An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-kappaB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-kappaB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-kappaB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.     

Enhanced expression of Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein induces resistance to Fas-mediated apoptosis in activated mast cells

Mast cells play a critical role in host immune responses and are implicated in the pathogenesis of allergic inflammation. Though mouse mast cell line MC/9 expresses cell surface Fas Ag and is sensitive to Fas-induced apoptosis, activated MC/9 cells are resistant to Fas-induced cell death by cross-linking of FcepsilonRI or FcgammaR. Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP), a caspase-8 inhibitor that lacks the cysteine domain, is one of the negative regulators of receptor-mediated apoptosis. In this report, we show that activation of mast cells by cross-linking of FcepsilonRI or FcgammaR can induce enhanced expression of FLIP and consequently a resistance to Fas-induced apoptosis, although the expression level of Fas Ag is not changed. Addition of antisense oligonucleotide for FLIP prevents resistance to Fas-induced apoptosis of activated mast cells, suggesting that endogenous FLIP inhibits Fas-mediated apoptosis in activated mast cells. Thus, the enhanced expression of FLIP in activated mast cells contributes to the resistance to Fas-induced apoptosis, which may result in the development and prolongation of allergic inflammation.     

Balance of apoptotic cell death and survival in allergic diseases

Allergic diseases result from over-reaction of the immune system in response to exogenous allergens, where inflammatory cells have constantly extended longevity and contribute to an on-going immune response in allergic tissues. Here, we review disequilibrium in the death and survival of epithelial cells and inflammatory cells in the pathological processes of asthma, atopic dermatitis, and other allergic diseases.     

Regulation of apoptosis in mast cells

Apoptosis is a physiological process of cell death that occurs in all multicellular organisms. Its dysregulation has been postulated as one of the main causes in the development of diseases such as cancer, AIDS, autoimmune diseases and allergy. Apoptosis has been mainly studied in the inflammatory cells that participate in the late and chronic stages of allergy (eosinophils, neutrophils, lymphocytes and macrophages) as a new way to elucidate the pathogenesis of this disease. Nevertheless, much less it is known about the regulation of apoptosis in the initiators of the allergic process: The Mast Cells. In normal conditions, mast cells are described as long-living cells that keep a constant number of cells in tissues. However, increased numbers of mast cells are observed in the late phase of asthma and in both the inflammatory and in the repair/remodeling stage of various inflammatory/fibrotic disorders. In this report, we discuss the possible mechanisms that regulate the apoptotic process in normal conditions and disease, such as survival factors and death receptors. A link between mast cell activation, during the early stages of the allergic process, and triggering of anti-apoptotic signaling pathways is also suggested as an important contributor to the extended life of mast cells.     

Human eosinophils: Apoptosis versus survival in the mediation of inflammation

Eosinophil-derived mediators are thought to make a major contribution to the inflammation underlying a number of allergic diseases, most notably asthma. The toxic potential of eosinophils at tissue sites of inflammation might be limited if they were cleared by the process of programmed cell death or apoptosis. In this review we have examined the relationship between the signal transduction pathways important in controlling cytokine-induced prolonged survival and the mechanisms responsible for the induction and control of apoptosis in the eosinophil. A greater understanding of these processes might result in the development of novel therapeutic agents which would promote the safe and rapid removal by apoptosis of this important pro-inflammatory cell.     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice

Helminths and their products can shape immune responses by modulating immune cells, which are dysfunctional in inflammatory diseases such as asthma. We previously identified SJMHE1, a small molecule peptide from the HSP60 protein of Schistosoma japonicum. SJMHE1 can inhibit delayed‐type hypersensitivity and collagen‐induced arthritis in mice. In the present study, we evaluated this peptide's potential intervention effect and mechanism on ovalbumin‐induced asthma in mice. SJMHE1 treatment suppressed airway inflammation in allergic mice, decreased the infiltrating inflammatory cells in the lungs and bronchoalveolar lavage fluid, modulated the production of pro‐inflammatory and anti‐inflammatory cytokines in the splenocytes and lungs of allergic mice, reduced the percentage of Th2 cells and increased the proportion of Th1 and regulatory T cells (Tregs). At the same time, Foxp3 and T‐bet expression increased, and GATA3 and RORγt decreased in the lungs of allergic mice. We proved that SJMHE1 can interrupt the development of asthma by diminishing airway inflammation in mice. The down‐regulation of Th2 response and the up‐regulation of Th1 and Tregs response may contribute to the protection induced by SJMHE1 in allergic mice. SJMHE1 can serve as a novel therapy for asthma and other allergic or inflammatory diseases.     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

