TNF-α plus IFN-γ induce expression of slaloadhesin receptor by stromal bone marrow macrophages (Mφ)

Recent research has found that Rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved satisfactory effect in clinical application. At present, many studies have confirmed that Eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then whether Rapa combined with Elt in the treatment of AA will be better than single drug application. In this study, we tested efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. Compared with AA group, the whole blood cell count of Rapa+Elt group increased significantly (Figure 1A) (P<0.05). Survival of mice of Rapa+Elt group was significantly higher than that in the Rapa group (p <0.01)(Figure 1B).There was no obvious difference in the numbers of NK cells and their subsets were noted in Rapa group,CsA group and Rapa+Elt group.The expression of NKG2D on peripheral functional NK cells was up-regulated in CsA group, Rapa group and Rapa+Elt group compared with AA group (P<0.05). But there was no significant difference between effect of Rapa and CsA on the function of NK cells (Figure 1C).When Rapa combined with Elt, the expression of CD80 and CD86 are down-regulated more compared to Rapa group, but there is no statistical significance. Although these results suggested that Rapa+Elt had no statistical significance effect on numbers of mDC and expression of its functional molecule CD80 and CD86, the combined therapy still indicated that there is a potential synergy with immunosuppressant on AA mice to improve its outcome (Figure 1D).The results showed that CD4+/CD8+ ratio in CsA group, Rapa group, Rapa + Elt group had an obvious elevation than AA group (all P<0.05). But there were no significant difference among the three groups on the CD4+/CD8+ ratio (Figure 1E,1F). As for INF-gamma, Rapa can reduce the secretion of IFN-γ from CD8+T cells with efficacy similar to that of the standard dose of CsA, and had a better outcome when combined with Elt in bone marrow failure mice (Figure 1E,1G).CsA group, Elt group, Rapa group, Rapa + Elt group showed notable increased ratio of Tregs compared with AA group, among which there were only Rapa group, Rapa + Elt group showed statistical significance(P<0.05). for IL-10/Tregs ratio, Rapa group and Rapa +Elt group were superior to than CsA group(P<0.05) (Figure 1H,1I).Rapa+Elt group and Rapa showed more lower level of IFN-γ compared with CsA group, and there was significant difference in Rapa+Elt group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa or Elt alone(Figure1J). Thus, Rapa+Elt significantly down-regulated cytokines related to Th1 immune responses, such as IFN-γ, and upregulated cytokines related to Th2 immune responses, such as IL-10. To some extent, Rapa combined with Elt has a synergistic effect with CsA and Rapa alone in AA treatment. Conclusions In this study, Although Rapa combined with Elt had no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice compared with Rapa alone, the Rapa+Elt can increase the secretion of IL-10 of Tregs and the number of Tregs, but has no significant effect on the number of Treg cells compared to with Rapa alone. Compared with AA group, the level of plasma IFN-γ, IL-2 and TNF-α decreased significantly (P<0.05), but IL-10, IL-4, IL-5 and IL-1β increased significantly in Rapa group(P<0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa alone. intervention treatment with Rapa in combination Elt in the AA mouse model more obviously ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. Combination therapy support potential clinical utility in aplastic anemia treatment, which may further improve the efficacy of AA patients. Keywords: Rapamycin, Eltrombopag, murine models, bone marrow failure Figure 1 Disclosures No relevant conflicts of interest to declare.

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Effects of bone marrow-derived mesenchymal stromal cells on gene expression in human alveolar type II cells exposed to TNF-α , IL-1β , and IFN-γ

Physiological Reports ◽

10.14814/phy2.13831 ◽

2018 ◽

Vol 6 (16) ◽

pp. e13831 ◽

Cited By ~ 2

Author(s):

Matthew Schwede ◽

Erin M. Wilfong ◽

Rachel L. Zemans ◽

Patty J. Lee ◽

Claudia dos Santos ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Tnf Α ◽

Ifn Γ

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Novel P38 MAP Kinase Inhibitor and Anti-P38 RNA Interference as Potential Therapeutic Approaches in Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v104.11.470.470 ◽

2004 ◽

Vol 104 (11) ◽

pp. 470-470

Author(s):

Mani Mohindru ◽

Perry Pahanish ◽

Efstratios Katsoulidis ◽

Robert Collins ◽

Thomas Rogers ◽

...

