IL-4 Augments IL-31/IL-31 Receptor Alpha Interaction Leading to Enhanced Ccl 17 and Ccl 22 Production in Dendritic Cells: Implications for Atopic Dermatitis

Severe pruritus is a characteristic feature of atopic dermatitis (AD) and is closely related to its activity. Recent studies have shown that IL-31 is a key determinant of pruritus in AD. Anti-IL-31 receptor alpha (IL-31RA) antibody treatment has also been reported to improve pruritus clinically, subsequently contributing to the attenuation of AD disease activity. Therefore, IL-31 has been thought to be an important cytokine for regulating pruritus and AD disease activity; however, how IL-31 is involved in the immune response in AD has remained largely unknown. Epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) derived from bone marrow cells have been reported to play a critical role in AD pathogenesis. LCs and DCs produce Ccl 17 and Ccl 22, which chemoattract Th2 cells, leading to AD development. Therefore, we aimed to clarify how IL-31/IL-31RA interaction affects Ccl 17 and Ccl 22 production. To test this, we analyzed murine bone marrow-derived DCs (BMDCs) stimulated with IL-4, an important cytokine in AD development. We found that IL-31RA expression was upregulated by IL-4 stimulation in a dose-dependent manner in BMDCs. Furthermore, IL-31 upregulates Ccl 17 and Ccl 22 production in the presence of IL-4, whereas IL-31 stimulation alone did not produce Ccl 17 and Ccl 22. These findings suggest that IL-4 mediates IL-31RA expression and IL-31/IL-31RA interaction augments Ccl 17 and Ccl 22 production in BMDCs, which promotes Th2-deviated immune response in AD. Since we previously reported that soybean tar Glyteer, an aryl hydrocarbon receptor (AHR) ligand, impairs IL-4/Stat 6 signaling in BMDCs, we examined whether Glyteer affects IL-31RA expression induced by IL-4 stimulation. Glyteer inhibited upregulation of IL-31RA expression induced by IL-4 stimulation in a dose-dependent manner. Glyteer also inhibited Ccl 17 and Ccl 22 production induced by IL-4 and IL-31 stimulation. Taken together, these findings suggest that Glyteer treatment may improve AD disease activity by impairing IL-31/IL-31RA interaction in DCs.

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Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.6.l1453 ◽

2001 ◽

Vol 281 (6) ◽

pp. L1453-L1463 ◽

Cited By ~ 67

Author(s):

Karen G. Brinker ◽

Emily Martin ◽

Paul Borron ◽

Elahe Mostaghel ◽

Carolyn Doyle ◽

...

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Dendritic Cells ◽

Antigen Presentation ◽

Surfactant Protein ◽

Innate Immune ◽

Surfactant Protein D ◽

Mouse Bone Marrow ◽

Bacterial Antigen ◽

Dependent Manner

Surfactant protein (SP) D functions as a soluble pattern recognition molecule to mediate the clearance of pathogens by phagocytes in the innate immune response. We hypothesize that SP-D may also interact with dendritic cells, the most potent antigen presenting cell, to enhance uptake and presentation of bacterial antigens. Using mouse bone marrow-derived dendritic cells, we show that SP-D binds to immature dendritic cells in a dose-, carbohydrate-, and calcium-dependent manner, whereas SP-D binding to mature dendritic cells is reduced. SP-D also binds to Escherichia coli HB101 and enhances its association with dendritic cells. Additionally, SP-D enhances the antigen presentation of an ovalbumin fusion protein expressed in E. coli HB101 to ovalbumin-specific major histocompatibility complex class II T cell hybridomas. The enhancement of antigen presentation by SP-D is dose dependent and is not shared by other collectin-like proteins tested. These studies demonstrate that SP-D augments antigen presentation by dendritic cells and suggest that innate immune molecules such as SP-D may help initiate an adaptive immune response for the purpose of resolving an infection.

