Epithelial-mesenchymal transition of ovarian tumor cells induces an angiogenic monocyte cell population

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14⁺/KDR⁺ angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14⁺/KDR⁺ cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14⁺/KDR⁺ cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14⁺/KDR⁺ cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14⁺/KDR⁺ population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.     

Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.     

Naïve rat umbilical cord matrix stem cells significantly attenuate mammary tumor growth through modulation of endogenous immune responses

Background aimsUn-engineered human and rat umbilical cord matrix stem cells (UCMSCs) attenuate growth of several types of tumors in mice and rats. However, the mechanism by which UCMSCs attenuate tumor growth has not been studied rigorously.MethodsThe possible mechanisms of tumor growth attenuation by rat UCMSCs were studied using orthotopic Mat B III rat mammary tumor grafts in female F344 rats. Tumor-infiltrating leukocytes were identified and quantified by immunohistochemistry analysis. Potential cytokines involved in lymphocyte infiltration in the tumors were determined by microarray and Western blot analysis. The Boyden chamber migration assay was performed for the functional analysis of identified cytokines.ResultsRat UCMSCs markedly attenuated tumor growth; this attenuation was accompanied by considerable lymphocyte infiltration. Immunohistochemistry analysis revealed that most infiltrating lymphocytes in the rat UCMSC-treated tumors were CD3⁺ T cells. In addition, treatment with rat UCMSCs significantly increased infiltration of CD8⁺ and CD4⁺ T cells and natural killer (NK) cells throughout tumor tissue. CD68⁺ monocytes/macrophages and Foxp3⁺ regulatory T cells were scarcely observed, only in the tumors of the phosphate-buffered saline control group. Microarray analysis of rat UCMSCs demonstrated that monocyte chemotactic protein-1 is involved in rat UCMSC-induced lymphocyte infiltration in the tumor tissues.ConclusionsThese results suggest that naïve rat UCMSCs attenuated mammary tumor growth at least in part by enhancing host anti-tumor immune responses. Naïve UCMSCs can be used as powerful therapeutic cells for breast cancer treatment, and monocyte chemotactic protein-1 may be a key molecule to enhance the effect of UCMSCs at the tumor site.     

Anaphylatoxins orchestrate Th17 response via interactions between CD16+ monocytes and pleural mesothelial cells in tuberculous pleural effusion

The complement system is activated in tuberculous pleural effusion (TPE), with increased levels of the anaphylatoxins stimulating pleural mesothelial cells (PMCs) to secrete chemokines, which recruit nonclassical monocytes to the pleural cavity. The differentiation and recruitment of naive CD4⁺ T cells are induced by pleural cytokines and PMC-produced chemokines in TPE. However, it is unclear whether anaphylatoxins orchestrate CD4⁺ T cell response via interactions between PMCs and monocytes in TPE. In this study, CD16⁺ and CD16^- monocytes isolated from TPE patients were cocultured with PMCs pretreated with anaphylatoxins. After removing the PMCs, the conditioned monocytes were cocultured with CD4⁺ T cells. The levels of the cytokines were measured in PMCs and monocyte subsets treated separately with anaphylatoxins. The costimulatory molecules were assessed in conditioned monocyte subsets. Furthermore, CD4⁺ T cell response was evaluated in different coculture systems. The results indicated that anaphylatoxins induced PMCs and CD16⁺ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16⁺ than in CD16⁻ monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16⁺ monocytes. Collectively, these data indicate that anaphylatoxins play a central role in orchestrating Th17 response mainly via interactions between CD16⁺ monocytes and PMCs in TPE.     

A Potential Inhibitory Profile of Liver CD68+ Cells during HCV Infection as Observed by an Increased CD80 and PD-L1 but Not CD86 Expression

