Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.     

Thymic stromal lymphopoietin is a key mediator of breast cancer progression

Inflammation is a double-edged sword that can promote or suppress cancer progression. In this study, we report that thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, is also expressed in human and murine cancers. Our studies with murine cancer cells indicate that TSLP plays an essential role in cancer escape, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP receptor on CD4(+) T cells, promoting Th2-skewed immune responses and production of immunosuppressive factors such as IL-10 and IL-13. Expression of TSLP therefore may be a useful prognostic marker, and its targeting could have therapeutic potential.     

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.     

CD99 is a key mediator of the transendothelial migration of neutrophils

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.     

Grb2 is a key mediator of helicobacter pylori CagA protein activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.     

Wnt signaling is a key mediator of Cdx1 expression in vivo

In the mouse, Cdx1 is essential for normal anteroposterior vertebral patterning through regulation of a subset of Hox genes. Retinoic acid (RA) and certain Wnts have also been implicated in vertebral patterning, although the relationship between these signaling pathways and the regulation of mesodermal Hox gene expression is not fully understood. Prior work has shown that Cdx1 is a direct target of both Wnt and retinoid signaling pathways, and might therefore act to relay these signals to the Hox genes. Wnt and RA are believed to impact on Cdx1 through an atypical RA-response element (RARE) and Lef/Tcf-response elements (LRE), respectively, in the proximal promoter. To address the roles of these regulatory motifs and pathways, we derived mice mutated for the LRE or the LRE plus the RARE. In contrast to RARE-null mutants, which exhibit limited vertebral defects, LRE-null and LRE+RARE-null mutants exhibited vertebral malformations affecting the entire cervical region that closely phenocopied the malformations seen in Cdx1-null mutants. Mutation of the LRE also greatly reduced induction of Cdx1 by RA, demonstrating a requirement for Wnt signaling in the regulation of this gene by retinoids. LRE and LRE+RARE mutants also exhibited vertebral fusions, suggesting a defect in somitogenesis. As Wnt signaling is implicated in somitogenesis upstream of the Notch pathway, it is conceivable that Cdx1 might play a role in this process. However, none of the Notch pathway genes assessed was overtly affected.     

Proteinase-activated receptor-2 promotes allergic sensitization to an inhaled antigen through a TNF-mediated pathway

The reason why particular inhaled Ags induce allergic sensitization while others lead to immune tolerance is unclear. Along with a genetic predisposition to atopy, intrinsic characteristics of these Ags must be important. A common characteristic of many allergens is that they either possess proteinase activity or are inhaled in particles rich in proteinases. Many allergens, such as house dust mite and cockroach allergens, have the potential to activate the proteinase-activated receptor (PAR)-2. In this study, we report that PAR-2 activation in the airways at the same time as exposure to inhaled Ags induces allergic sensitization, whereas exposure to Ag alone induces tolerance. BALB/c mice were administered OVA with a PAR-2 activating peptide intranasally. Upon allergen re-exposure mice developed airway inflammation and airway hyperresponsiveness, as well as OVA-specific T cells with a Th2 cytokine profile when restimulated with OVA in vitro. Conversely, mice given OVA alone or OVA with a PAR-2 control peptide developed tolerance. These tolerant mice did not develop airway inflammation or airway hyperresponsiveness, and developed OVA-specific T cells that secreted high levels of IL-10 when restimulated with OVA in vitro. Furthermore, pulmonary dendritic cell trafficking was altered in mice following intranasal PAR-2 activation. Finally, we showed that PAR-2-mediated allergic sensitization was TNF-dependent. Thus, PAR-2 activation in the airways could be a critical factor in the development of allergic sensitization following mucosal exposure to allergens with serine proteinase activity. Interfering with this pathway may prove to be useful for the prevention or treatment of allergic diseases.     

Breast milk-mediated transfer of an antigen induces tolerance and protection from allergic asthma

Allergic asthma is a chronic disease characterized by airway obstruction in response to allergen exposure. It results from an inappropriate T helper type 2 response to environmental airborne antigens and affects 300 million individuals. Its prevalence has increased markedly in recent decades, most probably as a result of changes in environmental factors. Exposure to environmental antigens during infancy is crucial to the development of asthma. Epidemiological studies on the relationship between breastfeeding and allergic diseases have reached conflicting results. Here, we have investigated whether the exposure of lactating mice to an airborne allergen affects asthma development in progeny. We found that airborne antigens were efficiently transferred from the mother to the neonate through milk and that tolerance induction did not require the transfer of immunoglobulins. Breastfeeding-induced tolerance relied on the presence of transforming growth factor (TGF)-beta during lactation, was mediated by regulatory CD4+ T lymphocytes and depended on TGF-beta signaling in T cells. In conclusion, breast milk-mediated transfer of an antigen to the neonate resulted in oral tolerance induction leading to antigen-specific protection from allergic airway disease. This study may pave the way for the design of new strategies to prevent the development of allergic diseases.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

