Co-culture Of Monocytes Research Articles

Interleukin-1 potently contributes to 25-hydroxycholesterol-induced synergistic cytokine production in smooth muscle cell-monocyte interactions

Objectives: Inflammation is essential for atherogenesis. Cholesterol, a cardiovascular risk factor, may activate inflammation in the vessel wall during this process. Cytokine-mediated interactions of human monocytes with vascular smooth muscle cells (SMCs) may perpetuate this process. Methods: We investigated the capacity of the cholesterol metabolite 25-hydroxycholesterol to induce inflammatory mediators in cocultures of freshly isolated monocytes with SMCs. We determined the role of interleukin-(IL)-1 in this interaction using qPCR, bioassays, ELISA and western blot. Cocultures with SMC to monocyte ratios from 1:4 to 1:20 were tested. Results: In separate SMC and monocyte cultures (monocultures) 25-hydroxycholesterol only poorly activated IL-1, IL-6 and MCP-1 production, whereas LPS stimulated much higher cytokine levels than unstimulated cultures. In contrast, cocultures of SMCs and monocytes stimulated with 25-hydroxycholesterol produced hundredfold higher cytokine levels than the corresponding monocultures. Blocking experiments with IL-1-receptor antagonist showed that IL-1 decisively contributed to the 25-hydroxycholesterol-induced synergistic IL-6 and MCP-1 production. The presence of intracellular IL-1β precursor, released mature IL-1β, and caspase-1 p10 indicated that the inflammasome was involved in this process. Determination of IL-1-mRNA in Transwell experiments indicated that the monocytes are the major source of IL-1, which subsequently activates the SMCs, the primary source of IL-6 in the coculture. Conclusion: Taken together, these interactions between local vessel wall cells and invading monocytes may multiply cholesterol-triggered inflammation in the vessel wall, and IL-1 may play a key role in this process. The data also indicate that lower cholesterol levels than expected from monocultures may suffice to initiate inflammation in the tissue.

Abstract 139: Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma

Abstract Multiple myeloma (MM) is an incurable haematological malignancy characterized by the clonal proliferation of malignant plasma cells within the bone marrow. The current data on MM disease progression indicate that bone marrow microenvironment plays crucial role in pathogenesis of MM. Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines, such as IL-6, insulin-like growth factor-1, inflammatory protein-1a in BMSCs promotes MM cell proliferation and protects against drug-induced cytotoxicity. MM lytic bone disease is caused by osteoclast activation and osteoblast inhibition. Disease-related bone complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for MM progression. Osteoclasts and BMSCs, along with extracellular matrix and cytokines stimulate myeloma cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions among tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells impact both the establishment and progression of MM. In current study, monocyte can directly promote osteogenic differentiation of mesenchymal stem cells through cell contact interactions and production of osteogenic factors. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the downregulation of osteoblast inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocyte, a component of bone marrow microenvironment, in the MM progression. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BMSCs and/or monocytes of MM patients. Consistently, we observed increased proliferation of MM cell lines in the presence of either BMSCs or monocytes compared to cell line-only control. Furthermore, the co-culture of BMSCs plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the bone marrow microenvironment. Citation Format: Hiroshi Ikeda, Yuka Aoki, Toshiaki Hyayashi, Yumiko Maruyama, Tadao Ishida, Takashi Tokino, Yasuhisa Shinomura, Yasushi Sasaki. Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2014-139

Inhibition mechanism of Qingluo Tongbi Granule () on osteoclast differentiation induced by synovial fibroblast and monocytes co-culture in adjuvant-induced arthritic rats.

To study the mechanism underlying the inhibitory effect of Qingluo Tongbi Granule (, QTG) on osteoclast differentiation in rheumatoid arthritis in rats. Fibroblast and monocyte co-culture were used to induce osteoclast differentiation in adjuvant-induced arthritic (AIA) rats. Serum containing QTG was prepared and added to the osteoclasts, and activation of the tumor necrosis factor receptor-associated factor 6/mitogen-activated protein kinase/nuclear factor of activated T cells, cytoplasmic1 (TRAF6/MAPK/NFATc1) pathways was examined. The induced osteoclasts were multinucleated and stained positive for tartrate-resistant acid phosphatase (TRAP) staining. Serum containing QTG at 14.4, 7.2 or 3.6 g/kg inhibited the activation of TRAF6, extracellular regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 and decreased the percentage of cells with nuclear NFATc1 in a dose-dependent manner, the high and middle doses exhibited clear inhibitory activity (P<0.01 and P<0.05, respectively). After the addition of MAPK inhibitors, the NFATc1 expression showed no significant difference compared with the control group (P>0.05). Serum containing QTG could generally inhibit the TRAF6/MAPK pathways and possibly inhibit the NFATc1 pathway. In addition, QTG may regulate other signaling pathways that are related to osteoclast differentiation and maturation.

