Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3596-3596
Author(s):  
Lilach Lifshitz ◽  
Galit Tabak ◽  
Max Gassman ◽  
Moshe Mittelman ◽  
Drorit Neumann
Keyword(s):  
Mhc Class Ii ◽  
Phagocytic Activity ◽  
Surface Expression ◽  
Class Ii ◽  
Experimental Models ◽  
Splenic Macrophage ◽  
And Function ◽  
Inflammatory Macrophages

Abstract Abstract 3596 Poster Board III-533 The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response. Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREXR, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts. These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion. In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator. Disclosures: Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).

Dendritic Cells as a Novel Target for Erythropoietin: Studies in Murine Models.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2417-2417
Author(s):  
Lilach Lifchitz ◽  
Sari Prutchi Sagiv ◽  
Maayan Markovitz ◽  
Moshe Mittelman ◽  
Drorit Newmann
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Mhc Class Ii ◽  
Surface Expression ◽  
Class Ii ◽  
Mapk Signaling ◽  
Target Cells ◽  
Erythroid Progenitor

Abstract Erythropoietin (EPO) is the major hormone that promotes the proliferation and differentiation of erythroid progenitor cells. Unexpectedly, EPO receptor (EPO-R) was also found on non-erythroid cells; thus leading to the discovery of non-erythroid effects of EPO. Our own previous contribution to that issue was in demonstrating that the immune system is a target for EPO, including both the cellular and humoral immune response types. As yet, the direct target cells for EPO as well as the molecular mechanisms underlying its function as an immunomodulator remain unknown. We first examined lymphocytes as possible candidates, and could not detect any expression of EPO-Rs on these cells. Here, we focused on dendritic cells (DCs), known to initiate immune response as antigen presenting and T cell priming cells. We employed murine bone marrow DCs (BMDCs) and splenic DCs (SDCs) models to determine EPO-R expression, and delineate in-vitro and in-vivo effects of EPO via these cells. We found that BMDCs express EPO-R mRNA, as detected by RT-PCR. In vitro stimulation of the BMDCs with recombinant human EPO (rHuEPO) activated the NFkB and MAPK signaling pathways, and induced a higher surface expression of CD80, CD86 and MHC class II. These data are reinforced by in vivo experiments, showing that rHuEPO injection into naïve mice led to an increase in the SDC population and in the cell surface expression of CD80, CD86 and MHC class II markers. These novel findings implicate the significance of the multifunctional role of EPO in the hematopoietic and immune systems, and may lead to its further clinical applications as an immunomodulator.

Absence of STAT1 Signaling in Host Hematopoietic Cells Leads to Enhanced Gvhd Induction and Involves Increased Expression of MHC Class II and Reduced PD-L1 Expression on Dendritic Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4026-4026
Author(s):  
Caisheng Lu ◽  
Huihui Ma ◽  
Ailing Liu ◽  
MeiHua Jin ◽  
Shirong Li ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Cell Activation ◽  
Hematopoietic Cells ◽  
Class Ii ◽  
Cfse Dilution ◽  
Stat1 Signaling ◽  
Donor Lymphocytes

