Resident Monocytes Research Articles

Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models.

Severe Hyperhomocysteinemia Promotes Bone Marrow–Derived and Resident Inflammatory Monocyte Differentiation and Atherosclerosis in LDLr/CBS-Deficient Mice

Hyperhomocysteinemia (HHcy) accelerates atherosclerosis and increases inflammatory monocytes (MC) in peripheral tissues. However, its causative role in atherosclerosis is not well established and its effect on vascular inflammation has not been studied. The underlying mechanism is unknown. This study examined the causative role of HHcy in atherogenesis and its effect on inflammatory MC differentiation. We generated a novel HHcy and hyperlipidemia mouse model, in which cystathionine β-synthase (CBS) and low-density lipoprotein receptor (LDLr) genes were deficient (Ldlr(-/-) Cbs(-/+)). Severe HHcy (plasma homocysteine (Hcy)=275 μmol/L) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 μmol/L from 244 μmol/L by vitamin supplementation, which elevated plasma folate levels. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic mice after sublethal irradiation of the recipient. HHcy accelerated atherosclerosis and promoted Ly6C(high) inflammatory MC differentiation of both BM and tissue origins in the aortas and peripheral tissues. It also elevated plasma levels of TNF-α, IL-6, and MCP-1; increased vessel wall MC accumulation; and increased macrophage maturation. Hcy-lowering therapy reversed HHcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory MC (Ly6C(high+middle)) accumulation. Plasma Hcy levels were positively correlated with plasma levels of proinflammatory cytokines. In primary mouse splenocytes, L-Hcy promoted rIFNγ-induced inflammatory MC differentiation, as well as increased TNF-α, IL-6, and superoxide anion production in inflammatory MC subsets. Antioxidants and folic acid reversed L-Hcy-induced inflammatory MC differentiation and oxidative stress in inflammatory MC subsets. HHcy causes vessel wall inflammatory MC differentiation and macrophage maturation of both BM and tissue origins, leading to atherosclerosis via an oxidative stress-related mechanism.

M2b monocytes predominantly appear in patients with advanced hepatocellular carcinoma (HCC patients) (162.29)

Abstract In response to tumor environments, resident Mϕ differentiate into M1Mϕ or M2Mϕ. Recently, three different subsets of M2Mϕ (M2aMϕ, M2bMϕ and M2cMϕ) have been reported. Therefore, in this study, the properties and subsets of monocytes isolated from early and advanced HCC patients were characterized. CD14(+) monocytes were isolated from peripheral blood of HCC patients by Ficoll-Hypque sedimentation and magnetic beads. HCC patients, who were confirmed as carriers of hepatic cancer cells, were enrolled in this study. Monocytes (1 x 10(6) cells/ml) were cultured for 48 hrs and culture fluids harvested were assayed for cytokines by ELISA. Tumoricidal activity of monocytes against hepatic cancer cells was assessed using a standard chromium release assay. In the results, monocytes from advanced HCC patients were shown to be M2b monocytes, because they produced IL-10 (188 ± 21 pg/ml) and CCL1 (24 ± 0.6 ng/ml), and did not produce IL-12, CCL17 and CXCL13. Monocytes from early HCC patients were characterized as M1 monocytes, and healthy donor monocytes were determined as resident monocytes. Monocytes from early HCC patients killed syngeneic hepatic cancer cells (73 ± 7%), while monocytes from advanced HCC patients did not (10 ± 3%). These results indicate that advanced HCC patients are carriers of M2b monocytes and early HCC patients are M1 monocyte carriers. M2b monocytes detected in advanced HCC patients may play a role on the decreased host’s antitumor resistances.

