There is no single universally accepted hallmark of antigen presenting cell (APC) activation. Instead a variety of methods are used to identify APCs and assess their activation state. These activation measures include phenotypic methods [e.g., assessing the increased expression of surface markers such as major histocompatability (MHC) class II] and functional assays (e.g., evaluating the enhanced ability to take up and process antigen, or stimulate naïve T cells). Sipuleucel-T is an investigational autologous active cellular immunotherapy product designed to stimulate a T cell immune response against human prostatic acid phosphatase (PAP), an antigen highly expressed in prostate tissue. Sipuleucel-T consists of peripheral blood mononuclear cells (PBMCs), including activated APCs displaying epitopes of PAP. In order to develop a robust reproducible potency assay that is not hampered by MHC restriction we have developed a method to simply assess the biological activation of antigen presenting cells (APCs). In the course of sipuleucel-T characterization, we analyzed various phenotypic and functional parameters to define the activation state of APCs obtained from peripheral blood. Flow cytometric assays revealed that CD54+ cells are responsible for antigen uptake, and that expression of CD54 predominantly localizes to APCs. Costimulation, as measured by an allogeneic mixed lymphocytic reaction (alloMLR) assay, showed that activity was restricted to the CD54+ cell population. Similarly, CD54+ cells harbor all of the PAP-specific antigen presentation activity, as assayed using a PAP-specific HLA-DRbeta1-restricted T cell hybridoma. Finally we show that CD54 expression is substantially and consistently upregulated on APCs during culture with a GM-CSF fusion protein, and that this upregulation activity can be quantified. Thus these data support the use of CD54 upregulation as a surrogate for assessing human APC activation and validates its utility as a potency measure of sipuleucel-T.

BACKGROUND

Amniotic membrane is a highly promising cell source for tissue engineering. Being part of the placenta, this tissue is abundantly available. It can be processed easily to yield large amounts of epithelial and mesenchymal cells that have shown broad differentiation potential. For tissue-engineering purposes, cells may be applied either directly after isolation from the tissue or after a period of in vitro expansion to obtain higher cell numbers. In order to investigate the advantages and drawbacks of these strategies we compared freshly isolated and cultivated human amniotic epithelial cells (hAEC) regarding their surface antigen (Ag) expression profile and osteogenic differentiation capacity.

METHODS

Expression of surface Ag that are characteristic for mesenchymal stromal and embryonic stem cells was analyzed by flow cytometry. Different protocols for osteogenic and adipogenic differentiation were compared.

RESULTS

We have demonstrated that expression of surface Ag changes dramatically during cultivation of hAEC. While not or only weakly expressed on primary isolates, the mesenchymal markers CD13, CD44, CD49e, CD54, CD90 and CD105 are strongly up-regulated during in vitro propagation. In contrast, expression of the embryonic markers TRA-1-60 and TRA-1-81, but not SSEA-4, rapidly decreases upon cultivation. This phenotypic shift is associated with a reduction in osteogenic differentiation.

CONCLUSIONS

Our results suggest that phenotypic alterations of hAEC during in vitro cultivation might be responsible for a functional reduction of the differentiation potential, which has to be considered for the potential application of these cells in regenerative medicine.

(1) The vasculature of the blood-brain barrier allows only comparatively few leukocytes to enter and survey the healthy central nervous system (CNS). However, during pathological CNS inflammation, the number of leukocytes adhering to and penetrating the CNS vasculature increases strongly. (2) Endothelial adhesion molecules do not only mediate firm adhesion of leukocyte to vascular beds but also trigger signaling cascades within the endothelial cell, which play a crucial role in modulating subsequent leukocyte diapedesis. (3) Signaling through endothelial intercellular adhesion molecule-1 (ICAM-1, CD54) has been shown to induce changes of the endothelial cytoskeleton, transcription, and interendothelial junctions, all of which may be important in modulating endothelial disposition to infiltrating leukocytes. Furthermore, a number of recent reports document that drugs interfering with endothelial ICAM-1 signaling, efficiently reduce leukocyte migration both in vitro and in animal models of CNS inflammation. (4) These approaches are novel in as much as they target vascular beds rather than the penetrating leukocytes. Since endothelial ICAM-1 signaling appears to differ between different vascular beds we propose that such compounds could potentially be used as exquisite drugs in the treatment of neuroinflammatory diseases.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.

Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.

Asthma is characterized by appearance of eosinophils in the airway. Eosinophils purified from the airway 48 h after segmental antigen challenge are described as exhibiting greater adhesion to albumin-coated surfaces via an unidentified beta2 integrin and increased expression of alphaMbeta2 (CD11b/18) compared with purified blood eosinophils. We have investigated the determinants of this hyperadhesive phenotype. Airway eosinophils exhibited increased reactivity with the CBRM1/5 anti-alphaM activation-sensitive antibody as well as enhanced adhesion to VCAM-1 (CD106) and diverse ligands, including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. Purified blood eosinophils did not adhere to the latter diverse ligands. Enhanced adhesion of airway eosinophils was blocked by anti-alphaMbeta2. Podosomes, structures implicated in cell movement and proteolysis of matrix proteins, were larger and more common on airway eosinophils adherent to VCAM-1 when compared with blood eosinophils. Incubation of blood eosinophils with IL-5 replicated the phenotype of airway eosinophils. That is, IL-5 enhanced recognition of alphaM by CBRM1/5; stimulated alphaMbeta2-mediated adhesion to VCAM-1, albumin, ICAM-1, fibrinogen, and vitronectin; and increased podosome formation on VCAM-1. Thus, the hyperadhesion of airway eosinophils after antigen challenge is mediated by upregulated and activated alphaMbeta2.

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice, only tumors of the blood vessel endothelium (haemangiomas) have been thus far reported. Here we describe a highly reproducible method for the induction of benign tumors of the lymphatic endothelial cells (lymphangiomas) in mice by intraperitoneal injection of incomplete Freund's adjuvant. Morphological and histopathological studies of the lesions revealed the presence of cells at various levels of vascular development. The lymphangiomas developed in the peritoneal cavity and expressed the endothelial markers CD31/PECAM (platelet endothelial cell adhesion molecule), CD54/ICAM-1 (InterCellular Adhesion Molecule-1), and CD102/ICAM-2, as well as the vascular endothelial growth factor (VEGF) receptor Flk-1, the endothelial cell specific receptors Tie-1 and Tie-2 and the lymphatic endothelial cell specific Flt4 receptor as shown by in situ hybridization. The Flk-1 and Flt4 receptors were also identified in immunoblots of the tumors and in cells cultured from them. When induced in beta-galactosidase knock-in Flt4(+/-) mice, the tumor endothelia could be stained blue in a number of tumor cells although the staining was of lower intensity than in normal lymphatic vessels. The tumor-derived cells could be propagated in vitro and they spontaneously differentiated, forming vessel-like structures. Murine lymphangiomas thus represent a highly reproducible and convenient source of lymphatic endothelial cells.

BACKGROUND

Polyclonal antithymocyte globulins (ATG) induce T-cell depletion and functional impairment of nondeleted lymphocytes. Interference of ATG with the main leukocyte surface molecules involved in cellular adhesion and leukocyte-endothelium interaction was investigated in the present study.

METHODS

In three rabbit ATG, the authors measured antibodies to integrins, beta2-integrin ligands, and chemokine receptors by flow cytometry; chemotactic responses; and down-modulation of cell surface expression on lymphocytes, monocytes, and neutrophils.

RESULTS

Antibodies to CD11a/CD18 (leukocyte function-associated antigen-1 [LFA-1]) present in ATG induced a dose-dependent down-modulation of cell surface expression of this beta2 integrin on lymphocytes, monocytes, and neutrophils. In contrast, anti-LFA-1 monoclonal antibodies did not induce LFA-1 modulation unless cross-linked by a second antibody. ATG also contained functional antibodies to the beta1 integrin CD49d/CD29 (VLA-4), the alpha4beta7 integrin, CD50, CD54, and CD102 but not to CD62L. ATG were shown to bind to CXCR4 and CCR7 on lymphocytes, CXCR4, and CCR5 on monocytes; to down-modulate cell surface expression of CCR7; and to decrease monocyte chemotactic response to CCL5 (RANTES) and lymphocyte chemotactic response to CCL19 (MIP-3beta).

