VLA-4 Expression Research Articles

Abstract 1536: UVB-irradiation regulates VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 under flow conditions

Abstract The major aspect contributing to the mortality of melanoma is its ability to spread, or metastasize. Ultraviolet B light (UVB) is considered an indirect cause of melanoma formation. However, little is known about the potential effects of UVB to melanoma metastasis. Integrins, a large family of cell adhesion molecules expressed on the melanoma cell surface, are important for cell signaling, growth, and migration during metastasis. Most critically, tumor cell tissue invasion is dependent on the initial interaction of tumor cells with vascular endothelium at the target organ, and there is increasing evidence for a prominent role of melanoma very late antigen-4 integrin (VLA-4, α4β1 integrin) binding to its endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) in this process. This research focuses on the quantitative modulation of VLA-4 integrin expression and function on melanoma cells after UVB irradiation. The present data show that at 3, 12, and 18 hours post-UVB irradiation, VLA-4 expression was unchanged relative to untreated cells, but adhesion to VCAM-1 decreased significantly. Immunofluorescence studies implied that the spatial organization of α4 integrin on the melanoma cell surface contributed to the changes in avidity for VCAM-1 upon UVB irradiation. Further analysis suggested that the surface level of α4 integrin was maintained by Akt activity after UVB irradiation. The level of cell surface α4 integrin was significantly increased after UVB-irradiation when Akt was inhibited. Inhibition of Akt also reversed the reduction of avidity of cells after the irradiation. Our data also show that UVB reduces the level of Akt. The inhibition of Akt activity correlated with a reduced amount of coupled cNOS and reduced amount of iNOS after UVB-irradiation. However the effect of NOSs on melanoma cell adhesion appears due to their roles in regulation of apoptosis after UVB-irradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2011-1536

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48h. 1400W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.

Evaluation of Mechanisms of Resistance to Arsenic Trioxide In Patients with Acute Promyelocytic Leukemia

AbstractAbstract 2746 Introduction:Newly diagnosed and relapsed acute promyelocytic leukemia (APL) patients respond to therapy with arsenic trioxide (ATO) based regimens. Significantly more patients with relapsed APL have disease recurrence after ATO based therapy than newly diagnosed cases. We undertook a series of experiments to evaluate the potential mechanisms to explain this increased recurrence rate in patients with relapsed APL.Patients and methods: From April 2007 to March 2009 bone marrow samples from newly diagnosed and relapsed cases admitted at our center were utilized for these studies. If required the bone marrow blasts and promyelocyte component was enriched to above 90% using a lineage depletion cocktail and VarioMACS (Miltenyi Biotec, Germany). For in-vitro intracellular ATO concentration measurement, 2 × 107 cells were washed and suspended in RPMI media with 0.5 μM concentration of ATO and incubated for 24 hours. Cells were then washed, made into a pellet and solubilized with HNO3 and H2O2 and ATO content measured using an atomic absorption spectrophotometer. An in-vitro sensitivity assay of malignant cells as previously reported was standardized using an MTT assay system. The impact of co-culture of mesenchymal stromal cells (MSC) and malignant promyelocytes on ATO induced apoptosis was studied with 7AAD and Annexin staining using a flowcytometer. A gene expression array using 44k human microarray chip analysis (Agilent technologies) was done on 8 newly diagnosed and 8 relapsed cases. Results:Sixty five patients were included in this study. Of these 47 (72%) were newly diagnosed and 18 (28%) were relapsed cases. On immunophenotyping, CD34 was positive (>20%) in 3.6% of newly diagnosed and 50% of relapsed cases (P=0.001). The mean MFI of the relapsed cases for expression of CD38, VLA-5 and CD13 was significantly lower in the relapsed group. The ability of both newly diagnosed and relapsed primary APL cells to concentrate ATO intracellular was not significantly different (15.2±9 nG/107 cell Vs. 16.3±9.7 nG/107 cell). Similarly the in-vitro IC-50 assay was not significantly different between the two groups (5.5±3.8 Vs. 4.7±4.5 μM). Neither of these assays correlate with clinical parameters such as relapse, event free (EFS) or overall survival (OS). Evaluation of the impact of MSC on ATO induced apoptosis demonstrates a protective effect in newly diagnosed and relapsed cases (Fig 1). This effect was mediated partly by the MSC conditioning media and could not be overcome by addition of VLA-4 or VLA-5 blocking antibodies (data not shown). Gene expression studies comparing the two cohorts revealed 1744 genes that were differentially expressed (>2 fold) between samples at diagnosis and at relapse. Quantitative Real-time RT-PCR using SYBR- Green detection system was done to confirm the gene expression results obtained from microarray analysis. Using ΔΔCT method the fold difference was calculated for five selected genes which validated the microarray data. Conclusion:Relapsed patients have significant immuno-phenotypic differences from newly diagnosed cases. Mechanisms of resistance to ATO are probably multi-factorial but are unlikely to be related to intra-cellular ATO concentration. In-vitro IC-50 does not appear to predict clinical outcomes. Stromal interaction protects malignant promyelocytes from the apoptotic action of ATO which appears more pronounced in relapsed than in newly diagnosed cases. Further evaluation of parameters that enhance such stromal interaction and protect malignant promyelocytes from the apoptotic effect of ATO along with evaluation of dysregulated genes and pathways are required. [Display omitted] Disclosure:No relevant conflicts of interest to declare.