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α-Lipoic acid suppresses osteoclastogenesis despite increasing the receptor activator of nuclear factor κB ligand/osteoprotegerin ratio in human bone marrow stromal cells

Journal of Endocrinology ◽

10.1677/joe.1.05995 ◽

2005 ◽

Vol 185 (3) ◽

pp. 401-413 ◽

Cited By ~ 18

Author(s):

Jung-Min Koh ◽

Young-Sun Lee ◽

Chang-Hyun Byun ◽

Eun-Ju Chang ◽

Hyunsoo Kim ◽

...

Keyword(s):

Bone Marrow ◽

Lipoic Acid ◽

Nuclear Factor Κb ◽

Human Bone ◽

Human Bone Marrow ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Nuclear Factor ◽

Receptor Activator ◽

Dose Dependent

Growing evidence has shown a biochemical link between increased oxidative stress and reduced bone density. Although α-lipoic acid (α-LA) has been shown to act as a thiol antioxidant, its effect on bone cells has not been determined. Using proteomic analysis, we identified six differentially expressed proteins in the conditioned media of α-LA-treated human bone marrow stromal cell line (HS-5). One of these proteins, receptor activator of nuclear factor κB ligand (RANKL), was significantly up-regulated, as confirmed by immunoblotting with anti-RANKL antibody. ELISA showed that α-LA stimulated RANKL production in cellular extracts (membranous RANKL) about 5-fold and in conditioned medium (soluble RANKL) about 23-fold, but had no effect on osteoprotegerin (OPG) secretion. Despite increasing the RANKL/OPG ratio, α-LA showed a dose-dependent suppression of osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts and in a mouse bone marrow cell culture system, and reduced bone resorption in a dose-dependent manner. In addition, α-LA-induced soluble RANKL was not inhibited by matrix metalloprotease inhibitors, indicating that soluble RANKL is produced by α-LA without any posttranslational processing. In contrast, α-LA had no significant effect on the proliferation and differentiation of HS-5 cells. These results suggest that α-LA suppresses osteoclastogenesis by directly inhibiting RANKL–RANK mediated signals, not by mediating cellular RANKL production. In addition, our findings indicate that α-LA-induced soluble RANKL is not produced by shedding of membranous RANKL.

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Polarization of Allogeneic T-Cell Responses by Influenza Virus-Infected Dendritic Cells

Journal of Virology ◽

10.1128/jvi.74.17.7738-7744.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7738-7744 ◽

Cited By ~ 25

Author(s):

Sangkon Oh ◽

Maryna C. Eichelberger

Keyword(s):

Immune Response ◽

T Cells ◽

Dendritic Cells ◽

Influenza Virus ◽

T Cell ◽

Interleukin 2 ◽

Dependent Manner ◽

Ifn Γ ◽

High Doses ◽

Type Response

ABSTRACT The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-γ) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-γ levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.

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Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation

Blood ◽

10.1182/blood.v75.3.596.596 ◽

1990 ◽

Vol 75 (3) ◽

pp. 596-602 ◽

Cited By ~ 112

Author(s):

JR Keller ◽

IK Mcniece ◽

KT Sill ◽

LR Ellingsworth ◽

PJ Quesenberry ◽

...

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Bone Marrow Cells ◽

Dependent Manner ◽

Tgf Beta ◽

Hematopoietic Progenitor ◽

Growth Factor Beta ◽

Dose Dependent

Abstract We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1- negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5- fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.