CONCLUSIONS

These results show that ATG may interfere with leukocyte responses to chemotactic signals but mostly inhibit the expression of integrins required for firm cellular adhesion. The latter property of inhibition is not shared by monoclonal antibodies, and it may contribute to decreasing graft cellular infiltration during acute rejection and possibly after postischemic reperfusion.

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8(+) T cells by cultivation of OVA-pulsed dendritic cells (DC(OVA)) with naive CD8(+) T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8(+) T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DC(OVA)-mediated CD8(+) CTL responses and antitumor immunity. We found that DC(OVA) uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I-TCR and CD54-LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DC(OVA) via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8(+) T cell EXOs, can inhibit DC(OVA)-stimulated CD8(+) CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DC(OVA)-mediated CD8(+) CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti-LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8(+) CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8(+) TCRs have a similar inhibitory effect as T cell EXOs in DC(OVA)-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8(+) T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial-mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

OBJECTIVE

As mesenchymal stromal cells (MSCs) have been proposed as a tool for management or prevention of graft-vs-host disease, we investigated their immunoregulatory properties, their expression of adhesion molecules and galectin-1, and the impact of environment context on these functions.

METHODS

The effects of MSCs on T-cell proliferation were analyzed using carboxyfluorescein diacetate N-succinimidyl ester labeling. We evaluated the expression of adhesion molecules and galectin-1 by MSCs and the impact of an inflammatory or infectious environment on these expressions. Using neutralizing antibodies against adhesion molecules and a galectin-1 inhibitor, we assessed the role of these molecules in MSC functions.

RESULTS

MSCs inhibition of T-cell proliferation depended on MSC concentrations, cell contact, and culture environment. Expression of adhesion molecules and secretion of galectin-1 by MSCs are tightly regulated. Coculture with activated T cells upregulated expression of CD54 (intercellular adhesion molecule 1) and CD58 (lymphocyte function-associated antigen 3) and secretion of galectin-1 by MSCs. Interestingly, in an inflammatory or infectious environment, expression of adhesion molecules and galectin-1 by MSCs was differentially modulated. Furthermore, blocking galectin-1 activity prevented the suppressive potential of MSCs. Neutralization of adhesion molecule activity had no effect on MSC inhibition.

CONCLUSIONS

Galectin-1 plays an important role in MSC immunoregulatory functions, which are depending on cell environment. The present study provides new insights concerning MSC physiology and will increase the safety and efficiency of MSCs in clinical settings.

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

BACKGROUND

The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. To gain a better understanding of this process the expression by these cells of cell surface activation markers, co-stimulatory molecules, and adhesion molecules was analysed.

METHODS

CD4+ and CD8+ T lymphocytes from peripheral blood (PBL) or bronchoalveolar lavage (BAL) fluid, as well as paired peripheral blood monocytes and alveolar macrophages from 27 patients with sarcoidosis were analysed by flow cytometry.

RESULTS

CD26, CD54, CD69, CD95, and gp240 were all overexpressed in T cells from BAL fluid compared with those from PBL in both the CD4+ and CD8+ subsets, while CD57 was overexpressed only in BAL CD4+ cells. In contrast, CD28 tended to be underexpressed in the BAL T cells. Monocyte/macrophage markers included CD11a, CD11b, CD11c, CD14, CD16, CD54, CD71, CD80 and CD86 and HLA class II. CD11a expression in alveolar macrophages (and peripheral blood monocytes) was increased in patients with active disease and correlated positively with the percentage of BAL lymphocytes. Expression of CD80 in macrophages correlated with the BAL CD4/CD8 ratio.