CCR2 and CCR5 Co-Expressing CD11b+ Monocytes Affect Neovascularization in An Ischemic Hind Limb Mouse Model,

Abstract 3270Monocyte/myeloid -lineage cells are known to be involved in neovascularization. However, the specific monocyte /myeloid population that participates in neovascularization has not been clearly identified. Several subsets of monocyte/myeloid cells have been proposed to be important for this process. For example, CD11b+CXCR1hiCCR5+ resident monocytes regulate tissue healing by increasing angiogenesis, and CD11b+CXCR1loCCR2+ inflammatory monocytes are mobilized to the inflammation site. Interestingly, we found that CD11b+ myeloid cells isolated from ischemic muscle of post-femoral artery dissected C57BL/6 mice are positive for both CCR2 and CCR5. To study the relationship between CD11b+CCR2+CCR5+ monocytes and neovascularization in an ischemic hind-limb model of C57BL/6 mice, we addressed the following questions:(1) Are the number of tissue-resident CD11b+CCR2+CCR5+ monocytes increased in ischemic muscle, post-ischemic injury?; (2) Are CD11b+CCR2+CCR5+ monocytes recruited to the ischemic muscle and do they incorporate into the blood vessels in the ischemic site?; (3) Do CD11b+CCR2+CCR5+ monocytes modulate neovascularization in ischemic hind-limbs of C57BL/6 mice upon direct injection of these cells into ischemic muscle. Results demonstrate that numbers of CD11b+CCR2+CCR5+ monocytes markedly increase in ischemic muscle by day 4 after the surgery (n=5 each; 0.8±0.4×105 cells/g tissue in non-surgically treated mice vs 9.5±1.7×105 cells/g tissue in the ischemic muscle, p<0.05) then return to basal levels at day 21. Following immunostaining of ischemic muscles, CD11b+CCR2+CCR5+ monocytes localize in perivascular areas of non-surgically treated C57BL/6 mice. At day 4 after the surgery, these cells infiltrate into ischemic muscles. Infiltrated CD11b+CCR2+CCR5+ cells make numerous vessel-like structures 21 days after the surgery. Following direct injection of purified CD11b+CCR2+CCR5+ monocytes from ischemic muscle at day 4, an increase in blood flow was manifest by 28 days in the CD11b+CCR2+CCR5+ monocyte-injected group (n=7 each, 67.6±5.7% in the CD11b+CCR2+CCR5+ monocyte-injected group vs 39.9±6.7% in the control group, p<0.05). Thus, we found that CD11b+CCR2+CCR5+ monocytes infiltrated and formed numerous vessel-like structures in the ischemic muscle, and additional injection of CD11b+CCR2+CCR5+ monocytes increased the blood flow. These findings strongly suggest that CD11b+CCR2+CCR5+ monocytes play an important role in neovascularization. Disclosures:No relevant conflicts of interest to declare.

The role of resident monocytes and vascular pericytes in the stem cell niche and regenerative medicine

The perivascular tissue-specific stem cell niche (SCN) determines nuclear reprogramming of multipotent stem cells into particular cell types in distinct tissues in vivo . It represents a morphostatic (homeostatic) mechanism executed by the so-called tissue control system (TCS), which is associated with postcapillary venules and consists of vascular pericytes regulated by the autonomic nervous system and of resident perivascular monocytederived cells. Tissue morphostasis executed by the TCS is a complex procedure consisting of three processes: i) Stem cell and tissue renewal by asymmetric division of stem cells, ii) Preservation of tissue cells in a properly differentiated (functional) state, and iii) Maintenance of tissue quantity. The TCSmediated functional preservation of tissuespecific cells is established during the morphogenetic critical developmental period, such as developmental immune adaptation, as a stop effect at the level of resident self-renewing tissue monocyte-derived cells (MDC), and its function declines with advancing age. The functional stop effect differs between the tissues, and an inhibition or acceleration of tissue differentiation during the critical developmental period can result in persistent functional immaturity or premature tissue aging. In addition, a retardation of tissue development can cause an earlier failure of that tissue function, like a premature ovarian failure. A promising approach in regenerative medicine is a chemical approach, such as the combination of sex steroids stimulating growth of endogenous stem cells for neuronal, vascular, and cardiac repair. Sex steroid combinations and doses for clinical applications are suggested. Regenerative medicine may be more successful in acute/traumatic disorders with intact morphostatic SCN compared to the chronic and degenerative diseases caused by an altered SCN. If we attain the capacity to repair the function of altered SCN and the tissue- specific stop effect by trascriptional therapy, we may be able to provide novel treatments for early postnatal tissue disorders, improve regenerative medicine, and delay aging.

