Resident Monocytes Research Articles

Amniotic Mesenchymal Tissue Cells Inhibit Dendritic Cell Differentiation of Peripheral Blood and Amnion Resident Monocytes

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G(0) phase and abolishes the production of inflammatory cytokines such as TNF-alpha, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood “resident” monocytes, and embryonic macrophages suggests common functions and developmental relationships

AbstractWe previously showed that Tie2-expressing monocytes (TEMs) have nonredundant proangiogenic activity in tumors. Here, we compared the gene expression profile of tumor-infiltrating TEMs with that of tumor-associated macrophages (TAMs), spleen-derived Gr1+Cd11b+ neutrophils/myeloid-derived suppressor cells, circulating “inflammatory” and “resident” monocytes, and tumor-derived endothelial cells (ECs) by quantitative polymerase chain reaction–based gene arrays. TEMs sharply differed from ECs and Gr1+Cd11b+ cells but were highly related to TAMs. Nevertheless, several genes were differentially expressed between TEMs and TAMs, highlighting a TEM signature consistent with enhanced proangiogenic/tissue-remodeling activity and lower proinflammatory activity. We validated these findings in models of oncogenesis and transgenic mice expressing a microRNA-regulated Tie2-GFP reporter. Remarkably, resident monocytes and TEMs on one hand, and inflammatory monocytes and TAMs on the other hand, expressed coordinated gene expression profiles, suggesting that the 2 blood monocyte subsets are committed to distinct extravascular fates in the tumor microenvironment. We further showed that a prominent proportion of embryonic/fetal macrophages, which participate in tissue morphogenesis, expressed distinguishing TEM genes. It is tempting to speculate that Tie2+ embryonic/fetal macrophages, resident blood monocytes, and tumor-infiltrating TEMs represent distinct developmental stages of a TEM lineage committed to execute physiologic proangiogenic and tissue-remodeling programs, which can be coopted by tumors.

Atherosclerosis is an inflammatory disease

During the last decades, emerging evidence has shown that atherosclerosis is characterised by a complex endocrine, paracrine and juxtacrine cross-talk between immune and vascular cells as well as several tissues and organs, including the liver, heart, kidney, adipose tissue, adrenal, pancreatic, pituitary and sex glands [1]. Thus, atherosclerosis has to be considered as a “systemic disease”, characterised by arterial inflammatory lesions that mature and modify themselves with disease progression. Atherosclerotic plaques are the battlefield among an unbalanced immune response and lipid accumulation in the intima of arteries. Although the “first event” favouring early lipid deposition within the arterial sub-intimal space has not been identified, the infiltration of low-density lipoproteins (LDL) should be considered as the pivotal step in plaque formation [2]. Oxidative and enzymatic modifications of retained LDL increase the expression of adhesion molecules on endothelial cell surface membrane, thus favouring leukocyte recruitment from the blood stream [3]. Leukocytes recruitment within atherosclerotic plaques is also orchestrated by the chemokine binding to their receptors on cell membrane. This is a redundant system which regulates all inflammatory phases of atherogenesis in both humans and animals [4]. Monocytes have been defined by Weber et al. as the “crucial force” to drive atherogenesis [3]. In vivo monocyte inflammatory functions and differentiation are influenced by the different expression of chemokine receptors, CD14 and CD16. Two different monocyte subsets have been identified. Inflammatory monocyte subset (CXC3CR1 CCR2Ly6CCD14CD16) represents the main population recruited within the plaque from the blood stream and display both phagocytic and proteolytic activities. On the other hand, resident monocyte subset (CXC3CR1 CCR2Ly6CCD14CD16) is the main population involved in the initiation of tissue inflammation, angiogenesis and atheroprogression [3]. Modified LDL are uptaked by scavenger receptors of resident macrophages that differentiate into different inflammatory cell types, such as dendritic cells, foam cells, osteoclastand osteoblast-like cells. These processes are modulated by several cytokines and growth factors (such as receptor activator of nuclear factor [NF]kappaB ligand [RANKL], osteoprotegerin [OPG], tumour necrosis factor [TNF]alpha, granulocyte macrophage-colony stimulating factor [GM-CSF], macrophage-colony-stimulating factor [MCSF] and interleukin [IL]-4). On the other hand, connexins, which mediate gap junction formation between adjacent cells (vascular and immune cells), have been recently shown to playing a crucial role in cell differentiation and growth. Three different connexins (Cx37, Cx40 and Cx43) are differently expressed in macrophage and endothelial cells during atherosclerotic plaque maturation [5]. At this step, the non-specific immune response, mainly characterised by local monocyte/macrophage-mediated inflammatory processes has started. In a second phase, resident inflammatory and vascular cells further produce chemokines for the recruitment of T lymphocytes within the plaque. This induces the deleterious vicious circle of the Semin Immunopathol (2009) 31:1–3 DOI 10.1007/s00281-009-0146-7

