Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Annexin A3 as a Marker Protein for Microglia in the Central Nervous System of Rats.

The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.

Myeloid-like γδ T cell subset in the immune response to an experimental Rift Valley fever vaccine in sheep.

γδ T cells are a numerically significant subset of immune cells in ruminants, where they may comprise up to 70 % of all peripheral blood mononuclear cells (PBMCs) in young animals and 25 % in adults. These cells can be activated through traditional TCR-dependent mechanisms, or alternatively in a TCR-independent manner by pattern recognition receptors and have been shown to uptake antigen, as well as process and present it to αß T cells. We have identified a novel CD11b+ subset of γδ T cells in normal sheep peripheral blood. An increase in the frequency of these cells in sheep peripheral blood in response to immunization with an experimental recombinant subunit Rift Valley fever (RVF) vaccine was observed. However, injection of the vaccine adjuvant ISA-25VG alone without the recombinant RVF virus antigens demonstrated the same effect, pointing to an antigen-independent innate immune function of CD11b+ γδ T cells in response to the adjuvant. In vitro studies showed repeatable increases of CD11b-, CD14-, CD86-, CD40-, CD72-, and IFNγ- expressing γδ T cells in PBMCs after 24 h of incubation in the absence of a mitogen. Moreover, the majority of these myeloid-like γδ T cells were demonstrated to process exogenous antigen even in the absence of mitogen. ConA activation increased CD25- and MHCII- expression in γδ T cells, but not the myeloid associated receptors CD14 or CD11b or co-stimulatory molecules such as CD86 and CD40. Considering the role of CD11b and CD14 in the activation of innate immunity, we hypothesize that this subpopulation of sheep γδ T cells may function as innate antigen presenting and pro-inflammatory cells during immune responses. The results presented here also suggest that stress molecules and/or damage-associated molecular patterns may be involved in triggering antigen presenting and pro-inflammatory functions of γδ T cells, given their appearance in vitro in the absence of specific stimulation. Taken together, these data suggest that the early appearance of γδ T cells following adjuvant administration and their possible role in early activation of αß T cell subsets may non-specifically contribute to augmented innate immunity and may promote strong initiation of the adaptive immune response to vaccines in general.

Translocator protein localises to CD11b+ macrophages in atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression. METHODS: ApoE-/- mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and established plaques by light sheet microscopy. RESULTS: While tissue resident F4/80 + macrophages were evident in the arteries of animals without atherosclerosis, no CD11b + macrophages were observed in these animals. In contrast, established plaques had high CD11b and low F4/80 expression. A ∼3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls. CONCLUSIONS: Imaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

Macranthol attenuates lipopolysaccharide-induced depressive-like behaviors by inhibiting neuroinflammation in prefrontal cortex.

Macranthol is a lignans natural product isolated from Illicium dunnianum Tutch. Our previous studies have shown that BDNF dependent signaling pathway activation was involved in the antidepressant-like effects of macranthol. However, it is not clear whether neuro-inflammation suppression is involved in the effects of macranthol. Therefore, the aim of this present study was to determine whether macranthol affected the neuro-inflammation system in lipopolysaccharide (LPS)-induced mice by measuring pro-inflammatory cytokines and CD11b. Macranthol was orally administrated for successive seven days before a single LPS injection. The behavioral evaluation showed that macranthol prevented LPS-induced depressive-like deficits both in sucrose preference test and forced swimming test. The elevation of serum and prefrontal cortex pro-inflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was decreased by macranthol pretreatment. In addition, LPS induced the elevation of CD11b in the prefrontal cortex, which was also inhibited by macranthol. Last but not the least, the immunofluorescence found that the number of positive iba-1 cells was also decreased by macranthol. These findings suggest that macranthol could alleviate depressive-like behaviors in mice induced by LPS that are mediated, at least by suppressing microglia-related neuro-inflammation in the prefrontal cortex.

Neutrophil priming that turns natural FFA2R agonists into potent activators of the superoxide generating NADPH-oxidase.

Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists.

Leishmania amazonensis induces modulation of costimulatory and surface marker molecules in human macrophages.