Dietary plasma proteins attenuate the innate immunity response in a mouse model of acute lung injury

We examined whether oral plasma protein supplements affect the innate immune response in a model of acute lung inflammation. Mice were fed diets supplemented with 8 % spray-dried plasma (SDP) or 2 % plasma Ig concentrate (IC) from day 19 (weaning) until day 34. The mice were challenged with intranasal lipopolysaccharide (LPS) at day 33 (and killed 24 h later for cytokine and leucocyte analyses) or at day 34 (and killed 6 h later for cytokine determinations). In bronchoalveolar lavage fluid (BALF), LPS increased the number of leucocytes by twenty-sevenfold, an effect that was partly prevented by both SDP and IC, and by twentyfold the percentage of activated monocytes, which was partly prevented by SDP. In the lung tissue, LPS increased the infiltrated leucocytes, and this effect was prevented in part by SDP. In unchallenged mice, both SDP and IC diets reduced the percentage of resident neutrophils and monocytes (P < 0·05). In the blood, both SDP and IC completely prevented LPS-dependent monocyte activation (CD14⁺; P < 0·05). LPS dramatically increased the concentration of cytokines (TNF-α, IL-1α, IL-6, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor) and chemokines (CXCL1, CCL2, CCL3 and CCL4) in BALF. The acute response of cytokine production was reduced by 20-80 % by both SDP and IC. For chemokines, plasma supplements had no effect on LPS-induced CXCL1 expression but significantly reduced CCL2, CCL3 and CCL4 production (P < 0·05). The results support the view that dietary plasma proteins can be used to attenuate endotoxin-associated lung inflammation.

Microparticles from apoptotic platelets promote resident macrophage differentiation

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PMap). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PMap formation. Flow cytometry and scanning electron microscopy demonstrated that PMap display activated integrins and interact to form microparticle aggregates. PMap were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PMap promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin αMβ2), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PMap polarized the cells into resident M2 monocytes. Cells treated with PMap actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PMap stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PMap with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes.

M2b Monocytes Predominated in Peripheral Blood of Severely Burned Patients

Severely burned patients were shown to be carriers of M2 monocytes, and all of the monocytes isolated from peripheral blood of severely burned patients (19 of 19 patients) were demonstrated as M2b monocytes (IL-12(-)IL-10(+)CCL1(+) monocytes). Low levels of M2a (IL-12(-)IL-10(+)CCL17(+) monocytes) and M2c monocytes (IL-12(-)IL-10(+)CXCL13(+) monocytes) were demonstrated in peripheral blood of severely burned patients (M2a, 2 of 19 patients; M2c, 5 of 19 patients). M2b, M2a, and M2c monocytes were not detected in peripheral blood of healthy donors. However, M2b monocytes appeared when healthy donor monocytes were cultured in media supplemented with burn patient serum (15%). CCL2 was detected in sera of all burn patients, and M2b monocytes were not generated from healthy donor monocytes cultured with media containing 15% burn patient sera that were previously treated with anti-CCL2 mAb. In addition, M2b monocytes were generated from healthy donor monocytes in cultures supplemented with rCCL2. These results indicate that M2b monocytes are predominant in peripheral blood of severely burned patients who are carriers of CCL2 that functions to stimulate monocyte conversion from resident monocytes to M2b monocytes.

Eotaxin-3/CC Chemokine Ligand 26 Is a Functional Ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.

Patrolling Murine Monocytes Are Defined by Their Expression of the Orphan Nuclear Receptor, Nur77 (nr4a1)

AbstractAbstract 4723‘Resident’ or ‘patrolling’ monocytes have recently been described as a unique subset of myeloid cells that differ in phenotype and function from classic inflammatory monocytes. Resident monocytes are distinguished by their ‘crawling’ migration on endothelial surfaces, their immunosuppressive activity, and their unique pattern of surface protein expression: Ly6Clo, CX3CR1hi, CCR2-, LFA-1+, and PDL-1+. Using a Nur77-eGFP reporter mouse, we show that all murine patrolling monocytes, but not conventional monocytes express Nur77+ (also known as NR4A1), a member of the Nur orphan nuclear receptor family that regulates cell fate decisions several cell types and inhibits leukemogenesis. Nur77 is an immediate early response gene, and we have recently determined that it is a sensitive indicator of antigen receptor signaling among T cells. We therefore propose that its expression among patrolling monocytes is an indication that these cells are actively receiving signals, perhaps via interactions with the endothelium. We are currently working to identify these signals and our preliminary data suggest that adhesion molecules play an influential role.We thank the Arnold and Mabel Beckman Foundation (HH), and NSF (MCB-0744570 (JP and NC)) for their support for this work. Disclosures:No relevant conflicts of interest to declare.