Abstract Abstract 4026 Interferon-g/STAT1 signaling plays a critical role in regulating dendritic cell activation and function. Blockade of IFN-g signaling leads to reduced DC activation and impaired anti-tumor and acquired adaptive immunity. We recently reported that lack of IFN-g/STAT1 in donor lymphocytes leads to reduced GVHD induction in both MHC- and mHA-mismatched mouse BMT models. In this study, we addressed the role of host STAT1 in the regulation of GVHD. Wildtype or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad). GVHD was induced using either BALB/c or B6 donor spleen cells. We unexpectedly observed that absence of STAT1 in recipient mice led to increased GVHD-associated mortality in both MHC-mismatched (MST 5 vs. 8, p=0.01) and mHA-mismatched (MST 11 vs. 23, p<0.01) BMT settings. The enhanced GVHD induction was found to be associated with increased activation (expression of CD69 and CD25) and allo-antigen driven proliferation of donor CD4 and CD8 T cells as determined by CFSE-dilution. As host APCs have been reported to being crucial for induction of GVHD, we phenotypically and functionally characterized STAT1 deficient DCs. Our studies revealed that STAT1-deficient bone marrow-derived dendritic cells (BMDCs) which were maturated in the presence of LPS showed significantly increased MHC class II, CD86, CD80 and CD40 expression compared with wildtype BMDCs. Furthermore, STAT1-deficient BMDC showed significantly increased direct allo-stimulatory capacity resulting in increased responder cell proliferation as determined by standard MLR assays using 3H-Thymidine uptake assays as well as CFSE-dilution studies. STAT1−/− BMDCs significantly promoted CD44+CD62L- expression in responder CD4 and CD8 T cells compared to wild type BMDCs (all p<0.001). The increased MHC II expression in STAT1-deficient DC was further confirmed in host CD11b+ and CD11c+ cells following GVHD induction in vivo. To determine whether non-hematopoietic cells in STAT1−/− host contribute to the increased GVHD induction, we created radiation chimeras in which STAT1 was only deficient in the hematopoietic compartment by transplanting 129.STAT1−/− BMC into 129.STAT1+/+ recipients following lethal irradiation. 120 days later GVHD was induced using fully MHC-mismatched BALB/c donor splenocytes. Similar to STAT1-deficient recipients STAT1−/− ®WT chimeras showed enhanced GVHD induction compared to STAT1+/+®WT chimeras (MST 11 vs. 5, p<0.05). To determine the mechanism underlying the enhanced expansion of donor T cells in response to stimulation with STAT1-deficient APC, we hypothesized that STAT-deficiency may impair expression of the T cell inhibitory molecules Programed Cell Death-Ligand1 or-2 (PD-L1,-L2) on APC. We therefore studied the expression of PD-L1 and PD-L2 expression on wildtype and STAT1-deficient DC. Indeed, were able to demonstrate that absence of STAT1 significantly suppressed PD-L1 expression on BMDCs upon in vitro LPS stimulation (Mean Fluorescence Intensity 167.2± 15.9 vs. 532.5±7.6, p<0.001) and also in vivo tested on day+ 6 post-BMT in the mHA-mismatched setting. In line with these results using in vitro stimulation we could demonstrate significantly reduced Activation Induced Cell Death (AICD) in activated B6.SJL CD69+ CD4 and CD8 cells stimulated with 129.STAT1−/− BMDCs compared to cells stimulated with 129.STAT1+/+ BMDCs (10.6±1.5% vs. 28.2±1.9 % for CD4; 13.0±0.7% vs. 30.5±1.1% for CD8 respectively, p<0.001 for all). Importantly, blocking IFN-g with neutralizing antibodies significantly increased MHC class II, CD86 expression and reduced reduced PD-L1 expression on BMDCs upon LPS stimulation. In summary, our data suggest two mechanisms how the absence of STAT1 signaling in host hematopoietic cells may promote the development of GVHD: First, increased expression of MHC II and co-stimulatory molecule in STAT1-deficient APC may lead to enhanced activation and proliferation of donor lymphocytes. Second, absence of STAT1 in maturated host DC inhibits PD-L1 expression thus leading to reduced AICD of activated donor lymphocytes. These findings suggest that STAT1-signaling modulates host APC function and shapes the GVH-response by causing increased allo-antigen-specific donor T cell activation, survival and proliferation. Disclosures: Lentzsch: Centocor Ortho Biotech: Research Funding; Genzyme: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Research Funding.

scholarly journals Paracrine Transfer of Mouse Mammary Tumor Virus Superantigen

1997 ◽  
Vol 185 (3) ◽  
pp. 471-480 ◽  
Author(s):  
Marc Delcourt ◽  
Jacques Thibodeau ◽  
Francois Denis ◽  
Rafick-Pierre Sekaly
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Cell Contact ◽  
Molecular Evidence ◽  
In Vitro System ◽  
Hla Dr ◽  
Mouse Mammary Tumor ◽  
Tumor Virus

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II− to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive Vβs. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II− murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific Vβs led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II–specific mAbs. Moreover, injection of vSAG7+ class II− cells in mice led to expansion of Vβ6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II− and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the α1 domain of HLA-DR.

scholarly journals ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling

Blood ◽  
2010 ◽  
Vol 116 (17) ◽  
pp. 3208-3218 ◽  
Author(s):  
Daniel B. Graham ◽  
Holly M. Akilesh ◽  
Grzegorz B. Gmyrek ◽  
Laura Piccio ◽  
Susan Gilfillan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Mhc Class Ii ◽  
Autoimmune Encephalitis ◽  
Class Ii ◽  
T Helper Cell ◽  
Helper Cell ◽  
Nucleotide Exchange ◽  
T Helper

Abstract Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

scholarly journals Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

2021 ◽  
Vol 8 ◽  
Author(s):  
Jamal Hussen
Keyword(s):  
Heat Stress ◽  
Adhesion Molecules ◽  
Mhc Class Ii ◽  
Whole Blood ◽  
Dromedary Camel ◽  
Macrophage Phenotype ◽  
Cell Vitality ◽  
And Function

