clathrin-dependent NKG2D endocytosis. NK cells dressed with Rae-1 were lysed by neighboring NK cells through the NKG2D-induced perforin pathway in vitro and in vivo. These results provide the unique NKG2D function in negative regulation of activated NK cells.

A novel autoantibody against fibronectin leucine-rich transmembrane protein 2 expressed on the endothelial cell surface identified by retroviral vector system in systemic lupus erythematosus

IntroductionAnti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.MethodsAECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated.ResultsTwo distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells.ConclusionsWe identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.

Activation of the NLRP3 inflammasome by cellular labile iron

Cellular labile iron, which contains chelatable redox-active Fe(2+), has been implicated in iron-mediated cellular toxicity leading to multiple organ dysfunction. Iron homeostasis is controlled by monocytes/macrophages through their iron recycling and storage capacities. Furthermore, iron sequestration by monocytes/macrophages is regulated by pro-inflammatory cytokines including interleukin-1, highlighting the importance of these cells in the crosstalk between inflammation and iron homeostasis. However, a role for cellular labile iron in monocyte/macrophage-mediated inflammatory responses has not been defined. Here we describe how cellular labile iron activates the NLRP3 inflammasome in human monocytes. Stimulation of lipopolysaccharide-primed peripheral blood mononuclear cells with ferric ammonium citrate increases the level of cellular Fe(2+) levels in monocytes and induces production of interleukin-1β in a dose-dependent manner. This ferric ammonium citrate-induced interleukin-1β production is dependent on caspase-1 and is significantly inhibited by an Fe(2+)-specific chelator. Ferric ammonium citrate consistently induced interleukin-1β secretion in THP1 cells, but not in NLRP3-deficient THP1 cells, indicating a requirement for the NLRP3 inflammasome. Additionally, activation of the inflammasome is mediated by potassium efflux, reactive oxygen species-mediated mitochondrial dysfunction, and lysosomal membrane permeabilization. Thus, these results suggest that monocytes/macrophages not only sequestrate iron during inflammation, but also mediate inflammation in response to cellular labile iron, which provides novel insights into the role of iron in chronic inflammation.

NKG2D⁺ IFN-γ⁺ CD8⁺ T cells are responsible for palladium allergy.

Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8+ T cells are responsible for the disease as CD8+ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8+ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8+ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D+ CD8+ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.

A novel regulatory role of gp49B on dendritic cells in T-cell priming

Dendritic cells (DC) play pivotal roles in the induction and regulation of both innate and acquired immunity. DC express several cell‐surface immune inhibitory receptors. However, little is known about their potential immunoregulatory functions in the context of T‐cell activation. Here we report that murine gp49B, a member of the immunoglobulin superfamily, harboring immunoreceptor tyrosine‐based inhibitory motifs, is expressed on DC and downregulates cellular activity to prevent the excessive activation of T cells in vitro and in vivo. Bone marrow‐derived DC (BMDC) from newly generated gp49B‐deficient (gp49B−/−) mice induced enhanced proliferation and IL‐2 release of antigen‐specific CD4+ and CD8+ T cells compared with BMDC from wild‐type mice, in a cell–cell contact manner. The enhanced proliferation by gp49B−/− BMDC was also observed in allogeneic CD4+ and CD8+ T cells. Moreover, the transfer of allogeneic BALB/c splenocytes into C57BL/6 gp49B−/− mice induced severe acute graft‐versus‐host disease with an augmented upregulation of CD86 on CD11c+ splenic gp49B−/− DC, while transfer of C57BL/6 gp49B−/− splenocytes into BALB/c mice did not, suggesting the exacerbation of the disease was due, at least in part, to augmented activation of recipient gp49B−/− DC. These findings demonstrate a novel regulatory role of gp49B in the function of DC.

Generalized laxity of connective tissue as a possible syndrome in systemic lupus erythematosus

We report a case of systemic lupus erythematosus (SLE) with Jaccoud’s arthropathy, presenting atlantoaxial subluxation and multiple joint hypermobility. Radiological studies showed degenerative changes, but no destructive changes like rheumatoid arthritis (RA) were observed. Since these are rare complications of SLE, the coexistence of these complications suggests that they have a common pathogenesis. Generalized laxity of connective tissues may be a novel syndrome in SLE.

NK-cell fratricide: Dynamic crosstalk between NK and cancer cells

The intercellular transfer of plasma membrane patches, also known as trogocytosis, has a strong impact on the function and fate of immune cells. We have recently shown that natural killer (NK) cells undergo fratricide following the trogocytosis-mediated acquisition of tumor-derived NKG2D ligands. Malignant cells may therefore employ trogocytosis to escape NKG2D-mediated immune responses.

Chronic Lupus Peritonitis Is Characterized by the Ascites with a Large Content of Interleukin-6

Systemic lupus erythematosus (SLE) is an autoimmune disease and can cause multi-organ damage. Peritoneal involvement, also called lupus peritonitis, is a rare but sometimes fatal manifestation. Deposition of immune complexes consisting of immunoglobulin G and complement is considered to be involved in the pathogenesis of lupus peritonitis; however, it remains unknown whether inflammatory cytokines contribute to the pathology of this manifestation. Here we present two patients with treatment-resistant lupus peritonitis: a 37-year-old woman with a 26-year history of SLE who had been treated with prednisolone and cyclophosphamide followed by azathioprine and a 65-year-old woman with a 33-year history of SLE who had been treated with prednisolone alone. Both patients were admitted to our department because of abdominal distention. Computed tomography scans showed massive ascites. Ascitic fluid examinations of both patients showed leukocytosis with no evidence of malignancy or infection. After eliminating other causes for ascites, they were diagnosed with lupus peritonitis. Despite the intensified immunosuppressive therapy, they died of uncontrolled peritonitis several months after admission. Examinations of the ascites at admission also revealed a large content of interleukin (IL)-6, compared with other inflammatory cytokines, IL-1β and tumor necrosis factor-α. In fact, the ascitic IL-6 levels of these two patients were 12,389 pg/mL and 5,486 pg/mL, much higher than their serum IL-6 levels of 36 pg/mL and 140 pg/mL, respectively. We therefore suggest that IL-6 may contribute to the pathogenesis of lupus peritonitis and that the inhibition of IL-6 signaling may provide a novel therapeutic strategy for lupus peritonitis.