Keyword(s):

Bone Marrow ◽

Map Kinase ◽

P38 Mapk ◽

Colony Formation ◽

P38 Map Kinase ◽

Hematopoietic Progenitors ◽

P38 Inhibitor ◽

Tnf Α ◽

Ifn Γ

Abstract Cytokines such as TNF α, IFN γ and others have been implicated in the pathogenesis of ineffective hematopoiesis in MDS and are thought to lead to the high rate of apoptosis in hematopoietic progenitors. The p38 Mitogen Activated Protein Kinase (MAPK) is an evolutionary conserved enzyme that is involved in many cellular processes including stress signaling. We have previously shown that the p38 MAP kinase is strongly activated by IFNs, TNF α, TGF β and other inhibitory cytokines in normal primary hematopoietic progenitors and plays an important role in the negative regulation of normal hematopoiesis. In the present study, we determined the role of the p38 MAPK in the pathogenesis of MDS evaluated its inhibition as a potential therapeutic strategy in this disease. p38 MAPK inhibition was achieved by the use of a novel p38 inhibitor - SD-282, a specific inhibitor of p38α MAP kinase. SD-282 performs very similarly in animal and cell models to a p38 inhibitor now in the clinic. We also transfected primary hematopoietic cells with flurescent labeled siRNAs against p38 and successfully downregulated the levels of the protein. Using these approaches, we demonstrate that pharmacological inhibition of the p38 MAPK can reverse the growth inhibitory effects of TNF α and IFN γ on erythroid and myeloid colony formation. This reversal of TNF α mediated inhibition correlates with significant reduction of apoptosis seen in human hematopoeitic progenitors pretreated with p38 inhibitor SD-282. Having established the importance of p38 MAPK in cytokine mediated inhibition of normal hematopoiesis, we performed colony forming assays with bone marrow CD34+ cells from 8 patients with MDS in the presence of either pharmacologic or siRNA based inhibitors of p38. All patients had refractory cytopenias with multilineage dysplasia. Our data indicates that SD-282 treatment strongly enhances both erythroid and myeloid colony formation in MDS CD34+ bone marrow cells in vitro. This increase was not observed when these progenitors were grown in the presence of negative controls - SB 202474 and the MEK inhibitor PD 98059. Similarly, an increase in hematopoietic colony formation, though of a lesser magnitude was seen when MDS bone marrow progenitors were transfected with siRNAs against p38 MAPK. To further determine the role of cytokines in the pathogenesis of MDS, we also used bone marrow derived sera from the same MDS patients. Our studies show exposure to patient derived sera led to the phosphorylation/activation of p38 MAPK in normal hematopoietic progenitors when compared to sera from healthy volunteers. Our studies also demonstrate that bone marrow derived sera from MDS patients can inhibit erythroid and myeloid colony formation of normal hematopoietic progenitors. This inhibition can be reversed by blocking p38 MAPK using SD-282, other p38 inhibitors and siRNAs. This finding confirms the role of marrow cytokine /serum factors in the ineffective hematopoiesis seen in MDS and suggests the importance of p38 MAPK activation in this phenomenon. Thus our studies show the p38 MAPK may be a common effector of inhibitory cytokine signaling in normal and MDS hematopoietic cells. These results provide a strong rationale for using p38 inhibition as a novel treatment strategy for MDS. Supported by Harris Methodist Foundation Grant, VISN-17 New Investigator Grant and VA Research Corp Grant to AV.

