Presence Of Monocytes Research Articles

Daclizumab reduces CD25 levels on T cells through monocyte-mediated trogocytosis

Daclizumab is a humanized monoclonal antibody that prevents interleukin-2 (IL-2) binding to CD25, blocking IL-2 signaling by cells that require high-affinity IL-2 receptors to mediate IL-2 signaling. The phase 2a CHOICE study evaluating daclizumab as a treatment for multiple sclerosis (MS) included longitudinal analysis of activated T cell counts. Whereas an exposure-dependent relationship was observed between daclizumab and reductions in HLA-DR+-activated T cells, a similar relationship was not observed for reductions in CD25 levels. The objective of this report is to determine the mechanism by which daclizumab reduces CD25 levels on peripheral blood mononuclear cells (PBMCs) using cytometric techniques. Daclizumab reduced T cell CD25 levels through a mechanism that required the daclizumab-Fc domain interaction with Fc receptors (FcR) on monocytes, but not on natural killer (NK) cells, and was unrelated to internalization or cell killing. Activated CD4+ T cells and FoxP3+ Treg cells showed evidence of trogocytosis of the CD25 antigen in the presence of monocytes. A daclizumab variant that retained affinity for CD25 but lacked FcR binding did not induce trogocytosis and was significantly less potent as an inhibitor of IL-2-induced proliferation of PBMCs. In conclusion, Daclizumab-induced monocyte-mediated trogocytosis of CD25 from T cells appears to be an additional mechanism contributing to daclizumab inhibition of IL-2 signaling.

FRI0029 IL-7-driven T cell activation and toll-like receptor 7 triggering cause additive B cell activation that is further enhanced by monocytes

BackgroundIL-7 is a potent T cell activating cytokine that has been shown to cause proliferation, survival and differentiation of T cells as well as T cell-dependent activation of myeloid cells...

SAT0158 IL-7 and IL-7 receptor blockade selectively inhibit TLR7-induced B cell activation in primary sjögren’s syndrome

BackgroundToll-like receptors (TLRs) have been implicated in several auto-immune diseases, especially those involved in the recognition of nucleic acids (viral, bacterial, and possibly self). In primary Sjögren’s syndrome (pSS) TLR7-induced...

The Immunologically Active Oligosaccharides Isolated from Wheatgrass Modulate Monocytes via Toll-like Receptor-2 Signaling

Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. Herein, wheatgrass-derived oligosaccharides (WG-PS3) were isolated and found to induce CD69 and Th1 cytokine expression in human peripheral blood mononuclear cells. In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Maltoheptaose activated monocytes via Toll-like receptor 2 (TLR-2) signaling, as discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and also determined by click chemistry. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms.

IFN-γ production by human natural killer cells in response to HCV-infected hepatoma cells is dependent on accessory cells

Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells (NK) and T lymphocytes, is an important regulator of innate and adaptive immunity during hepatitis C virus (HCV) infection. However, the cellular sources and mechanisms of IFN-γ induction in HCV-infection are not fully understood. We cultured normal human peripheral blood mononuclear cells (PBMCs) with different populations of immune cells and JFH-1 HCV-infected HuH7.5 (JFH-1/HuH7.5) cells. We found that PBMCs produced large amounts of IFN-γ after co-culture with JFH-1/HuH7.5 cells. Using intracellular cytokine staining, we confirmed that NK cells and NKT cells (to a lesser extent) were the major IFN-γ producers within PBMCs. Purified NK/NKT cells did not produce IFN-γ in response to JFH-1/HuH7.5 cells and depletion of accessory (HLA-DR(+)) cells prevented IFN-γ induction in PBMCs. Through selective cell depletion of dendritic cells or monocytes from PBMCs, we determined that plasmacytoid dendritic cells (pDCs) were indispensable for NK-IFN-γ induction and the presence of monocytes was needed for maximal NK-IFN-γ induction. We further revealed that NK-IFN-γ induction depended on pDC-derived IFN-α while other IFN-γ inducing cytokines, IL-12, and IL-18, played minimal roles. Close contact between JFH-1/HuH7.5 cells and NK cells was required for IFN-γ production and monocyte-derived IL-15 significantly augmented IFN-γ induction. We discovered a novel mechanism where NK cells interact with pDCs and monocytes, efficiently producing IFN-γ in response to HCV-infected cells. This indicates that co-operation between NK cells and accessory cells is critical for IFN-γ production and regulation of immunity during HCV infection.

