Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47(-/-) red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/α2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor-inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47(-/-) red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis.     

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE^−/− mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.     

TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells

The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.     

Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1

Apoptotic cells are swiftly phagocytosed by macrophages and immature dendritic cells. In this study, we found that one mouse macrophage cell line (BAM3) engulfed apoptotic thymocytes, but not a lymphoma cell line (WR19L). mAbs that inhibited the phagocytosis of apoptotic thymocytes by BAM3 were identified. Purification of the Ag revealed that it was Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1 (SHPS-1). CD47, the ligand for SHPS-1, was expressed in mouse thymocytes, but not in WR19L. When WR19L was transformed with CD47, the transformants, after induction of apoptosis, could be phagocytosed by BAM3. The WR19L transformants expressing CD47 were more efficiently engulfed in vivo by splenic dendritic cells than the parental WR19L. Masking of the phosphatidylserine exposed on apoptotic thymocytes inhibited the engulfment, whereas the anti-SHPS-1 mAb inhibited not only the engulfment, but also the binding of apoptotic cells to phagocytes. These results indicate that macrophages require CD47 and phosphatidylserine on apoptotic cells for engulfment, and suggest that the interaction between CD47 and SHPS-1 works as a tethering step in the phagocytosis.     

Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.     

β 2 -Glycoprotein I (Apolipoprotein H) Modulates Uptake and Endocytosis Associated Chemiluminescence in Rat Kupffer Cells

&#103 ₂-Glycoprotein I (&#103 ₂GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. &#103 ₂GPI influence upon the reactive species production by Kupffer cells was evaluted in order to investigate whether &#103 ₂GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolmyristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25mol% phosphatidylserine (PS) or 50mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). &#103 ₂GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat &#103 ₂GPI. Albumin (500 &#119 g/ml) showed no effect upon chemiluminescence. &#103 ₂GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of &#103 ₂GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of &#103 ₂GPI. At a concentration of 125 &#119 g/ml, &#103 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of &#103 ₂GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of &#103 ₂GPI as a mediator of senescent cell removal.     

Neutrophils Regulate Tissue Neutrophilia in Inflammation via the Oxidant-modified Lipid Lysophosphatidylserine

Resolution of neutrophilia characteristic of acute inflammation requires cessation of neutrophil recruitment and removal of tissue neutrophils. Based on in vitro studies, a role in these events was hypothesized for oxidant-generated lysophosphatidylserine (lyso-PS) on recruited neutrophils signaling via the G2A receptor on macrophages. Peritoneal exudate neutrophils harvested from wild type (WT) mice had 5-fold more lyso-PS (lyso-PS^high) than those of gp91^phox−/− (lyso-PS^low) mice. Ex vivo engulfment of lyso-PS^high neutrophils (95% viable) by WT peritoneal macrophages was quantitatively similar to UV-irradiated apoptotic blood neutrophils, although the signaling pathway for the former was uniquely dependent on macrophage G2A. In contrast, lyso-PS^low neutrophils were poorly engulfed unless presented with exogenous lyso-PS. Enhanced clearance of lyso-PS^high neutrophils was also seen in vivo following their adoptive transfer into inflamed peritonea of WT but not G2A^−/− mice, further supporting a requirement for signaling via G2A. To investigate downstream effects of lyso-PS/G2A signaling, antibody blockade of G2A in WT mice reduced macrophage CD206 expression and efferocytosis during peritonitis. Conversely, adoptive transfer of lyso-PS^high neutrophils early in inflammation in gp91^phox−/− mice led to accelerated development of efferocytic^high and CD206^high macrophages. This macrophage reprogramming was associated with suppressed production of pro-inflammatory mediators and reduced neutrophilia. These effects were not seen if G2A was blocked or lyso-PS^low neutrophils were transferred. Taken together, the results demonstrate that oxidant-generated lyso-PS made by viable tissue neutrophils is an endogenous anti-inflammatory mediator working in vivo to orchestrate the “early” and rapid clearance of recruited neutrophils as well as the reprogramming of “resolving” macrophages.     

Selective Depletion of Eosinophils or Neutrophils in Mice Impacts the Efficiency of Apoptotic Cell Clearance in the Thymus

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b⁺ cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b⁺ cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b⁺ cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal.     

Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: A possible link between the nervous system and atherosclerosis development

Objective

The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the “cholinergic anti-inflammatory reflex” (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (α7^−/−) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in α7^−/− mice is associated with enhanced serum and macrophage atherogenicity.