On the Use of Micron-Sized Iron Oxide Particles (MPIOS) to Label Resting Monocytes in Bone Marrow

The use of MRI to monitor immune cell infiltration into various pathologies is well established. In an effort to boost the magnetic material within immune cells, this work attempted to label resting monocytes within bone marrow, in mice, by intravenous administration of micron-sized iron oxide particles (MPIOs), similar in fashion to the administration of (U)SPIO. MPIOs were incubated with various immune cells both in culture, and in whole blood. Flow cytometry and histology were used to analyze magnetic cell labeling. Also, MPIOs were injected intravenously into mice. In vivo, high-resolution 3-D MRI was performed on mouse legs, and signal changes were quantified. Flow cytometry and histology were used to analyze magnetic cell labeling of bone marrow resident cells. It is demonstrated here that monocytes and neutrophils can indeed endocytose MPIOs both in cell culture and ex vivo in whole blood. However, despite rapid accumulation of MPIOs within the bone marrow following injection, MPIOs did not label monocytes or any other hematopoietic cell type in the marrow. Hypotheses are drawn to explain these results in light of recent usage of MPIOs for immune cell tracking. Systemic administration of various MPIO formulations showed that MPIOs arrive in bone marrow rapidly following injection and remain there for at least 7 days. Data also shows slow clearance of some particles from the tissue over this period. While MPIOs can efficiently label monocytes in culture and in whole blood ex vivo, they were not found to label bone marrow resident monocytes.

P33 Treatment with Toll-like receptor 4 antagonist restores the inflammatory cellular immune dysfunction in acetaminophen induced acute liver failure mice

IntroductionAcute liver failure (ALF) is a rare, catastrophic syndrome commonly caused by acetaminophen (APAP) that results in the death of about 40% patients. In addition to liver dysfunction, its outcome...

Immune Inhibitory Ligand CD200 Induction by TLRs and NLRs Limits Macrophage Activation to Protect the Host from Meningococcal Septicemia

Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation.

Blood monocytes from mammary tumor-bearing mice: Early targets of tumor-induced immune suppression?

We have previously shown that peritoneal macrophages from mice bearing advanced D1-DMBA3 mammary tumors are impaired in their inflammatory functions but are not alternatively activated either and are less differentiated than the ones from normal mice. However, little is known about whether similar defects exist in their precursor stages as blood monocytes. We examined if blood monocytes from mammary tumor-bearing mice are already altered in their activation profiles before becoming macrophages and whether they correspond to inflammatory or resident monocyte subtypes. Much effort is currently devoted to reversing macrophage adverse traits in tumor hosts; as these cells reside within tissues, access is limited. Blood monocytes could be better targeted and manipulated by less invasive means. In the present study, mononuclear cells were isolated from whole blood of D1-DMBA-3 mammary tumor-bearing and normal BALB/c mice and CD115(+) monocytes were analyzed. Our results show that there is an increase in circulating monocytes in tumor hosts; these monocytes exhibit a reduced expression of several myeloid differentiation markers such as CD115, F4/80, CD68 and CD11b. Moreover, downregulation of MHC II, CD62L and the proangiogenic marker Tie-2 are observed in these cells, whereas Gr-1 and Ly6C are upregulated. Furthermore, gene microarray analysis performed for the first time in blood monocytes from tumor hosts indicates that they express a mixture of pro-inflammatory and anti-inflammatory cytokines and chemokines. Interestingly, CCR2 and CX3CR1, which are crucial in monocyte definition as inflammatory or resident, respectively, are both upregulated. Importantly, complement proteins are enhanced whereas nitric oxide production is decreased and there is no measurable arginase activity detected in these cells. Collectively, our study represents the first comprehensive analysis of blood monocytes from tumor-bearing mice; we conclude that these cells are neither completely inflammatory nor suppressive and are less differentiated, similar to the macrophages they later become.