Dual functions of natural killer cells in selection and differentiation of stem cells; role in regulation of inflammation and regeneration of tissues.

Accumulated evidence from our laboratory indicates that conditioned or anergized NK cells have the ability to induce resistance of healthy stem cells and transformed cancer stem cells through both secreted factors and direct cell-cell contact by inducing differentiation. Cytotoxic function of NK cells is suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. Furthermore, decreased peripheral blood NK cell function has been documented in many cancer patients. We have previously shown that NK cells mediate significant cytotoxicity against primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs), human dental pulp stem cells (hDPSCs) and induced human pluripotent stem cells (hiPSCs) were all significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or gene deletion of COX2 significantly augmented NK cell function. Furthermore, the induction of resistance of the stem cells to NK cell mediated cytotoxicity and their subsequent differentiation is amplified when either the stem cells or the NK cells were cultured in the presence of monocytes. Therefore, we propose that the two stages of NK cell maturation namely CD16+CD56dimCD69- NK cells are important for the lysis of stem cells or poorly differentiated cells whereas the CD16dim/-CD56dim/+CD69+NK cells are important for differentiation and eventual regeneration of the tissues and the resolution of inflammation, thus functionally serving as regulatory NK cells (NKreg). CD16 receptor on the NK cells were found to be the receptor with significant potential to induce NK cell anergy, however, our recent data indicated that NKp46 but not NKp30 or NKp44 were also able to induce significant anergy in NK cells, although the levels were less when compared to CD16 receptor triggering. The concept of split anergy in NK cells and generation of NKreg and its contribution to cell differentiation, tissue repair and regeneration and in tumor resistance will be discussed in this review.

Monocytes and the 38kDa-antigen of mycobacterium tuberculosis modulate natural killer cell activity and their cytolysis directed against ovarian cancer cell lines

BackgroundDespite strong efforts to improve clinical outcome of ovarian cancer patients by conventional and targeted immuno-based therapies, the prognosis of advanced ovarian cancer is still poor. Natural killer (NK) cells mediate antibody-dependent cellular cytotoxicity (ADCC), release immunostimulatory cytokines and thus function as potent anti-tumour effector cells. However, tumour cells developed mechanisms to escape from an effective immune response. So highly immunogenic substances, like the 38 kDa-preparation of M. tuberculosis, PstS-1, are explored for their potential to enhance cancer-targeted immune responses. In this study we examined the modulation of different NK cell functions by accessory monocytes and PstS-1. We focussed on NK cell activation as well as natural and antibody-dependent cellular cytotoxicity directed against epidermal-growth-factor-receptor (EGFR)-positive ovarian cancer cell lines.MethodsActivation, cytokine release and cytotoxicity of NK cells stimulated by monocytes and PstS-1 were determined by FACS-analysis, ELISA, Bioplex assay and quantitative polymerase-chain reaction (qPCR). Transwell assays were used to discriminate cell-cell contact-dependent from contact-independent mechanisms. Five ovarian cancer cell lines (A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3) with different EGFR-expression were used as target cells for natural and antibody-dependent cellular cytotoxicity assays. Cetuximab (anti-EGFR-antibody) was used for ADCC studies.ResultsOur data show that monocytes effectively enhance activation as well natural and antibody-dependent cytolytic activity of NK cells. PstS-1 directly stimulated monocytes and further activated monocyte-NK-co-cultures. However, PstS-1 did not directly influence purified NK cells and did also not affect natural and antibody-dependent cellular cytotoxicity directed against EGFR-positive ovarian cancer cells, even in presence of monocytes. Direct cell-cell contact between NK cells and monocytes was required for NK activation, while released cytokines seemed to play a minor role.ConclusionsOur data suggest that monocytes enhance natural and antibody-dependent cytotoxic activity of NK cells in a cell-cell contact dependent manner. The TLR-agonist PstS-1 provides additional monocyte activation and induces NK activation markers, while NK cytotoxicity remains unaffected. We conclude that monocytes provide accessory function for ADCC exerted by NK during antibody-based cancer immunotherapy directed against EGFR-positive ovarian cancer cells.

Simple Monitoring of Cancer Cells Using Nanoparticles

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.

Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

BackgroundFunctional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry.MethodsBoth asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay.ResultsMitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry.ConclusionsA flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.

