Surface Levels Of CXCR4 Research Articles

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

Analyses of the Kinetics and Phenotype of Multiple Intraclonal CXCR4/CD5 B Cell Subsets Suggest Differences in Life Cycle Transitioning in CLL

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease so that defining the dynamic features of the clone and its intraclonal subpopulations are essential to understand disease pathogenesis and to develop novel, effective therapies. For instance, because cell division is linked with new mutations, the ability to preferentially select cells that recently divided allows studying the subpopulation(s) most likely responsible for disease progression and resistance to therapies.The intraclonal kinetics of CLL B cells have been studied in clonal subgroups defined by reciprocal surface levels of CXCR4 and CD5. In that model, three fractions are identified: recently divided “proliferative” (PF; CXCR4 DimCD5 Bright); “intermediate” (IF; CXCR4 IntCD5 Int) and “resting” (RF; CXCR4 BrightCD5 Dim). Here, we have expanded the examination of subpopulations differing for time since last division (“age”).Unmanipulated CLL cells studied ex vivo from 10 patients who drank 2H 2O for 4 weeks were sorted by the relative densities of CXCR4 and CD5 to isolate the formerly identified PF, IF and RF as well as two fractions not previously characterized, “Double Dim” (DDF: CXCR4 DimCD5 Dim) and “Double Bright” (DBF; CXCR4 BrightCD5 Bright). For each fraction, the amount of deuterium incorporated into cellular DNA in vivo was measured. Consistently, the PF contained significantly higher levels of 2H-labeled DNA and higher calculated cell division rates when compared with the RF and IF. Interestingly, the DDF also contained significantly more 2H-labeled DNA compared to the RF; in contrast, the DBF resembled more closely the RF fraction. The overall 2H-incorporation gradient was: PF>DDF>IF>DBF>RF.In CLL, BCR signaling is fundamental, with the amount of membrane (m) IgM associating with signaling competence and disease aggressiveness. Additionally, when engaged independently, mIgM and mIgD can lead to different signaling sequelae. Therefore, we analyzed the 5 subpopulations for the densities of mIgM and mIgD. This showed a distribution similar to that of 2H-DNA incorporation: for IgM: PF=DDF>IF=DBF=RF, and for IgD: PF>DDF>IF=DBF>RF.Accordingly, we next measured 2H-DNA in subpopulations with low, intermediate and high levels of IgM and IgD. This revealed a direct correlation between IG densities and in vivo DNA synthesis, consistent with intraclonal subpopulations with high IGs having divided more recently than those with low IGs.However, these findings are not in line with cell division being primarily initiated by BCR engagement since that would lower mIgM levels. Therefore, we tested if engagement of TLR9 would affect mIG densities on CLL cells. After stimulation of 32 CLL clones with CpG+IL15, anti-IgM+IL4, anti-IgD+IL4, or anti-IgM-IgD+IL4, there was a significant increase in mIGs only after CpG+IL15 activation; each anti-IG stimulation led to downregulation of mIGs.Finally, we questioned the subclonal responsiveness to BTK inhibition in vivo. CLL samples taken from the same patients, before and during ibrutinib treatment, displayed intraclonal changes in mIG densities and cell size, the latter a marker of cellular and metabolic activation also linked with CLL in vivo birth rates. Ibrutinib treatment normalized mIgM and mIgD intraclonal densities and lead to an overall cell size decrease with larger, 2H-enriched and higher mIG density cells being more affected (PF>DDF>IF>DBF>RF).Collectively, these findings suggest that the most recently born cells enter the circulation as the PF from which they transition to either lower CD5 (DDF) or higher CXCR4 (IF and DBF) phenotypes. Each eventually converge as the RF. Moreover, since mIG densities on the more recently divided populations (PF and DDF) are high, the data imply that successful cell division is not solely a consequence of BCR engagement; the involvement of the TLR pathways, concomitantly or in series with BCR signaling, is more consistent with the higher mIG levels. Finally, ibrutinib treatment appears to preferentially target more recently divided cells with high mIG levels. DisclosuresAllen: Alexion: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees.

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4R334X). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4R334X receptor variant. Transfection of CXCR4R334X-coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.