Manipulation of costimulatory and surface molecules that shape the extent of immune responses by Leishmania is suggested as one of the mechanisms of evading the host's defences. The experiments reported here were designed to evaluate the expressions of CD11b, CD11c, CD14, CD18, CD54, CD80, CD86, CD206, MHC class II and TLR-2 (Toll-like receptor 2) in human macrophages infected with L. amazonensis. Phenotypic evaluation revealed a negative modulation in CD11b, CD11c, CD14, CD18, CD54 and MHC class II molecules, depending on the level of infection. The results showed that as early as 1 hour after infection no reduction in marker expression occurs, whereas after 24 hours, downregulation of these molecules was observed in macrophages. No significant changes were observed in the expressions of CD80, CD86, CD206 and TLR2. Evidence of the differential modulation of markers expression and that after parasite uptake no reduction in surface marker expression occurs indicates that parasite internalization is not involved in the phenomena of down-modulation.

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

The role of MyD88- and TRIF-dependent signaling in monophosphoryl lipid A-induced expansion and recruitment of innate immunocytes.

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.

Melatonin Pretreatment Enhances the Homing of Bone Marrow-derived Mesenchymal Stem Cells Following Transplantation in a Rat Model of Liver Fibrosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has been considered as a promising milestone in liver fibrosis treatment. However, low amounts of homing are a major obstacle. We aimed to investigate the role of melatonin pretreatment in BMMSC homing into experimental liver fibrosis. METHODS: BMMSCs were obtained, grown, propagated and preconditioned with 5 µM melatonin and analyzed for multipotency and immunophenotypic features at passage three. The cells were labelled with CM-Dil and infused into the rats received the i.p. injection of carbon tetrachloride (CCl4) for five weeks to induce liver fibrosis. Animals were divided into two groups: One group received BMMSCs, whereas the other group received melatonin-pretreated BMMSCs (MT-BMMSCs). After cell injection at 72 h, animals were sacrificed, and the liver tissues were assessed for further evaluations: fibrosis using Masson's trichrome and hematoxylin and eosin staining and homing using fluorescent microscopy and flow cytometry. RESULTS: BMMSCs and MT-BMMSCs expressed a high level of CD44 but low levels of CD11b, CD45 and CD34 (for all P≤0.05) and were able to differentiate into adipocytes and Schwann cells. CCl4 induction resulted in extensive collagen deposition, tissue disruption and fatty accumulation with no obvious difference between the two groups. There was a significant increase in homing of MT-BMMSCs in both florescent microscopy (P≤0.001) and flow cytometry (P≤0.01) assays, as compared with non-treated BMMSCs. CONCLUSION: This study indicates the improved homing potential of BMMSCs in pretreatment with melatonin. Therefore, this strategy may represent an applied approach for improving the stem cell therapy of liver fibrosis.

99mTc-labelled anti-CD11b SPECT/CT imaging allows detection of plaque destabilization tightly linked to inflammation.

It remains challenging to predict the risk of rupture for a specific atherosclerotic plaque timely, a thrombotic trigger tightly linked to inflammation. CD11b, is a biomarker abundant on inflammatory cells, not restricted to monocytes/macrophages. In this study, we fabricated a probe named as (99m)Tc-MAG3-anti-CD11b for detecting inflamed atherosclerotic plaques with single photon emission computed tomography/computed tomography (SPECT/CT). The ApoE-knockout (ApoE(-/-)) mice were selected to establish animal models, with C57BL/6J mice used for control. A higher CD11b(+)-cell recruitment with higher CD11b expression and more serious whole-body inflammatory status were identified in ApoE(-/-) mice. The probe showed high in vitro affinity and specificity to the Raw-264.7 macrophages, as well as inflammatory cells infiltrated in atherosclerotic plaques, either in ex vivo fluorescent imaging or in in vivo micro-SPECT/CT imaging, which were confirmed by ex vivo planar gamma imaging, Oil-Red-O staining and CD11b-immunohistochemistry staining. A significant positive relationship was identified between the radioactivity intensity on SPECT/CT images and the CD11b expression in plaques. In summary, this study demonstrates the feasibility of anti-CD11b antibody mediated noninvasive SPECT/CT imaging of inflammatory leukocytes in murine atherosclerotic plaques. This imaging strategy can identify inflammation-rich plaques at risk for rupture and evaluate the effectiveness of inflammation-targeted therapies in atheroma.