Abstract 113: Monocytes Embedded in Plasma Clots Synthesize Coagulation Factors: Balancing Procoagulant and Anticoagulant Signals

Objective: Previous studies have demonstrated that vascular cells acutely regulate fibrin clot structure and stability. However, the mechanisms by which fibrin clots push vascular cells, in particular leukocytes, to synthesize pro-and anti-coagulant proteins is unknown. Here, we characterize monocyte-derived pro-and anti-coagulant activity in clots. Methods: Human monocytes were isolated from whole blood using CD14 microbeads. In parallel, cell-free plasma was isolated and clot formation induced in the presence of monocytes by the addition of tissue factor and calcium. RNA was isolated using RNAzol. Cell surface tissue factor (TF), tissue factor pathway inhibitor (TFPI), urokinase plasminogen activator (uPA) and its receptor (uPAR) as well as plasminogen activator inhibitor-1 (PAI-1) was measured by ELISA. TF activity was measured using a chromogenic factor Xa assay. Results: Monocytes embedded within plasma clots transcribed mRNA for uPAR (2.3-fold), uPA (9.5-fold), TF (9.2-fold), TFPI, and PAI-1 (6.2-fold) in a time-dependent manner. Consistent with this transcriptional response, increased levels of TF, TFPI, and uPAR protein were visually detected on the monocyte surface 18-hours after plasma clot formation. Accumulation of TF, TFPI and uPAR protein was blocked by actinomycin D and cycloheximide, indicating protein synthesis was dependent on transcription of new mRNA. TF-activity was similarly increased (8.9-fold) on the surface monocytes (i.e., increase in plasma-clot versus suspension monocytes). Lastly, active PAI-1 (11.1-fold increase), but not uPA, increased in the culture supernatant over time. Conclusions: Plasma clot formation induced robust increases in coagulation-related mRNAs and their corresponding proteins. The balance between the transcription of pro- and anti-coagulant mRNAs and their subsequent translation into bioactive protein likely plays key roles in clot development, stability and resolution.

The effect of lipopolysaccharide structure on the synthetic activity of human lymphocytes

258 Lipopolysaccharide (LPS) is the major component of the outer membrane of Gramnegative bacteria. It activates monocytes, macrophages, and neutrophils, which may lead to an uncontrolled immune response. To prevent hyperactivation of the target cell, it is nec� essary to know the mechanisms which can be influ� enced, thereby preventing the development of patho� logical process. For a long time it was believed that human lymphocytes, unlike B cells in mice, are not activated in response to LPS. Studies in the last few years showed that LPS is able to stimulate prolifera� tion of T cells in the presence of monocytes. The acti� vation of lymphocytes by lipopolysaccharides (endot� oxins) is associated with direct contact of the CD28 receptor on responsive T cells with the B7 receptor on monocytes (1), which is indicative of a relationship

Microparticles of Pregnant Women and Preeclamptic Patients Activate Endothelial Cells in the Presence of Monocytes

Preeclampsia is a pregnancy-specific disorder that may result from an adverse maternal response to circulating placenta-derived factors, causing a systemic inflammation including endothelial activation. Plasma from preeclamptic patients was shown to induce endothelial activation in the presence of monocytes. We investigated whether microparticles (MP) are the plasma factors causing this activation of endothelial cells. Monocultures and co-cultures of monocytes and endothelial cells were incubated with plasma, MP-poor plasma or isolated MP from non-pregnant and pregnant women and preeclamptic patients (each n = 8). ICAM-1 expression was analyzed with flow cytometry. The expression of ICAM-1 was significantly increased in monocytes and endothelial cells in co-cultures after the addition of isolated MP from preeclamptic patients (P = 0.017) and to a lesser extent in pregnant women (P = 0.012) compared to non-pregnant controls. Microparticles from preeclamptic patients activate endothelial cells in the presence of monocytes. Whether all MP have the same effect on monocytes and endothelial cells or only a specific subgroup is the focus of future research.

Enhanced neutrophil longevity and recruitment contribute to the severity of oviduct pathology during Chlamydia muridarum infection.