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Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2012/271606 ◽

2012 ◽

Vol 2012 ◽

pp. 1-15 ◽

Cited By ~ 26

Author(s):

Isabel Andújar ◽

José Luis Ríos ◽

Rosa María Giner ◽

José Miguel Cerdá ◽

María del Carmen Recio

Keyword(s):

Disease Activity ◽

Activity Index ◽

Disease Activity Index ◽

Experimental Colitis ◽

Myeloperoxidase Activity ◽

Dependent Manner ◽

Inflammatory Bowel ◽

Activity Index Score ◽

Bloody Stools ◽

Dose Dependent

The naphthoquinone shikonin, a major component of the root ofLithospermum erythrorhizon, now is studied as an anti-inflammatory agent in the treatment of ulcerative colitis (UC). Acute UC was induced in Balb/C mice by oral administration of 5% dextran sodium sulfate (DSS). The disease activity index was evaluated, and a histologic study was carried out. Orally administered shikonin reduces induced UC in a dose-dependent manner, preventing the shortening of the colorectum and decreasing weight loss by 5% while improving the appearance of feces and preventing bloody stools. The disease activity index score was much lower in shikonin-treated mice than in the colitic group, as well as the myeloperoxidase activity. The expression of cyclooxygenase-2 was reduced by 75%, activation of NF-κB was reduced by 44%, and that of pSTAT-3 by 47%, as well as TNF-α, IL-1β, and IL-6 production. Similar results were obtained in primary macrophages culture. This is the first report of shikonin’s ability to attenuate acute UC induced by DSS. Shikonin acts by blocking the activation of two major targets: NF-κB and STAT-3, and thus constitutes a promising potential therapeutic agent for the management of the inflammatory bowel disease.

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Differential modulation of innate immune response by epinephrine and estradiol

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2016-0046 ◽

2017 ◽

Vol 30 (3) ◽

Cited By ~ 3

Author(s):

Sona Margaryan ◽

Armenuhi Hyusyan ◽

Anush Martirosyan ◽

Shushan Sargsian ◽

Gayane Manukyan

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Inflammatory Responses ◽

Innate Immune ◽

Dependent Manner ◽

Induced Expression ◽

Short Term Exposure ◽

Tnf Α ◽

Vitro Effect ◽

Dose Dependent

AbstractBackgroundAlthough it is widely accepted that catecholamines and estrogens influence immunity and have consequences for health, their effect on innate immunity (e.g. monocytes and neutrophils) is still not fully investigated.Materials and methodsOur study aimed to analyze the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1 and IL-8 by whole blood cells following short-term exposure to epinephrine (Epi) and 17β-estradiol (E2) in the presence or absence of lipopolysaccharide (LPS). We also evaluated the in vitro effect of these hormones on expression of β2 integrin (CD11b/CD18) and L-selectin (CD62L) by circulating neutrophils and monocytes in the blood of healthy subjects.ResultsEpi has shown a potential to modulate the production of pro-inflammatory mediators. Its exposure resulted in significantly increased production of IL-8 in a dose-dependent manner. On the contrary, a dose-dependent suppression of LPS-induced production of IL-1β, IL-8, and MCP-1 by Epi was observed. In neutrophils, a modest rise in CD11b expression was observed after Epi exposure. Simultaneously, Epi suppressed LPS-induced expression of CD11b and CD18. In monocytes, Epi suppressed LPS-induced expression of C11b. E2 inhibited LPS-induced TNF-α production and caused a significant decrease in CD62L expression in both cell populations. No significant changes were observed after double exposure of cells with Epi and E2.ConclusionsThus, our results show that Epi and E2 differentially modulate the innate immune response and have a dual effect on cytokine modulation. The findings suggest that the observed immunoregulatory role of Epi and E2 may influence the outcome in endotoxin responses and can be critical in the regulation of inflammatory responses.

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Pasteurella multocida Toxin Activates Human Monocyte-Derived and Murine Bone Marrow-Derived Dendritic Cells In Vitro but Suppresses Antibody Production In Vivo

Infection and Immunity ◽

10.1128/iai.73.1.413-421.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 413-421 ◽

Cited By ~ 22

Author(s):

