Impaired neonatal macrophage phagocytosis is not explained by overproduction of prostaglandin E2

<p>Abstract</p> <p>Background</p> <p>Neonates and young infants manifest increased susceptibility to bacterial, viral and fungal lung infections. Previous work has identified a role for eicosanoids in mediating host defense functions of macrophages. This study examines the relationship between alveolar macrophage (AM) host defense and production of lipid mediators during the neonatal period compared to adult AMs.</p> <p>Methods</p> <p>AMs were harvested from young (day 7 and day 14) and adult (~10 week) rats. The functionality of these cells was assessed by examining their ability to phagocytose opsonized targets, produce cytokines, eicosanoids and intracellular cAMP measured by enzyme immunoassays, and gene expression of proteins, enzymes and receptors essential for eicosanoid generation and phagocytosis measured by real time RT-PCR.</p> <p>Results</p> <p>AMs from young animals (day 7 and 14) were defective in their ability to phagocytose opsonized targets and produce tumor necrosis factor (TNF)- α. In addition, young AMs produce more prostaglandin (PG) E₂, a suppressor of host defense, and less leukotriene (LT) B₄, a promoter of host defense. Young AMs express higher levels of enzymes responsible for the production of PGE₂and LTB₄; however, there was no change in the expression of E prostanoid (EP) receptors or LT receptors. Despite the similar EP profiles, young AMs are more responsive to PGE₂as evidenced by their increased production of the important second messenger, cyclic AMP. In addition, young AMs express higher levels of PDE3B and lower levels of PDE4C compared to adult AMs. However, even though the young AMs produced a skewed eicosanoid profile, neither the inhibition of PGE₂by aspirin nor the addition of exogenous LTB₄rescued the defective opsonized phagocytosis. Examination of a receptor responsible for mediating opsonized phagocytosis showed a significant decrease in the gene expression levels of the Fcgamma receptor in young (day 7) AMs compared to adult AMs.</p> <p>Conclusion</p> <p>These results suggest that elevated production of PGE₂and decreased production of LTB₄do not contribute to impaired opsonized macrophage phagocytosis and highlight an important difference between young and adult AMs.</p

Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

Topographical and Biological Evidence Revealed FTY720-Mediated Anergy-Polarization of Mouse Bone Marrow-Derived Dendritic Cells In Vitro

Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies were used. FTY720 induced significant alterations of surface markers, as well as decline of shape indices, cell volume, surface roughness in LPS-activated mature BMDCs. These phenotypic, morphological and topographical changes were accompanied by FTY720-mediated down-regulation of proinflammatory cytokines, including IL-6, TNF-α, IL-12 and MCP-1. Together with suppressed nitric oxide (NO) production and CCR7 transcription in FTY720-treated BMDCs with or without LPS activation, an inhibitory mechanism of NO and cytokine reciprocal activation was suggested. This implication was supported by the impaired phagocytotic, endocytotic and specific antigen presentation abilities observed in the FTY720-treated BMDCs. In conclusion, we demonstrated FTY720 can induce anergy-polarization in both immature and LPS-activated mature BMDCs. A possible mechanism is FTY720-mediated reciprocal suppression on the intrinsic activation pathway and cytokine production with endpoint exhibitions on phagocytosis, endocytosis, antigen presentation as well as cellular morphology and topography

Altered maturation of peripheral blood dendritic cells in patients with breast cancer

Tumours have at least two mechanisms that can alter dendritic cell (DC) maturation and function. The first affects the ability of haematopoietic progenitors to differentiate into functional DCs; the second affects their differentiation from CD14+ monocytes, promoting an early but dysfunctional maturation. The aim of this study was to evaluate the in vivo relevance of these pathways in breast cancer patients. For this purpose, 53 patients with invasive breast cancer were compared to 68 healthy controls. To avoid isolation or culture procedures for enrichment of DCs, analyses were directly performed by flow cytometry on whole-blood samples. The expression of surface antigens and intracellular accumulation of regulatory cytokines upon LPS stimulation were evaluated. The number of DCs, and in particular of the myeloid subpopulation, was markedly reduced in cancer patients (P < 0.001). Patient DCs were characterized by a more mature phenotype compared with controls (P = 0.016), and had impaired production of IL-12 (P < 0.001), These alterations were reverted by surgical resection of the tumour. To investigate the possible role of some tumour-related immunoactive soluble factors, we measured the plasmatic levels of vascular endothelial growth factor, IL-10 and spermine. A significant inverse correlation between spermine concentration and the percentage of DCs expressing IL-12 was found. Evidence was also obtained that in vitro exposure of monocyte-derived DCs to spermine promoted their activation and maturation, and impaired their function. Taken together, our results suggest that both the above-described mechanisms could concomitantly act in breast cancer to affect DC differentiation, and that spermine could be a mediator of dysfunctional maturation of DCs

Co-Housing Rodents with Different Coat Colours as a Simple, Non-Invasive Means of Individual Identification:Validating Mixed-Strain Housing for C57BL/6 and DBA/2 Mice

Standard practice typically requires the marking of laboratory mice so that they can be individually identified. However, many of the common methods compromise the welfare of the individuals being marked (as well as requiring time, effort, and/or resources on the part of researchers and technicians). Mixing strains of different colour within a cage would allow them to be readily visually identifiable, negating the need for more invasive marking techniques. Here we assess the impact that mixed strain housing has on the phenotypes of female C57BL/6 (black) and DBA/2 (brown) mice, and on the variability in the data obtained from them. Mice were housed in either mixed strain or single strain pairs for 19 weeks, and their phenotypes then assessed using 23 different behavioural, morphological, haematological and physiological measures widely used in research and/or important for assessing mouse welfare. No negative effects of mixed strain housing could be found on the phenotypes of either strain, including variables relevant to welfare. Differences and similarities between the two strains were almost all as expected from previously published studies, and none were affected by whether mice were housed in mixed- or single-strain pairs. Only one significant main effect of housing type was detected: mixed strain pairs had smaller red blood cell distribution widths, a measure suggesting better health (findings that now need replicating in case they were Type 1 errors resulting from our multiplicity of tests). Furthermore, mixed strain housing did not increase the variation in data obtained from the mice: the standard errors for all variables were essentially identical between the two housing conditions. Mixed strain housing also made animals very easy to distinguish while in the home cage. Female DBA/2 and C57BL/6 mice can thus be housed in mixed strain pairs for identification purposes, with no apparent negative effects on their welfare or the data they generate. This suggests that there is much value in exploring other combinations of strains

Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity

The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions

Cysteinyl leukotrienes: multi-functional mediators in allergic rhinitis

Cysteinyl leukotrienes (CysLTs) are a family of inflammatory lipid mediators synthesized from arachidonic acid by a variety of cells, including mast cells, eosinophils, basophils, and macrophages. This article reviews the data for the role of CysLTs as multi-functional mediators in allergic rhinitis (AR). We review the evidence that: (1) CysLTs are released from inflammatory cells that participate in AR, (2) receptors for CysLTs are located in nasal tissue, (3) CysLTs are increased in patients with AR and are released following allergen exposure, (4) administration of CysLTs reproduces the symptoms of AR, (5) CysLTs play roles in the maturation, as well as tissue recruitment, of inflammatory cells, and (6) a complex inter-regulation between CysLTs and a variety of other inflammatory mediators exists.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75432/1/j.1365-2222.2006.02498.x.pd