NK cells are effectors for resolvin E1 in the timely resolution of allergic airway inflammation

Immune responses are pathologically sustained in several common diseases, including asthma. To determine endogenous proresolving mechanisms for adaptive immune responses, we used a murine model of self-limited allergic airway inflammation. After cessation of allergen exposure, eosinophils and T cells were cleared concomitant with the appearance of increased numbers of NK cells in the lung and mediastinal lymph nodes. The mediastinal lymph node NK cells were activated, expressing CD27, CD11b, CD69, CD107a, and IFN-γ. NK cell depletion disrupted the endogenous resolution program, leading to delayed clearance of airway eosinophils and Ag-specific CD4(+) T cells. NK cell trafficking to inflamed tissues for resolution was dependent upon CXCR3 and CD62L. During resolution, eosinophils and Ag-specific CD4(+) T cells expressed NKG2D ligands, and a blocking Ab for the NKG2D receptor delayed clearance of these leukocytes. Of interest, NK cells expressed CMKLR1, a receptor for the proresolving mediator resolvin E1, and depletion of NK cells decreased resolvin E1-mediated resolution of allergic inflammation. Resolvin E1 regulated NK cell migration in vivo and NK cell cytotoxicity in vitro. Together, these findings indicate new functions in catabasis for NK cells that can also serve as targets for proresolving mediators in the resolution of adaptive immunity.     

Association of AIM, a novel apoptosis inhibitory factor, with hepatitis via supporting macrophage survival and enhancing phagocytotic function of macrophages

A hallmark of many inflammatory diseases is the destruction of tissue cells by infiltrating hematopoietic cells including lymphocytes, neutrophils, and macrophages. The regulation of apoptosis of both target tissue cells and the infiltrating cells is one of the key events that defines the initiation and the progression of inflammation. However, the precise picture of the apoptosis regulation of the cells at the inflammatory sites is still unclear. We recently isolated a novel apoptosis inhibitory factor, termed AIM, which is secreted exclusively by tissue macrophages. In this report, we present unique characteristics of AIM associated with liver inflammation (hepatitis), identified by introducing an experimental hepatitis in both AIM-transgenic mice, which overexpress AIM in the body, and normal mice. First, endogenous AIM expression in macrophages is rapidly increased in response to inflammatory stimuli. Second, AIM appears to inhibit the death of macrophages in the inflammatory regions, judging by the remarkably increased number of macrophages observed in the liver from transgenic mice. In addition, we show that AIM also enhances the phagocytosis by macrophages, which emphasizes the multifunctional character of AIM. All these findings strongly provoke an idea that AIM may play an important role in hepatitis pathogenesis in a sequential manner; first AIM expression is up-regulated by inflammatory stimuli, and then in an autocrine fashion, AIM supports the survival of infiltrating macrophages as well as enhances phagocytosis by macrophages, which may result in an efficient clearance of dead cells and infectious or toxic reagents.     

Neuroglial response after induction of experimental allergic encephalomyelitis in susceptible and resistant rat strains

Experimental allergic encephalomyelitis (EAE), the animal model for multiple sclerosis in humans, a T-cell mediated disease of the central nervous system is characterized by inflammatory infiltrates of myelin antigen(s)-specific T cells and consecutive demyelination. Spinal cord tissue emulsified in complete Freund's adjuvant clinical disease in the genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although similar inflammatory infiltrates in the CNS are observed in both strains 10-12 days after induction. We have shown that the resistance to clinical disease of AO rats is associated with rapid clearance of infiltrating mononuclear cells by a mechanism of apoptosis. Here, we demonstrate by immunohistochemical and FACS analyses of the expression of CD11b/c that microglial cells respond differently to disease induction in the two strains. Whereas microglial cells are activated throughout the period of day 10-28 days after EAE induction in AO rats they are only activated at the inception and resolution phases but not at the peak of clinical disease in DA rats when there is the highest level of CD4+ T cell infiltration. Our findings are compatible with the notion that microglia terminate effector T cells by apoptosis and that lack of this mechanism as evidenced by the lack of CD11b/c expression, support T cell survival and clinical expression of disease.     

