Production Of Soluble Factors Research Articles

Abstract 4676: Understanding progenitor cells in the peritumor region of glioblastoma: GD3 ganglioside and NG2 proteoglycan expression

Abstract Invasion is a defining hallmark of glioblastoma (GBM) and its infiltrative capacity is supported by the presence of tumor cells at a distance greater than 4 cm from the tumor margin. Several studies have suggested that the space demanding growth, the production of soluble factors and the peculiar vasculature of GBM induce changes in surroundings. We previously reported that the presence of activated MAP kinases and stem cell marker nestin in peritumor tissue of GBM, even in the absence of tumor cells, carries a prognostic significance, and that neoangiogenesis occurs in peritumor areas. GD3 ganglioside is linked with malignant transformation that promotes migration, invasion and neoangiogenesis. Neuro-glial proteoglycan 2 (NG2) is also involved in invasion and angiogenesis; it is expressed by pericytes that have multipotent stem cell properties. Both markers are present in several precursor cells. To gain a deeper insight into the characteristics of GBM peritumor tissue, we investigated GD3 and NG2 expression in peritumor areas compared to the enhanced lesion. Patients (n=40) recruited in this study had a supratentorial GBM and underwent “en bloc” surgery. Tissue samples from the enhanced lesion (1st area), white matter at a distance <1 cm (2nd area) and between 1 and 3.5 cm from the tumor margin (3rd area) were obtained using adopted surgical technique. Immunohistochemical analysis using anti-GD3 (n=40) monoclonal and anti-NG2 (n=15) polyclonal antibody was performed in paraffin-embedded sections and frozen sections, respectively. Results showed cell membrane and cytoplasmic GD3 and NG2 expression in both GBM and peritumor tissue. Specifically, GD3 was expressed in tumor and endothelial cells; it was present in reactive astrocytes and in apparently normal cells in peritumor areas. Percentage of GD3 positive cells was higher in GBM than in 2nd and 3rd areas (P< 0.001); GD3 was higher in peritumor areas when tumor cells were present (P< 0.005). NG2 was expressed in GBM and in cells of various morphology in peritumor tissue. Double labelled confocal microscopy analysis showed that NG2 was co-expressed with alpha-smooth muscle actin in pericytes while CD105 was present in endothelium of the vessel walls in tumor and peritumor tissue. Percentage of NG2 positive cells was higher in GBM than in peritumor areas (P< 0.001), and was lower in the 3rd compared to the 2nd area (P< 0.05). Our findings confirm that peritumor tissue undergoes a significant transformation. Apparently normal glial cells expressing GD3 and NG2 might represent precursor cells in peritumor tissue. Marker expression in both these cells and pericytes may indicate the acquisition of properties linked to proliferation, motility and neovascularization contributing to tumor progression. Importantly, our results provide a useful tool to develop informed and unified approaches to diagnosis and therapy of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4676.

Abstract 538: Mesenchymal stem cell subset prevents cycling of KG1a leukemic cells by cell-cell interaction

Abstract The interactions that occur between leukemic cells and the microenvironment are currently poorly understood. The definition of novel pathways that deliver survival and drug resistance signaling from the milieu to leukemic cells may lead to the discovery of new drugs that are able to target microenvironmental cues perpetuating the leukemic clones. Since the bone marrow (BM) environment is comprised of a complex assortment of different cell types, matrices and soluble and membrane-bound factors, it is crucial to start delineating specific cell-cell interactions and their effects on leukemic cell survival. Mesenchymal stem cells (MSC) are a fundamental component of the BM niche as they support hematopoiesis and are able to differentiate into neighboring cellular types. Therefore, in these studies we investigated the effect of Stro-1+ MSC on the growth and survival of KG1a, a primitive, differentiation-resistant subtype of acute myeloid leukemia. MSC and KG1a cells were co-cultured for 3 days under contact or non-contact conditions, at incremental ratios of 1:1, 1:5, 1:10 and 1:100 respectively, while maintaining a constant initial total number of cells for each dilution (n=3). In transwell non-contact cultures, KG1a cells proliferated at the same rate as KG1a alone with a 3 to 5 fold increase in cell numbers, regardless of the ratio. By contrast, when KG1a were cultured in direct contact with MSC, KG1a growth inhibition was directly proportional to the MSC:KG1a ratio. Specifically, MSC cultured with KG1a cells at ratios of 1:1, 1:5, 1:10, and 1:100 inhibited the growth of KG1a cells by 84%, 76%, 74%, and 45%, respectively while remaining 99-100% viable. Thus, we next performed cell cycle analysis using flow cytometry and found that while the cell cycle distribution of KG1a cultures were: 23% in G0/G1; 19% in G2/M; and 58% in S phase, KG1a co-cultured with MSC had undergone cell cycle arrest in S-phase (98%). Although our results contrast with prior studies showing that MSC inhibited KG1a in G1phase through the production of soluble factors, it is possible that the differences seen are due to the population of MSC used. Within the MSC, Stro-1+ cells constitute a primitive population of cells in intimate relation with the osteoblastic niche, the role of which is to retain stem cells in a quiescent state. Therefore, studies are currently underway to identify molecules responsible cell cycle arrest upon KG1a/MSC interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 538.

Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

Inhibition of hepatic stellate cells proliferation by mesenchymal stem cells and the possible mechanisms

During fibrosis, hepatic stellate cells (HSCs) undergo a complex activation process characterized by increased proliferation and extracellular matrix deposition. Previous studies have suggested that mesenchymal stem cells (MSCs) may ameliorate fibrogenesis and represent a promising strategy for cell therapy. However, the underlying mechanisms are not fully understood. Hepatic stellate cells were treated with or without MSCs. Then cell proliferation and cell cycle were analyzed. Production of soluble factors by MSCs and its relation with cell proliferation suppression was evaluated by transwell co-culture and RNA interference. Effects of MSCs on the gene expression of collagen were also evaluated. MSCs induced G(0)/G(1) arrest of HSCs growth partly through secreting soluble factors TGF-beta3 and HGF, which resulted in up-regulation of p21(Cip1) and p27(Kip1) expression and down-regulation of cyclinD1. MSCs inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and reduced gene expression of collagen type I and III. MSCs did not reverse the proliferation and collagen type I gene expression of HSCs provoked by PDGF. The growth inhibition of HSCs induced by MSCs through an arrest in the G(0)/G(1) phase of the cell cycle is partially mediated by secretion of TGF-beta3 and HGF. MSCs inhibit HSCs activation through decreasing phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. These results further support MSCs may be used as a novel therapy for treating fibrotic diseases in human.

The enigmatic role of mast cells in dominant tolerance.

The role of regulatory T cells (Treg) in peripheral tolerance has been studied extensively in transplantation research. Recently, mast cells have been shown to play an indispensable role in allograft tolerance. The purpose of this review is to inform the reader on the current standings of the role of mast cells in dominant tolerance with an emphasis on the interaction of mast cells with Treg. Mast cells are required to sustain peripheral tolerance via Treg. Treg can stabilize mast cells degranulation by contact-dependent mechanisms through the interaction of OX40 and its ligand OX40L, and by production of soluble factors, such as interleukin-10 and transforming growth factor-beta. Conversely, the activation and subsequent degranulation of mast cells break peripheral tolerance. Both mast cells and Treg are needed to create a local immunosuppressive environment in the transplant. Treg are not only necessary to suppress effector T-cell responses but also to stabilize mast cells. Mast cells in return could contribute to the immunosuppressive state by release of transforming growth factor-beta, interleukin-10 and specific proteases. However, the molecular basis for mast cells control of Treg suppression in organ transplantation is still unresolved.