Methods

We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from α7^−/− mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE^−/−) mice.

Results

α7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, α7-deficiency significantly increased serum CRP and IL-6 (p < 0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the α7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the α7 receptor (p < 0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, α7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p < 0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the α7^−/− mice.

Conclusion

The above results suggest an anti-atherogenic role for the macrophage α7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.     

CD4(+) T-cells are important in regulating macrophage polarization in C57BL/6 wild-type mice

During activation, macrophages undergo physiological changes affecting their surface protein expression and cytokine production and have been subsequently categorized into M1 (classically-activated) and M2 (alternatively-activated) macrophages. It remains unclear which lymphocyte population provides the immune microenvironment to regulate macrophage polarization. In this study, we establish a functional and phenotypic profile of peritoneal macrophages from C57BL/6 wild-type mice. We also showed that Rag1^−/− and Rag2^−/−γc^−/− mice have similar, exaggerated M1 characteristics in comparison to control mice, suggesting that NK and/or NK-T cells may not be essential in this process. By controlling for environmental factors, we determine that lymphocyte-derived cytokines, rather than inherent properties of macrophages themselves, are crucial for their regulation. Lastly, we report that macrophages from CD4^−/− mice display an M1 profile, suggesting that CD4⁺ T-cells play a dominant role over other lymphocyte populations in providing the cytokine environment for regulating macrophages towards an M2 profile under normal wild-type conditions.     

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PS^POS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PS^POS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNEL^POS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PS^POS and PS-negative (PS^NEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PS^NEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PS^POS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.     

CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes failed to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.     

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14⁺ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P₃ antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP⁺ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP⁺ cells represented more significant populations in tissues of Cd14^−/− than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14⁺ phagocytes.Subject terms: Phospholipids, Cell death and immune response     

TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) signals through Toll-like receptor 2 (TLR2) to regulate antigen presenting cells (APCs). Mtb lipoproteins, including LpqH, LprA, LprG and PhoS1, are TLR2 agonists, but their co-receptor requirements are unknown. We studied Mtb lipoprotein-induced responses in TLR2^−/−, TLR1^−/−, TLR6^−/−, CD14^−/− and CD36^−/− macrophages. Responses to LprA, LprG, LpqH and PhoS1 were completely dependent on TLR2. LprG, LpqH, and PhoS1 were dependent on TLR1, but LprA did not require TLR1. None of the lipoproteins required TLR6, although a redundant contribution by TLR6 cannot be excluded. CD14 contributed to detection of LprA, LprG and LpqH, whereas CD36 contributed only to detection of LprA. Studies of lung APC subsets revealed lower TLR2 expression by CD11b^high/CD11c^low lung macrophages than CD11b^low/CD11c^high alveolar macrophages, which correlated with hyporesponsiveness of lung macrophages to LpqH. Thus, lung APC subsets differ in TLR expression, which may determine differences in responses to Mtb.     

Dose-dependent inhibitory effect of CD47 in macrophage uptake of IgG-opsonized murine erythrocytes

The cell surface glycoprotein CD47 on target cells can bind to the inhibitory receptor SIRPalpha on macrophages to inhibit phagocytosis of antibody sensitized blood cells. The aim of this study was to determine if CD47 dose-dependently can regulate macrophage uptake of IgG-opsonized RBCs. CD47(+/-) RBCs express about 50% of the CD47 level found on CD47(+/+) RBCs. When injected into CD47(+/+) mice, CD47(+/-) RBCs showed a significantly faster antibody-mediated clearance as compared with CD47(+/+) RBCs injected into the same recipient. In vitro phagocytosis experiments confirmed that CD47(+/-) RBCs were taken up significantly more than CD47(+/+) RBCs, but significantly less than CD47(-/-) RBCs. A reduction in RBC CD47 expression just below 50% of that in normal RBCs can significantly accelerate RBC clearance by macrophages in the presence of RBC autoantibodies. This may have relevance for transfusion of stored RBCs, where loss of CD47 is seen over time, and in clearance of these cells by antibody-dependent phagocytosis.     

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

Steady-state and transient polarized absorption spectroscopy of photosytem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosytem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(−), 683-685(−), 696-697(−), and 711(−) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700⁺A₁⁻ or ³P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P_A, whereas the cation is localized most likely on P_B.