Engulfment of Hb‐activated platelets differentiates monocytes into pro‐inflammatory macrophages in PNH patients

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)‐activated platelets, classical monocytes differentiated into pro‐inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb‐activated platelets, monocytes differentiate into M1‐macrophages under M1‐polarization stimulus (GM‐CSF, IFN‐γ + LPS). When grown under M2‐polarization stimulus (M‐CSF, IL‐4 + IL13), the cells exhibited an M1‐like phenotype, secreted elevated levels of pro‐inflammatory cytokines including TNF‐α and IL‐1β, and displayed loss of the secretion of cytokine such as IL‐10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho‐STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte–platelet aggregates in circulation and accumulation of CD80^high macrophages in thioglycollate‐induced inflamed peritoneum. The spleen of the mice was also populated by CD80^high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb‐activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.     

CD13 is a novel mediator of monocytic/endothelial cell adhesion

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept is strengthened by the fact that activated monocytic cells adhere to immobilized recombinant CD13. Furthermore, treatment with anti-CD13 antibodies in a murine model of peritonitis results in a decrease in leukocyte infiltration into the peritoneum, suggesting a potential role for CD13 in leukocyte trafficking in vivo. Therefore, this work supports a new direction for CD13 biology, where these cell surface molecules act as true molecular interfaces that induce and participate in critical inflammatory cell interactions.     

Wnt5a Inhibits Human Monocyte‐Derived Myeloid Dendritic Cell Generation

Wnt5a is a non‐canonical Wnt protein that is expressed at elevated levels in inflammatory conditions. Its role in inflammation remains unclear, although it is known that Wnt5a is expressed at a higher level in monocyte‐derived myeloid dendritic cells (Mo‐mDCs) than in monocytes and macrophages. The function of Wnt5a in dendritic cells (DCs) remains relatively unexplored. Here, we found that under Mo‐mDC culture conditions, Wnt5a inhibited the generation of CD14^+/low Mo‐mDCs while promoting the generation of CD14^+/++ CD16⁺ monocytes. We could further show that stimulation of monocytes with rWnt5a induced a rapid IL‐6 production and that the rWnt5a treated Mo‐mDC differentiation was restored upon blocking of IL‐6. Also, conditioned media from Wnt5a stimulated human breast cancer cells producing IL‐6, specifically inhibited Mo‐mDC differentiation. These observations are strengthened by our finding that patients with sepsis, a disease involving elevated Wnt5a and IL‐6 levels, also showed a significant increase in the CD14⁺ CD16⁺⁺/CD14^+/++ CD16⁺ monocyte populations, which was accompanied by a significant decrease in circulating mDCs. We finally show that under typical Mo‐mDC culture conditions, monocytes isolated from patients with sepsis as compared to healthy controls, preferentially differentiated into CD14^+/++ HLA‐DR⁺⁺ cells. We suggest that Wnt5a is a possible candidate mediator for the CD14^+/++ CD16⁺ monocyte accumulation seen in patients with infectious disease and cancer.     

Investigating the role of proinflammatory CD16+ monocytes in the pathogenesis of inflammatory bowel disease

Infiltrating monocytes and macrophages contribute to the initiation and perpetuation of mucosal inflammation characteristic for human inflammatory bowel disease (IBD). Peripheral blood monocytes expressing the low‐affinity Fcγ receptor CD16 have been identified previously as a major proinflammatory cell population, based on their unique cytokine secretion profile. However, the contribution of these cells to the pathogenesis of inflammatory bowel disease remains to be elucidated. Thus, in this study we investigated whether the peripheral CD16⁺ monocyte count correlates with common IBD disease parameters, and whether these cells infiltrate the intestinal mucosa under inflammatory conditions. We observed that CD16⁺ peripheral blood monocytes are increased significantly in active Crohn's disease, particularly in patients with high Crohn's disease activity index and colonic involvement. Furthermore, we found that CD16⁺ cells are a major contributor to the inflammatory infiltrate in Crohn's disease mucosa, although their spontaneous migration through primary human intestinal endothelial cells is limited. Our data suggest that lamina propria, but not peripheral blood, CD16⁺ monocytes are a crucial proinflammatory cell population in IBD, and a potential target for anti‐inflammatory therapy.     