Characterization of folate receptor positive macrophages and monocytes and folate receptor-mediated specific targeting in inflammation (92.10)

Abstract Folate receptor (FR) expressing macrophages have been shown to play an integral role in a number of inflammatory diseases. (1) In this report, we characterize these FR+ macrophages and further investigate whether monocytes, the precursors to these macrophages, also express FR. We demonstrate that FR expression is observed on a subset of highly activated peritoneal and synovial macrophages, but not on other infiltrating immune cells or resident macrophages. We further find that a subset of human peripheral blood monocytes also expresses FR, albeit at a lower level than activated macrophages. Surface antigen characterization of the FR+ monocytes suggests markers characteristic of an inflammatory phenotype (CD14hiCD16), while the resident monocytes (CD14loCD16+) express few or no FR. Preliminary clinical data also suggest that the numbers of peripheral FR+ monocytes can be reduced by a folate-targeted immunotherapy. These results suggest that activated macrophages and inflammatory monocytes express a FR that can be targeted with folate-linked drugs without promoting drug uptake by non-activated macrophages and resident monocytes. 1. Xia W, Hilgenbrink AR, et al. Blood. 2008 Oct 24. [Epub ahead of print] This work was supported in part by a grant from Endocyte, Inc.

CCR2+ and CX3CR1+ Bone Marrow‐Derived Cells Differentially Regulate Microvascular Remodeling in an Inflammation/Injury Model Without Transdifferentiating into Smooth Muscle

Monocytes are generally known to regulate vascular remodeling. However, monocyte subpopulations with distinct functional roles exist, and their relative contributions to remodeling are poorly understood. Here, the relative expression of CX3CR1 and CCR2, which mark inflammatory (CCR2high/CX3CR1low) and resident (CCR2low/CX3CR1high) monocytes, was used to study the roles of these cells in regulating microvascular remodeling during injury/inflammation. Dorsal skin‐fold window chambers were implanted into wild‐type (WT) c57bl/6 mice engrafted with bone marrow‐derived cells (BMCs) from WT (control), CCR2−/−, CX3CR1−/−, or EGFP+ mice. WT control chimeras exhibited significant increases in maximum arteriolar (68%) and venular (40%) diameters from day 7 to 14. In contrast, arteriolar remodeling (‐0.8%) was completely blocked in CCR2−/− chimeras, but no change in venular remodeling was observed compared to control (37%). Interestingly, CX3CR1−/− chimeras exhibited only a partial attenuation of arteriolar (32%) and venular (14%) remodeling. EGFP+ chimeras revealed no evidence that recruited EGFP+ BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. We conclude that CCR2+ and CX3CR1+ BMCs differentially regulate microvascular remodeling during the injury/inflammation response. Because these cells do not transdifferentiate into vascular cells, it is likely that both subpopulations serve as paracrine growth factor sources. Supported by NIH HL74082.