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Prophylactic Amotosalen-Treated Donor T-Cells Prevent Late CMV Infection in Allogeneic Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v106.11.3024.3024 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3024-3024

Author(s):

Mohammad S. Hossain ◽

John D. Robak ◽

Edmund K. Waller

Keyword(s):

Bone Marrow ◽

T Cells ◽

Viral Infection ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Chronic Gvhd ◽

Mcmv Infection ◽

Tnf Α ◽

Donor T Cells ◽

Ifn Γ

Abstract A major problem in allogeneic BMT is post transplant immunodeficiency leading to opportunistic infection and relapse. Previously we showed that amotosalen-treated allogeneic donor T cells given at the time of BMT and experimental murine cytomegalovirus (MCMV) infection could prevent lethal MCMV disease without producing GvHD. In this study we have focused on a more clinically applicable model where prophylactic amotosalen-treated allogeneic donor splenocytes are given at the time of BMT, followed by MCMV infection 100 days later. We observed that amotosalen-treated donor T-cells significantly expanded and responded well in presence of viral infection without inducing any GvHD, protected recipients against viral disease, and were associated with significantly improved hematopoietic engraftment and immune reconstitution. Methods: Using a parent to F1 mouse BMT model, splenocytes (3x106 untreated or 10x106 amotosalen-treated) from MCMV immunized C57BL/6 donors were transplanted along with 5x106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C). Recipient mice were infected i.p. with a sublethal dose (5x104 pfu per mouse) of MCMV 100 days or more after transplant. Clinical chronic GvHD was monitored by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. Flow cytometry was used to quantitate T cell chimerism (in recipient PBMC, spleen, liver and thymus) and MCMV-peptide specific CD8+ T-cells (tetramer+ and IFN-γ producing). Serum IFN-γ and TNF-α were determined by ELISA. Liver and spleen viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: Recipients of untreated control donor splenocytes suffered from chronic GvHD within 100 days of transplant, while those that received amotosalen-treated splenocytes experienced no GvHD. In response to MCMV infection at 100 days post transplant, residual amotosalen-treated donor T-cells rapidly expanded over 25-fold within 10 days, but did not cause lethality or detectable GvHD. Expanded amotosalen-treated T-cells showed activated anti-viral responses and developed a memory phenotype at late phases of viral infection. PBMC, spleen and liver showed elevated levels of MCMV specific tetramer+, IFN-γ+, and TNF-α+ CD8+ T-cells that were associated with accelerated viral clearance within day 3 after viral infection. While expansion and generation of amotosalen-treated donor T-cells mostly occurred in the liver, the generation of donor bone marrow-derived new T-cells occurred through both the thymus and the liver. In contrast, recipients of untreated donor splenocytes had reduced thymic function, resulting in severely impaired immune reconstitution and decreased anti-viral immunity. Conclusion: Prophylactically administered amotosalen-treated allogeneic donor T cells 1) were almost completely devoid of GvHD activity, 2) promoted hematopoietic engraftment and improved immune reconstitution, and 3) persisted long-term (>100 days) and successfully protected recipients from sublethal MCMV infection. Thus, infusion of amotosalen-treated donor T-cells at the time of transplantation is a clinically-attractive approach to adoptive anti-viral immunotherapy without chronic GvHD following hematopoietic progenitor cell transplantation.

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Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽

10.1182/blood.v110.11.4099.4099 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4099-4099

Author(s):

Zhenhua Qiao ◽

Xiujuan Zhao

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Aplastic Anemia ◽

Hematopoietic Cell ◽

Control Group ◽

Rt Pcr ◽

Tnf Α ◽

Ifn Γ ◽

Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

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Multiple Myeloma Cell-Derived Interleukin-32gamma Increases the Immunosuppressive Function of Macrophages By Promoting Indoleamine 2,3-Dioxygenase (IDO) Expression

Blood ◽

10.1182/blood-2018-99-114316 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3179-3179

Author(s):

