Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK^+/+). TK-deficient (TK^?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK^+/? revertants arising in TK^?/? cultures should possess TK enzyme identical with one of those present in the original TK^+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK^+/? lines, each derived in turn from separate TK^?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) K_m′s; and (4) ATP-Mg²⁺ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Capacity for tumor cell implantation as a function of in vitro cell density

Implantation properties of two melanoma cell lines, line 26 (low rate of implantation) and line 37 (high rate of implantation) were studied as a function of the cell density of the cells grown in monolayer in vitro. Sparse cultures (collected at a density of 0.8 × 10³ cells cm^?2) of line 37 produced 7.7 times as many lung tumor foci as those of line 26. Confluent cultures (collected at a density of 40 × 10³ cells cm^?2) resulted in greater numbers of tumor foci for both cell lines, but line 37 produced only 3.1 times as many tumor foci as did line 26 cells. Thus the high implantation line (37) has a much greater ability to implant when grown in the sparse state and injected than the low implantation line (26), but both lines have high implantation rates when injected as confluent cells.     

Dual Benefit of Reduced Cx43 on Atherosclerosis in LDL Receptor-Deficient Mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR^?/?) mice were intercrossed with mice heterozygous for Cx43 (Cx43^+/?mice). Male mice with normal (Cx43^+/+LDLR^?/?) or reduced (Cx43^+/?LDLR^?/?) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43^+/?LDLR^?/?mice compared with Cx43^+/+LDLR^?/?littermate controls. In addition, atheroma in Cx43^+/?LDLR^?/?mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Spontaneous and induced osteoclastogenic behaviour of human peripheral blood mononuclear cells and their CD14(+) and CD14(-) cell fractions

Objectives: Osteoclasts are descended from the CD14⁺ monocyte/macrophage lineage, but influence of other haematopoietic cells on osteoclastic commitment of their precursors has remained poorly understood. In this study, osteoclastogenic behaviour of peripheral blood mononuclear cells (PBMC) and their CD14⁺ and CD14^? subpopulations has been accessed, in the absence or presence of M‐CSF and RANKL. Materials and Methods: Cell cultures were characterized for presence of actin rings and vitronectin and calcitonin receptors, TRAP activity and calcium phosphate resorbing activity, expression of osteoclast‐related genes and secretion of M‐CSF and RANKL. Results: In the absence of growth factors, PBMC and CD14⁺ cultures had some degree of cell survival, and some spontaneous osteoclastogenesis was observed, only on cultures of the former. Supplementation with M‐CSF and RANKL significantly increased osteoclastogenic behaviour of cell cultures, particularly CD14⁺ cell cultures. Nevertheless, PBMC derived a higher degree of osteoclastogenesis, either as absolute values or after normalization by protein content. It was observed that unlike CD14⁺ cells, PBMC were able to express M‐CSF and RANKL, which increased following growth factor treatment. Also, expression of TNF‐α, GM‐CSF, IL‐1β, IL‐6 and IL‐17 was higher in PBMC cultures. Finally, CD14^? cultures exhibited limited cell survival and did not reveal any osteoclast features. Conclusions: Results show that although osteoclastic precursors reside in the CD14⁺ cell subpopulation, other populations (such as CD14^? cells) derived from PBMC, have the ability to modulate osteoclastogenesis positively.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalisation by macrophages

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc–macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc–macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc–macrophage interaction. Using optical tweezers to study cell‐to‐cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide‐glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1^?/? mice) showed a deficient ability to interact with Hc. Coincubation of oligo‐GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1^?/? macrophages. In addition, macrophages with reduced CD18 expression (CD18^low) were associated with Hc at levels similar to wild‐type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc–macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc–macrophage adhesion and mediate efficient internalisation during histoplasmosis.     