TLR-Stimulated CD34 Stem Cell-Derived Human Skin-Like and Monocyte-Derived Dendritic Cells Fail to Induce Th17 Polarization of Naive T Cells but Do Stimulate Th1 and Th17 Memory Responses

Dendritic cells (DCs) are important in linking innate and adaptive immune responses by priming and polarizing naive CD4(+) Th cells, but little is known about the effect of different human DC subsets on Th cells, particularly Th17 cells. We have investigated the ability of TLR-stimulated human Langerhans cells (LC), dermal DCs (dDC), and monocyte-derived DCs (moDC) to affect naive and memory Th17 and Th1 responses. MoDCs stimulated greater memory T cell proliferation while LCs and dDCs more potently stimulated naive T cell proliferation, indicating functionally distinct subsets of DCs. TLR stimulation of all three DC types was unable to induce Th17 polarization from naive T cell precursors, despite inducing Th1 polarization. Dectin stimulation of DCs in IMDM was however able to produce Th17 cells. TLR-stimulated DCs were capable of inducing IL-17A and IFN-gamma production from memory T cells, although the mechanism used by each DC subset differed. MoDCs partially mediated this effect on memory Th1 and Th17 cells by the production of soluble factors, which correlated with their ability to secrete IL-12p70 and IL-23. In contrast, LCs and dDCs were able to elicit a similar memory response to moDCs, but in a contact dependent manner. Additionally, the influence of microbial stimulation was demonstrated with TLR3 and TLR7/8 agonists inducing a Th1 response, whereas TLR2 or dectin stimulation of moDCs enhanced the IL-17 response. This study emphasizes the differences between human DC subsets and demonstrates that both the DC subset and the microbial stimulus influence the Th cell response.

Disruption of Leukemia Stem Cell (LSC) Interactions with Bone Marrow Stromal Niche Enhances Efficacy of FLT3 Tyrosine Kinase Inhibitors (TKI) in Vivo

The bone marrow stromal microenvironment comprises “niches” that promote survival and quiescence of LSC and, together with molecular features intrinsic to LSC or bulk leukemic cells, may contribute to therapeutic resistance. AML patients treated with FLT3 inhibitors have had better clinical responses in the peripheral blood than in the bone marrow, suggesting that the marrow niche may represent an important resistance mechanism in this setting. We have shown that FLT3 is a valid therapeutic target in MLL-rearranged (MLL-R) ALL, and FLT3 TKIs are being tested in newly diagnosed infants with MLL-R ALL. We hypothesized that: 1) marrow stroma may provide a protective niche for MLL-R ALL LSCs, and 2) disrupting the LSC-stroma interaction may render MLL-R ALL LSCs more vulnerable to FLT3 TKI.N=28 primary childhood ALL samples (9 infant MLL-R, 21 other) were split into 2 aliquots, one cultured in medium only, one in medium + normal human bone marrow feeder layers. Survival of viable cells (defined as CD19+, annexin−) was quantified as: (# of viable cells at baseline)/(# at 48 hrs). Surviving fraction in medium was similar for MLL-R group (56%) vs. others (42%, p=0.23). On stroma, surviving fraction was greater for MLL-R group (106%) vs. others (75%, p=0.03). The effect of stroma co-culture on FLT3 TKI (lestaurtinib)-mediated 48 hr MTT cytotoxicity was determined on 3 MLL-R and 3 other samples. Stroma was protective for the MLL-R samples at all 6 dose levels (5 – 100 nM). At 50 nM, e.g., the mean OD (% control) was 25% (medium) vs. 47% (stroma, p=0.03). A protective effect was not seen for the other samples. Thus, stromal interactions selectively promote the survival of MLL-R ALL cells in absence and presence of FLT3 TKI.The relative contribution of physical interaction vs. production of soluble factors by stromal cells was addressed using N=3 MLL-R samples, each cultured in 3 conditions: in physical contact with stroma (S); with leukemia cells and stroma separated by a permeable Transwell membrane (T); and medium only (M). WST-1 and annexin binding assays were done @ 4, 24 and 48 hrs −/+ 5–100 nM TKI. Mean OD (% control) in untreated wells in S/T/M conditions were: 257%/172%/97% @ 24 hr, and 321%/229%/95% @ 48 hrs. Annexin+ fraction in S/T/M conditions (untreated) was: 12%/22%/21% @ 48 hrs. Mean OD (% control) in 50 nM TKI in S/T/M conditions were: 172%/86%/88% @ 24 hr, and 163%/110%/71% @ 48 hr. Thus: S conditions enhanced proliferation, reduced apoptosis, and abrogated TKI-mediated cytotoxicity. These effects in T conditions were generally less striking, and absent in the case of apoptois. While soluble factors are able to mediate some effects of stroma on MLL-R ALL cells, physical interaction is required for maximal effect.The effects of the S/T/M conditions on the LSC (as opposed to bulk leukemia cells) were then assessed: 48 hr S/T/M ex vivo culture (−/+ 20 nM TKI) was followed by in vivo xenografting in NOD/SCID mice (tail vein injection, 6 cohorts, N=6 per cohort). Marrow engraftment of ALL cells was assessed after 10 weeks (reflecting LSC activity). Mean engraftment (% human ALL cells in marrow) with S/T/M conditions was 96%/74%/34% (no TKI) and 87%/44%/7% (20 nM TKI) (p<0.05 for all comparisons except S @ 0 nM vs. S @ 20 nM), showing that S is superior to T, and T superior to M, in promoting survival of LSC ex vivo, an effect that is particularly pronounced under the pressure of FLT3 TKI.The combination of G-CSF and the CXCR4 antagonist AMD3100 potently mobilize CD34+ stem progenitors. We tested this combination as a therapeutic strategy to disrupt the stroma/LSC interaction and improve the efficacy of FLT3-targeted therapy in MLL-R ALL. Cohorts of 10 NOD/SCID mice were injected with N=2 primary MLL-R ALL cells (5 mice per sample), allowed to engraft for 2 weeks, then treated with 1 of 4 regimens: vehicle control (C), G-CSF-AMD3100 (G-A), lestaurtinib (L), or G-CSF-AMD3100 + lestaurtinib (G−A + L). At week 10, mice were sacrificed and assessed for marrow engraftment of ALL cells. Mean engraftment for C/G−A/L/G−A+L was 94%/90%/69%/23% (p<0.05 for all comparisons except C vs. G−A), showing that the G-CSF/AMD3100 combination potently enhance the efficacy of FLT3 inhibitors against MLL-R ALL LSCs.Thus, stromal-leukemia interactions are protective for MLL-R ALL bulk cells and LSCs and represent an important mechanism of resistance to FLT3-targeted therapy. Disruption of these interactions with stem cell mobilizing agents may be able to overcome therapeutic resistance and improve the efficacy of FLT3 TKI.