Kenneth C. Bagley ◽

Sayed F. Abdelwahab ◽

Robert G. Tuskan ◽

George K. Lewis

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Phospholipase C ◽

Cholera Toxin ◽

Pasteurella Multocida ◽

Human Monocyte ◽

Mouse Bone Marrow ◽

Dependent Manner

ABSTRACT Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts and osteoblastic cells. PMT activates phospholipase C-β through Gqα, and the activation of this pathway is responsible for its mitogenic activity. Here, we investigated the effects of PMT on human monocyte-derived dendritic cells (MDDC) in vitro and show a novel activity for PMT. In this regard, PMT activates MDDC to mature in a dose-dependent manner through the activation of phospholipase C and subsequent mobilization of calcium. This activation was accompanied by enhanced stimulation of naïve alloreactive T cells and dominant inhibition of interleukin-12 production in the presence of saturating concentrations of lipopolysaccharide. Surprisingly, although PMT mimics the activating effects of cholera toxin on human MDDC and mouse bone marrow-derived dendritic cells, we found that PMT is not a mucosal adjuvant and that it suppresses the adjuvant effects of cholera toxin in mice. Together, these results indicate discordant effects for PMT in vitro compared to those in vivo.

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Vibrio parahaemolyticus Flagellin Stimulates the Maturation of Human Monocyte-Derived Dendritic Cells and Elicits Th1-Type Immune Response.

Blood ◽

10.1182/blood.v106.11.3872.3872 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3872-3872

Author(s):

Hyun-Kyu Kang ◽

Myong-Suk Park ◽

Shee-Eun Lee ◽

Joon-Haeng Rhee ◽

Jung-Sun Park ◽

...

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Vibrio Parahaemolyticus ◽

Human Monocyte ◽

Phenotypic Change ◽

Target Cells ◽

Maximal Effect ◽

Dependent Manner ◽

Functional Maturation

Abstract Flagellin, the principal component of bacterial flagella, interacts with Toll-like receptor (TLR5) and induces the generation of a pro-inflammation response and activation of host dendritic cells (DCs) in vivo. In this study, we investigated the role of Vibrio parahaemolyticus (V. parahaemolyticus)-derived flagellin as a DC maturation-inducing molecule. V. parahemolyticus-derived flagellin (100–1,000 ng/ml) induced the maturation of human monocyte-derived dendritic cells in a concentration-dependent manner with maximal effect at 500 ng/ml of flagellin as determined by increased levels of surface markers, namely, CD1a, CD80, CD86, CD83, and HLA-DR, a response which could be compared with the phenotypic change in immature DCs (iDCs) treated with lipopolysaccharide (LPS) or cytokine cocktails (CC) with TNF-α, IL-1β, IL-6, and PGE2. Moreover, V. parahaemolyticus-derived flagellin also reduced phagocytic activity, and increased IL-12 production in a polymyxin B-insensitive manner and DC-mediated T cell proliferation, which is comparable with that of LPS- or CC-treated iDCs at several responder to stimulator ratios, suggesting the functional maturation of DCs by V. parahaemolyticus-derived flagellin. Maturation of DCs by V. parahaemolyticus-derived flagellin also elicited a significant increase in specific cytotoxic activity against target cells at several effector to target cells ratios as determined by 51Cr-release assay, and induced Th1-type immune response, such as increase in INF-γ producing cells, determined by ELISPOT assay and analysis of intracellular cytokine staining assay. Taken together, this study demonstrates the role of V. parahaemolyticus-derived flagellin in the functional maturation of DCs, and suggests that V. parahaemolyticus-derived flagellin as a useful molecule for the development of a DC-based immunotherapy against tumors.

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The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1844.1844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1844-1844 ◽

Cited By ~ 1

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Michelle Kuhne ◽

AbdelKareem Azab ◽

Patricia Maiso ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Mouse Models ◽

Research Funding ◽

Therapeutic Agents ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

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Monocytes Modulate Megakaryocyte-Mediated Fibrosis of Bone Marrow Stromal Cells in Vitro

Blood ◽

10.1182/blood.v120.21.1759.1759 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1759-1759

Author(s):

Emil Tom Kuriakose ◽

Jason Shieh ◽

Jae Hung Shieh ◽

Richard T. Silver ◽

Malcolm A.S. Moore

Keyword(s):