Progranulin haploinsufficiency causes biphasic social dominance abnormalities in the tube test

Loss‐of‐function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia (FTD), a neurodegenerative disorder in which social behavior is disrupted. Progranulin‐insufficient mice, both Grn^+/? and Grn ^?/? , are used as models of FTD due to GRN mutations, with Grn^+/? mice mimicking the progranulin haploinsufficiency of FTD patients with GRN mutations. Grn^+/? mice have increased social dominance in the tube test at 6 months of age, although this phenotype has not been reported in Grn ^?/? mice. In this study, we investigated how the tube test phenotype of progranulin‐insufficient mice changes with age, determined its robustness under several testing conditions, and explored the associated cellular mechanisms. We observed biphasic social dominance abnormalities in Grn^+/? mice: at 6–8 months, Grn^+/? mice were more dominant than wild‐type littermates, while after 9 months of age, Grn^+/? mice were less dominant. In contrast, Grn ^?/? mice did not exhibit abnormal social dominance, suggesting that progranulin haploinsufficiency has distinct effects from complete progranulin deficiency. The biphasic tube test phenotype of Grn^+/? mice was associated with abnormal cellular signaling and neuronal morphology in the amygdala and prefrontal cortex. At 6–9 months, Grn^+/? mice exhibited increased mTORC2/Akt signaling in the amygdala and enhanced dendritic arbors in the basomedial amygdala, and at 9–16 months Grn^+/? mice exhibited diminished basal dendritic arbors in the prelimbic cortex. These data show a progressive change in tube test dominance in Grn^+/? mice and highlight potential underlying mechanisms by which progranulin insufficiency may disrupt social behavior.     

Role of CD69 in acute lung injury

AimsCD69 is an early activation marker in lymphocytes and an important signal transmitter in inflammatory processes. However, its role in acute lung injury (ALI) is still unknown. We used a lipopolysaccharide (LPS)-induced mouse model of ALI to study the role of macrophage-surface CD69 in this condition.Main methodsWe investigated bronchoalveolar lavage fluid (BALF) cell subpopulations, myeloperoxidase levels in lung homogenates, lung pathology, and lung oedema in CD69-deficient (CD69^?/?) mice 24 h after LPS instillation. We also determined cytokine/chemokine expression levels in BALF and macrophage culture supernatant from CD69^?/? and wild type (WT) mice. Also, we investigated CD69, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 localization in the lungs after LPS administration. Furthermore, we examined the effect of anti-CD69 antibody on LPS-induced cytokine/chemokine release from cultured macrophages.Key findingsOur study shows that intratracheal instillation of LPS-induced neutrophilic infiltration, histopathological changes, myeloperoxidase positivity, and oedema in the lung to a lower degree in CD69^?/? mice than in WT mice. The immunoreactivities for CD69, KC and MIP2 were induced in the lung of WT mice instilled with LPS and were predominantly localized to the macrophages. Moreover, the cytokine/chemokine expression profile between the two genotypes of cultured macrophages in response to LPS was similar to that observed in the BALF. In addition, anti-CD69 antibody inhibited the LPS-induced cytokine/chemokine expression.SignificanceThese results suggest that CD69 on macrophages plays a crucial role in the progression of LPS-induced ALI and may be a potentially useful target in the therapy for ALI.     

Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

TIPE2 knockout reduces myocardial cell damage by inhibiting IFN-γ-mediated ferroptosis

Acute rejection of the transplanted heart is mediated by oxidative programmed cell death through the synergistic effects of the innate and adaptive immune systems. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been widely evaluated. Tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2), also known as TIPE2, is required for maintaining immune homeostasis. To characterize the role of TIPE2 in mediating heart allografts, BALB/c hearts were transplanted into C57BL/6 wild-type (WT) and TIPE2^?/? recipient mice. In TIPE2^?/? recipient mice, allograft injury in BALB/c allograft hearts was significantly reduced through the inhibition of allograft ferroptosis. On day 3 and day 6 post-transplantation, the numbers of CD3⁺, CD4⁺, and CD8⁺ cells among splenocytes and draining lymph node cells were significantly decreased, and the activation of CD4⁺ and CD8⁺ cells in grafts was decreased in TIPE2^?/? recipient mice compared with WT mice. Moreover, CD4⁺ and CD8⁺ T cells in TIPE2^?/? recipient mice were characterized by deficient capacities for interferon-γ (IFN-γ) production through the TBK1 signaling axis and increased glutathione peroxidase 4 (GPX4). In cell experiments, treatment with IFN-γ enhanced ferroptosis-specific lipid peroxidation in myocardial cells and correlated inversely with GPX4 expression. Mechanistically, IFN-γ administration decreased the expression of GPX4 by inhibiting MEK/ERK phosphorylation. In summary, our findings demonstrated that TIPE2 deficiency inhibits T-cell production of IFN-γ to reduce ferroptosis in allografts by restraining lipid peroxidation.     