AimThe lack of potent innate immune responses during HCV infection might lead to a delay in initiating adaptive immune responses. Kupffer cells (KCs) and liver-infiltrating monocytes/macrophages (CD68⁺ cells) are essential to establish effective anti-HCV responses. They express co-stimulatory molecules, CD80 and CD86. CD86 upregulation induces activator responses that are then potentially regulated by CD80. The relative levels of expression of CD80, CD86 and the inhibitory molecule, PD-L1, on CD68⁺ cells modulate T cell activation. A few studies have explored CD80 and PD-L1 expression on KCs and infiltrating monocytes/macrophages in HCV-infected livers, and none investigated CD86 expression in these cells. These studies have identified these cells based on morphology only. We investigated the stimulatory/inhibitory profile of CD68⁺ cells in HCV-infected livers based on the balance of CD80, CD86 and PD-L1 expression.MethodsCD80, CD86 and PD-L1 expression by CD68⁺ cells in the lobular and portal areas of the liver of chronic HCV-infected (n = 16) and control (n = 14) individuals was investigated using double staining immunohistochemistry.ResultsThe count of CD68⁺ KCs in the lobular areas of the HCV-infected livers was lower than that in the control (p = 0.041). The frequencies of CD68⁺CD80⁺ cells and CD68⁺PD-L1⁺ cells in both lobular and total areas of the liver were higher in HCV-infected patients compared with those in the control group (p = 0.001, 0.031 and 0.007 respectively). Moreover, in the lobular areas of the HCV-infected livers, the frequency of CD68⁺CD80⁺ cells was higher than that of CD68⁺CD86⁺ and CD68⁺PD-L1⁺ cells. In addition, the frequencies of CD68⁺CD80⁺ and CD68⁺CD86⁺ cells were higher in the lobular areas than the portal areas.ConclusionsOur results show that CD68⁺ cells have an inhibitory profile in the HCV-infected livers. This might help explain the delayed T cell response and viral persistence during HCV infection.     

Increased expression levels of monocyte CCR2 and monocyte chemoattractant protein-1 in patients with diabetes mellitus

Increased monocyte recruitment into subendothelial space in atherosclerotic lesions is one of the hallmarks of diabetic angiopathy. The aim of this study was to determine the state of peripheral blood monocytes in diabetes associated with atherosclerosis. Diabetic patients treated with/without an oral hypoglycemic agent and/or insulin for at least 1 year were recruited (n=106). We also included 24 non-diabetic control subjects. We measured serum levels of monocyte chemoattractant protein (MCP)-1, fasting plasma glucose (FPG), HbA1c, total cholesterol, triglyceride, body mass index (BMI), high sensitivity CRP (hs-CRP) and evaluated CCR2, CD36, CD68 expression on the surface of monocytes. Serum MCP-1 levels were significantly (p<0.05) higher in diabetic patients than in normal subjects. In diabetic patients, serum MCP-1 levels correlated significantly with FPG, HbA1c, triglyceride, BMI, and hs-CRP. The expression levels of CCR2, CD36, and CD68 on monocytes were significantly increased in diabetic patients and were more upregulated by MCP-1 stimulation. Our data suggest that elevated serum MCP-1 levels and increased monocyte CCR2, CD36, CD68 expression correlate with poor blood glucose control and potentially contribute to increased recruitment of monocytes to the vessel wall in diabetes mellitus.     

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14⁺ peripheral blood mononuclear cells (CD14⁺ PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14⁺ PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14⁺ PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.     

Recruitment of CD16+ monocytes to endothelial cells in response to LPS-treatment and concomitant TNF release is regulated by CX3CR1 and interfered by soluble fractalkine

Fractalkine (FKN, CX3CL1) is a regulator of leukocyte recruitment and adhesion, and controls leukocyte migration on endothelial cells (ECs). We show that FKN triggers different effects in CD16⁺ and CD16⁻ monocytes, the two major subsets of human monocytes. In the presence of ECs a lipopolysaccharide (LPS)-stimulus led to a significant increase in tumor necrosis factor (TNF)-secretion by CD16⁺ monocytes, which depends on the interaction of CX3CR1 expressed on CD16⁺ monocytes with endothelial FKN. Soluble FKN that was efficiently shed from the surface of LPS-activated ECs in response to binding of CD16⁺ monocytes to ECs, diminished monocyte adhesion in down-regulating CX3CR1 expression on the surface of CD16⁺ monocytes resulting in decreased TNF-secretion. In this process the TNF-converting enzyme (TACE) acts as a central player regulating FKN-shedding and TNFα-release through CD16⁺ monocytes interacting with ECs. Thus, the release and local accumulation of sFKN represents a mechanism that limits the inflammatory potential of CD16⁺ monocytes by impairing their interaction with ECs during the initial phase of an immune response to LPS. This regulatory process represents a potential target for therapeutic approaches to modulate the inflammatory response to bacterial components.     

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.     

Myosin IIa activation is crucial in breast cancer derived galectin-1 mediated tolerogenic dendritic cell differentiation

Background

Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.

Methods

Circulating CD14⁺CD16⁺ of breast cancer patients and induced CD14⁺CD16⁺ DCs were identified as tDCs by treating CD14⁺ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.