Haimeng Yan ◽

Donghua He ◽

Xi Huang ◽

Zhang En Fan ◽

He Huang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

T Cell ◽

Rna Sequencing ◽

Cd4 T Cell ◽

T Cell Proliferation ◽

Indoleamine 2,3 Dioxygenase ◽

Tnf Α ◽

Ifn Γ

Abstract Background: The interaction of multiple myeloma (MM) cells with macrophages (MΦs) in the bone marrow microenvironment contributes to the pathophysiology of MM. In addition to promoting angiogenesis through vasculogenic mimicry, MM-associated MΦs (mMΦs) protect MM cells from spontaneous and chemotherapy-induced apoptosis. mMΦs therefore represent a potential target for myeloma treatment and it is essential to explore the mechanisms underlying normal MΦ polarization to mMΦs. We previously showed that IL-32 is overexpressed in MM patients and is mainly derived from MM cells. The present study was designed to explore the clinical significance of IL-32 in MM and to further elucidate the molecular mechanisms underlying the IL-32-mediated immune function of MΦs. Methods: We examined the expression of IL-32 in bone marrow biopsy samples using immunohistochemistry. Quantitative real-time PCR, western blot analysis and immunofluorescence were applied to measure the expression of IL-32, IDO and proteinase 3 (PR3). We obtained the global transcriptional profile of the IL-32γ-treated MΦs by RNA sequencing (RNA-Seq). Immunoprecipitation (IP) and GST pulldown experiments was applied to confirm the binding affinity of PR3 for IL-32. We created IL-32-knockdown MM cells by transfection of IL-32 shRNA and silenced PR3 expression in MΦs using siRNA targeting PR3. CD4+ T cell proliferation and IL-2, IFN-γ and TNF-α production were measured by flow cytometry. Results: We found that high IL-32 expression in MM patients was associated with advanced clinical stage and high serum β2-microglobulin levels. Several isoforms of IL-32 were detected in MM cells and IL-32γ was the most active subtype. RNA sequencing revealed that IL-32γ significantly induced the production of the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO) in MΦs and this effect was verified at the protein level. Furthermore, IL-32-knockdown MM cells showed less ability than control MM cells to promote IDO expression. As a binding protein for IL-32, PR3 was universally expressed on the surface of MΦs and knockdown of PR3 or inhibition of the STAT3 and nuclear factor κB (NF-κB) pathways hindered the IL-32γ-mediated stimulation of IDO expression. Finally, IDO-positive IL-32γ-educated MΦs inhibited CD4+ T cell proliferation and IL-2, IFN-γ and TNF-α production in response to activation. Conclusion: Our study showed that MM cell-derived IL-32γ induced IDO production in MΦs through PR3 and the downstream STAT3 and NF-κB pathways, resulting in the suppression of the proliferation and effector function of CD4+ T cells. High IL-32 expression in MM may contribute to an immunosuppressive microenvironment by upregulating IDO production in MΦs and promote MM progression. Disclosures No relevant conflicts of interest to declare.

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Anti-IFN-γ Antibody Promotes Osteoclastogenesis in Human Bone Marrow Monocyte-Derived Macrophages Co-Cultured with Tuberculosis-Activated Th1 Cells

Cellular Physiology and Biochemistry ◽

10.1159/000493455 ◽

2018 ◽

Vol 49 (4) ◽

pp. 1512-1522

Author(s):

Jiezhong Deng ◽

Dong Sun ◽

Fei Luo ◽

Qiang Zhang ◽

Feifan Chen ◽

...

Keyword(s):