PF396 HISTONE DEACETYLASE INHIBITOR PANOBINOSTAT TARGETS CALCINEURIN/NFATC1 SIGNALING AND ABROGATES THE RESISTANCE OF PH+ LEUKEMIA CELLS TO DASATINIB

Background:BCR‐ABL‐targeted tyrosine kinase inhibitors (TKI) revolutionized the outcome of patients inflicted with CML and Ph+ B‐ALL. However, TKI resistance continues to pose challenges for both CML and ALL.Aims:We hypothesized that overcoming the BM microenvironment‐mediated protection of Ph+ leukemic cells from TKI‐mediated apoptosis may further enhance the responsiveness to TKI therapy.Methods:In vitro experiments using BCR‐ABL‐positive ALL (BV‐173, NALM1 and NALM20) and CML (K562 and LAMA84) cell lines.Results:Dasatinib treatment significantly suppressed the growth of BCR‐ABL‐positive ALL and CML cell lines (p < 0.01). However, co‐culture with bone marrow stromal cells (BMSCs) protected both ALL and CML cells from dasatinib‐mediated toxicity. Furthermore, dasatinib treatment promoted significant upregulation of the chemokine receptor CXCR4, on both mRNA and cell surface levels. Elevated CXCR4 expression was accompanied by increased responsiveness of ALL and CML cells to CXCL12 stimulation, resulting in strong and sustained phosphorylation of Erk1/2 and Akt and increased adhesion capacity to BMSCs. Consequently, dasatinib‐induced upregulation of CXCR4 promotes the stroma‐mediated survival advantage of Ph+ leukemia cells upon TKI therapy.Next, in order to overcome the CXCR4‐mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete CXCR4 in AML cells. Single‐agent treatment with panobinostat demonstrated significant inhibition of Ph+ ALL and CML cell growth at low nanomolar concentrations (p < 0.01). Importantly, combination of panobinostat with dasatinib synergized (CI<0.5), effectively overcoming the protection provided by BMSCs and inducing apoptosis of Ph+ leukemia cells, as demonstrated by phosphatidylserine externalization and mitochondrial depolarization. Moreover, panobinostat/dasatinib treatment suppressed cell cycle progression, inducing subG0/G1 DNA fragmentation. Furthermore, combining panobinostat with dasatinib significantly reduced CXCR4 surface levels and CXCL12‐mediated responses, including Erk1/2 and Akt activation in Ph+ leukemic cells. Notably, panobinostat/dasatininb combination significantly inhibited soft agar colony formation of K562 cells.To elucidate the molecular mechanisms, we performed gene and protein expression analysis. Panobinostat, alone or in combination with dasatinib, significantly down‐regulated the protein levels of calcineurin, a serine–threonine protein phosphatase previously implicated in ALL and CML pathogenesis, as well as of NFATc1, a critical effector of the calcineurin signaling cascade, and NFATc1‐regulated target genes. Additionally, inhibiting calcineurin activity with cyclosporin A (CsA) decreased both basal and dasatinib‐induced CXCR4 surface levels and sensitized leukemic cells to dasatinib, overcoming the protection of the BMSCs. In addition, combining CsA with panobinostat demonstrated synergistic (CI<0.5) anti‐leukemic effect that results in deeper suppression of NFATc1‐regulated target genes. We thus link the effect of panobinostat with calcineurin‐dependent downregulation of CXCR4 and NFTAc1 signaling, blocking the ability of the leukemic cells to respond to CXCL12‐mediated stromal support and enhancing their response to BCR‐ABL inhibition.Summary/Conclusion:Taken together, our results identify calcineurin/NFATc1 signaling pathway as a novel target of panobinostat in Ph+ leukemia cells. Based on these findings, HDAC inhibition with panobinostat may be effective strategy for facilitating the anti‐leukemic activity of TKI therapy.

Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

High Peripheral Blood Circulating BDNF Levels Are Associated with Good Prognosis in CLL Patients; A CXCR-4 Dependent Effect?