The dromedary camel (Camelus dromedarius) is well-adapted to the desert environment with the ability to tolerate increased internal body temperatures rising daily to 41–42°C during extreme hot. This study was undertaken to assess whether in vitro incubation of camel blood at 41°C, simulating conditions of heat stress, differently alters cell vitality, phenotype, and function of leukocytes, compared to incubation at 37°C (normothermia). Using flow cytometry, the cell vitality (necrosis and apoptosis), the expression of several cell markers and adhesion molecules, and the antimicrobial functions of camel leukocytes were analyzed in vitro. The fraction of apoptotic cells within the granulocytes, lymphocytes, and monocytes increased significantly after incubation of camel whole blood at 41°C for 4 h. The higher increase in apoptotic granulocytes and monocytes compared to lymphocytes suggests higher resistance of camel lymphocytes to heat stress. Functionally, incubation of camel blood at 41°C for 4 h enhanced the phagocytosis and ROS production activities of camel neutrophils and monocytes toward S. aureus. Monocytes from camel blood incubated at 41°C for 4 h significantly decreased their expression level of MHC class II molecules with no change in the abundance of CD163, resulting in a CD163high MHC-IIlow M2-like macrophage phenotype. In addition, heat stress treatment showed an inhibitory effect on the LPS-induced changes in camel monocytes phenotype. Furthermore, in vitro incubation of camel blood at 41°C reduced the expression of the cell adhesion molecules CD18 and CD11a on neutrophils and monocytes. Collectively, the present study identified some heat-stress-induced phenotypic and functional alterations in camel blood leukocytes, providing a paradigm for comparative immunology in the large animals. The clinical relevance of the observed changes in camel leukocytes for the adaptation of the camel immune response to heat stress conditions needs further in vitro and in vivo studies.

scholarly journals Differentiation and function of human monocyte-derived dendritic cells under the influence of leflunomide

2011 ◽  
Vol 63 (2) ◽  
pp. 353-364
Author(s):  
Z. Stojic-Vukanic ◽  
M. Colic ◽  
A. Backovic ◽  
J. Antic-Stankovic ◽  
B. Bufan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Flow Cytometric Analysis ◽  
Human Monocyte ◽  
Surface Expression ◽  
Immunosuppressive Drugs ◽  
Experimental Models ◽  
Immunosuppressive Drug ◽  
Adhesive Molecules ◽  
And Function

Leflunomide is an immunosuppressive drug effective in experimental models of transplantation and autoimmune diseases and in the treatment of active rheumatoid arthritis (RA). Having in mind that it has been shown that some other immunosuppressive drugs (glucocorticoids, mycophenolate mofetil, sirolimus etc.) impair dendritic cell (DC) phenotype and function, we investigated the effect of A77 1726, an active metabolite of leflunomide, on the differentiation and function of human monocyte-derived dendritic cells (MDDC) in vitro. Immature MDDC were generated by cultivating monocytes in medium supplemented with GM-CSF and IL-4. To induce maturation, immature MDDC were cultured for 2 additional days with LPS. A77 1726 (100 ?M) was added at the beginning of cultivation. Flow cytometric analysis showed that MDDC differentiated in the presence of A77 1726 exhibited an altered phenotype, with a down-regulated surface expression of CD80, CD86, CD54 and CD40 molecules. Furthermore, the continuous presence of A77 1726 during differentiation and maturation prevented successful maturation, judging by the decreased expression of maturation marker CD83, costimulatory and adhesive molecules on A77 1726-treated mature MDDC. In addition, A77 1726-pretreated MDDC exhibited a poor stimulatory capacity of the allogeneic T cells and a low production of IL-10 and IL-18. These data suggest that leflunomide impairs the differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

scholarly journals Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH

Open Biology ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 190235 ◽  
Author(s):  
Stephanie J. Crowley ◽  
Patrick T. Bruck ◽  
Md Aladdin Bhuiyan ◽  
Amelia Mitchell-Gears ◽  
Michael J. Walsh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Class Ii ◽  
Tumour Immunity ◽  
Tumour Rejection

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

scholarly journals HLA-DM Interactions with Intermediates in HLA-DR Maturation and a Role for HLA-DM in Stabilizing Empty HLA-DR Molecules

1996 ◽  
Vol 184 (6) ◽  
pp. 2153-2166 ◽  
Author(s):  
Lisa K. Denzin ◽  
Craig Hammond ◽  
Peter Cresswell
Keyword(s):  
Mhc Class Ii ◽  
Antigen Processing ◽  
Class Ii ◽  
Physical Interaction ◽  
Antigenic Peptides ◽  
Histocompatibility Complex ◽  
Chain Complexes ◽  
Hla Dr

Major histocompatibility complex (MHC) class II–positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments (class II–associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II–CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II molecules entered the MHC class II compartment. HLA-DR αβ dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species, but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR αβCLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free αβ dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules may be an important property of HLA-DM in facilitating antigen processing.

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