CXCR4 antagonist AMD3100 redistributes leukocytes from primary immune organs to secondary immune organs,lung, and blood in mice

AMD3100 (plerixafor), is a specific CXCR4 antagonist approved by the FDA for mobilizing hematopoietic stem cells from bone marrow to blood for transplantation in cancer. AMD3100 also mobilizes most mature leukocyte subsets to blood; however, their source and trafficking potential have not been fully delineated. Here, we show that a single injection of AMD3100 10 mg/kg into C57Bl/6 mice rapidly mobilizes (peak ～ 2.5 h) the same leukocyte subsets to blood as in humans. Using this model, we found that AMD3100 mobilization of neutrophils, lymphocytes, and monocytes to blood is not reduced by splenectomy or by blockade of lymphocyte egress from lymph node with FTY720, but is coupled to (i) reduced content of each of these cell types in the bone marrow; (ii) reduced T‐cell numbers in thymuses; (iii) increased lymphocytes in lymph nodes; and (iv) increased neutrophil and monocyte content in the lung. Direct intrathymic labeling showed that AMD3100 selectively mobilizes naïve thymic CD4⁺ and CD8⁺ T cells to blood. Finally, AMD3100‐induced neutrophil mobilization to blood did not reduce neutrophil trafficking to thioglycollate‐inflamed peritoneum. Thus, AMD3100 redistributes lymphocytes, monocytes, and neutrophils from primary immune organs to secondary immune organs, peripheral tissues, and blood, without compromising neutrophil trafficking to inflamed sites.     

Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice

Foxp3⁺ regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss‐of‐function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X‐linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue‐specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M‐CSF and other inflammatory cytokines, including monocyte‐recruiting chemokines. Adoptive transfer of WT CD4⁺ cells and in vivo neutralization of M‐CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte‐related cytokines in Sf mice, while Treg cell transfer to RAG2^?/? mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.     

Monocyte infiltration into obese and fibrilized tissues is regulated by PILRα

Paired immunoglobulin‐like type 2 receptor α (PILRα) is an inhibitory receptor that is mainly expressed on myeloid cells, and negatively regulates neutrophil infiltration during inflammation. However, PILRα role on monocyte has not been described. Under both steady‐state and inflammatory conditions, monocytes migrate into tissues and differentiate into macrophages. Macrophages in adipose and liver tissues play important roles in tissue homeostasis and pathogenesis of metabolic diseases. Here, we found that PILRα controls monocyte mobility through regulating integrin signaling and inhibiting CD99–CD99 binding. Moreover, we found that Pilra^?/? mice developed obesity and hepatomegaly with fibrosis, and the numbers of macrophages in adipose and liver tissues are significantly increased in Pilra^?/? mice. These data suggest that immune inhibitory receptor, PILRα, plays an important role in the prevention of obesity and liver fibrosis.     

The early monocytic response to cytomegalovirus infection is MyD88 dependent but occurs independently of common inflammatory cytokine signals

Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M‐CSF receptor. Activated bone marrow monocytes were more sensitive to M‐CSF and less sensitive to granulocyte‐monocyte colony stimulating factor in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88‐sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88 independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL‐6, TNF‐α and IL‐1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine‐like protein MCK‐2 (m131/129) expressed by murine CMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection.     

Circulating CD14+CD16+ monocyte levels predict tissue invasive character of cholangiocarcinoma

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro‐tumorigenic activity. In this study we identified elevation in CD14⁺CD16⁺, a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14⁺CD16⁺ monocytes in CCA patient blood correlated with degree of MAC387‐positive (recent blood‐derived macrophage migrant‐specific marker) tumour‐associated macrophage infiltration as determined by immunohistochemistry. These CD14⁺CD16⁺ monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor‐related genes (epiregulin, VEGF‐A and CXCL3). Elevation of peripheral CD14⁺CD16⁺ monocyte levels was associated with features associated with poor prognosis CCA parameters (non‐papillary type and high number of tissue macrophages). These data indicate that the CD14⁺CD16⁺ monocytes from CCA patients with pro‐tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14⁺CD16⁺ monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.     