PGE2 Promotes Apoptosis Induced by Cytokine Deprivation through EP3 Receptor and Induces Bim in Mouse Mast Cells

Increased mast cell numbers are observed at sites of allergic inflammation and restoration of normal mast cell numbers is critical to the resolution of these responses. Early studies showed that cytokines protect mast cells from apoptosis, suggesting a simple model in which diminished cytokine levels during resolution leads to cell death. The report that prostaglandins can contribute both to recruitment and to the resolution of inflammation together with the demonstration that mast cells express all four PGE₂ receptors raises the question of whether a single PGE₂ receptor mediates the ability of PGE₂ to regulate mast cell survival and apoptosis. We report here that PGE₂ through the EP3 receptor promotes cell death of mast cells initiated by cytokine withdrawal. Furthermore, the ability of PGE₂ to limit reconstitution of tissues with cultured mast cells is lost in cell lacking the EP3 receptor. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨ_m), increased caspase-3 activation, chromatin condensation, and low molecular weight DNA cleavage. PGE₂ augmented cell death is dependent on an increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE₂ can contribute to the resolution of inflammation in part by augmenting the removal of inflammatory cells in this case, mast cells.     

Apoptosis and inflammation

During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972) introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.     

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H₂O₂)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H₂O₂-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator’s expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.     

Identification of a common gene expression response in different lung inflammatory diseases in rodents and macaques

To identify gene expression responses common to multiple pulmonary diseases we collected microarray data for acute lung inflammation models from 12 studies and used these in a meta-analysis. The data used include exposures to air pollutants; bacterial, viral, and parasitic infections; and allergic asthma models. Hierarchical clustering revealed a cluster of 383 up-regulated genes with a common response. This cluster contained five subsets, each characterized by more specific functions such as inflammatory response, interferon-induced genes, immune signaling, or cell proliferation. Of these subsets, the inflammatory response was common to all models, interferon-induced responses were more pronounced in bacterial and viral models, and a cell division response was more prominent in parasitic and allergic models. A common cluster containing 157 moderately down-regulated genes was associated with the effects of tissue damage. Responses to influenza in macaques were weaker than in mice, reflecting differences in the degree of lung inflammation and/or virus replication. The existence of a common cluster shows that in vivo lung inflammation in response to various pathogens or exposures proceeds through shared molecular mechanisms.     

Gp130 signaling promotes development of acute experimental colitis by facilitating early neutrophil/macrophage recruitment and activation

IL-6 is known to play a crucial role in the pathogenesis of chronic intestinal inflammation by modulating T cell functions. In this study, we investigated the role of gp130, the common signal transducer for all IL-6 cytokines, in a murine model of acute T cell independent colitis to better characterize the impact of gp130 on innate immune cells and the early stages of inflammation. Experimental colitis was induced by dextran sulfate sodium treatment of mice with inducible systemic deletion of gp130 (MxCre/gp130(-/-)), macrophage/neutrophil-specific gp130-deficiency (LysCre/gp130(-/-)), or bone marrow chimeric mice and compared with wild-type controls (gp130(f/f)). Systemic deletion of gp130 (MxCre/gp130(-/-)) protected mice from severe colitis and wasting and attenuated the mucosal inflammatory infiltrate as well as local cytokine, chemokine, and adhesion molecule expression. Experiments in newly generated macrophage/neutrophil-specific gp130-deleted animals (LysCre/gp130(-/-)) and gp130 bone marrow chimeric mice, revealed a dual mechanism of proinflammatory effects mediated by gp130. Leukocyte recruitment was impaired in gp130-deleted animals and gp130-deleted recipients of wild-type bone marrow, demonstrating a central role of gp130-dependent signals in nonmyeloid cells for directing leukocytes to sites of inflammation, which was further confirmed in a model of sterile peritonitis. In contrast, macrophage/neutrophil-specific gp130 deficiency delayed and attenuated the disease but only marginally affected the inflammatory infiltrate, indicating a defective activation of mucosal leukocytes. We provide evidence that IL-6 cytokines acting via gp130 are required in the acute stages of intestinal inflammation by modulating the dynamics of innate immune cell recruitment and activation.     

Induction of eosinophil apoptosis by the cyclin-dependent kinase inhibitor AT7519 promotes the resolution of eosinophil-dominant allergic inflammation

Background

Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo.

Methodology/Principal Findings

Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk).

Conclusions

Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.