Introduction: BDNF is a neuronal growth factor that previously showed to exert survival effect in B cells. The role of BDNF in CLL is unknown.Objectives: To study the circulating levels of BDNF in CLL patients and their association with disease prognosis. We also looked for CXCR-4 as a possible mechanisms underlying BDNF effect in CLL.Design and methods: The total BDNF levels and the levels of the BDNF precursor; proBDNF were quantified in the serum of 36 CLL patients and 5 healthy controls by commercial ELISA kits (DY248 and DY3175 DuoSet, respectively, from R&D Systems, Minneapolis, MN, USA). The patients’ BDNF levels were correlated to the disease characteristics and clinical course. mRNA and protein expression of the high affinity receptor for BDNF; TrkB were evaluated by real time PCR and flow cytometry respectively. The effect of BDNF on CXCR4 surface expression and migration of CLL cells towards SDF-1 were studied in-vitro.Results: The total serum levels of BDNF in CLL patients was not statistically different compared to healthy controls (mean±SD; 19.9±15.1ng/ml vs. 10.5±9.5ng/ml respectively, p=0.19). Serum proBDNF levels in both CLL patients and healthy controls were under the detection limit of the kit. Within the CLL group we found higher total BDNF levels in patients with mutated immunoglobulin variable heavy-chain (IGHV) status than in patients with unmutated IGHV (25.2±14.6ng/ml vs. 14.1±13.0 in mutated and unmutated status, respectively, p=0.028). We also found higher serum levels of total BDNF in CLL patients in Binet stage A compared to those with a more advance clinical stage (mean±SD ; 24.5±14.6ng/ml vs. 17.0±16.9 and 10.2±9.6 in A,B and C, respectively, p=0.013). Accordantly, higher BDNF levels were detected in patients who had a clinically stable disease compared to patients who had disease progression and required treatment (23.1±14.7ng/ml vs. 13.4±14.5, respectively, p=0.013). The mRNA levels of the BDNF receptor; TrkB, were twofold lower in CLL cells compared to normal B cells. However, the overall TrkB mRNA transcripts were very low in CLL cells and normal B cells compared to the normalized housekeeping genes of Beta-2-microglobulin and CD19. Likewise, surface expression of TrkB in CLL was undetectable using several commercial monoclonal Abs by flow cytometry. In vitro culture of CLL cells in serum free media for 24h resulted in an increased CXCR4 surface expression and greater apoptosis. Culture of CLL cells in the presence of recombinant BDNF (50ng/ml) resulted in downregulation CXCR-4 surface levels and protection from spontaneous apoptosis, irrespectively of the IGHV mutational status. The effect of BDNF on CXCR4 downregulation was also confirmed at 1h culture as it shows to induce similar effect to SDF-1 (50ng/ml) and additive effect when combined with SDF-1. Finally, possible competition between BDNF and SDF-1 was indicated as pretreatment of CLL cells with BDNF inhibit their migration toward SDF-1.Discussion: Our findings show association between high BDNF levels and favorable disease prognosis in CLL. Undetected TrkB expression in CLL cells is compatible with previous reports showing sequestration of TrkB in normal and malignant B cells and suggests TrkB independent effect of BDNF in CLL. The effect of BDNF on the survival and migration of CLL cells via CXCR-4 needs to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity.

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4+ T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48–72 h after activation of CD4+ T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4+ T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4+ T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4+ T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

The Spiegelmer® Nox-A12 Abrogates Homing Of Human CLL Cells To Bone Marrow and Mobilizes Murine CLL Cells In The Eμ-TCL1 Transgenic Mouse Model Of CLL