Human β defensin‐3 induces chemokines from monocytes and macrophages: diminished activity in cells from HIV‐infected persons

Human β defensin‐3 (hBD‐3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD‐3 and, for comparison, Pam3CSK4 and LL‐37 to induce co‐stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including monocyte chemoattractant protein 1 (MCP‐1), Gro‐α, macrophage‐derived chemokine (MDC) and macrophage inflammatory protein 1β (MIP1β), while only hBD‐3 and Pam3CSK4 significantly induced the angiogenesis factor, vascular endothelial growth factor (VEGF). Human BD‐3 induced similar chemokines in monocyte‐derived macrophages and additionally induced expression of Regulated upon activation normal T‐cell expressed and presumably secreted (RANTES) in these cells. Comparison of monocytes from HIV⁺ and HIV^– donors indicated that monocytes from HIV⁺ donors were more likely to spontaneously express certain chemokines (MIP‐1α, MIP‐1β and MCP‐1) and less able to increase expression of other molecules in response to hBD‐3 (MDC, Gro‐α and VEGF). Chemokine receptor expression (CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV⁺ donors compared with cells from HIV^– donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset (CD14⁺ CD16⁺⁺) of HIV⁺ donors. These observations implicate chemokine induction by hBD‐3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV‐infected persons could influence cell migration and modify the effects of hBD‐3 at sites of inflammation.     

Macrophage‐mediated psoriasis can be suppressed by regulatory T lymphocytes

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis‐like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro‐inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis‐like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF‐mediated psoriasis, ihTNFtg mice were crossbred into RAG1^KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1^KO or wild‐type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild‐type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3‐positive cells into RAG1^KO or wild‐type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro‐inflammatory activity of macrophages, which are the major immune effector cells in hTNF‐mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4⁺ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4⁺ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4⁺ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4⁺ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4⁺ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4⁺ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4⁺ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology.     

NR4A1‐dependent Ly6Clow monocytes contribute to reducing joint inflammation in arthritic mice through Treg cells

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1^?/? mice, which lack patrolling lymphocyte antigen 6C (Ly6C^low) monocytes, we found that inflammatory Ly6C^high monocytes contribute to rapid development of arthritis in a serum transfer‐induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1^?/? mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn‐B), a NR4A1‐specific agonist, significantly reduces severity of disease. Effects of Csn‐B were absent in monocyte‐depleted mice treated with clodronate until Ly6C^low monocytes were restored. Adoptive transfer of Ly6C^low monocytes in arthritic NR4A1^?/? mice treated with Csn‐B reduces joint inflammation, supporting the regulatory role of Ly6C^low subset on disease development. Our results also reveal that administration of Csn‐B to arthritic mice enhances levels of circulating CD4⁺CD25⁺FoxP3⁺ Treg cells, a process requiring the presence of Ly6C^low monocytes. Together, these data indicate that Ly6C^high monocytes are involved in the initiation and progression of arthritis and Ly6C^low monocytes contribute to reduce joint inflammation through the mobilization of Treg cells.     