CONCLUSIONS

Our data indicate substantial activation of both CD4+ and CD8+ lung T cells in sarcoidosis. There were also increased numbers of BAL lymphocytes whose phenotypic characteristics have earlier been associated with clonally expanded, replicatively senescent cells of the Th1 type.

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO(•)). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO(•) to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration. Effects of NO(•), either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO(•) down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO(•) was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression. In conclusion, during angiogenesis and leukocyte recruitment, NO(•) regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO(•), reflecting the functions of the tissue they originate from.

The beta 2-integrin (CD18) family members bind to their ligands subsequent to activation of a number of well defined and diverse signal transduction pathways. The precise molecular changes associated with activation of the integrin family members have remained elusive. Here, we characterize a monoclonal, CBR LFA-1/2, that binds to the beta 2-subunit and is able to mimic activation induced upon stimulation by phorbol esters. The Ab induces binding of the LFA-1-expressing cell line, JY, to ICAM-1 (CD54) and ICAM-3 (CD50). Activation of binding by this Ab is independent of Fc interactions and does not occur through cross-linking at the cell surface, because the Fab fragment of the Ab is able to modulate the same effect. Stimulation of neutrophils with CBR LFA-1/2 induces binding to ICAM-1 through activation of both LFA-1 and Mac-1. Activation of Mac-1 by CBR LFA-1/2 was further confirmed by stimulation of neutrophil binding to fibrinogen, a ligand for Mac-1. CBR LFA-1/2 lowers by 10-fold the concentration of Mg2+ required to achieve maximal binding of LFA-1 to ICAM-1. It therefore appears that CBR LFA-1/2 induces a conformational change that directly increases the avidity of beta 2-integrins for ligands.

The natural exposure to house dust mites causes sensitization in genetically susceptible patients. Persistent exposure of sensitized patients causes chronic inflammation, and consequently, hyperreactivity, thus promoting the development of clinical features. Recently, intercellular adhesion molecule-1 (ICAM-1)/CD54 expression on epithelial cells triggered by allergen has been demonstrated and related to the inflammation caused by the allergic reaction. Therefore we evaluated the possible presence of inflammation (i.e., inflammatory cell infiltrate and ICAM-1/CD54 expression on epithelium) at conjunctival and nasal levels in patients with asymptomatic allergic rhinitis caused by mites, in their relatives living in the same environment, and in healthy volunteers. In addition, the possible relationship between inflammation and house dust mite allergen exposure was evaluated. Conjunctival and nasal scrapings of allergic subjects enrolled in the study showed many inflammatory cells. A mild ICAM-1/CD54 expression on conjunctival and nasal epithelium was detectable in allergic subjects, whereas relatives and healthy volunteers showed few inflammatory cells and no ICAM-1/CD54 expression on epithelial cells. A detectable level of house dust mite, sufficient to cause sensitization, was found in all houses. This study demonstrates a minimal persistent inflammation at conjunctival and nasal levels constantly detectable in patients without symptoms who are sensitized to mites and continuously exposed to the natural allergens.

Infection of B cells with Epstein-Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV-latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (Delta CTAR1) or 232 and 386 (Delta CTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NF kappa B activation by constitutive repressive I kappa B-alpha only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 microM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.

The molecular mechanisms involved in regulating the balance between cellular proliferation and differentiation remain poorly understood. Members of the Ets-domain family of transcription factors are candidates for proteins that might differentially regulate cell cycle control and cell type-specific genes during the differentiation of myeloid progenitor cells. The Ets repressor PE-1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation. An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Here, we present evidence that the PE-1/METS gene is a transcriptional target of the cyclic AMP response element-binding protein-1 (CREB-1). CREB-1 expression is dramatically up-regulated during macrophage differentiation and phosphorylation of CREB-1 and the related factor CREM-1 are stimulated by M-CSF in a SAPK2/p38-dependent manner. Chromatin immunoprecipitation experiments demonstrate that CREB-1/CREM-1 are recruited to the PE-1/METS promoter as well as to the promoters of other genes that are up-regulated during terminal macrophage differentiation. Overexpression of CREB-1 stimulates the activities of the PE-1/METS, and macrosialin promoters, while expression of a dominant negative form of CREB-1 during macrophage differentiation inhibits expression of the PE-1/METS and macrosialin genes. Inhibition of CREB function also results in reduced expression of CD54 and impaired cell adhesion. Taken together, these findings reveal new roles of CREB-1/CREM-1 as regulators of macrophage differentiation.

