Microvesicles released by apoptotic human neutrophils suppress proliferation and IL‐2/IL‐2 receptor expression of resting T helper cells

Membrane‐coated microvesicles (MVs) have been identified as important mediators in intercellular communication. During the process of apoptosis, dying cells dynamically release MVs. Neutrophils are the most abundant type of leukocytes in the circulation. Due to their very short lifespan, it is likely that they are the source of large amounts of apoptotic cell‐derived MVs. Here, we show that MVs released by apoptotic human polymorphonuclear neutrophils (apoPMN‐MVs), but not the apoptotic neutrophils themselves, selectively suppress the proliferation of CD25 (IL‐2Rα)^neg CD127 (IL‐7Rα)^pos Th cells in a dose‐dependent manner. In contrast, the proliferation of total T cells is not affected by MVs. Importantly, apoPMN‐MVs suppress the secretion of IL‐2 as well as the expression of and signaling via the IL‐2 receptor (IL‐2R) by CD25^negCD127^pos Th cells. Addition of IL‐7 strongly reduced the suppression of T‐cell proliferation by MVs and the addition of IL‐2 completely abrogated the suppressive effect. Thus, apoPMN‐MVs suppressed a subset of Th cells by downregulating IL‐2 and IL‐2R expression and signaling. This may represent an important mechanism to prevent the activation and expansion of resting T cells in the absence of sufficient cytokine stimulation, and thereby maintaining immune tolerance.     

Extracellular vesicles in autoimmune vasculitis - Little dirts light the fire in blood vessels

Systemic vasculitis is diverse group of autoimmune disorders which are characterized by inflammation of blood vessel walls with deep aching and burning pain. Their underlying etiology and pathophysiology still remain poorly understood. Extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, are membrane vesicular structures that are released either during cell activation, or when cells undergo programmed cell death, including apoptosis, necroptosis, and pyroptosis. Although EVs were thought as cell dusts, but now they have been found to be potently active since they harbor bioactive molecules, such as proteins, lipids, nucleic acids, or multi-molecular complexes. EVs can serve as novel mediators for cell-to-cell communications by delivery bioactive molecules from their parental cells to the recipient cells. Earlier studies mainly focused on MVs budding from membrane surface. Recent studies demonstrated that EVs may also carry molecules from cytoplasm or even from nucleus of their parental cells, and these EVs may carry autoantigens and are important in vasculitis. EVs may play important roles in vasculitis through their potential pathogenic involvements in inflammation, autoimmune responses, procoagulation, endothelial dysfunction/damage, angiogenesis, and intimal hyperplasia. EVs have also been used as specific biomarkers for diagnostic use or disease severity monitoring. In this review, we have focused on the aspects of EV biology most relevant to the pathogenesis of vasculitis, discussed their perspective insights, and summarized the exist literature on EV relevant studies in vasculitis, therefore provides an integration of current knowledge regarding the novel role of EVs in systemic vasculitis.     

The Regulation of FasL Expression—A Distinguishing Feature Between Monocytes and T Lymphocytes/NK Cells with Possible Implications for SLE

Monocytes and lymphocytes from patients with systemic lupus erythematosus (SLE) had a higher cell surface expression of FasL than the corresponding cells from healthy individuals. Inhibitors of metalloproteases upregulated the surface expression of FasL in peripheral blood lymphocytes (PBL), indicating that a metalloprotease is responsible for the cleavage of FasL. The level of sFasL in serum was slightly increased in the patient group compared to the controls. Therefore, the possible contribution of various mononuclear cell types to the release of FasL was analyzed. Isolated NK cells and T lymphocytes released FasL into the medium and the release was prevented by inhibitors of metalloproteases. In contrast, isolated monocytes did not release FasL. FasR expression was elevated in patients with inverted CD4/CD8 ratio, while FasL expression showed no relationship to CD4/CD8 ratio. The absence of FasL release by isolated cells and a high level of surface expression of FasL distinguish monocytes and T lymphocytes/NK cells.     