▪NOX-A12 is a novel mirror-image oligonucleotide (Spiegelmer®) that specifically antagonizes the chemokine stromal cell-derived factor 1 (SDF-1, CXCL12). SDF-1 is highly expressed by stromal and endothelial cells, and binding to its classical receptor CXCR4 leads to the activation of multiple signaling pathways regulating cell migration, retention and survival. SDF-1-induced inside-out activation of the integrin VLA-4 (a4ß1, CD49d/CD29) via CXCR4 is essential for homing of various normal and malignant hematopoietic cells to bone marrow (BM). We previously found that VLA-4 is indispensable for BM homing of chronic lymphocytic leukemia (CLL) cells. Hence, CXCR4 binding to SDF-1 may be a prerequisite for VLA-4 mediated trafficking of CLL cells into the protective BM microenvironment and for their retention in lymphoid organs, favoring their survival and chemotherapy-resistance.In this project we compared the efficacy of NOX-A12 and the CXCR4 antagonist AMD3100 to mobilize CLL cells from their protective niches to the peripheral blood, thereby sensitizing them to cytotoxic agents such as fludarabine, and to prevent their recirculation into the protective lymphoid microenvironment.To assess the contribution of SDF-1/CXCR4 signals to homing of CLL cells, we performed in vivo short term adoptive transfers of human VLA-4+ CLL cells into immune deficient mice. CLL cells (5-10 x 106) were injected into the tail vein of NOD/SCID mice that were treated either with NOX-A12 (20 mg/kg) or AMD3100 (5 mg/kg). After a homing period of 3 hours lymphatic organs were analyzed for human CLL cell count. We found that for the majority of investigated CLL samples (except patient samples bearing the chromosomal aberration trisomy 12), CXCR4/SDF-1 inhibition reduced CLL cell recirculation into BM, thereby redirecting them into the spleen. The mean (± SD) relative homing rate (homed human CLL cells relative to injected cells per 1 x 106 acquired murine cells) of CLL cells to BM decreased upon NOX-A12 treatment from 137 (± 87) to 30 (± 17) (p = 0.047), and upon AMD3100 treatment from 137 (± 71) to 38 (± 24) (p = ns). Further, we determined CXCR4 surface levels of CLL cells before injection and after cells had homed to BM. We noticed that homed CLL cells expressed elevated CXCR4 surface levels as mean MFIR (± SD) rose from 25 (± 13) to 93 (± 11) and that CXCR4 levels were further increased upon CXCR4/SDF-1 inhibition with NOX-A12 to MFIR 225 (± 2) and with AMD3100 to MFIR 213 (± 7).Next, we used the Tcl1 transgenic transplantation model that mimics human CLL to test NOX-A12 and AMD3100-induced CLL cell mobilization. C57BL/6J wild type mice engrafted with splenocytes (1 x 107) from Tcl1tg mice with established disease were treated with separate doses of NOX-A12 (0, 10, 20 and 50 mg/kg) by single intravenous injections. Separate doses of AMD3100 (0, 3, 5, and 10 mg/kg) were administered subcutaneously. Blood was collected from the tail vein before treatment, 1 hr, 3 hrs, 6 hrs, and 24 hrs post treatment and analyzed for absolute leukocyte counts and numbers of CD5+/CD19+cells. NOX-A12 demonstrated a considerable potential to mobilize CLL cells and other lymphocytes as the relative CLL cell and absolute lymphocyte blood count raised within 1 hour by 50% when using mice with high tumor load and by 200% when using mice with lower tumor load. Mobilization reached a peak after 6 hours with 100% and 220% relative increase to 0 hrs value. Compared to untreated control mice, CLL cell blood count raised from 80% to 120% within 6 hours. Even low doses (10 mg/kg) of NOX-A12 showed a high degree of mobilization of CLL cells and total lymphocytes. Higher doses lead to a prolonged mobilization duration. AMD3100 treatment did not lead to a significant increase in CLL cell or total lymphocyte blood count in peripheral blood.In summary our data demonstrate a high efficiency of NOX-A12 to mobilize CLL cells and to prevent their recirculation into protective lymphatic organs. Abrogating SDF-1/CXCR4 signaling in combination with chemotherapy may provide an attractive approach for treatment of CLL. Currently, the effect of combined NOX-A12 and fludarabine treatment on progression and overall survival is evaluated using the Tcl1 transgenic transplantation model. Disclosures:Kruschinski:Noxxon: Employment. Greil:NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

New insights into lipid raft function regulating myocardial vascularization competency in human idiopathic dilated cardiomyopathy