Bone Marrow ◽

Bone Resorption ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Th1 Cells ◽

Actin Ring ◽

Tnf Α ◽

Ifn Γ ◽

Trap Staining

Background/Aims: Tuberculosis induces bone loss and activates Th1 cells that play an important role in the host defense of Bacille Calmette-Guérin tuberculosis vaccine. However, the role of tuberculosis-activated Th1 cells in differentiation of osteoclast precursors to osteoclasts is unclear. As secretion of IFN-γ in Th1 cells is induced by tuberculosis, we aimed to investigate the role of anti-IFN-γ antibody on the differentiation and activation of osteoclasts in bone marrow monocyte-derived macrophages (BMMs). Methods: BMMs were isolated and co-cultured with CD4+T helper 1 cells (Th1 cells), pretreated with anti-IFN-γ antibody. Then, cell proliferation, expression and release of cytokines, formation of actin ring, differentiation of osteoclasts and bone resorption function were measured by CCK8 assay, qRT-PCR/Western blot/flow cytometry, ELISA, immunofluorescence, tartrate-resistant acidic phosphatase (TRAP) staining and bone absorbance assay, respectively. Results: Anti-IFN-γ antibody inhibited the cell viability of BMMs, and induced the expressions of RANKL, TNF-α, NF-κB and TRAF6 in BMMs. In addition, it led to increased expression levels of RANK on cell surfaces, and increased production of RANKL, TNF-α, MCP-1 and SDF-1. Anti-IFN-γ antibody also induced the expression of osteoclast differentiation factor and actin ring formation, but inhibited the expression of osteoprotegerin. TRAP staining and bone resorption assays showed that anti-IFN-γ antibody induced an increase in osteoclast formation and bone resorption. Conclusion: The anti-IFN-γ antibody induced osteoclast formation, and is probably mediated by RANKL-induced activation of NF-κB, that induces TRAF6 in the RANKL-RANK signaling pathway. Our data suggest an inhibitory role for IFN-γ in osteoclast formation induced by tuberculosis.

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Abstract 5551: Double Deficiency of Tumor Necrosis Factor-α and Interferon-γ in Bone Marrow-Derived Cells Prevents Neointimal Formation in a Murine Model of Vascular Injury

Circulation ◽

10.1161/circ.118.suppl_18.s_573-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Hideki Murayama ◽

Masafumi Takahashi ◽

Yuji Shiba ◽

Masaya Takamoto ◽

Hirohiko Ise ◽

...

Keyword(s):

Bone Marrow ◽

Vascular Injury ◽

Tumor Necrosis ◽

Interferon Γ ◽

Neointimal Formation ◽

Tnf Α ◽

Bone Marrow Derived Cells ◽

Ifn Γ ◽

Factor Α ◽

Necrosis Factor

Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Recent evidence indicates that inflammatory responses induced by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are involved in the progression of neointimal formation. However, the role of TNF-α and IFN-γ in the restenosis after PCI has not been fully understood. The purpose of this study is to examine the impact of TNF-α and IFN-γ in bone marrow-derived cells in the development of neointimal formation after vascular injury in mice. Wild-type (WT), TNF-α-deficient (TNF-α −/− ), IFN-γ-deficient (IFN-γ −/− ), and TNF-α/IFN-γ double-deficient (DKO) mice were subjected to wire-mediated vascular injury of the right femoral artery. Immunohistochemical analysis showed the expression of TNF-α and IFN-γ was detected in the neointimal lesion of WT mice, but these cytokines were not detected in the lesion of the corresponding deficient mice. Neointimal formation was significantly reduced after the injury in the DKO mice, compared to that in the WT, TNF-α −/− , and IFN-γ −/− mice (I/M ratio, WT: 2.28±0.17, TNF-α −/− : 2.13±0.20, IFN-γ −/− : 2.37±0.16, DKO: 1.32±0.10, p<0.05, each n=14–17). No significant difference in reendothelialization (CD31 staining) was observed among these groups. Further, vascular smooth muscle cell (α-SMA) and macrophage (F4/80) contents in the neointimal area also did not differ among the groups. The number of proliferating cell nuclear antigen (PCNA) and Ki-67 positive cells in the neointimal lesion was significantly decreased in DKO mice. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow chimeric (BMT Wild→Wild , BMT DKO→Wild , and BMT Wild→DKO ) mice. The neointimal formation in BMT DKO→Wild mice was significantly reduced as compared to that in BMT Wild→Wild (I/M ratio, p<0.05, each n=7) and BMT Wild→DKO mice (p<0.05). These results suggest that the lack of TNF-α and IFN-γ in bone marrow-derived cells synergistically prevents neointimal formation after vascular injury and provide new insights into the mechanisms underlying the restenosis after PCI.

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