Technical Advance: Autofluorescence as a tool for myeloid cell analysis

Cellular AF is usually considered a hindrance to flow cytometric analysis. Here, we incorporate AF into analysis of complex mixtures of leukocytes. Using a mouse model, we examined cellular AF at multiple excitation and emission wavelengths, and populations with discrete patterns were gated and examined for surface marker expression. In the spleen, all major myeloid populations were identified. In particular, the approach allowed simultaneous characterization of RPM and resident monocytes. When monocytes and RPM were compared, RPM exhibited a phenotype that was consistent with involvement in physiological processes, including expression of genes involved in lipid and iron metabolism. The presence of large amounts of stored ferric iron within RPM enabled purification of these cells using a magnetic-based approach. When adapted for use on leukocytes isolated from a range of other organs, incorporation of AF into analysis allowed identification and isolation of biologically important myeloid populations, including subsets that were not readily identifiable by conventional cytometric analysis.

Abstract SY27-02: Heterogeneity of tumor-associated macrophages: Implications for antiangiogenic therapy

Abstract SY27-02: Heterogeneity of tumor-associated macrophages: Implications for antiangiogenic therapy

Inflammatory Monocytes Are a Reservoir for Merkel Cell Polyomavirus

Merkel cell polyomavirus (MCPyV) is a recently discovered virus that is implicated in the oncogenesis of Merkel cell carcinoma (MCC). The route of dissemination and the reservoir(s) of MCPyV within the human body have not yet been identified. In this study we describe two patients with multiple MCPyV-positive inflammatory and neoplastic skin lesions at different anatomic sites. Patient 1 was suffering from psoriasis for many years and was diagnosed with MCC 7 years before this study. Patient 2 had developed numerous non-melanoma skin cancer lesions under post-transplant immunosuppression. In both patients, MCPyV DNA was detected in whole blood and in urine using PCR and direct sequencing of PCR products. When we analyzed different blood compartments, we found MCPyV exclusively in cell-free serum and in blood monocytes, but not in lymphocytes or granulocytes. Upon separate analysis of resident (CD14(lo)CD16(+)) and inflammatory (CD14(+)CD16(-)) monocytes, we detected MCPyV exclusively in inflammatory, but not in resident monocytes. Our findings raise the possibility that MCPyV persists in inflammatory monocytes and spreads along the migration routes of inflammatory monocytes. This points to intervention strategies to contain MCPyV. Moreover, blood or urine tests may serve as ancillary tests to confirm MCPyV infection in a clinical setting.

Systemic levels of G-CSF and interleukin-6 determine the angiogenic potential of bone marrow resident monocytes

There is considerable interest in the potential of cell-based approaches to mediate therapeutic angiogenesis for acute and chronic vascular syndromes. Using a mouse model of HLI, we showed previously that adoptive transfer of a small number of donor monocytes enhanced revascularization significantly. Herein, we provide data suggesting that the BM resident monocytes sense systemic signals that influence their future functional capacity. Specifically, following induction of distant ischemia, the angiogenic capacity of BM resident monocytes is reduced markedly. We provide evidence that G-CSF and IL-6 represent such "conditioning" signals. Systemic levels of G-CSF and IL-6 are increased significantly following induction of HLI. Accordingly, BM resident monocytes from ischemic mice exhibited increased pSTAT3 and STAT3 target gene expression. Finally, G-CSFR(-/-) and IL-6(-/-) mice were resistant to the deleterious effects of ischemic conditioning on monocyte angiogenic potential. RNA expression profiling suggested that ischemia-conditioned monocytes in the BM up-regulate the well-described M2 polarization markers Chi3l4 and Lrg1. Consistent with this observation, M2-skewed monocytes from SHIP(-/-) mice also had impaired angiogenic capacity. Collectively, these data show that G-CSF and IL-6 provide signals that determine the angiogenic potential of BM resident monocytes.