Our previous studies revealed that intravaginal infection of mice with a plasmid-deficient strain of Chlamydia muridarum, CM3.1, does not induce the development of oviduct pathology. In this study, we determined that infection with CM3.1 resulted in a significantly reduced frequency and absolute number of neutrophils in the oviducts during acute infection. This reduction in neutrophils was associated with significantly lower levels of neutrophil chemokines in the oviducts and decreased production of neutrophil chemokines by oviduct epithelial cells infected with CM3.1 in vitro. Infection with CM3.1 also resulted in an increased frequency of late apoptotic/dead neutrophils in the oviduct. Examination of the ability of Chlamydia trachomatis to prevent neutrophil apoptosis in vitro revealed that C. trachomatis strain D/UW-3/Cx exhibited an enhanced ability to prevent neutrophil apoptosis compared to plasmid-deficient CTD153, and this effect was dependent on the presence of CD14(high) monocytes. The presence of monocytes also resulted in enhanced neutrophil cytokine production and increased production of tissue-damaging molecules in response to D/UW-3/Cx relative to results with CTD153. Attempts to use antibody-mediated depletion to discern the specific role of neutrophils in infection control and pathology in vivo revealed that although Ly6G(high) neutrophils were eliminated from the blood and oviducts with this treatment, immature neutrophils and high levels of tissue-damaging molecules were still detectable in the upper genital tract. These data support the role of neutrophils in chlamydia-induced pathology and reveal that novel methods of depletion must be developed before their role can be specifically determined in vivo.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Grey zone lymphoid neoplasms with features overlapping between splenic marginal zone lymphoma and hairy cell leukaemia: splenic B-cell lymphoma/leukaemia, unclassifiable

The best characterized primary splenic B-cell lymphoma/leukaemias include splenic marginal zone lymphoma (SMZL) and hairy cell leukaemia (HCL). However, there are other primary splenic B-cell neoplasms that do not have the characteristic features of SMZL or HCL. The WHO classification has proposed a provision entity—splenic B-cell lymphoma/leukaemia, unclassifiable (SBLLu) to include the better-defined small B-cell clonal lymphoproliferations involving spleen that do not fall into any of the other types of B-cell lymphoid neoplasms. The recognized provisional entities among SBLLu include splenic diffuse red pulp small B-cell lymphoma (SDRPSBL) and hairy cell leukaemia variant (HCLv). The possibility of SBLLu arises when the spleen shows diffuse red pulp involvement, or when the bone marrow trephine biopsy shows extensive intra-sinusoidal involvement by a small B-cell lymphoma with marked splenomegaly, or when a patient with marked splenomegaly presents with a high white cell count in the peripheral blood without monocytopenia, and with presence of atypical lymphocytes with villous or hairy projections and with prominent nucleoli, and the cells are CD25 negative. These clonal B cells lack IgD and frequently express IgG with or without IgM. They are consistently negative for CD25, Annexin A1, cyclin D1, CD38 and cytoplasmic Ig, and are variably positive for CD76, tartrate-resistant acid phosphatase, CD11c, CD103 and CD123. Distinguishing SDRPSBL and HCLv can pose diagnostic problems. Features favouring SDRPSBL include relatively low lymphocytosis along with leucopenia and thrombocytopenia, lack of a paraprotein and a relatively monomorphic cytology with only a proportion of cells being nucleolated. Features that favour HCLv include high white cell count (average ∼35 × 109/L) and presence of monocytes, prominent and obvious intra-sinusoidal infiltrates in spleen and bone marrow and prominence of cells with nucleoli resembling prolymphocytes.

Interplay between CD8α+ Dendritic Cells and Monocytes in Response to Listeria monocytogenes Infection Attenuates T Cell Responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α+ dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8+ T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α+ DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α+ DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b+ DCs primarily secrete low levels of TNFα while CD8α+ DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α+ DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.

Plasma from preeclamptic women activates endothelial cells via monocyte activation in vitro