Cyclic dinucleotides modulate human T‐cell response through monocyte cell death

Cyclic dinucleotides, a class of microbial messengers, have been recently identified in bacteria, but their activity in humans remains largely unknown. Here, we have studied the function of cyclic dinucleotides in humans. We found that c‐di‐AMP and cGAMP, two adenosine‐based cyclic dinucleotides, activated T lymphocytes in an unusual manner through monocyte cell death. c‐di‐AMP and cGAMP induced the selective apoptosis of human monocytes, and T lymphocytes were activated by the direct contact with these dying monocytes. The ensuing T‐cell response comprised cell‐cycle exit, phenotypic maturation into effector memory cells and proliferation arrest, but not cell death. This quiescence was transient since T cells remained fully responsive to further restimulation. Together, our results depict a novel activation pattern for human T lymphocytes: a transient quiescence induced by c‐di‐AMP‐ or cGAMP‐primed apoptotic monocytes.     

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells

Summary.  Bovine herpesvirus 1 (BHV-1), a member of the it Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral Blymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro. Accepted November 17, 1997 Received September 2, 1997     

Autologous tolerogenic dendritic cells derived from monocytes of systemic lupus erythematosus patients and healthy donors show a stable and immunosuppressive phenotype

Systemic lupus erythematosus (SLE) is an autoimmune disease with unrestrained T‐cell and B‐cell activity towards self‐antigens. Evidence shows that apoptotic cells (ApoCells) trigger an autoreactive response against nuclear antigens in susceptible individuals. In this study, we focus on generating and characterizing tolerogenic dendritic cells (tolDCs) to restore tolerance to ApoCells. Monocyte‐derived dendritic cells (DCs) from healthy controls and patients with SLE were treated with dexamethasone and rosiglitazone to induce tolDCs. Autologous apoptotic lymphocytes generated by UV irradiation were given to tolDCs as a source of self‐antigens. Lipopolysaccharide (LPS) was used as a maturation stimulus to induce the expression of co‐stimulatory molecules and secretion of cytokines. TolDCs generated from patients with SLE showed a reduced expression of co‐stimulatory molecules after LPS stimulation compared with mature DCs. The same phenomenon was observed in tolDCs treated with ApoCells and LPS. In addition, ApoCell‐loaded tolDCs stimulated with LPS secreted lower levels of interleukin‐6 (IL‐6) and IL‐12p70 than mature DCs without differences in IL‐10 secretion. The functionality of tolDCs was assessed by their capacity to prime allogeneic T cells. TolDCs displayed suppressor properties as demonstrated by a significantly reduced capacity to induce allogeneic T‐cell proliferation and activation. ApoCell‐loaded tolDCs generated from SLE monocytes have a stable immature/tolerogenic phenotype that can modulate CD4⁺ T‐cell activation. These properties make them suitable for an antigen‐specific immunotherapy for SLE.     

MicroRNA‐21 expression in neonatal blood associated with antenatal immunoglobulin E production and development of allergic rhinitis