Regulation of triggering receptor expressed on myeloid cells 1 expression on mouse inflammatory monocytes

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor involved in inflammatory diseases and septic shock. The TREM-1 ligand(s) (TREM-1L) have not yet been identified. In this study, we performed a detailed analysis of the expression of mouse TREM-1 and its ligand(s). Our results demonstrate that TREM-1 is expressed on bone-marrow-derived dendritic cells (BMDC). On bone-marrow-derived macrophages (BMM) its expression is induced in vitro after stimulation by granulocyte-macrophage colony-stimulating factor, interleukin-3 or by myeloid differentiation primary response gene 88 (MyD88)-dependent Toll-like receptor (TLR) ligands. Under steady-state conditions mouse TREM-1 is detectable on a Gr-1(-) F4/80(+) monocyte subpopulation bearing markers of resident monocytes, but not on Gr-1(+) F4/80(+) inflammatory monocytes. During lipopolysaccharide (LPS)-induced endotoxaemia TREM-1 was also up-regulated on inflammatory Gr-1(+) F4/80(+) cells in vivo. In tumour-bearing mice, TREM-1 was up-regulated on Gr-1(+) F4/80(+) monocytes, which phenotypically and functionally resembled mononuclear myeloid-derived suppressor cells. Using a soluble TREM-1 fusion protein, we demonstrate that after intravenous injection of LPS TREM-1L was induced on Gr-1(+) granulocytes and monocytes but not on other cell populations in peripheral blood. This up-regulation on granulocytes was directly mediated by TLR ligands and required the adapter protein MyD88. In contrast to human, mouse platelets expressed TREM-1L neither under steady-state conditions nor after LPS injection in vivo. Our study reveals differential regulation of TREM-1 expression on mouse monocyte subpopulations and improves our understanding of the biological role of TREM-1 during disease.

Immune System and Atherosclerotic Disease

Atherosclerosis is an inflammatory disease in which a systemic inflammatory reaction is combined with an accumulation of immune cells, such as monocytes/macrophages, dendritic cells (DCs), and numerous lymphocytes, in atherosclerotic plaques. The immune system, comprising innate immunity and adaptive immunity, has been implicated in all stages of atherosclerosis, from initiation through progression and in atherothrombotic complications. It is clear that different subpopulations of leukocytes are involved in the pathogenesis of atherosclerosis and plaque instability. Recent studies have also demonstrated that each heterogeneity of immune-associated cells contributes to the atherogenic and atheroprotective axis. This review highlights recent advances in research and explores the role of the complex heterogeneity of leukocyte subsets, especially monocytes/macrophages (inflammatory monocytes, resident monocytes, M1, and M2), DCs (myeloid DCs, plasmacytoid DCs, pre DCs, conventional DCs, inflammatory DCs), and CD4(+) cells (T-helper 1, T-helper 2, regulatory T, and T-helper 17 cells), in the initiation and development of atherosclerotic disease and its complications.

Extranodal Rosai-Dorfman disease of the kidney

Extranodal Rosai-Dorfman disease of the kidney

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Neutrophil secretion products pave the way for inflammatory monocytes

AbstractThe leukocyte response in inflammation is characterized by an initial recruitment of polymorphonuclear leukocytes (PMN) preceding a second wave of monocytes to the site of injury or infection. In the mouse, 2 populations of monocytes have been identified, Gr1−CCR2−CX3CR1hi resident monocytes and Gr1+CCR2+CX3CR1lo inflammatory monocytes. Here, intravital microscopy of the musculus cremaster and a subcutaneous air pouch model were used to investigate a possible link between PMN extravasation and the subsequent emigration of inflammatory monocytes in response to local stimulation with PAF. In mice that were made neutropenic by injection of a PMN-depleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell count but was restored by local treatment with secretion of activated PMN. Components of the PMN secretion were found to directly activate inflammatory monocytes and further examination revealed PMN-derived LL-37 and heparin-binding protein (HBP/CAP37/azurocidin) as primary mediators of the recruitment of inflammatory monocytes via activation of formyl-peptide receptors. These data show that LL-37 and HBP specifically stimulate mobilization of inflammatory monocytes. This cellular cross-talk functionally results in enhanced cytokine levels and increased bacterial clearance, thus boosting the early immune response.