ObjectiveIdiopathic dilated cardiomyopathy (IDCM) affects myocardial vascularization. Whether a lack of demand for increased myocardial vascularization and/or an impaired response of circulating angiogenic-supportive cells are responsible for the vascular derangements found in IDCM is unknown. Methods and resultsLeft ventricle (LV) samples obtained at transplant from IDCM hearts were compared to control hearts from non-cardiac decedents. Peripheral colony-forming myeloid cells were extracted from age- and sex-matched IDCM patients and healthy volunteers. At the tissue level, no differences were detected in stromal cell-derived factor (SDF)-1α expression, but integrin-linked kinase (ILK) levels and activity were increased in IDCM. A marked co-localization of SDF-1α and the specific marker of cholesterol-enriched lipid rafts Flotillin (Flot)-1 was found in IDCM. SDF-1α was also highly distributed into IDCM lipid rafts. Non-adherent pro-angiogenic cells from both groups, which were found increased in patients but showed similar surface levels of CXCR-4, equally supported Matrigel-mediated cell network formation. However, SDF-1-mediated migration was reduced in IDCM-derived cells, which also exhibited decreased ILK activity and downstream ERK activation. ConclusionsTaken together, our results point out that myocardial competency to increase vascularization is not altered in IDCM, but dysfunctional SDF-1-mediated migration by peripheral pro-angiogenic cells through ILK and downstream ERK signaling may compromise endothelial recovery in patients. We provide new insights into lipid raft function in human IDCM and envision more effective treatments.

88 : CXCR3 modulates CXCL12-mediated cellular responses in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of B lymphocytes that accumulate in blood, bone marrow, and secondary lymphoid organs. Signals from the lymphoid tumor microenvironment are known to critically influence the pathogenesis of CLL by stimulating tumor cell proliferation and survival. In consequence, localization of CLL cells within this environment contributes to residual disease after therapy and to the development of a chemoresistant phenotype. The chemokine stromal-cell derived factor 1 (SDF-1, CXCL12), produced by stromal cells and other accessory cells in the bone marrow and lymph nodes, has a key role in protective CLL cell-microenvironment interactions. We found that CXCL12-induced integrin activation is essential for the capacity of CLL cells to home to the bone marrow. Ligation of the corresponding chemokine receptor CXCR4, which is highly expressed in CLL, rapidly activates VLA-4 on CLL cells resulting in their arrest on VCAM-1 substrates under shear flow. Here, we found that CLL cells express CXCR3. CXCR3 engagement by its ligands CXCL9, CXCL10 or CXCL11 diminishes CLL cell interactions with the VCAM-1/CXCL12 substrate under shear conditions. Moreover, chemotaxis to CXCL12 is significantly reduced in the presence of the CXCR3 ligands whereby the CXCR4 surface levels are not altered. Notably, CXCR3 does not affect CCR7-mediated chemotaxis to CCL19, suggesting a specific CXCR3-CXCR4 crosstalk in CLL migration and adhesion. CLL cell proliferation is not influenced by the presence of CXCR3 ligands, and since CXCR4 is entirely downregulated in proliferating CLL cells, a CXCR4-CXCR3 crosstalk in this process is unlikely. However, survival benefits provided by CXCL12 are partially abolished by co-incubation of the CLL cells with CXCL11. Collectively, our data point to a crosstalk of CXCR3 and CXCR4 that might be based on their heterodimerization, which is currently investigated.

Expression of Nef from unintegrated HIV-1 DNA downregulates cell surface CXCR4 and CCR5 on T-lymphocytes

BackgroundTranscription of HIV-1 cDNA prior to, or in the absence of, integration leads to synthesis of all classes of viral RNA transcripts. Yet only a limited range of viral proteins, including Nef, are translated in this context. Nef expression from unintegrated HIV-1 DNA has been shown to reduce cell surface CD4 levels in T-cells. We wished to determine whether Nef expressed from unintegrated DNA was also able to downregulate the chemokine coreceptors CXCR4 and CCR5.Viral integration was blocked through use of an inactive integrase or by using the integrase inhibitor raltegravir. Infected cells bearing unintegrated DNA were assayed by flow cytometry in the GFP reporter cell line, Rev-CEM, for cell surface levels of CD4, CXCR4 and CCR5.ResultsIn cells bearing only unintegrated HIV-1 DNA, we found that surface levels of CXCR4 were significantly reduced, while levels of CCR5 were also diminished, but not to the extent of CXCR4. We also confirmed the downregulation of CD4. Similar patterns of results were obtained with both integrase-deficient virus or with wild-type infections of cells treated with raltegravir. The Alu-HIV qPCR assay that we used for detection of proviral DNA did not detect any integrated viral DNA.ConclusionsOur results demonstrate that Nef can be expressed from unintegrated DNA at functionally relevant levels and suggest a role for Nef in downregulation of CXCR4 and CCR5. These findings may help to explain how downregulation of CXCR4, CCR5 and CD4 might restrict superinfection and/or prevent signal transduction involving HIV-1 infected cells.