Redirected Activity of Human Antitumor Chimeric Immune Receptors is Governed by Antigen and Receptor Expression Levels and Affinity of Interaction

Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.

Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption

Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 ± 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-κB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N G -nitro- l-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.

Interactions of primary neuroepithelial progenitor and brain endothelial cells: distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.

NF-kB activation and p38 phosphorilation in microglial cells infected with Leptospira or exposed to partially purified leptospiral lipoproteins

Recently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response.

Activation of Nuclear Factor κB by Different Agents

The transcription factor nuclear factor kappaB (NF-kappaB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-kappaB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-kappaB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-kappaB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-kappaB activation by TNF-alpha were examined. NF-kappaB was activated by TNF-alpha, IL-1beta, PMA, and camptothecin in a dose-dependent manner, but not by LPS. TNF-alpha results in the strongest induction of NF-kappaB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20-35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB activation or direct communication via gap junctions in the cell layer diminishing the TNF-alpha response.

Myeloma Microenvironment Controls T Regulatory Cell Activity: Potential Target for Therapeutic Interventions.

Multiple myeloma (MM) is associated with significant immune dysfunction. The biological basis of this dysfunction remains ill defined. We have previously observed significantly decreased number of T regulatory (Treg) cells, as measured by Foxp3 expression, in both MGUS and MM compared to normal donors. Moreover, Treg cells in MM and MGUS are unable to suppress anti-CD3-mediated T cell proliferation. Here, we have further analyzed elements of bone marrow (BM) microenvironment that may be responsible for dysfunctional Treg cells in MM. Both cellular and soluble components of the BM microenvironment directly, by cell-cell interactions, as well as by production of soluble factors not only affect MM cell growth, survival, migration and drug resistance but also modulate function of immune components. Interactions between MM cells and bone marrow stromal cells trigger production of IL-6, as well as a number of other cytokines and chemokines including TNF-α , VGEF, IGF-1, SDF-1α , IL-1β , TGF-β , and MIP-1α /β with immunomodulatory activity. We and others have in fact demonstrated significantly elevated levels of serum IL-6 as well as soluble IL-6 (sIL-6) receptor in MM. Based on in vivo data that IL-6 can modulate Treg cell number and function in a murine model of asthma, we evaluated its role in Treg cell dysfunction in myeloma. We performed T cell proliferation assays with soluble anti-CD3 antibody with or without Treg cells from normal donors in presence or absence of IL-6/sIL-6R. We observed that the presence of IL-6 and/or sIL-6 receptor leads to loss of normal Treg cell suppressive activity on normal donor T cells (from inhibition of proliferation by 27% to increase in proliferation by 6% in presence of IL-6/sIL-6R, p=0.01). The Treg cell dysfunction observed here is similar to that observed in MM patients. Conversely, when Treg cells from MM patients were treated with IL-6 receptor super antagonist, Sant 7, prior to their addition to T cell proliferation assay, the suppressive activity of Treg cells was restored. Presence of untreated Treg cells was associated with 28% increase in T cell proliferation while Sant 7-treated Treg cells led to 35% inhibition of T cell proliferation (p=0.01). Additionally, we