S100A8 Production in CXCR2-Expressing CD11b+Gr-1high Cells Aggravates Hepatitis in Mice Fed a High-Fat and High-Cholesterol Diet.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with a spectrum of presentations. S100A8 has been suggested to play a pivotal role as an endogenous immune-activator in inflammatory diseases. In this study, we investigated the involvement of S100A8 in the development of NAFLD. We used a diet model of NAFLD, in which mice were fed either a high-fat and high-cholesterol diet (HFHCD) or a normal diet (ND) as a control. We also assessed liver tissues from patients with NAFLD, including patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). HFHCD-fed mice, but not ND-fed mice, developed steatohepatitis. S100A8 expression was significantly elevated in the livers of HFHCD-fed mice compared with the controls. S100A8 was exclusively expressed in CXCR2-expressing CD11b(+)Gr-1(high) cells, which significantly increased in the livers of HFHCD-fed mice. These cells were F4/80 negative and did not possess a suppressor function. TNF-α expression was enhanced by S100A8 in primary liver leukocytes or a hepatocyte cell line and significantly elevated in the livers of HFHCD-fed mice. TNF-α was primarily produced from CD11b(+)F4/80(+) cells in liver leukocytes in response to S100A8. TNF-α deficiency attenuated hepatitis in HFHCD-fed mice. S100A8 was significantly more expressed in the liver tissues of patients with NASH than in those of patients with NAFL. In conclusion, these results suggest that S100A8 is primarily produced from CXCR2-expressing CD11b(+)Gr-1(high) cells, and it upregulates TNF-α production in CD11b(+)F4/80(+) cells through cellular cross-talk, which is an important mechanism in the development of NAFLD.

TNF-α-mediated microRNA-136 induces differentiation of myeloid cells by targeting NFIA.

Immune cell-lineage specification and function are influenced by progenitor origin and environmental factors. The mechanism of differentiation of immune cells, such as neutrophils, monocytes, and myeloid-derived suppressor cells, in inflammatory environments has not been elucidated completely. In this study, we have identified human microRNA-136 as a positive regulator of the differentiation of granulocytes and monocytes. Ectopic microRNA-136 induced cells to express higher levels of CD11b, CD14, and C/EBPε, secrete more cytokines, and synthesize higher levels of reactive oxygen species and H(2)O(2). microRNA-136 was shown to target and degrade multiple differentiation-associated molecules, such as the transcription factor NFIA, which induced the release of another microRNA, microRNA-223, with the ability to promote CD11b expression. Furthermore, microRNA-136 expression was remarkably increased by TNF-α, which activated NF-κB to bind to the DNA-promoter region controlling microRNA-136 expression. Additionally, TNF-α may alter NFIA expression through its modulation of microRNA-136 expression. Thus, TNF-α-mediated microRNA-136 may play a critical role in the generation and differentiation of inflammatory immune cells.

Role of Toll like receptor 4 signaling pathway in the secondary damage induced by experimental spinal cord injury.