Zoledronic acid causes γδ T cells to target monocytes and down‐modulate inflammatory homing

Zoledronic acid (ZA) is a potential immunotherapy for cancer because it can induce potent γδ T‐cell‐mediated anti‐tumour responses. Clinical trials are testing the efficacy of intravenous ZA in cancer patients; however, the effects of systemic ZA on the activation and migration of peripheral γδ T cells remain poorly understood. We found that γδ T cells within ZA‐treated peripheral blood mononuclear cells were degranulating, as shown by up‐regulated expression of CD107a/b. Degranulation was monocyte dependent because CD107a/b expression was markedly reduced in the absence of CD14⁺ cells. Consistent with monocyte‐induced degranulation, we observed γδ T‐cell‐dependent induction of monocyte apoptosis, as shown by phosphatidylserine expression on monocytes and decreased percentages of monocytes in culture. Despite the prevailing paradigm that ZA promotes tumour homing in γδ T cells, we observed down‐modulation of their tumour homing capacity, as shown by decreased expression of the inflammatory chemokine receptors CCR5 and CXCR3, and reduced migration towards the inflammatory chemokine CCL5. Taken together our data suggest that ZA causes γδ T cells to target monocytes and down‐modulate the migratory programme required for inflammatory homing. This study provides novel insight into how γδ T cells interact with monocytes and the possible implications of systemic use of ZA in cancer.     

A two-step human culture system replicates intestinal monocyte maturation cascade: Conversion of tissue-like inflammatory monocytes into macrophages

Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte “waterfall” process in vitro by first, generating tissue-like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14⁺CD16⁻ monocytes, cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ and IL23, differentiated into CD14⁺CD163⁻ cells that displayed a monocyte-like morphology. In vitro generated inflammatory CD14⁺CD163⁻ (inflammatory monocyte-like cells) cells promoted IL-1β-dependent memory Th17 and Th17/Th1 responses, like the CD14⁺CD163⁻ mo-like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte-like cells converted to functional CD163⁺ MΦ following exposure to TGF-β and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163⁺ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two-step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.     

Elevated circulating CD14<Superscript>low</Superscript>CD16<Superscript>+</Superscript> monocyte subset in primary biliary cirrhosis correlates with liver injury and promotes Th1 polarization

Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease in which monocytes/macrophages infiltration and skewed T helper type (Th) 1 and Th17 cell responses participate in the development of the disease. Human peripheral blood monocytes are heterogeneous and can be divided into classical CD14^highCD16^?, intermediate CD14^highCD16⁺, and nonclassical CD14^lowCD16⁺ monocyte subsets. Compared to classical monocytes, CD16⁺ monocytes are generally termed pro-inflammatory monocytes and play an important pathogenic role in autoimmune diseases. However, little is known about the immunophenotype and immunopathogenic role of peripheral blood CD16⁺ monocytes in PBC. Thus, we investigated the phenotype and function of these circulating monocyte subsets from PBC patients. The frequencies of circulating CD14^highCD16⁺ and CD14^lowCD16⁺ subpopulation were increased in disease compared with healthy controls. Among them, CD14^lowCD16⁺ monocyte subset positively correlated with disease progress, liver damage indicators and serum C-reactive protein, respectively. Furthermore, the frequencies of Th1 and Th17 cells were upregulated and CD14^lowCD16⁺ monocyte subset was also positively associated with Th1 cell frequency in PBC. Using a vitro coculture model, we further found that CD14^lowCD16⁺ monocytes promoted Th1 cell polarization compared to classical monocytes. Interleukin-12 (IL-12) and direct contact of patient CD4⁺T cell and CD14^lowCD16⁺ monocytes, were responsible for CD14^lowCD16⁺ monocytes promotion of Th1 cells polarization in PBC. Our study demonstrated that the enhanced CD14^lowCD16⁺ monocyte subset participated in fostering liver damage and inflammatory responses, and promoted Th1 cells skewing in PBC.     

CD16+ monocytes in breast cancer patients: expanded by monocyte chemoattractant protein-1 and may be useful for early diagnosis