TIM-3 shuttled by MV3 cells-secreted exosomes inhibits CD4+ T cell immune function and induces macrophage M2 polarization to promote the growth and metastasis of melanoma cells

This study is sought to determine the physiological mechanisms by which exosomes-encapsulated TIM-3 derived from melanoma cells might mediate CD4⁺ T cell immune function and macrophage M2 polarization in melanoma. Initially, exosomes were isolated from the human skin-derived melanoma cell line MV3for analysis of TIM-3 expression pattern. Next, the exosomes sourced from MV3 cells manipulated with sh-TIM-3 were co-incubated with CD4⁺ T cells to detect CD4⁺ T cell proliferation and MV3 cell migration and invasion, to observe the macrophage M2 polarization, and to determine levels of several EMT-related factors. Finally, melanoma nude mouse models were established to study the in vivo modulatory effects of TIM-3 from MV3 cells-derived exosomes. MV3 cells-derived exosomes inhibited CD4⁺ T cell immune function and promoted macrophage M2 polarization in melanoma. Our results revealed the abundance of TIM-3 in MV3 cells-derived exosomes. Of importance, silencing of TIM-3 shuttled by MV3 cells-derived exosomes improved CD4⁺ T cell immune function and inhibited macrophage M2 polarization to attenuate the growth and metastasis of melanoma cells. Collectively, MV3 cells-derived exosomes-loaded TIM-3 suppressed CD4⁺ T cell immune function and induced macrophage M2 polarization to improve occurrence and development of melanoma, therefore providing us with a potential therapeutic target for effectively combating melanoma.     

GABA concentration and GABAergic neuron populations in limbic areas are differentially altered by brain serotonin deficiency in Tph2 knockout mice

While tryptophan hydroxylase-2 (Tph2) null mutant (Tph2 ^?/?) mice are completely deficient in brain serotonin (5-HT) synthesis, the formation of serotonergic neurons and pathfinding of their projections are not impaired. However, 5-HT deficiency, during development and in the adult, might affect morphological and functional parameters of other neural systems. To assess the influence of 5-HT deficiency on γ-amino butyric acid (GABA) systems, we carried out measurements of GABA concentrations in limbic brain regions of adult male wildtype (wt), heterozygous (Tph2 ^+/?) and Tph2 ^?/? mice. In addition, unbiased stereological estimation of GABAergic interneuron numbers and density was performed in subregions of amygdala and hippocampus. Amygdala and prefrontal cortex displayed significantly increased and decreased GABA concentrations, respectively, exclusively in Tph2 ^+/? mice while no changes were detected between Tph2 ^?/? and wt mice. In contrast, in the hippocampus, increased GABA concentrations were found in Tph2 ^?/? mice. While total cell density in the anterior basolateral amygdala did not differ between genotypes, the number and density of the GABAergic interneurons were significantly decreased in Tph2 ^?/? mice, with the group of parvalbumin (PV)-immunoreactive (ir) interneurons contributing somewhat less to the decrease than that of non-PV-ir GABAergic interneurons. Major morphological changes were also absent in the dorsal hippocampus, and only a trend toward reduced density of PV-ir cells was observed in the CA3 region of Tph2 ^?/? mice. Our findings are the first to document that life-long reduction or complete lack of brain 5-HT transmission causes differential changes of GABA systems in limbic regions which are key players in emotional learning and memory processes. The changes likely reflect a combination of developmental alterations and functional adaptations of emotion circuits to balance the lack of 5-HT, and may underlie altered emotional behavior in 5-HT-deficient mice. Taken together, our findings provide further insight into the mechanisms how life-long 5-HT deficiency impacts the pathogenesis of anxiety- and fear-related disorders.     

MMP-9 gene ablation mitigates hyperhomocystenemia-induced cognition and hearing dysfunction