Human Peritoneal Macrophages From Ascitic Fluid Can be Infected by a Broad Range of HIV-1 Isolates

Macrophages are major HIV target cells. They support both productive and latent HIV-1 infection. Susceptibility of primary macrophages to HIV depends on the anatomical location and activation state of the cells. We demonstrate that peritoneal macrophages (PMs) are abundant in ascitic fluid of patients with liver cirrhosis and are susceptible to HIV-1 infection. PMs expressed CD68, a differentiation marker, exhibited phagocytic activity, and survived in culture for 2 months without additional growth factors. Freshly isolated PMs were susceptible to HIV-1 R5 strains but not to X4-T-cell line-adapted strains. Interestingly, after 7 days in culture, PMs acquired susceptibility to X4-T-cell line-adapted strains. HIV entry inhibitors, TAK779 and AMD3100, blocked HIV infection of PMs, indicating that infection by R5 and X4 strains was mediated by CCR5 and CXCR4, respectively. Although PMs did not express detectable cell surface levels of CXCR4 and CCR5, they did express mRNAs of these HIV coreceptors and responded to stimulation by their natural ligands, SDF-1alpha and RANTES. PMs were susceptible to HIV-1 X4, R5, and X4R5 primary isolates. PMs after 7 days in culture produced greater amounts of X4 and X4R5 HIV than freshly isolated PMs. The day-7 PMs were more susceptible to R5 infection in a single-cycle infection assay, but there was no increase in viral production in a multiple-round infection assay. The level of CXCR4 mRNA and production of CC-chemokines (MIP-1alpha, MIP-1beta, and RANTES) increased significantly during 7 days in culture. Our results indicate that PMs are susceptible to receptor-mediated infection by a broad range of HIV strains. These primary macrophages could provide a valuable system for investigating the role of primary macrophages in HIV pathogenesis.

The Interaction of CXCR4/SDF-1 and VLA-4 Regulates Adhesion and Transendothelial Migration in Waldenstrom Macroglobulinemia.

Background. Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM). Adhesion of malignant cells to the BM microenvironment induces proliferation and resistance to therapy. We previously showed that SDF-1/CXCR4 axis regulates migration and adhesion of WM cells in vitro indicating a potential role in homing. Homing of malignant cells to the BM niches requires active navigation through the endothelial cell barrier where tumor cells must adhere, roll and transmigrate. We hypothesized that CXCR4 and its interaction with VLA-4 is critical for this 3-step process.Methods. The level of CXCR4 and VLA-4 was determined using flow cytometry and RT-PCR in patient samples and WM cell lines (BCWM.1 and WM-WSU). The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO), Gi protein inhibitor pertussis toxin, PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. These studies were confirmed using CXCR4 knockdown using shRNA with lentivirus infection. Adhesion assay was done with either plates coated with the VLA-4 ligand fibronectin for adhesion of tumor cells in presence and absence of SDF1 (30nM), or coated with endothelial cells (HUVEC) and BMSCs for cell-cell adhesion experiments. Transmigration assay was performed using the transwell system with either SDF1 in the lower chamber to study migration towards SDF1, or with the upper chamber coated overnight with endothelial cells (HUVEC) and BMSCs to study transmigration. Experiments with HUVEC cells were performed in absence or presence of TNF-alpha that activates the HUVEC cells.Results. WM tumor cells from patients and cell line expressed high surface levels of CXCR4 (mean 70%) and VLA-4 (mean 95%). Adhesion of WM cells to fibronectin was significantly increased with SDF1 compared to BSA control, and AMD3100 20uM inhibited adhesion by 50% compared to control. Similar results were obtained with PTX 200ng/mL and using CXCR4 knockdown in WM cells. Furthermore, WM cells adhesion was greatly enhanced in co-culture with BMSCs and endothelial cells, and was significantly inhibited using AMD3100 and PTX. To further dissect the mechanisms of interaction of CXCR4 and VLA-4, we showed that AMD3100 inhibited surface expression of CXCR4 but not of VLA-4. Immunoprecipitation studies showed that CXCR4 and VLA-4 directly interact in response to SDF-1. Downstream signaling pathways showed that CXCR4 regulates migration and adhesion through the PI3K/Akt and ERK/MAPK pathways. We next studied whether CXCR4 was involved in transmigration through the endothelial barrier. We demonstrated that BCWM.1 cells transmigrated through the barrier of endothelial cells, that was increased when SDF1 was added to the lower chamber, and that AMD3100 inhibited the transmigration of BCWM.1 cells. Studies of the role of CXCR4 in rolling on endothelial cells is ongoing.Conclusion. We showed CXCR4/SDF1 axis regulates migration and adhesion of WM cells to the BM microenvironment indicating a potential role in homing. Moreover, we demonstrate that CXCR4/SDF1 axis is critical for transendothelial migration, a critical step of homing and egression of tumor cells in and out the BM niches.

Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration

Synaptotagmins regulate vesicle trafficking and fusion of vesicles with membranes - processes that have been implicated in cell migration. We therefore hypothesized that synaptotagmins play a role in T-cell migration. Amongst synaptotagmins 1-11, we found synaptotagmin 3 (SYT3) to be the only one that is expressed in T cells. CXCR4-triggered migration was inhibited by antisense synaptotagmin 3 mRNA and by the isolated C2B domain, known to impair oligomerization of all synaptotagmins, but not by a C2B mutant that binds Ca(2+) but does not block oligomerization. The C2B domain also blocked CXCR4-triggered actin polymerization and invasion. However, CXCR4-dependent adhesion in flow was not affected. Surprisingly, we found that little or no SYT3 is present near the plasma membrane but that it is mainly localized in multivesicular bodies, which also contained much of the CXCR4. Impaired SYT3 function blocked CXCR4 recycling and thus led to reduced surface levels of CXCR4. Migration was restored by overexpression of CXCR4. We conclude that STT3 is essential for CXCR4 recycling in T cells and thereby for the maintenance of high CXCR4 surface levels required for migration.

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.

Induction of CXCR4 in Murine Hematopoietic Cells To Improve Progenitor Homing and Overcome the Intrinsic Stem Cell Repopulation Defect in Fancc−/− Mice.

Adhesive interaction between chemokine receptor (CXCR) 4 and its ligand, stromal cell derived factor -1 alpha (SDF-1, CXCL12), is principally responsible for the homing of bone marrow progenitor cells to sites of hematopoiesis. Recent evidence in murine models suggests that engraftment and donor chimerism after transplantation are responsive to changes in progenitor CXCR4 expression. We hypothesized that this mechanism might also serve to overcome the repopulation deficiency in a murine model of Fanconi Anemia (FA), where an pro-apoptotic stem cell phenotype compromises overall repopulation efficiency. We investigated potential CXCR4 upregulation alternatively by cytokine stimulation, hypoxia, or CD26 (dipeptidylpeptidase IV) inactivation. Initial studies ascertained comparable baseline frequency and proliferation kinetics among c-kit, sca-1 and thy-1 expressing lineage depleted (lin−) subsets of bone marrow cells from wild-type (wt) and FA transgenic (Fancc−/−, ko) animals. Evaluation of CXCR4 expression by flow-cytometry revealed low baseline levels and substantial induction in sca-1, c-kit and thy-1 progenitors after stem cell factor and interleukin 3 supplemented culture, in both wt and ko animals. CXCR4 is also upregulated in response to hypoxia, and FA proteins are involved in oxidative stress signaling. We therefore reasoned that culture under hypoxic conditions may avoid some of the exaggerated apoptotic responses of FA hematopoietic cells during conventional culture and further improve Fancc−/− progenitor homing. Results in lin− bone marrow progenitor subsets from wt and Fancc−/− animals confirmed that CXCR4 could be upregulated following overnight hypoxia (1% O2 tension). No additional improvement was seen after extended culture. In murine competitive repopulation studies using CD45.1 and CD45.2 cells we confirmed the improved post transplantation chimerism in myeloablated recipients that received CD45.2/Fancc−/− cells cultured in low-O2 environment, compared to untreated wt cells. We also investigated the inactivation of cell surface peptidase CD26 (dipeptidylpeptidase IV) by Diprotin A (DA, Ile-Pro-Ile) which has recently been shown to improve progenitor homing. Our initial competitive transplantation experiments in cohorts of wt recipients confirmed improved chimerism from DA treated cells versus PBS control. In apparent contrast however, cells treated with DA after cytokine or hypoxia culture showed substantially decreased cell surface levels of CXCR4. This may suggest a more complex mechanism for the improved homing after DA exposure than previously thought. In summary, we show that Fancc−/− mice share similar progenitor compartment composition and dynamics in culture when compared with wt animals. Their hematopoietic cells express normal levels of CXCR4, and retain the ability to respond to stimulation by diverse stimuli with upregulation, resulting in improved donor engraftment and chimerism. These are the first reported studies to apply homing directed strategies to overcome the intrinsic qualitative defect in an established model of a stem cell repopulation deficiency (Fancc). The proposed strategies, individually or combined, may also offer a novel approach to overcome the repopulation disadvantages in other stem cell transplantation settings.