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA-5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.

Activation of the aryl hydrocarbon receptor (AhR) in immune cells, such as dendritic cells (DCs), can lead to suppressed immune responses. Although AhR activation is most recognized for mediating the effects of its prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), many compounds existing in dietary sources can also bind the AhR. Because the immunomodulatory effects of indole-3-carbinol (I3C) and indirubin-3'-oxime (IO) have yet to be investigated in DCs, we evaluated the potential immunomodulatory effects of these compounds on murine DCs. We hypothesized that I3C and IO suppress immune and inflammatory responses in DCs. We found that both I3C and IO decreased the expression of CD11c, CD40, and CD54 while they increased expression of MHC2 and CD80. Following lipopolysaccharide (LPS)-activation, I3C and IO suppressed the production of pro-inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-12, and nitric oxide but increased IL-10 levels. These effects of I3C and IO were partially mediated by the AhR. Additionally, immunoregulatory genes, such as ALDH1A, IDO and TGFB, were upregulated following treatment with I3C or IO. Both I3C and IO decreased basal levels of nuclear factor-kappa B p65, but only I3C suppressed the LPS-induced activity of RelB. Finally, when cultured with naïve T cells, bone marrow-derived dendritic cells treated with the dietary AhR ligands increased the frequency of Foxp3+ Tregs in an antigen-specific manner. Taken together, these results indicate that I3C and IO exhibit immunosuppressive and anti-inflammatory effects on DCs. Because I3C and IO are significantly less toxic than TCDD, these natural products may ultimately become useful therapeutics for the treatment of autoimmune and inflammatory diseases.

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.

Complex clinical outcomes, such as adverse reaction to vaccination, arise from the concerted interactions among the myriad components of a biological system. Therefore, comprehensive etiological models can be developed only through the integrated study of multiple types of experimental data. In this study, we apply this paradigm to high-dimensional genetic and proteomic data collected to elucidate the mechanisms underlying the development of adverse events (AEs) in patients after smallpox vaccination. As vaccination was successful in all of the patients under study, the AE outcomes reported likely represent the result of interactions among immune system components that result in excessive or prolonged immune stimulation. In this study, we examined 1442 genetic variables (single nucleotide polymorphisms) and 108 proteomic variables (serum cytokine concentrations) to model AE risk. To accomplish this daunting analytical task, we employed the Random Forests (RF) method to filter the most important attributes, then we used the selected attributes to build a final decision tree model. This strategy is well suited to integrated analysis, as relevant attributes may be selected from categorical or continuous data. Importantly, RF is a natural approach for studying the type of gene-gene, gene-protein and protein-protein interactions we hypothesize to be involved in the development of clinical AEs. RF importance scores for particular attributes take interactions into account, and there may be interactions across data types. Combining information from previous studies on AEs related to smallpox vaccination with the genetic and proteomic attributes identified by RF, we built a comprehensive model of AE development that includes the cytokines intercellular adhesion molecule-1 (ICAM-1 or CD54), interleukin-10 (IL-10), and colony stimulating factor-3 (CSF-3 or G-CSF) and a genetic polymorphism in the cytokine gene interleukin-4 (IL4). The biological factors included in the model support our hypothesized mechanism for the development of AEs involving prolonged stimulation of inflammatory pathways and an imbalance of normal tissue damage repair pathways. This study shows the utility of RF for such analytical tasks, while both enhancing and reinforcing our working model of AE development after smallpox vaccination.

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10(-7) M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1alpha by immature DC and also up-regulates IL-1beta, RANTES, and macrophage-inflammatory protein 1beta but not TNF-alpha and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.