Neoadjuvant immunotherapy for prostate cancer with GM-CSF and tumor infiltration by antigen presenting cells

3063 Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for antigen presenting cells that may enhance anti-tumor immune responses. Although this cytokine is being evaluated in immunotherapy trials for different malignancies, the impact of GM-CSF dose and schedule has not been directly addressed. Because systemic GM-CSF could potentiate anti-tumor immunity by enhancing presentation of endogenous tumor antigens, we examined whether increasing duration of GM-CSF treatment prior to radical prostatectomy could alter the composition of antigen presenting cells within the prostate tissue. Methods: A phase I trial in patients with localized prostate cancer was performed in subjects who were planned to undergo radical prostatectomy (RP). Two cohorts of 6 patients were sequentially accrued and treated with either 2 weeks or 3 weeks of daily GM-CSF (sargramostim, Bayer) 250 mg/m2/d SC. Patients were monitored for toxicity as well as for changes in circulating immune cells. RP tissues were assessed for immune cells by flow cytometry or immunohistochemistry (IHC) and digital slide scanning. In addition, IHC was also performed on RPs from untreated control Gleason grade- matched patients. Results: 12 patients were treated with neoadjuvant GM-CSF for either 2 weeks or 3 weeks. While significant leukocytosis was observed with treatment, no dose-limiting toxicities were encountered. As expected, circulating monocytes and dendritic cells (DC) were expanded following treatment. Compared to RPs from 11 untreated control patients, there was an increase in prostate-resident CD1a+ interstitial DC (Kruskal-Wallis test: p = 0.04). The increase was most pronounced in the 3-week treatment cohort. Moreover, tumor-resident DC expressed higher levels of the activation markers CD83 and 86 compared to DC residing in adjacent normal prostate and benign prostatic hypertrophy tissue. Conclusions: Systemically administered neoadjuvant GM-CSF can enhance the frequency of DC in prostate cancer. These effects appear to be dose dependent as they were most pronounced with 3 weeks of treatment. The composition of resident DC and monocytes also differs between malignant, benign, and hypertrophic prostate tissue. No significant financial relationships to disclose.

Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

Sialic acid–binding Ig‐like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus

Type I interferon (IFN) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE) and is therefore considered a potential therapeutic target. This study was undertaken to establish a feasible biomarker for IFN effects with respect to disease activity and effectiveness of IFN-suppressive therapy in SLE patients. Transcriptomes of purified monocytes from 9 SLE patients and 7 healthy controls were analyzed by Affymetrix GeneChip technology. Levels of sialic acid-binding Ig-like lectin 1 (Siglec-1) (sialoadhesin, CD169) in inflammatory and resident monocytes were determined at the protein level in 38 healthy controls and 52 SLE patients, using multicolor flow cytometry. Transcriptomes of peripheral monocytes from SLE patients revealed a dominant type I IFN signature. Siglec-1 was identified as one of the most prominent type I IFN-regulated candidate genes. At the protein level, the frequency of Siglec-1-expressing monocyte subsets was correlated with disease activity (as measured by the SLE Disease Activity Index) and was inversely correlated with levels of complement factors. Most interestingly, levels of anti-double-stranded DNA (anti-dsDNA) antibodies were highly correlated with the percentage of resident monocytes, but not inflammatory monocytes, expressing Siglec-1. High-dose glucocorticoid treatment resulted in a dramatic reduction of Siglec-1 expression in cells from patients with active SLE. Our findings indicate that Siglec-1 expression in resident blood monocytes is a potential biomarker for monitoring disease activity, displaying type I IFN responses, and estimating levels of anti-dsDNA antibodies. Moreover, our results suggest that resident and inflammatory monocytes contribute differently to the process of autoantibody formation in SLE.

A Vicious Cycle Involving Release of Heat Shock Protein 60 from Injured Cells and Activation of Toll-Like Receptor 4 Mediates Neurodegeneration in the CNS

Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

THERMAL INJURY ELEVATES THE INFLAMMATORY MONOCYTE SUBPOPULATION IN MULTIPLE COMPARTMENTS

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b LY6C), IMs (CD11b LY6C), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-alpha was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-alpha. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1alpha. After burn injury, mice deficient in the receptor for soluble TNF-alpha had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-alpha.

The Inflammatory Subset of Monocytes Stimulates Angiogenesis at Sites of Ischemia by Altering the Balance of Pro- and Anti-Inflammatory Cytokines.