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is specific immunologic response to systemic bacterial infection and injury. TLRs contribute to the initial induction of neuroinflammation in the CNS. In spinal cord injury (SCI) intricate immune cell interactions are triggered, typically consisting of a staggered multiphasic immune cell response, which can become deregulated. The present study aims to evaluate the role of TLR4 signaling pathway in the development of secondary damage in a mouse model of SCI using TLR4-deficient (TLR4-KO) mice such as C57BL/10ScNJ and C3H/HeJ mice. We evaluated behavioral changes, histological, immunohistochemistry and molecular assessment in TLR4-KO after SCI. SCI was performed on TLR4-KO and wild-type (WT) mice by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. Mice were sacrificed at 24h after SCI to evaluate the various parameters. SCI TLR4 KO mice developed severer hind limb motor dysfunction and neuronal death by histological evaluation, myeloid differentiation primary response 88 (Myd88) expression as well as an increase in nuclear factor NF-κB activity, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß levels, glial fibrillary acidic protein (GFAP), microglia marker (CD11ß), inducible nitric oxide synthases (iNOS), poly-ADP-ribose polymerase (PARP) and nitrotyrosine expression compared to WT mice. Moreover, the absence of TLR4 also caused a decrease in phosphorylated interferon regulatory transcription factor (p-IRF3) and interferon (IFN-ß) release. In addition, SCI TLR4 KO mice showed in spinal cord tissues a more pronounced up-regulation of Bax and a down-regulation of Bcl-2 compared to SCI WT mice. Finally, we clearly demonstrated that TLR4 is important for coordinating post-injury sequel and in regulating inflammation after SCI.

Inhibition of microglial activation contributes to propofol-induced protection against post-cardiac arrest brain injury in rats.

It has been suggested that propofol can modulate microglial activity and hence may have potential roles against neuroinflammation following brain ischemic insult. However, whether and how propofol can inhibit post-cardiac arrest brain injury via inhibition of microglia activation remains unclear. A rat model of asphyxia cardiac arrest (CA) was created followed by cardiopulmonary resuscitation. CA induced marked microglial activation in the hippocampal CA1 region, revealed by increased OX42 and P2 class of purinoceptor 7 (P2X7R) expression, as well as p38 MAPK phosphorylation. Morris water maze showed that learning and memory deficits following CA could be inhibited or alleviated by pre-treatment with the microglial inhibitor minocycline or propofol. Microglial activation was significantly suppressed likely via the P2X7R/p-p38 pathway by propofol. Moreover, hippocampal neuronal injuries after CA were remarkably attenuated by propofol. In vitro experiment showed that propofol pre-treatment inhibited ATP-induced microglial activation and release of tumor necrosis factor-α and interleukin-1ß. In addition, propofol protected neurons from injury when co-culturing with ATP-treated microglia. Our data suggest that propofol pre-treatment inhibits CA-induced microglial activation and neuronal injury in the hippocampus and ultimately improves cognitive function. We proposed a possible mechanism of propofol-mediated brain protection after cardiac arrest (CA). CA induces P2X7R upregulation and p38 phosphorylation in microglia, which induces release of TNF-α and IL-1ß and consequent neuronal injury. Propofol could inhibit microglial activation and alleviate neuronal damage. Our results suggest propofol-induced anti-inflammatory treatment as a plausible strategy for therapeutic intervention in post-CA brain injury.

Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers.

BACKGROUND: Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues. METHODS: RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC. RESULTS: We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells. CONCLUSIONS: These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

[Monocytes of human blood as a target of Streptococcus pyogenes components].

AIM: Study the effect of components of destroyed streptococci on human blood monocyte functions related to processes of trans-endothelial migration in vitro. MATERIALS AND METHODS: Mononuclear leukocytes, isolated from blood of healthy donors, endothelial cells of EA.hy 926 line and supernatant of ultrasound disintegrated Streptococcus pyogenes (DSS) were the objects of the study. Evaluation of adhesion and monocyte migration, level of expression of adhesion molecules and phosphokinases on monocytes was carried out by flow cytometry using monoclonal antibodies. Cytokine concentration was determined by using standard commercial test systems in enzyme immunoassay. RESULTS: Under the effect of DSS, expression of adhesion molecules CD162 and CD11b, as well as phospho-p38 MAPK changed, IL-6 and IL-8 secretion induction took place. DSS caused enhancement of migration and adhesive activity of monocytes, however, inhibited intensity of trans-endothelial migration. CONCLUSION: Products of destroyed streptococci have a multi-directional effect on human blood monocytes, that could be explained by the presence of components with varying biological activity in DSS.

Circulating TNF and mitochondrial DNA are major determinants of neutrophil phenotype in the advanced-age, frail elderly.

Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age ("inflammaging") and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA. Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b(++) neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR(+) neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors. In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly.

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle.

M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.