observed expansion of Foxp3+ Treg cell number in PBMC from MM patients following in vitro treatment with anti-IL-6 antibody. Furthermore, TGF-β , a known stimulator of Treg cell function was less effective in stimulating FOXP3+ Treg cells in PBMCs from MM patients (15.2%) compared to normal donor PBMCs (24%). Since Treg cells require interaction with antigen presenting cells (APC), we evaluated effects of IL-6 and TGF-β on the ability of APC to help suppressive activity of Treg cells. Prior exposure of mature dendritic cells to IL-6 and TGF-β abrogated Treg cell suppressive activity from 47% inhibition to 23% increase in T cell proliferation (p=0.01). In conclusion, we have observed that combination of IL6/sIL6R and TGF-β significantly affects Treg cell number and function. These cytokines are significantly expressed in the MM BM microenvironment and may be responsible for the observed Treg cell dysfunction. These cytokines thus may be targets to modulate immune responses in myeloma to enhance immune function and devise effective vaccination strategies in the future.

Toll-Like Receptor-Stimulated CD14+ Monocytes Induce Soluble Factors That Activate Human Mesenchymal Stem Cells To Inhibit T-Cell Alloreactivity.

Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and alloreactivity. We have previously shown that MSCs require activation by CD14+ monocytes in cell culture. Toll-like receptors (TLRs) critically modulate antigen-presenting cell (APC) activation, maturation and function. Therefore, we aimed to determine whether TLR agonists could enhance CD14-mediated MSC activation and subsequent MSC-mediated T-cell inhibition. TLR agonists and human IL-1β were used to stimulate CD14+ cells isolated from peripheral blood of normal donors. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), and R848 (Resiquimod, TLR7/8) in complete RPMI media (heat-inactivated FBS, glutamine, and antibiotics). TLR-stimulated CD14+ cells and supernatants from TLR-stimulated CD14+ cells were then used to stimulate third- and fourth-passage human MSCs (CD45−CD105+CD90+CD80−CD73+HLA-I+) expanded from normal volunteer bone marrow aspirate specimens. After a 24-hour culture with stimulant cells or supernatants, stimuli were removed, MSCs were washed twice with sterile PBS, and then were cultured for an additional 24 h in FBS-free RPMI media. Supernatants from TLR-stimulated CD14+ cell cultures and from washed MSC cultures were used to measure cytokine and chemokine production and to inhibit T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists and media alone. TLR stimulation resulted in high-level soluble factor induction (IL-1β, IL-6, TNF-α, and RANTES) from CD14+ cells. For example, levels of inducible IL-6 measured in CD14+ cell culture supernatants following LPS, SAC and R848 stimulation were 5.2 (70.3 ± 26.2 ng/ml), 4.7 (64.1 ± 16.3 ng/ml), and 4.6 (63.4 ± 13.1 ng/ml)-fold higher than after IL-1β stimulation (13.7 ± 4.0 ng/ml) (Mean ± SEM, 4 independent experiments). Supernatants of TLR-stimulated CD14+ cells induced higher levels of soluble factors from MSCs than stimulation with CD14+ cells themselves, suggesting that soluble factors from CD14+ cells activate MSCs. Not all supernatants of TLR-stimulated CD14+ cells were similar in their capacity to activate MSC-mediated inhibition of T-cell alloreactivity. For example, supernatants of Pam3Cys-, R848- and SAC-stimulated CD14+ cells all induced high levels of TNF-α in washed MSC cultures; but only supernatant from Pam3Cys-stimulated CD14+ cells resulted in MSC-mediated inhibition of T-cell alloreactivity (63.7 ± 12.9 % inhibition, Mean ± SEM, 4 independent ELISPOTs). Ongoing studies are being performed to define these immunomodulatory factors. Together, these results suggest that ex vivo TLR stimulation enhances soluble factor production from CD14+ cells, which, in turn, increases MSC production of immunomodulatory factors that mediate inhibition of T-cell alloreactivity.

Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins

Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces. These forces are significantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic overload. Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems. Two immediate early gene-encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2), are highly expressed in several mechanical stress-related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton. Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events. In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibrotic diseases. At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated by various components of the signaling machinery, i.e., small Rho GTPases, mitogen-activated protein kinases, and actin binding proteins. This review discusses the mechanical regulation of the Cyr61 and CTGF in various tissues and cell culture models with a special attention to the cytoskeletally based mechanisms involved in such regulation.

An Introductory Review of Cell Mechanobiology

Mechanical loads induce changes in the structure, composition, and function of living tissues. Cells in tissues are responsible for these changes, which cause physiological or pathological alterations in the extracellular matrix (ECM). This article provides an introductory review of the mechanobiology of load-sensitive cells in vivo, which include fibroblasts, chondrocytes, osteoblasts, endothelial cells, and smooth muscle cells. Many studies have shown that mechanical loads affect diverse cellular functions, such as cell proliferation, ECM gene and protein expression, and the production of soluble factors. Major cellular components involved in the mechanotransduction mechanisms include the cytoskeleton, integrins, G proteins, receptor tyrosine kinases, mitogen-activated protein kinases, and stretch-activated ion channels. Future research in the area of cell mechanobiology will require novel experimental and theoretical methodologies to determine the type and magnitude of the forces experienced at the cellular and sub-cellular levels and to identify the force sensors/receptors that initiate the cascade of cellular and molecular events.

Reconstitution of human immunodeficiency virus–induced neurodegeneration using isolated populations of human neurons, astrocytes, and microglia and neuroprotection mediated by insulin-like growth factors

Primary human neuron cultures are an important in vitro model system for studies on mechanisms involved in human immunodeficiency virus (HIV)-associated dementia (HAD) and other neurological disorders. Here, more than 80 cell surface antigens were screened to identify a marker that could readily distinguish between neurons and astrocytes and found that neurons lack CD44 surface expression, whereas astrocytes and other cell types in brain are CD44+. Neurons and astrocytes were isolated from human fetal brain based on differential expression of CD44. Using purified neurons cocultured with astrocytes and/or microglia, it was demonstrated that HIV infection of microglia induces cellular activation and production of soluble factors that activate uninfected microglia and astrocytes and induce neuronal cell death. Activated astrocytes promoted HIV replication in microglia, thereby amplifying HIV-induced neurotoxicity. A screen for 120 cytokine/proteins detected upregulation of insulin-like growth factor (IGF)-binding protein (IGFBP)-2, interleukin (IL)-6, and CCL8/MCP-2 (monocyte chemoattractant protein 2) in supernatants of HIV-infected brain cell cultures. IGF-1 and -2 increased neuronal survival in HIV-infected brain cell cultures, whereas IGFBP-2 inhibited prosurvival effects of these growth factors. These findings identify CD44 as a marker that can be used to sort neurons from other cell types in brain, suggest the importance of microglia-astrocyte interactions in neurodegenerative mechanisms associated with HIV infection, and indicate a role for insulin-like growth factors in neuroprotection from HIV-induced neurodegeneration. The ability to reconstitute brain cultures using isolated populations of neurons, astrocytes, and microglia will be valuable for studies on pathogenic mechanisms in HAD and other neurological disorders, and will also facilitate neuroactive drug discovery.