Hyperhomocysteinemia (HHcy) is associated with cognitive decline and hearing loss due to vascular dysfunction. Although we have shown that HHcy-induced increased expression of matrix metalloproteinase-9 (MMP-9) is associated with cochlear pathology in cystathionine-β-synthase heterozygous (CBS^+/?) mice, it is still unclear whether MMP-9 contributes to functional deficit in cognition and hearing. Therefore, we hypothesize that HHcy-induced MMP-9 activation causes vascular, cerebral and cochlear remodeling resulting in diminished cognition and hearing. Wildtype (WT), CBS^+/?, MMP-9^?/? and CBS^+/?/MMP-9^?/? double knock-out (DKO) mice were genotyped and used. Doppler flowmetry of internal carotid artery (ICA) was performed for peak systolic velocity [PSV], pulsatility index [PI] and resistive index [RI]. Cognitive functions were assessed by Novel Object Recognition Test (NORT) and for cochlear function Auditory brainstem response (ABR) was elicited. Peak systolic velocity, pulsatility and resistive indices of ICA were decreased in CBS^+/? mice, indicating reduced perfusion. ABR threshold was increased and maximum ABR amplitude and NORT indices (recognition, discrimination) were decreased in CBS^+/? mice compared to WT and MMP-9^?/?. All these parameters were attenuated in DKO mice suggesting a significant role of MMP-9 in HHcy-induced vascular, neural and cochlear pathophysiology. Regression analysis of PSV with ABR and cognitive parameters revealed significant correlation (0.44–0.58). For the first time, MMP-9 has been correlated directly to functional deficits of brain and cochlea, and found to have a significant role. Our data suggests a dual pathology of HHcy occurring due to a decrease in blood supply (vasculo-neural and vasculo-cochlear) and direct tissue remodeling.     

Decreased aggression and increased repetitive behavior in Pten haploinsufficient mice

Aggression is an aspect of social behavior that can be elevated in some individuals with autism spectrum disorder (ASD) and a concern for peers and caregivers. Mutations in Phosphatase and tensin homolog (PTEN), one of several ASD risk factors encoding negative regulators of the PI3K–Akt–mTOR pathway, have been reported in individuals with ASD and comorbid macrocephaly. We previously showed that a mouse model of Pten germline haploinsufficiency (Pten^+/?) has selective deficits, primarily in social behavior, along with broad overgrowth of the brain. Here, we further examine the social behavior of Pten^+/? male mice in the resident–intruder test of aggression, using a comprehensive behavioral analysis to obtain an overall picture of the agonistic, non‐agonistic and non‐social behavior patterns of Pten^+/? mice during a free interaction with a novel conspecific. Pten^+/? male mice were involved in less aggression than their wild‐type littermates. Pten^+/? mice also performed less social investigation, including anogenital investigation and approaching and/or attending to the intruder, which is consistent with our previous finding of decreased sociability in the social approach test. In contrast to these decreases in social behaviors, Pten^+/? mice showed increased digging. In summary, we report decreased aggression and increased repetitive behavior in Pten^+/? mice, thus extending our characterization of this model of an ASD risk factor that features brain overgrowth and social deficits.     

M2 differentiation of MonoMac-1 cell line induced by M-CSF and glucocorticoid pathways

Models of macrophage subtypes require molecular characterization to reliably facilitate their differentiation. Although CD16⁺ (Fc-gamma III receptor) monocytes that express CD163 (a hemoglobin/haptoglobin receptor) have been implicated in a variety of disease states, the conditions necessary to establish lineages of these cell subtypes remains unknown. The current investigations utilize a cell line derived from acute myelogenous leukemia lineage, MonoMac-1, to interrogate the factors that promote the development of CD16⁺ macrophages that express CD163. Results implicate the glucocorticoid pathway as well as c-fms signaling based on the action of dexamethasone and macrophage colony-stimulating factor-1 in promoting CD16⁺ expression, in addition to phorbol myristate acetate and lipopolysaccharides treatment. The ability of glucocorticoid and c-fms receptor antagonists to inhibit CD16⁺ cell formation further establishes the role of these pathways in CD16 expression in this cell line. In view of the inherent difficulty in working with primary cells as well as donor variation, cell lines may be preferable to primary cells for their consistency. We envision that the process we use to induce CD16 expression in this cell type will be useful for screening and identification of drug candidates potentially useful for the treatment of diseases where the etiology involves the expansion of the CD16⁺ monocytes subset or the accumulation of CD163⁺ tissue macrophages.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Liraglutide dictates macrophage phenotype in apolipoprotein E null mice during early atherosclerosis

Background

Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response.

Methods

Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE^?/?) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE^?/? mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 μg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE^?/? mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry.

Results

Liraglutide decreased atherosclerotic lesion formation in ApoE^?/? mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 μg/kg liraglutide treatment in ApoE^?/? mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells.

Conclusions

This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.