Cell death is an important biological process that is believed to have a central role in intestinal ischaemia/reperfusion (I/R) injury. While the apoptosis inhibition is pivotal in preventing intestinal I/R, how necrotic cell death is regulated remains unknown. Necroptosis represents a newly discovered form of programmed cell death that combines the features of both apoptosis and necrosis, and it has been implicated in the development of a range of inflammatory diseases. Here, we show that receptor‐interacting protein 1/3 (RIP1/3) kinase and mixed lineage kinase domain‐like protein recruitment mediates necroptosis in a rat model of ischaemic intestinal injury in vivo. Furthermore, necroptosis was specifically blocked by the RIP1 kinase inhibitor necrostatin‐1. In addition, the combined treatment of necrostatin‐1 and the pan‐caspase inhibitor Z‐VAD acted synergistically to protect against intestinal I/R injury, and these two pathways can be converted to one another when one is inhibited. In vitro, necrostatin‐1 pre‐treatment reduced the necroptotic death of oxygen‐glucose deprivation challenged intestinal epithelial cell‐6 cells, which in turn dampened the production of pro‐inflammatory cytokines (tumour necrosis factor‐α and interleukin‐1β), and suppressed high‐mobility group box‐1 (HMGB1) translocation from the nucleus to the cytoplasm and the subsequent release of HMGB1 into the supernatant, thus decreasing the activation of Toll‐like receptor 4 and the receptor for advanced glycation end products. Collectively, our study reveals a robust RIP1/RIP3‐dependent necroptosis pathway in intestinal I/R‐induced intestinal injury in vivo and in vitro and suggests that the HMGB1 signalling is highly involved in this process, making it a novel therapeutic target for acute ischaemic intestinal injury.     

Granulocyte-macrophage colony-stimulating factor is a key mediator in experimental osteoarthritis pain and disease development

Introduction

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be important in the development of inflammatory models of rheumatoid arthritis and there is encouraging data that its blockade may have clinical relevance in patients with rheumatoid arthritis. The aims of the current study were to determine whether GM-CSF may also be important for disease and pain development in a model of osteoarthritis.

Methods

The role of GM-CSF was investigated using the collagenase-induced instability model of osteoarthritis. We studied both GM-CSF-/- mice and wild-type (C57BL/6) mice treated prophylactically or therapeutically with a monoclonal antibody to GM-CSF. Disease development (both early and late) was evaluated by histology and knee pain development was measured by assessment of weight distribution.

Results

In the absence of GM-CSF, there was less synovitis and matrix metalloproteinase-mediated neoepitope expression at week 2 post disease induction, and less cartilage damage at week 6. GM-CSF was absolutely required for pain development. Therapeutic neutralization of GM-CSF not only abolished the pain within 3 days but also led to significantly reduced cartilage damage.

Conclusions

GM-CSF is key to the development of experimental osteoarthritis and its associated pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abolished existing arthritic pain and suppressed the degree of arthritis development. Our results suggest that it would be worth exploring the importance of GM-CSF for pain and disease in other osteoarthritis models and perhaps clinically for this form of arthritis.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

The neuropeptide substance P is a critical mediator of burn-induced acute lung injury

The classical tachykinin substance P (SP) has numerous potent neuroimmunomodulatory effects on all kinds of airway functions. Belonging to a class of neuromediators targeting not only residential cells but also inflammatory cells, studying SP provides important information on the bidirectional linkage between how neural function affects inflammatory events and, in turn, how inflammatory responses alter neural activity. Therefore, this study aimed to investigate the effect of local burn injury on inducing distant organ pulmonary SP release and its relevance to lung injury. Our results show that burn injury in male BALB/c mice subjected to 30% total body surface area full thickness burn augments significant production of SP, preprotachykinin-A gene expression, which encodes for SP, and biological activity of SP-neurokinin-1 receptor (NK1R) signaling. Furthermore, the enhanced SP-NK1R response correlates with exacerbated lung damage after burn as evidenced by increased microvascular permeability, edema, and neutrophil accumulation. The development of heightened inflammation and lung damage was observed along with increased proinflammatory IL-1beta, TNF-alpha, and IL-6 mRNA and protein production after injury in lung. Chemokines MIP-2 and MIP-1alpha were markedly increased, suggesting the active role of SP-induced chemoattractants production in trafficking inflammatory cells. More importantly, administration of L703606, a specific NK1R antagonist, 1 h before burn injury significantly disrupted the SP-NK1R signaling and reversed pulmonary inflammation and injury. The present findings show for the first time the role of SP in contributing to exaggerated pulmonary inflammatory damage after burn injury via activation of NK1R signaling.     

Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

Differentiation of neuronal from non-neuronal Substance P

Substance P (SP) originally found as a neuropeptide in capsaicin-sensitive sensory neurons, had more recently been identified in non-neuronal cells, especially under pathological conditions. Neuronal and non-neuronal SP may perform distinct functions. A simple technique to differentiate different SP sources is currently unavailable. Herein, we describe a two-step sequential acetic acid extraction to differentiate SP source. The efficiency of this two-step extraction in differentiating SP in capsaicin-sensitive neurons was verified by using capsaicin as a tool to deplete SP in sensory neurons. Specifically, Balb-c mice were treated with high dose capsaicin (200 mg/kg). Skin was removed two weeks after treatment. In a separate experiment, lung and skin tissues from control animals (untreated) were incubated in-vitro with capsaicin, and sequential acetic acid extraction was performed. Following capsaicin treatment, both in-vivo and in-vitro, SP recovered in first extraction decreased significantly in lung and skin. Lastly, presence of capsaicin solvent (10% methanol and 10% Tween 80) or protease inhibitor cocktail in solution altered SP EIA test, yielding false positive results. These results demonstrated that SP in capsaicin sensitive sensory neurons was extracted in initial extraction of 15 min while non-neuronal SP was present in second extraction. Because SP in non-neuronal tissues may possibly be more important in pathological conditions, this technique could be useful in determining effects of various treatments on neuronal and non-neuronal SP levels and their consequences.