Our laboratory has previously shown that early delivery of monocytes to ischemic tissue by mobilization from the bone marrow to blood or by adoptive transfer enhances revascularization after acute vascular injury. There is accumulating evidence that monocytes represent a heterogeneous population, containing subsets with distinct functional properties. Herein, we show that the inflammatory subset of monocytes mediates the angiogenic response in a murine model of hindlimb ischemia induced by femoral artery excision (FAE). Inflammatory (CX3CR1loGr-1+) and resident (CX3CR1hiGr-1−) monocytes were sorted from the bone marrow of CX3CR1+/GFP mice and injected intravenously into recipient mice 24 after FAE. Revascularization was monitored by laser Doppler imaging. A marked improvement in blood flow was observed after adoptive transfer of inflammatory but not resident monocytes [ratio of blood flow in ischemic to nonischemic limb 7 days after surgery: 0.75±0.05 (inflammatory monocytes); 0.40±0.07 (resident monocytes); 0.40±0.03 (saline control); P <0.01]. Consistent with this finding, inflammatory monocytes are selectively recruited to sites of ischemia. Finally, we show that the bone marrow is a rich reservoir of inflammatory monocytes that is readily mobilized into the blood by ischemia. To begin to elucidate the mechanisms by which inflammatory monocytes stimulate angiogenesis, we determined the effect of the adoptive transfer of monocytes on the local production of key cytokines/chemokines in the ischemic tissue. Specifically, a non-biased multiplex immunoassay-based screen for soluble factors present in the cell-free muscle homogenates was performed. Data revealed decreased levels of the pro-inflammatory cytokines IL-6, KC, and mip-1beta and increased levels of the anti-inflammatory cytokine IL-10 in mice receiving an adoptive transfer of monocytes. In keeping with this observation, levels of free myoglobin were dramatically decreased in mice receiving an adoptive transfer of monocytes, indicating a reduction in inflammation-mediated tissue damage [myoglobin levels at 28 hours post-ischemia: 18,580±879 ng/mL (no adoptive transfer); 158±112 ng/mL (adoptive transfer)]. Interestingly, known regulators of angiogenesis, MCP-1, MMP-9, VEGF, and FGF-basic were also significantly increased. Taken together, these data indicate that adoptive transfer of a small number of monocytes shifts the balance of pro- and anti-inflammatory signals in the ischemic environment, which limits tissue damage and enhances revascularization. This finding has important therapeutic implications, since deliver of inflammatory monocytes to sites of ischemia may polarize the microenviroment towards tissue regeneration and angiogenesis.

Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo.

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate here that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− FL cells into lethally irradiated wild-type (wt) mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. While the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

Ly-6Chi INFLAMMATORY MONOCYTES GOVERN THE SEVERITY OF CAERULEIN- AND TAUROCHOLATE-INDUCED ACUTE PANCREATITIS IN MICE

Background: Recent reports have shown that circulating monocytes exist in at least 2 phenotypic subsets- i.e. resident (Ly-6Clo) and inflammatory (Ly-6Chi) monocytes. Inflammatory monocytes traffic to, and enter, sites of inflammation in response to appropriate stimuli and, as inflammatory tissue macrophages, they play key pro-inflammatory roles. We have previously shown that targeted and conditional monocyte/macrophage depletion reduces the severity of caerulein and taurocholate-induced pancreatitis. Aim: To identify the monocyte subset that regulates the severity of pancreatitis and to examine its route of trafficking. Methods: Mice expressing the human diphtheria toxin receptor (hDTR) coupled to the CD11b promoter were pre-treated with diphtheria toxin (DT) to deplete monocytes/ macrophages and, 24 hrs later, caerulein or taurocholate-induced pancreatitis was elicited. Twelve hrs after the start of pancreatitis induction, the mice were infused with peripheral blood mononuclear cells (PBMC) or bone marrow cells (BMC) obtained from naive donors. In trafficking studies, donor cells were pre-labeled by incubation with CFSE. Pancreatitis severity and the fate of adoptively transferred PBMC or BMC were evaluated using standard histological methods and flow cytometry. Results: Ly-6Chi cells were (a) depleted in the pancreas during pancreatitis, (b) present in the adoptive transfer mixture, and (c) found in circulating blood after adoptive transfer of either PBMC or BMC. Pancreatitis severity was restored by adoptive transfer of either PBMC or BMC but CFSE-labeled Ly-6Chi cells were found in the pancreas only after adoptive transfer of BMC. Removal of Ly-6Chi cells from the adoptive transfer pool prevented restoration of pancreatitis severity after adoptive transfer. Conclusions: Pancreatitis severity is governed by inflammatory Ly-6Chi monocytes that are mobilized from the bone marrow but act after trafficking to the pancreas.