Cholera Toxin Indirectly Activates Human Monocyte-Derived Dendritic Cells In Vitro through the Production of Soluble Factors, Including Prostaglandin E2and Nitric Oxide

Cholera toxin (CT) is a potent adjuvant that activates dendritic cells (DC) by increasing intracellular cyclic AMP (cAMP) levels. In vivo and in vitro, very small amounts of CT induce potent adjuvant effects and activate DC. We hypothesized that DC intoxicated by CT may release factors that enhance their own maturation and induce the maturation of toxin-free bystander DC. Through the use of mixed cultures and transwell cultures, we found that human monocyte-derived DC (MDDC) pulsed with CT or other cAMP-elevating agonists induce the maturation of bystander DC. Many DC agonists including CT increase the production of prostaglandin E(2) (PGE(2)) and nitric oxide (NO). For this reason, we determined whether the actions of PGE(2) or NO are involved in the maturation of MDDC induced by CT or dibutyryl-cAMP (d-cAMP). We found that blocking the production of PGE(2) or blocking prostaglandin receptors inhibited MDDC maturation induced by CT and d-cAMP. Likewise, sequestering NO or blocking the downstream actions of NO resulted in the inhibition of MDDC maturation induced by CT and d-cAMP. These results indicate that endogenously produced factors including PGE(2) and NO contribute to the maturation of DC induced by CT and that these factors participate in bystander DC maturation. The results of this study may help explain why bacterial toxins that elevate cAMP are such potent adjuvants.

Human mesenchymal stem cells modulate B-cell functions

Human mesenchymal stem cells (hMSCs) suppress T-cell and dendritic-cell function and represent a promising strategy for cell therapy of autoimmune diseases. Nevertheless, no information is currently available on the effects of hMSCs on B cells, which may have a large impact on the clinical use of these cells. hMSCs isolated from the bone marrow and B cells purified from the peripheral blood of healthy donors were cocultured with different B-cell tropic stimuli. B-cell proliferation was inhibited by hMSCs through an arrest in the G0/G1 phase of the cell cycle and not through the induction of apoptosis. A major mechanism of B-cell suppression was hMSC production of soluble factors, as indicated by transwell experiments. hMSCs inhibited B-cell differentiation because IgM, IgG, and IgA production was significantly impaired. CXCR4, CXCR5, and CCR7 B-cell expression, as well as chemotaxis to CXCL12, the CXCR4 ligand, and CXCL13, the CXCR5 ligand, were significantly down-regulated by hMSCs, suggesting that these cells affect chemotactic properties of B cells. B-cell costimulatory molecule expression and cytokine production were unaffected by hMSCs. These results further support the potential therapeutic use of hMSCs in immune-mediated disorders, including those in which B cells play a major role.

Growth kinetics of progenitor cell-enriched hematopoietic cell populations in long-term liquid cultures under continuous removal of mature cells

During long-term culture of primitive hematopoietic cells large numbers of mature cells are generated that, on the one hand, consume nutrients and cytokines present in the medium and, on the other hand, may produce or elicit the production of soluble factors that limit the growth of primitive cells. Thus it is possible that under standard culture conditions hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials. Hematopoietic cell populations, enriched for CD34+ cells, were obtained from both umbilical cord blood (UCB) and mobilized peripheral blood (MPB), and cultured in cytokine-supplemented liquid culture, under continuous removal of mature cells by means of weekly re-selection of primitive, lineage-negative (Lin-) cells. Proliferation and expansion capacities of such cells were determined weekly for a 42-day culture period. As expected, based on our previous studies in standard liquid cultures, throughout the culture period there was a continuous decrease in the proportion of progenitor cells; however, after every re-selection on days 7, 14 and 21, there was a significant enrichment for both CD34+ cells and colony-forming cells (CFC). As a result of such an enrichment, the cumulative increase in the numbers of total cells and CFC in cultures with two, three or four selections was significantly higher than the increments observed in standard cultures, in which only a single selection was performed on day 0. Cultures of UCB cells showed consistently higher levels of both total cells and CFC than cultures of MPB cells. Taken together, these results indicate that continuous removal of mature cells from liquid cultures of primitive progenitors results in higher increments in the levels of both total cells and CFC.