Results

The CD14⁺CD16⁺ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c⁺ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c⁺/Ly6G⁺/IL-10⁺ in the BALB/c mice.

Conclusions

Galectin-1 can induce tDCs after internalizing into CD14⁺ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.

General significance

Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.     

Mouse CD11b+Kupffer Cells Recruited from Bone Marrow Accelerate Liver Regeneration after Partial Hepatectomy

TNF and Fas/FasL are vital components, not only in hepatocyte injury, but are also required for hepatocyte regeneration. Liver F4/80⁺Kupffer cells are classified into two subsets; resident radio-resistant CD68⁺cells with phagocytic and bactericidal activity, and recruited radio-sensitive CD11b⁺cells with cytokine-producing capacity. The aim of this study was to investigate the role of these Kupffer cells in the liver regeneration after partial hepatectomy (PHx) in mice. The proportion of Kupffer cell subsets in the remnant liver was examined in C57BL/6 mice by flow cytometry after PHx. To examine the role of CD11b⁺Kupffer cells/Mφ, mice were depleted of these cells before PHx by non-lethal 5 Gy irradiation with or without bone marrow transplantation (BMT) or the injection of a CCR2 (MCP-1 receptor) antagonist, and liver regeneration was evaluated. Although the proportion of CD68⁺Kupffer cells did not significantly change after PHx, the proportion of CD11b⁺Kupffer cells/Mφ and their FasL expression was greatly increased at three days after PHx, when the hepatocytes vigorously proliferate. Serum TNF and MCP-1 levels peaked one day after PHx. Irradiation eliminated the CD11b⁺Kupffer cells/Mφ for approximately two weeks in the liver, while CD68⁺Kupffer cells, NK cells and NKT cells remained, and hepatocyte regeneration was retarded. However, BMT partially restored CD11b⁺Kupffer cells/Mφ and recovered the liver regeneration. Furthermore, CCR2 antagonist treatment decreased the CD11b⁺Kupffer cells/Mφ and significantly inhibited liver regeneration. The CD11b⁺Kupffer cells/Mφ recruited from bone marrow by the MCP-1 produced by CD68⁺Kupffer cells play a pivotal role in liver regeneration via the TNF/FasL/Fas pathway after PHx.     

Therapy for pneumonitis and sialadenitis by accumulation of CCR2-expressing CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cells in MRL/lpr mice

Adoptive transfer of CD4⁺CD25⁺ regulatory T cells has been shown to have therapeutic effects in animal models of autoimmune diseases. Chemokines play an important role in the development of autoimmune diseases in animal models and humans. The present study was performed to investigate whether the progression of organ-specific autoimmune diseases could be reduced more markedly by accumulating chemokine receptor-expressing CD4⁺CD25⁺ regulatory T cells efficiently in target organs in MRL/MpJ-lpr/lpr (MRL/lpr) mice. CD4⁺CD25⁺Foxp3⁺ T cells (Treg cells) and CD4⁺CD25⁺Foxp3⁺ CCR2-transfected T cells (CCR2-Treg cells) were transferred via retro-orbital injection into 12-week-old MRL/lpr mice at the early stage of pneumonitis and sialadenitis, and the pathological changes were evaluated. Expression of monocyte chemoattractant protein-1 (MCP-1)/CCL2 was observed in the lung and submandibular gland of the mice and increased age-dependently. The level of CCR2 expression and MCP-1 chemotactic activity of CCR2-Treg cells were much higher than those of Treg cells. MRL/lpr mice to which CCR2-Treg cells had been transferred showed significantly reduced progression of pneumonitis and sialadenitis in comparison with MRL/lpr mice that had received Treg cells. This was due to more pronounced migration of CCR2-Treg cells and their localization for a longer time in MCP-1-expressing lung and submandibular gland, resulting in stronger suppressive activity. We prepared chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression by accumulating in target organs. This method may provide a new therapeutic approach for organ-specific autoimmune diseases in which the target antigens remain undefined.     