Human peripheral blood monocytes are a heterogeneous population, including CD14⁺CD16^‐‘classical’ monocytes and CD14⁺CD16⁺‘proinflammatory’ monocytes. CD16⁺ monocytes are expanded in various inflammatory conditions. However, little is known about the CD14⁺CD16⁺ monocytes in patients with breast cancer. We detected CD14⁺CD16⁺ monocytes in 96 patients with breast cancer and 54 control subjects using flow cytometry. Receiver‐operating characteristic (ROC) curve analysis was used to determine the feasibility of CD14⁺CD16⁺ monocytes as an indicator for diagnosis of breast cancer. We found that the frequency of CD14⁺CD16⁺ monocytes showed a significantly greater increase in breast cancer patients than in controls (16·96% versus 10·84%, P < 0·0001). The area under the ROC curve for CD14⁺CD16⁺ monocytes was 0·805 [95% confidence interval (95% CI): 0·714–0·877, P = 0·0001]. Furthermore, the levels of CD16⁺ monocytes were significantly negatively associated with the tumour size and pathological staging. In vitro, we showed that CD14⁺CD16⁺ monocytes were expanded significantly when the purified CD14⁺ monocytes were exposed to Michigan Cancer Foundation (MCF)‐7 cells‐conditioned medium (MCF‐CM) or, separately, to monocyte chemotactic protein 1 (MCP‐1). Neutralizing antibodies against MCP‐1 inhibited the expansion of CD14⁺CD16⁺ monocytes by MCF‐CM. Collectively, our findings indicated that MCP‐1 can expand CD14⁺CD16⁺ monocytes in patients with breast cancer. Furthermore, the CD14⁺CD16⁺ monocyte may be a useful indicator in early diagnosis of breast cancer.     

Monocytes/macrophages infected with Toxoplasma gondii do not increase co‐stimulatory molecules while maintaining their migratory ability

Toxoplasma gondii is an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells and use them for dispersal throughout the body, but the activation state of those cells is unknown. We investigated the ability of human and murine cells from monocytic/macrophage lineages that had not previously been exposed to inflammatory cytokines to up‐regulate co‐stimulatory and adhesion molecules upon infection. Toxoplasma gondii‐infected human monocytes (freshly isolated and THP1 lineage) were unable to up‐regulate CD86, CD83, CD40 or CD1a. CD80 expression increased in infected cells but expression of l ‐selectin and β2 integrin was unaltered. We evaluated the ability of infected macrophages from wild type C57/BL/6 or CD14^?/? mice to migrate in 8 μm transwells. Infected cells from CD14^?/? mice were more likely to de‐adhere than infected cells from wild type mice but they did not show any increase in migratory ability. The non‐stimulatory profile of these infected cells may contribute to parasite spread throughout the lymphatic circulation in the initial phases of infection.     

Synergy‐inducing chemokines enhance CCR2 ligand activities on monocytes

The migration of monocytes to sites of inflammation is largely determined by their response to chemokines. Although the chemokine specificities and expression patterns of chemokine receptors are well defined, it is still a matter of debate how cells integrate the messages provided by different chemokines that are concomitantly produced in physiological or pathological situations in vivo. We present evidence for one regulatory mechanism of human monocyte trafficking. Monocytes can integrate stimuli provided by inflammatory chemokines in the presence of homeostatic chemokines. In particular, migration and cell responses could occur at much lower concentrations of the CCR2 agonists, in the presence of chemokines (CCL19 and CCL21) that per se do not act on monocytes. Binding studies on CCR2⁺ cells showed that CCL19 and CCL21 do not compete with the CCR2 agonist CCL2. Furthermore, the presence of CCL19 or CCL21 could influence the degradation of CCL2 and CCL7 on cells expressing the decoy receptor D6. These findings disclose a new scenario to further comprehend the complexity of chemokine‐based monocyte trafficking in a vast variety of human inflammatory disorders.     

Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever

Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus–target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14⁺ monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM‐1) were significantly increased during acute DF. A reduced number of CD14⁺ human leucocyte antigen (HLA)‐DR⁺ monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14⁺ monocytes expressing toll‐like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV‐2 infection up‐regulated only TLR2. Increased numbers of CD14⁺ CD16⁺ activated monocytes were found after in vitro and in vivo DENV‐2 infection. The CD14^high CD16⁺ monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and interleukin (IL)‐18 in dengue patients were inversely associated with CD14^high CD16⁺, indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL‐10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte‐activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in order to control both intense immunoactivation and virus replication.