Differential Functional Activation of Chemokine Receptor CXCR4 Is Mediated by G Proteins in Breast Cancer Cells

CXCR4 is a G protein-coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein alpha and beta subunits to form a heterotrimeric complex with CXCR4. Galpha and Gbeta were able to bind to CXCR4 independently in all cell lines, but the association of G protein alphabetagamma heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein-coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein-receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.

M-CSF Drives Human Cord Blood Monocyte Differentiation along an Alternative Pathway into Suppressive CXCR4highIL-10highIL-12absentTNF-adefective Dendritic Cells.

M-CSF Drives Human Cord Blood Monocyte Differentiation along an Alternative Pathway into Suppressive CXCR4highIL-10highIL-12absentTNF-αdefective Dendritic cellsGeling Li and Hal E. Broxmeyer. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Cancer Institute, Indianapolis, IN, USAMacrophage colony-stimulating factor (M-CSF) in cooperation with TGF-β1 induces CD34+ hematopoietic progenitor cells to differentiate into Langerhans cells, a subset of immature dendritic cells (DCs) specifically localized in the epidermis. M-CSF levels are dramatically elevated in immunosuppressive conditions and M-CSF deficient mice are osteopetrotic. Therefore, we hypothesized that M-CSF might be involved in the generation of a novel subtype of suppressive DCs which may be located primary in bone marrow where large amounts of SDF-1 are constitutively produced. We also speculated that M-CSF-induced DCs might express the only known ligand of SDF-1, CXCR4 and migrate towards SDF-1. Highly purified umbilical cord blood monocytes (> 98% purity) cultured in the presence of M-CSF, IL-4 and TGF-β1 differentiated into a highly homologous population of cells with typical dendrites and acquired a langerhan cell (LC) phenotype by up-regulated expression of CD1a and E-cadherin. We termed these cells M-LC to distinguish them from regular LCs cultured in the presence of GM-CSF, IL-4 and TGF-β1. In comparison with LC, M-LCs produced a much higher level of IL-10, no IL-12 (detection limit, 4 pg/ml) and a minimal level of TNF-α (21.6 ± 37.5 pg/ml) upon LPS stimulation. LPS-activated M-LCs induced lower allogeneic mixed lymphocyte reaction (MLR) than LPS-activated LCs. Furthermore, exposure to LPS-activated M-LCs rendered a state of low-responsiveness in cord blood naïve CD4+ T cells to further re-stimulation with alloantigens in secondary MLR. M-LCs expressed much higher surface levels of CXCR4 than LCs regardless of their maturation status. M-LCs started to migrate towards SDF-1 at a lower concentration (≥10 fold lower than that required for LCs) and displayed enhanced responsiveness to SDF-1-induced chemotaxis. Our results demonstrate that M-CSF drives monocyte differentiation along an alternative pathway into suppressive CXCR4highIL-10highIL-12absentTNF-αdefective DCs. These novel DCs may be of use to suppress unwanted immune responses in vivo.