Background The prevalence of allergic diseases has increased in the past decades. It is unknown whether expression of certain microRNAs (miRNAs) in neonatal leucocytes is correlated to IgE production and/or allergic diseases. Objective This study investigated the association of miRNA expression in neonatal leucocytes with cord blood IgE (CBIgE) elevation and development of allergic disease. Methods We screened for the expression of a panel of 157 miRNAs in mononuclear leucocytes from human umbilical cord blood (CB) samples with elevated CBIgE and tracked the association of down‐regulated miRNA expression to the miRNA‐targeted gene expression and to children with allergic rhinitis (AR). Results Among the initial screen of 10 CB samples with elevated CBIgE, expression of eight of the 157 miRNAs was low. Of these eight down‐expressed miRNAs, three remained down‐regulation in a validation with other 20 CB samples, and two of the three miRNAs, miR‐21 and miR‐126, were significantly lower in monocytes from AR children. Further analysis of mRNA expression of the miR‐21‐targeted genes identified that TGFBR2 expression on monocytes was significantly up‐regulated in CB with elevated CBIgE, and in AR patients. Transfection of miR‐21 precursor into monocytes from patients with AR increased miR‐21 expression and decreased TGFBR2 expression. Conclusion This study demonstrated the first in the literature that lower miR‐21 expression in CB and increased TGFBR2 expression is associated with antenatal IgE production and development of AR. Cite this as: R.‐F. Chen, H.‐C. Huang, C.‐Y. Ou, T.‐Y. Hsu, H. Chuang, J.‐C. Chang, L. Wang, H.‐C. Kuo and K. D. Yang, Clinical & Experimental Allergy, 2010 (40) 1482–1490.     

Invariant natural killer T‐cell development and function with loss of microRNA‐155

Invariant natural killer T (iNKT) cells are adaptive T cells with innate‐like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR‐155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon‐γ expression. We examined the role of miR‐155 in the development and function of iNKT cells. Using germline‐deficient miR‐155 mice, we showed that loss of miR‐155 resulted in unchanged iNKT cell frequency and cell number. Although miR‐155 was up‐regulated in iNKT cells upon activation with α‐galactosylceramide, loss of miR‐155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR‐155 expression.     

Application of talcum powder,trichloroacetic acid and silver nitrate in female rats for non‐surgical sterilization: evaluation of the apoptotic pathway mRNA and miRNA genes

There are several methods used for non‐surgical sterilization in birth control including quinacrine, trichloroacetic acid (TCA), erythromycin, tetracycline, silver nitrate and talcum powder. Among these, talcum powder, TCA and silver nitrate are the most commonly used. However, the toxic and carcinogenic activities of these chemicals in ovarian tissue have been poorly elucidated. This study demonstrates the expression levels of antioxidant, apoptotic and anti‐apoptotic genes after administration of talc powder, TCA and silver nitrate for non‐surgical sterilization in female rat models. The expression changes of some microRNAs (miR‐15b, miR‐21, miR‐34a and miR‐98) that play key roles in the apoptosis pathway were also included. All expression analyses were evaluated with real‐time PCR. The expression levels of all genes appeared to be upregulated in the talcum powder group, but the results were not statistically significant. Increased expression of Gsr and Sod1 genes was statistically significant in the talcum powder group. In TCA and silver nitrate group, expression of all genes was appeared to be elevated but only the Gsr expression was statistically significant in the TCA‐administrated group; there were no statistically significant changes in the silver nitrate group. miRNA expression levels were increased in talcum powder and TCA‐administrated groups, but these results were not significant. Expression levels of miR‐15b, miR‐21 and miR‐98 in the silver nitrate group were significantly increased. Consequently, these chemicals appear to be non‐carcinogenic agents for rat ovarian tissue which do not induce apoptosis. However, talcum powder and TCA can be considered as agents that are toxic to ovarian tissue.     

Induction of apoptosis in monocytes and lymphocytes by serum from patients with systemic lupus erythematosus − an additional mechanism to increased autoantigen load?