Bone Marrow ◽

Tissue Culture ◽

Positive Selection ◽

Peripheral Blood ◽

Normal Adult ◽

Dependent Manner ◽

Marrow Stroma ◽

Dose Dependent ◽

Selection Of

Abstract Abstract 1759 Myelofibrosis (MF) is a terminal feature of the chronic myeloproliferative neoplasms (MPNs), primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET).We and others have shown, using both in vitro and in vivo models, that proliferation of megakaryocytes (MK) and their pathologic interaction with marrow stroma plays a central role in MF. However, the marrows of patients with MPNs remain free of fibrosis for a substantial part of their clinical course, despite increased MK proliferation and turnover in the marrow, suggesting that additional factors may modulate the fibrotic effects of the MK on marrow stroma. Since monocytosis is often seen in patients with MF, we examined whether monocytes may play such a role in MF. Human hematopoietic stem cells (HSC), MK progenitors, and circulating monocytes were obtained from peripheral blood of 13 patients with MF (3 post PV MF, 10 PMF), G-CSF mobilized peripheral blood from normal adults (MPB), and cord blood (CB) using MACS column separation by positive selection of cells expressing CD34, CD41, and CD14 respectively. HSCs were cultured in serum free medium (SFM) on the murine bone marrow stromal cell line OP9 transduced with an adenoviral vector expressing the human thrombopoietin gene (OP9-adenoTPO). After 10–12 days in culture, mature MKs were harvested using MACS column by positive selection of cells expressing human CD41. Purity of cell fractions was more than 90% by flow cytometry. Isolated MKs and monocytes were seeded with trypsinized OP9 in SFM at various ratios on 96 well or 384 well tissue culture treated plates and incubated at 37° C. MKs formed focal aggregates on adherent OP9 cells within 24 hours, which by 48 hours, became round dark fibrotic nodules when seen using phase contrast microscopy. Formation of these focal fibrosis (FF) areas was more pronounced with higher MK:OP9 ratios, and was equally induced by MKs from MF patients, normal adult MKs, and CB MKs. FF was not observed with CD41 negative cells, nor in control OP9 wells. Time lapse photography revealed that FF formation involved migration of both MKs and OP9 cells, and that FF was enhanced by inhibition of CXCR4 using AMD3100. Peripheral blood monocytes from normal adult controls and CB did not induce formation of FF. Circulating monocytes from most MF patients induced FF, but to a lesser degree than MKs. Addition of monocytes to MK-OP9 FF showed that normal adult monoctyes inhibited FF formation in a dose dependent manner, whereas monocytes of MF patients had variable effects, with some inhibiting FF, and others not. To determine whether differential conditioning of monocytes can induce variable stromal changes, normal adult circulating monocytes were cultured in SFM with TGF- ß1, interferon alfa (IFNα), and TNFα in tissue culture flasks. Monocytes cultured in TNFα (MoTNF) became adherent and spindle shaped within 72 hours. Conditioned medium (CM) from MoTNF suppressed OP9 differentiation into adipocytes in a dose dependent manner. CM from monocytes cultured in IFNα (MoIFN) enhanced OP9 differentiation into adipocytes in a dose dependent manner. MoTGF caused proliferation of OP9 and suppressed adipocyte differentiation, but was not significantly different from control with TGFβ alone. CM from MoIFN decreased FF formation by MKs on OP9 and increased adipocyte number, but IFNα by itself had no such effect on FF formation. Both CM from MoTNF and TNFα increased FF formation by MKs in a dose dependent manner. Together, these results demonstrate that monocytes can enhance or hinder MK induced fibrosis depending on their conditioning by specific cytokines, with IFNα hindering and TNFα enhancing the fibrotic effect. Our data suggest that the known anti-megakaryocytic and anti-fibrotic activities of IFNα may be due to its conditioning of monocytes into an anti-fibrotic phenotype. Disclosures: No relevant conflicts of interest to declare.

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