Luminal Microbes Promote Monocyte–Stem Cell Interactions Across a Healthy Colonic Epithelium

The intestinal epithelium forms a vital barrier between luminal microbes and the underlying mucosal immune system. Epithelial barrier function is maintained by continuous renewal of the epithelium and is pivotal for gut homeostasis. Breaching of the barrier causes mobilization of immune cells to promote epithelial restitution. However, it is not known whether microbes at the luminal surface of a healthy epithelial barrier influence immune cell mobilization to modulate tissue homeostasis. Using a mouse colonic mucosal explant model, we demonstrate that close proximity of luminal microbes to a healthy, intact epithelium results in rapid mucus secretion and movement of Ly6C+7/4+ monocytes closer to epithelial stem cells. These early events are driven by the epithelial MyD88-signaling pathway and result in increased crypt cell proliferation and intestinal stem cell number. Over time, stem cell number and monocyte–crypt stem cell juxtapositioning return to homeostatic levels observed in vivo. We also demonstrate that reduced numbers of tissue Ly6C+ monocytes can suppress Lgr5EGFP+ stem cell expression in vivo and abrogate the response to luminal microbes ex vivo. The functional link between monocyte recruitment and increased crypt cell proliferation was further confirmed using a crypt–monocyte coculture model. This work demonstrates that the healthy gut epithelium mediates communication between luminal bacteria and monocytes, and monocytes can modulate crypt stem cell number and promote crypt cell proliferation to help maintain gut homeostasis.

SAT0558 Vegf Promotes RANKL Expression and Osteoclastogenesis in Rheumatoid Arthritis

BackgroundVascular endothelial growth factor (VEGF) is an angiogenic, inflammatory, and bony destructive molecule in the pathogenesis of rheumatoid arthritis (RA).ObjectivesThis study was aimed to determine the specific role of VEGF...

Involvement of the 4-1BB/4-1BBL Pathway in Control of Monocyte Numbers by Invariant NKT Cells

4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection.

AGEs‐induced pro‐atherosclerosis pathway: endothelium are regulated by the AGEs‐RAGE axis (832.12)

During hyperglycemia, reducing sugars react with the amino groups of proteins non‐enzymatically to form Schiff base and Amadori products, which undergo formation of advanced glycation end‐products (AGEs). Binding AGEs and receptor for AGEs (RAGE) generated oxidative stress and several vascular cells triggered secretion of inflammatory cytokine and abnormal signal transduction but the underlying mechanism remains unclear. We evaluate the hypothesis that the AGEs‐RAGE axis might mediate combined signals contributed to the local regulations in co‐cultures of human monocytes (THP‐1), endothelial cells (HUVEC) and aortic vascular smooth muscle cell (SMC). The results revealed that production of reactive oxygen species (ROS) and adhesion of monocytes to endothelium induced AGEs were decreased by RAGE knockdown HUVEC. We further discovered that TNF‐α and IL‐1β as pro‐inflammatory cytokines were markedly suppressed in AGEs‐stimulated HUVECs and THP‐1 simultaneously knockdown of RAGE. Besides, SMC were down‐regulated inflammatory cytokines and decreased proliferation in co‐culture system. In conclusion, AGEs stimulation of vascular cells causes binding of RAGE, enhances the production of ROS, adhesion of monocytes, and expression of cytokines eventually contributes to the progression of atherosclerosis.

Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold

Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC–monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC–monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5ngml−1) or IL-6 (1ngml−1) for 7days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC–monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC–monocyte co-cultures on D-PHI scaffolds for 7days, implying that IL-6 mediates VSMC response in monocyte–VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.

The Uncoupling of Monocyte–Platelet Interactions from the Induction of Proinflammatory Signaling in Monocytes

Induction of an inflammatory monocyte phenotype by activated platelets is implicated in the pathogenesis of inflammatory diseases, including atherosclerosis. In this study, we investigated the early signaling events associated with this platelet-induced inflammatory phenotype. We report that coculture of human monocytes with activated platelets induces phosphorylation of Akt, together with rapid mobilization of intracellular Ca(2+), and show that these signaling events can be uncoupled from monocyte binding to activated platelets. Specifically, Ab-inhibition studies and incubation of monocytes with activated platelet supernatant highlighted a role for secreted product(s) of activated platelets. We also identified a role for pertussis toxin-sensitive G protein-coupled receptors and excluded key candidates platelet-activating factor receptor and CCR5. Our results suggest that inhibition of monocyte-platelet interactions via PSGL-1 or P-selectin is not sufficient to prevent platelet-mediated monocyte activation in an inflammatory context. These findings have important implications for the development of therapeutics to treat diseases in which platelet-monocyte complexes are implicated in pathogenesis.

Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146(+) and CD146(-) stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146(+) and CD146(-) cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146(+) CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146(-) cells express markers associated with pre-dendritic cells. Importantly, when CD14(+) monocytes are cultured in contact with the CD146(+) cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146(-) cells does not differ from that obtained when monocytes are cultured with CD146(-) CM. These in vitro results show that the CD146(+) marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146(+) cells and CD68(+) cells associated with these upregulated proteins.

Tumor Monocyte Cross Talk Promotes Chemotherapy Resistance In Lymphoma

BackgroundImmune editing is a major mechanism used by tumors to promote their survival. We have previously reported the presence of immunosuppressive monocytes (CD14+HLA-DR low/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. Here we report an in vitro model to examine tumor monocyte cross talk and demonstrate that these monocytes may directly promote tumor cells' resistance to chemotherapy. MethodMonocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, Su-DHL2, Su-DHL6, Jeko-1, Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability, proliferation, and induced protein expression. Changes in vitro were confirmed in primary tissue by gene expression profile from 48 primary diffuse large B cell (DLBCL) lymphoma tumors. ResultsMonocytes converted CD14+HLA-DR+ to the CD14+HLA-DRlow/neg phenotype by co-culture with 5 of the 8 B cell lymphoma cell lines. The HLA-DR suppression was not due to the known regulatory effects of IL-10 nor was the effect seen in co-culture using B cells from healthy donors. Thus, the magnitude of DR loss is mediated by a yet undetermined mechanism. DOX incubation induced apoptosis in all of the cell lines. Co-culture with monocytes improved lymphoma cell survival in 3 of the 4 lymphoma cell lines. For example, untreated OCI-Ly3 had a 1.3±0.2 fold expansion that was reduced to 0.6±0.1 when treated with DOX (p<0.0001, n=13). However, when the cells were treated with DOX and co-cultured with monocytes the survival improved to 0.8±0.2 (p<0.0001, n=13). Having previously shown that CD14+HLA-DRlow/neg were capable of suppressing patient immunity, partially through arginase I, we determined if co-culture of monocytes with tumor cells increased arginase I expression in either monocytes or tumor cells. Not only did we see arginase I expression increases during co-culture(n=10, p<0.01), but surprisingly, the greatest increases were seen during co-culture and DOX treatment (n=10, p=0.01). To determine if our in vitro observations reflect human tumors, we used gene expression data obtained from 48 DLBCL tumors and found that increased CD14 gene expression was positively correlated with increased arginase I expression (n=48, p=0.02). ConclusionsWe have demonstrated that certain tumor cells induced phenotype changes in monocytes in an IL-10 independent and a tumor specific way. In those cells where tumors factors mediate monocyte phenotype, the monocytes can promote tumor cell resistance to cytotoxic killing by DOX, thus demonstrating a cross-talk between monocyte and tumor function. We found that in an environment of monocyte/tumor cross talk, arginase I expression is further increased in a cytotoxic environment. Together, this data demonstrates an active cross talk between monocytes and tumors resulting in multiple mechanisms of tumor resistance to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma (MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures:No relevant conflicts of interest to declare.

Thymosin β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide (H2O2) released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that Thymosin β4-Sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, lies downstream of H2O2 in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T cell/CD14+ monocyte co-cultures, Tβ4-SO inhibits IFN-γ and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, Tβ4-SO is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

Expansion of decidual CD45RO+ T cells with high expression of CEACAM1 in the early stage of pregnancy

The aim of the present study was to investigate the mechanism involved in the expansion of CD45RO+ T cells in the decidual microenvironment, and in the expression of the inhibitory carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on the surface of decidual CD45RO+ T cells. Twenty-one healthy nonpregnant females and seventeen healthy pregnant females in the first trimester were included in the study. Peripheral blood samples from nonpregnant and pregnant females, and decidual tissues from pregnant females following elective abortion, were obtained and analyzed by flow cytometry. The percentages of CD45RO+ T cells and CEACAM1-expressing CD45RO+ T cells were significantly higher in first trimester human decidua than in the peripheral blood. Conditioned medium from the coculture of monocytes and the human trophoblast HTR8/SVneo cell line (MHM) was added to the model for the generation of CD45RO+ T cells in vitro. MHM caused an increase in the percentage of CD45RO+ T cells in a monocyte chemoattractant protein-1 (MCP-1)‑dependent manner and an increase in the percentage of CEACAM1-expressing CD4+CD45RO+ T cells in the model. In conclusion, our results implied that trophoblast cells and monocytes may be involved in the increase of decidual CD45RO+ T cells and the high expression of CEACAM1 on their surfaces.

Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment

AbstractChronic lymphocytic leukemia (CLL) cells depend on microenvironmental stimuli for their survival, provided for example by monocyte-derived nurse-like cells (NLCs). The immunomodulatory drug lenalidomide shows therapeutic effects in subgroups of CLL patients, and is believed to act via the microenvironment. To investigate the effects of lenalidomide on the survival support of NLCs, cocultures of monocytes and CLL cells were treated for 14 days with lenalidomide, which resulted in significantly decreased viability of CLL cells. Among the changes induced by this drug, we observed reduced expression of HLA-DR in NLCs as well as increased secretion of interleukin-10 (IL-10), indicating an altered inflammatory milieu in the cocultures. The increase in IL-10 levels lead to an induction of STAT1 phosphorylation in CLL cells and to enhanced cell-surface expression of intercellular adhesion molecule 1 and altered expression of cytoskeletal and migration-related genes. Chemotaxis assays with lenalidomide-treated CLL cells revealed an impaired migration capability. Our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug affects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide acts on the migratory potential of CLL cells, which may affect circulation and homing of CLL cells in vivo.

Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6

Multipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14(-)CD1a(+)) and resulted in the generation of CD14(+)CD1a(-) cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory interleukin-10-producing cell type.

Granzyme A Produced by γ9δ2 T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

Suppression of Vaccine Immunity by Inflammatory Monocytes

Vaccine adjuvant-induced inflammation augments vaccine immunity in part by recruiting APCs to vaccine draining lymph nodes (LNs). However, the role of one APC subtype, inflammatory monocytes, in regulating vaccine immunity in healthy animals has not been fully examined in detail. Therefore, vaccine-mediated monocyte recruitment and subsequent immune responses were investigated using murine vaccination models and in vitro assays. Recruitment of inflammatory monocytes to vaccine draining LNs was rapid and mediated primarily by local production of MCP-1, as revealed by studies in MCP-1(-/-) mice. Interrupting monocyte recruitment to LNs by either transient monocyte depletion or monocyte migration blockade led to marked amplification of both cellular and humoral immune responses to vaccination. These results were most consistent with the idea that rapidly mobilized inflammatory monocytes were actually suppressing vaccine responses. The suppressive nature of vaccine-elicited monocytes was confirmed using in vitro cocultures of murine monocytes and T cells. Furthermore, it was determined that inflammatory monocytes suppressed T cell responses by sequestering cysteine, as cysteine supplementation in vitro and in vivo appreciably augmented vaccine responses. These findings indicated, therefore, that vaccination-elicited inflammation, although necessary for effective immunity, also generated potent counter-regulatory immune responses that were mediated primarily by inflammatory monocytes. Therefore, interrupting monocyte-mediated vaccine counterregulatory responses may serve as an effective new strategy for broadly amplifying vaccine immunity.

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8(+) T cells after co-culturing. The CD8(+) T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8(+) T cell responses. Interestingly, the allo- and self-HLA-restricted CD8(+) T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.

Peripheral compartment innate immune response to Haemophilus influenzae and Streptococcus pneumoniae in chronic obstructive pulmonary disease patients

Alterations in innate immunity that predispose to chronic obstructive pulmonary disease (COPD) exacerbations are poorly understood. We examined innate immunity gene expression in peripheral blood polymorphonuclear leukocytes (PMN) and monocytes stimulated by Haemophilus influenzae and Streptococcus pneumoniae. Thirty COPD patients (15 rapid and 15 non-rapid lung function decliners) and 15 smokers without COPD were studied. Protein expression of IL-8, IL-6, TNF-α and IFN-γ (especially monocytes) increased with bacterial challenge. In monocytes stimulated with S. pneumoniae, TNF-α protein expression was higher in COPD (non-rapid decliners) than in smokers. In co-cultures of monocytes and PMN, mRNA expression of TGF-β1 and MYD88 was up-regulated, and CD14, TLR2 and IFN-γ down-regulated with H. influenzae challenge. TNF-α mRNA expression was increased with H. influenzae challenge in COPD. Cytokine responses were similar between rapid and non-rapid decliners. TNF-α expression was up-regulated in non-rapid decliners in response to H. influenzae (monocytes) and S. pneumoniae (co-culture of monocytes and PMN). Exposure to bacterial pathogens causes characteristic innate immune responses in peripheral blood monocytes and PMN in COPD. Bacterial exposure significantly alters the expression of TNF-α in COPD patients, although not consistently. There did not appear to be major differences in innate immune responses between rapid and non-rapid decliners.