The most likely source of autoantigens in systemic lupus erythematosus (SLE) is apoptotic material. Because increased levels of circulating apoptotic cells are found in SLE we wanted to investigate the capacity of serum from patients with SLE or other autoimmune or infectious diseases and normal healthy donors (NHD) to induce apoptosis in normal monocytes, lymphocytes and corresponding cell lines, in relation to clinical and immunological data. Monocytes and lymphocytes from healthy donors were incubated with sera from 37 SLE patients, 37 sex- and age-matched NHD and sera from patients with rheumatoid arthritis, vasculitis, sepsis and mononucleosis. Sera from SLE patients were sampled at both active and inactive disease. The apoptosis-inducing effect (AIE) of these sera was monitored with flow cytometry using annexin V and propidium iodide (PI) binding. The AIE in monocytes and lymphocytes was significantly higher in sera from SLE patients than in other patient groups and NHD (P < 0.001) and was also higher when cell lines were used. Level of C5a in cell culture supernatant correlated with AIE in monocytes (r = 0.451, P = 0.005), suggesting involvement of complement. Heat-inactivation of sera did not affect the AIE, nor did depletion of IgG by protein G absorption of serum. Kinetic analyses showed a peak in apoptosis induction at 12-16 h, with a delayed PI positivity. AIE was equally high using sera from active and inactive SLE cases, and did not correlate with the SLE Disease Activity Index (SLEDAI). Thus, SLE serum has a strong and apparently disease-specific apoptosis-inducing capacity, which could contribute to a high load of potential autoantigen.     

MicroRNA profiling of human intermediate monocytes

Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes.By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of <10⁻¹⁰, of which two miRNAs – miR-6087 (upregulated) and miR‐150-5p (downregulated) – differed in their expression more than ten-fold. Pathway analysis of the 38 differentially expressed miRNAs linked intermediate monocytes to distinct biological processes such as gene regulation, cell differentiation, toll-like receptor signaling as well as antigen processing and presentation. Moreover, differentially expressed miRNAs were connected to those genes that we previously identified as markers of intermediate monocytes.In aggregation, we provide first genome-wide miRNA data in the context of monocyte heterogeneity, which substantiate the concept of monocyte trichotomy in human immunity. The identification of miRNAs that are specific for intermediate monocytes may allow to develop strategies, which particularly target this cell population while sparing the other two subsets.     

Microparticles shed from different antigen-presenting cells display an individual pattern of surface molecules and a distinct potential of allogeneic T-cell activation

Various cells such as platelets, lymphocytes, endothelial cells, red blood cells and monocytes do release surface-derived microparticles (mps). We analysed mp isolated from supernatants of cultured antigen-presenting human cells (APCs) and human cell lines. Particle sizing by dynamic light scattering revealed a characteristic size of the particles ranging from 80 nm to 300 nm in viable cells and from 400 nm to 1200 nm in irradiated cells. Employing flow-cytometry, we observed partly an altered surface protein composition of the mp compared to their cellular source. Mp originating from dendritic cells (DCs) differed in their surface composition from those released from monocytes and monocyte-derived macrophages. In functional assays, these mp stimulated alloreactive T-cells. The treatment of the cells with either UV-B or lipopolysaccharide strongly influenced the quantity, the immunostimulatory features and the surface composition of the mp. Mp from apoptotic macrophages were able to reduce the stimulatory capacity of vital macrophages but not of DC. Apoptotic mps from DC, on the other hand, were always stimulatory. This is the first report regarding the study of mp released from DC and compared with those released from other APC.     

Altered miR‐155 Expression in Allergic Asthmatic Airways

We and others have previously identified microRNAs (miRNAs) with pathological roles in animal models of asthma, where miR‐146a and miR‐155 have been described to play important roles in inflammatory responses. To date, few studies have investigated miRNA expression in human asthmatics. In the current study, significantly lower levels of miR‐155 were detected in cell‐free sputum from allergic asthmatics compared to healthy controls. Induced sputum isolated from allergic asthmatics in and out of pollen season revealed that miR‐155 expression, but not miR‐146a, is reduced in lymphocytes in season compared to post‐season. In contrast, miR‐155 was found to increase, whereas miR‐146a decreased in PBMCs and cell‐free PBMC culture media upon T cell receptor stimulation via αCD3/CD28 in both allergic asthmatics and healthy controls. Our findings suggest that miR‐155 is differentially expressed ex vivo in airways of allergic asthmatics compared to healthy controls, which may have implications in the local immune response in allergic asthma.     