In this study we tested whether plasma from preeclamptic women contains factors that can activate endothelial cells in the presence of monocytes in vitro. Plasma from preeclamptic women (n=6), healthy pregnant women (n=6) and nonpregnant women (n=6) was incubated with mono-cultures and co-cultures of human umbilical vein endothelial cells (HUVEC) and monomac-6 monocytes. Reactive oxygen species (ROS) production and ICAM-1 expression were measured using flow cytometry. Whether scavenging of ROS by superoxide dismutase and catalase inhibited HUVEC ICAM-1 expression was also investigated. We found that in HUVEC co-cultured with monomac-6 cells but not in HUVEC cultured alone, ICAM-1 was upregulated after incubation with plasma from preeclamptic women but not plasma from non-pregnant women. Also in co-cultures, monomac-6 ICAM-1 was upregulated by plasma from preeclamptic women, while in both mono- and co-cultures monomac-6 ROS production was upregulated by plasma from pregnant and preeclamptic women, compared with plasma from non-pregnant women. Scavenging of ROS by superoxide dismutase and catalase resulted in a further upregulation of HUVEC ICAM-1 after incubation with plasma from preeclamptic women, compared with incubation without superoxide dismutase and catalase. These results show that endothelial cells in vitro are activated by plasma of preeclamptic women only if they are co-cultured with monocytes. This upregulation appeared not to be due to extracellular ROS production by monocytes or HUVEC, pointing to involvement of other mechanisms. Our data suggest that plasma of preeclamptic women activates monocytes, and that these monocytes subsequently activate endothelial cells.

Dendritic cells previously exposed to mannan-binding lectin enhance cytokine production in allogeneic mononuclear cell cultures

Mannan (or mannose)–binding lectin (MBL) can bind to monocytes and dendritic cells, but the significance of such interactions is unknown. We hypothesized that the presence of MBL might prevent the differentiation of monocytes into monocyte-derived dendritic cells or interfere with the development of dendritic cells in some way. We therefore investigated the influence of recombinant human MBL on surface antigen expression and on secretion of selected cytokines. By these means, no direct influence of rhMBL on dendritic cell differentiation or maturation was detected. However, mature dendritic cells prepared in the presence of rhMBL and subsequently co-cultured with allogeneic mononuclear cells, markedly promoted production of interleukin-1β, interleukin-6, and tumor necrosis factor–α in vitro. In most dendritic cell–mononuclear cell combinations, IFN-γ production was also enhanced. This influence required the presence of rhMBL during dendritic cell maturation and was critically dependent on the presence of monocytes. This observation provides evidence that MBL can influence cellular immunity in addition to its established role as an opsonin.

Wound healing effect of collagen-hyaluronic acid implanted in partially injured anterior cruciate ligament of dog

The purpose of this study was to evaluate the cell compatibility of collagen-hyaluronan (HA) substrate in vitro by assessing ACL cell cultures. Additionally, the use of collagen-HA substrate as a dressing material for partial ACL defects as well as its effect on collagen synthesis and angiogenesis were evaluated in vivo. The initial attachment and proliferation of dog ACL cells on silk matrix covered with collagen-HA substrate (SMCH) was greater than that observed on silk matrix alone. Silk matrix and SMCHs were implanted as dressing materials into partial ACL defects located at the knees of dogs, and they were harvested six weeks after implantation. A histological evaluation of the collagen-HA substrates revealed the presence of monocytes and the absence of giant cells in all cases. MT staining of the SMCH-grafted group showed a higher level of granulation tissue formation consisting of fibroblasts and collagen fibers compared to the silk matrix-grafted group. In addition, CD31 staining revealed that the SMCH-grafted area showed more blood vessel formation than the silk matrix-grafted area. These results suggest that the collagen-HA substrate was cell-compatible in vitro and enhanced collagen synthesis and new blood vessel formation in vivo.

Diesel exhaust particles override natural injury-limiting pathways in the lung

Induction of effective inflammation in the lung in response to environmental and microbial stimuli is dependent on cooperative signaling between leukocytes and lung tissue cells. We explored how these inflammatory networks are modulated by diesel exhaust particles (DEP) using cocultures of human monocytes with epithelial cells. Cocultures, or monoculture controls, were treated with DEP in the presence or absence of LPS or flagellin. Production of cytokines was explored by Western blotting and ELISA; cell signaling was analyzed by Western blotting. Here, we show that responses of epithelial cells to DEP are amplified by the presence of monocytes. DEP amplified the responses of cellular cocultures to very low doses of TLR agonists. In addition, in the presence of DEP, the responses induced by LPS or flagellin were less amenable to antagonism by the physiological IL-1 antagonist, IL-1ra. This was paralleled by the uncoupling of IL-1 production and release from monocytes, potentially attributable to an ability of DEP to sequester or degrade extracellular ATP. These data describe a model of inflammation where DEP amplifies responses to low concentrations of microbial agonists and alters the nature of the inflammatory milieu induced by TLR agonists.