Functional Phenotype of Synovial Monocytes Modulating Inflammatory T-Cell Responses in Rheumatoid Arthritis (RA)

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14⁺CD16⁺, but not CD14^dimCD16⁺, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14⁺ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14⁺CD16⁺ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Increased CD14+HLA-DR-/low myeloid-derived suppressor cells correlate with extrathoracic metastasis and poor response to chemotherapy in non-small cell lung cancer patients

Accumulating evidence has demonstrated that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells, play an important role in the subversion, inhibition, and downregulation of the immune response to cancer. However, the characteristics of these cells, particularly clinical relevance, in malignant tumors remain unclear due to a lack of specific markers. In this study, we characterized peripheral CD14⁺HLA-DR^-/low cells, a new human MDSC subpopulation, in 89 patients with non-small cell lung cancer (NSCLC). As expected, both frequency and absolute number of CD14⁺HLA-DR^-/low cells were significantly increased in the peripheral blood of NSCLC patients compared with that of the healthy controls and indicated an association with metastasis, response to chemotherapy, and progression-free survival. These cells showed decreased expression of CD16 and CD86 compared with HLA-DR⁺ monocytes. Unlike classical monocytes, these populations showed significantly decreased allostimulatory activity and showed the ability to inhibit autologous T cell proliferation and IFN-γ production in a cell-contact-dependent manner. Furthermore, we demonstrated that CD14⁺HLA-DR^-/low cells expressed the NADPH oxidase component gp91^phox and generated high level of reactive oxygen species (ROS). Moreover, inactivation of ROS reversed their immunosuppressive capacity on T cell response. These results prove, for the first time, the existence of ROS-producing CD14⁺HLA-DR^-/low myeloid-derived suppressor cells in NSCLC patients, which mediate tumor immunosuppression and might thus represent a potential target for therapeutic intervention.     

Expression and anatomical distribution of TIM-containing molecules in Langerhans cell sarcoma

Signals from the T cell immunoglobulin and mucin-domain (TIM)-containing molecules have been demonstrated to be involved in regulating the progress of carcinoma. However, the expression and anatomical distribution of TIMs in Langerhans cell sarcoma (LCS), which is a rare malignancy derived from dendritic cells of the epidermis, has yet to be determined. In this study, the expression of TIM-1, TIM-3 and TIM-4 in LCS samples were detected by immunohistochemistry. Our results showed that these three molecules were found in LCS sections. At the cellular level, these molecules were found on the cell membrane and in the cytoplasm. Immunofluorescence double-staining demonstrated that these TIMs were co-expressed with Langerin, a potential biomarker for detecting LCS. In addition, TIM-1 was also expressed on CD68⁺ macrophages and CK-18⁺ epithelial cells, while TIM-3 and TIM-4 were expressed on all cell types investigated, including CD3⁺T cells, CD68⁺ macrophages, CD11c⁺ dendritic cells, CD16⁺ NK Cells, CD31⁺ endothelial cells and CK-18⁺ epithelial cells. Interestingly, TIMs were also co-expressed with some members of the B7 superfamily, including B7-H1, B7-H3 and B7-H4 on sarcoma cells. Our results clearly showed the characteristic expression and anatomical distribution of TIMs in LCS, and a clear understanding of their functional roles may further elucidate the pathogenesis of this carcinoma and potentially contribute to the development of novel immunotherapeutic strategies.     

Bone marrow stromal cells transplantation promotes the resolution and recanalization of deep vein thrombosis in rabbits through regulating macrophage infiltration and angiogenesis

This study aims to validate whether bone marrow stromal cells (BMSCs) transplantation could promote the resolution and recanalization of deep vein thrombosis (DVT) and to explore the underlying mechanism. The right hind femoral vein was embolized to establish the DVT rabbit model. BMSCs from New Zealand white rabbits were isolated and identified, and then injected into DVT rabbits. After that, the extent of angiogenesis was determined by the amount of capillaries that were positive for antibody against vWF. Macrophage infiltration was measured by immunohistochemistry with F4/80 antibody. M1 or M2 macrophages were identified as F4/80 + CD11c ⁺ or F4/80 + CD206 ⁺ cells by using flow cytometry analysis, respectively. BMSCs were successfully isolated and identified. BMSCs transplantation promotes macrophage infiltration and angiogenesis in DVT rabbits. BMSCs transplantation causes M1/M2 polarization, altered cytokine production and increased monocyte chemotactic protein 1 (MCP-1) protein expression in DVT rabbits. However, injection of MCP-1 protein not only reversed the effects of BMSCs transplantation on macrophage infiltration and angiogenesis, but also reversed the effects of BMSCs transplantation on M1/M2 polarization and cytokine production in DVT rabbits. BMSCs transplantation promotes the resolution and recanalization of DVT in rabbits through regulating macrophage infiltration and angiogenesis, the underlying mechanism is associated with MCP-1 expression.     

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition

Purpose To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. Experimental design Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. Results Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors’ monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P < 0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68− CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. Conclusions Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.