SLE serum induces classical caspase-dependent apoptosis independent of death receptors

The main source of autoantigens in systemic lupus erythematosus (SLE) is most likely apoptotic material. We have previously shown that sera from SLE patients can induce apoptosis in monocytes and lymphocytes, and here we characterized mechanisms of apoptosis induced by SLE serum. SLE serum seems to induce caspase-dependent classical apoptosis since cells exposed to SLE serum displayed morphology consistent with classical apoptosis as demonstrated by confocal microscopy, and pan-caspase inhibitor Z-VAD.fmk significantly reduced SLE serum-induced apoptosis. Death-receptor-independent pathways seemed to be involved since SLE serum induced apoptosis equally in FADD-mutant and wild-type Jurkat cell lines, and blocking of Fas and TNFR1 did not reduce apoptosis induction. Importantly, apoptosis was significantly reduced in a Bcl-2 overexpressing Jurkat cell line indicating involvement of mitochondrial pathways. Thus, based on morphology and caspase inhibition experiments, we have demonstrated that SLE serum induce classical caspase-dependent apoptosis, and this was independent of death receptor pathways.     

H2O2 induces monocyte apoptosis and reduces viability of Mycobacterium avium-M. intracellulare within cultured human monocytes.

Mycobacterium avium-M. intracellulare, an intracellular parasite of mononuclear phagocytes, rarely causes disease in immunocompetent individuals. In contrast, in human immunodeficiency virus type 1-infected patients, M. avium-M. intracellulare can infect almost every tissue and organ. This suggests that immunocompetent individuals have a protective mechanism to control or prevent the infection. How mycobacterial may be killed by the host immune response is unclear. We have recently reported that induction of apoptosis of Mycobacterium bovis BCG-infected macrophages with ATP4- was associated with killing of the intracellular mycobacteria. In the present study, a long-term culture of M. avium-M. intracellulare-infected monocytes was used to further evaluate the interaction between M. avium-M. intracellulare and primary human monocytes. In our system, M. avium-M. intracellulare parasitized the human monocytes and appeared to replicate slowly over 14 days within the host cells. To examine the role of apoptotic mechanisms in survival or death of intracellular mycobacteria, M. avium-M. intracellulare-infected human monocytes were treated with a monoclonal antibody to Fas receptor (APO-1/CD95) or with various concentrations of H2O2. Although both of these exogenous agents induced monocyte apoptosis, optimal killing (65% reduction in CFU) of intracellular M. avium-M. intracellulare was observed only when M. avium-M. intracellulare-infected cells were treated with 10 mM H2O2. Fas-induced apoptosis did not affect M. avium-M. intracellulare viability. Our results suggest that not all stimuli of monocyte apoptosis induce killing of intracellular M. avium-M. intracellulare. Since release of H2O2 following phagocytosis of mycobacteria has been documented, H2O2-induced apoptotic death of M. avium-M. intracellulare-infected monocytes and its association with killing of the intracellular bacilli may be a physiological mechanism of host defense against M. avium-M. intracellulare.     

FSAP‐mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE

Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII‐activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP‐mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma‐purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross‐linking is involved. In conclusion, FSAP‐mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.     

Ectosomes as modulators of inflammation and immunity

Vesicles released by cells have been described using various names, including exosomes, microparticles, microvesicles and ectosomes. Here we propose to differentiate clearly between ectosomes and exosomes according to their formation and release. Whereas exosomes are formed in multi-vesicular bodies, ectosomes are vesicles budding directly from the cell surface. Depending upon the proteins expressed, exosomes activate or inhibit the immune system. One of the major properties of exosomes released by antigen-presenting cells is to induce antigen-specific T cell activation. Thus, they have been used for tumour immunotherapy. By contrast, the major characteristics of ectosomes released by various cells, including tumour cells, polymorphonuclear leucocytes and erythrocytes, are the expression of phosphatidylserine and to have anti-inflammatory/immunosuppressive activities similarly to apoptotic cells.