New observations on the trafficking and diapedesis of monocytes

Monocytes play multiple roles in immune system functions and inflammatory diseases such as atherosclerosis. These roles are coupled to diverse trafficking and cellular migration behaviors. Here, we review recent advances in our understanding of such behaviors with emphasis on broad scale trafficking patterns and the cellular and molecular mechanisms regulating diapedesis, a central aspect of trafficking. Monocytes consist of 'inflammatory' and 'resident' subsets, which exhibit differential functions and trafficking properties. Notably, the spleen has recently been identified as a reservoir of inflammatory monocytes, which are readily recruited to injured myocardium and possibly other tissues. Resident monocytes have been shown to undergo long-range crawling within the lumen of the microvasculature, which facilitates immune surveillance and rapid response to infection. Monocyte diapedesis has been demonstrated to utilize both para and transcellular migration routes facilitated by endothelial 'transmigratory cups'. A significant number of new adhesion molecules and signaling pathways have recently been uncovered as functional mediators and modulators of these processes. Our improving understanding of monocyte trafficking and migration mechanisms has begun to shed light on the functions of these often enigmatic cells. Continued progress in this area will be critical for elucidating roles of monocytes in disease and for developing therapeutics that target monocytes.

Abstract 5813: Hyperhomocysteinemia Enhances Bone Marrow-derived and Vessel Resident Ly6C Inflammatory Monocyte Differentiation Contributing to Atherogenesis

Background: We recently reported that hyperhomocysteinemia (HHcy) enriched circulating Ly6C monocytes (MCs) independent of hyperlipidemia. Here we studied the effect and mechanism of HHcy on Ly6C MC function and differentiation in aorta. Methods and Results: We generated a mouse model with cystathionine β -synthase (CBS) and LDLr deficiencies. LDLr − / − CBS − /+ mice (8wk) received BM transplantation from EGFP mice. The chimeric mice were fed a high fat (HF) diet as control (Hcy, 10.1 μ M), HF/high methionine (HM) diet to induce severe HHcy (275.6 μ M), or HF/HM/high vitamin B-6/12 and folic acid diet as Hcy-lowering therapy (39.0 μ M) for another 8wk. Morphological analysis exhibited that severe HHcy accelerated atherosclerosis and increased BM-derived Ly6C MC accumulation in the lesion. FACS analysis on whole aortic cell suspension showed that severe HHcy increased BM-derived and resident putative MC (CD11b + Ly6G − ) to 4872 and 3761 from 1332 and 599 cells/aorta in the control. The putative MC was further divided into 4 distinct subsets, severe HHcy, compared with the control, increased BM-derived and resident Ly6C hi MC (Ly6C hi F4/80 − , 816 vs 43 and 842 vs 115), differentiating MC (Ly6C hi F4/80 + , 558 vs 43 and 784 vs 28), differentiated macrophage (Ly6C low F4/80 − , 1874 vs 766 and 1095 vs 92), and Ly6C low MC (Ly6C low F4/80 − , 1624 vs 480 and 1040 vs 365) in aorta. Plasma Hcy levels were significantly correlated with plasma TNF α (r=0.650), IL-6 (r=0.594) and MCP-1 (r=0.591) levels. Hcy-lowering therapy significantly reduced atherosclerotic lesion, plasma inflammatory cytokine levels and aortic MC differentiation. In vitro, L-Hcy (100 –500 μ M) enhanced IFN γ -induced Ly6C hi+mid MC differentiation, which was abolished by Tiron, SOD/catalase, and NADPH oxidase inhibitor apocynin. L-Hcy (500 μ M) increased the TNFa/IL-6 cytokine-producing Ly6C hi+mid MC by 3-fold in IFN γ -treated cells and significantly enhanced transendothelial migration of IFN γ -treated THP1 cells. Conclusions: Severe HHcy induces systemic inflammation and promotes BM-derived and vessel resident Ly6C inflammatory MC differentiation leading to exacerbated atherosclerosis. NADPH oxidase mediated oxidative stress participates in Ly6C inflammatory MC differentiation induced by Hcy.