Abstract 810: Recruitment of the Inflammatory Subset of Monocytes to Sites of Ischemia Induces Angiogenesis in a Monocyte Chemoattractant Protein-1 Dependent Fashion

Our lab has previously shown that mobilization of monocytes from the bone marrow to the blood by G-CSF and/or AMD3100 administration stimulated angiogenesis and enhanced revascularization following the induction of hindlimb ischemia in mice. Recent data suggest that monocytes can be divided into two distinct subsets based upon the expression of the fractalkine receptor CX3CR1. The inflammatory subset of monocytes express low levels of CX3CR1 but high levels of CCR2 and Gr-1. In contrast, the resident subset of monocytes express high levels of CX3CR1 but no CCR2 or Gr-1. Using transgenic mice expressing GFP under the control of the 2.1kb CX3CR1 promoter, we sorted inflammatory and resident monocytes and adoptively transferred them into mice 24 hours after the induction of hindlimb ischemia. Whereas inflammatory monocytes markedly improved revascularization, no effect was observed with resident monocytes. These data suggest that the inflammatory monocyte subset may be an attractive cell population to study in the therapeutic setting. The mechanism by which monocytes stimulate angiogenesis at sites of inflammation is not fully understood. Herein, we show that adoptively transferred CFSE-labeled monocytes are rapidly and specifically recruited from the blood to the ischemic hindlimb. These recruited donor monocytes induce a massive secondary recruitment of host monocytes that is associated with local production of MCP-1. To further explore the role of MCP-1 in this process, we repeated these adoptive transfer experiments in MCP-1 −/ − mice. Adoptive transfer of wild type monocytes into wild type mice markedly improved revascularization while no angiogenic effect was observed after adoptive transfer into MCP-1 −/ − mice. Moreover, whereas recruitment of adoptively transferred CFSE-labeled monocytes into the ischemic limb in MCP-1 −/ − mice was normal, the induction of MCP-1 expression and secondary recruitment of endogenous monocytes from the blood was significantly attenuated. Furthermore, MCP-1 −/ − mice showed no enhancement of angiogenesis following mobilization with G-CSF and AMD3100. These data suggest that MCP-1 plays a key role in the inflammatory angiogenic response by recruiting host monocytes from the blood into the ischemic tissue.

Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection

Neutrophils, monocytes, and dendritic cells (DC) are phenotypically and functionally related phagocytes whose presence in infected tissues is critical to host survival. Their overlapping expression pattern of surface molecules, the differentiation capacity of monocytes, and the presence of monocyte subsets underscores the complexity of understanding the role of these cells during infection. In this study we use five- to seven-color flow cytometry to assess the phenotype and function of monocytes recruited to Peyer's patches (PP) and mesenteric lymph nodes (MLN) after oral Salmonella infection of mice. The data show that CD68(high)Gr-1(int) (intermediate) monocytes, along with CD68(int)Gr-1(high) neutrophils, rapidly accumulate in PP and MLN. The monocytes have increased MHC-II and costimulatory molecule expression and, in contrast to neutrophils and DC, produce inducible NO synthase. Although neutrophils and monocytes from infected mice produce TNF-alpha and IL-1beta upon ex vivo culture, DC do not. In addition, although recruited monocytes internalize Salmonella in vitro and in vivo they did not induce the proliferation of OT-II CD4(+) T cells after coincubation with Salmonella expressing OVA despite their ability to activate OT-II cells when pulsed with the OVA(323-339) peptide. We also show that recruited monocytes enter the PP of infected mice independently of the mucosal address in cell adhesion molecule-1 (MAdCAM-1). Finally, recruited but not resident monocytes increase in the blood of orally infected mice, and MHC-II up-regulation, but not TNF-alpha or iNOS production, occur already in the blood. These studies are the first to describe the accumulation and function of monocyte subsets in the blood and GALT during oral Salmonella infection.