Anti-inflammatory defense mechanisms of Entamoeba histolytica

The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures, may contribute to the delayed inflammation observed in amoebic hepatic abscess. This factor was isolated by ultra-filtration and high powered liquid chromatography, obtaining a primary Met-Gln-Cys-Asn-Ser structure, identified afterwards as the carboxyl-terminal (…Cys-Asn-Ser) active site. The selective anti-inflammatory effects of the pentapeptide have been observed in both in vitro and in vivo models, using a synthetic pentapeptide to maintain the same anti-inflammatory conditions during the experimental assays. Anti-inflammatory effects observed include inhibition of human monocyte locomotion and the respiratory burst in monocytes and neutrophils, increasing expression of anti-inflammatory cytokines and inhibiting expression of the adhesion molecules VLA-4 and VCAM, among others. In this review, we will describe the effects of MLIF detected so far and how it might be used as a therapeutical agent against inflammatory diseases.

Aberrant expression of c-met and HGF/c-met pathway provides survival advantage in B-chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of CD5(+) B lymphocytes. Decreased VLA-4 (Cd49d/CD29) and CD11a expression and defective adhesion in B-CLL have been previously shown, although there was no substantial data about its importance in immunobiology of B-CLL. The hepatocyte growth factor (HGF) receptor, c-met, plays a role in adhesion by acting on VLA-4. c-met and VLA-4 share crucial signaling molecules in cell survival. In this study, relationship between expressions of c-met and CD49d, CD11a, and additional common signaling molecules in B-CLL was investigated. White blood cells from 24 patients with CLL were studied by flow cytometry and/or western blotting prior to and after culturing with recombinant HGF. HGF level from sera was measured with a bead-based flow cytometric assay. c-metα and c-metβ were expressed on B-CLL cells, while no expression was observed on normal donor CD19+ cells. This increase was inversely correlated with decreased expression of adhesion molecules. Serum level of HGF in B-CLL was found to be increased. In vitro experiments showed that HGF supported survival in B-CLL cells supporting the possible function of HGF/c-met pathway in B-CLL. Furthermore, expressions of critical signaling molecules shared by both VLA-4 and HGF/c-met systems including Bcl-XL, Akt, PI3K, and phospho-bad(136) following HGF stimulations of B-CLL cells have been found to be increased. Increased expression of c-met and HGF may bypass the importance of expression of critical adhesion molecules and support survival of B-CLL cells. c-met, being one of the surface tyrosine kinases, may serve as a target for future therapies in B-CLL meriting more attention.

Protective effects of dehydroepiandrosterone on atherosclerosis in ovariectomized rabbits via alleviating inflammatory injury in endothelial cells

ObjectiveThe risk for atherosclerosis is increased in postmenopausal women. Dehydroepiandrosterone (DHEA) is postulated to have anti-atherogenic properties, but the mechanism remains unclear. The aim of this study was to elucidate the protective effect of DHEA on atherosclerosis in ovariectomized rabbits. MethodsThe lipid status and atherosclerotic lesions were examined in vivo in ovariectomized rabbits. The effects of DHEA on expression of inflammatory molecules were evaluated in vitro, such as nitric oxide (NO), malondialdehyde (MDA), monocyte chemoattractant protein-1 (MCP-1), adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in the human umbilical vein endothelial cells (HUVECs) injured by oxidized low-density lipoproteins (ox-LDL). The adhesion of the monocytic U937 cells to HUVECs was treated with supernatants of ox-LDL treated HUVECs with or without DHEA, and then the expressions of CCR2, LFA-1, VLA-4 were analyzed in U937 cells. The HUVECs with or without LPS treatment were then treated with DHEA, and NF-κB activity was measured by luciferase activity. ResultsDHEA administration alleviates efficiently the early pathologic damage of atherosclerosis, increases the serum NO level, and up-regulates the endothelial cell estrogen receptor (ER) expression of ovariectomized rabbits. DHEA in vitro significantly promotes NO synthesis, suppresses MDA and MCP-1 secretion of endothelial cells, and decreases ICAM-1, VCAM-1 and E-selectin expression in HUVECs; neither selective ERα antagonist (methyl-piperidino-pyrazole, MPP) nor ERβ antagonist (R,R-tetrahydrochrysene, R,RTHC) can abolish these effects. Furthermore, DHEA reduces CCR2, LFA-1 and VLA-4 expression in U937 cells, which in turn inhibits the adherence of monocytes to the injured endothelial cells. DHEA significantly decreased the LPS-induced NF-κB transcription. ConclusionsOur findings suggest that DHEA can alleviate inflammation in endothelial cells. The effects of DHEA on endothelial cells are independent of ERα or ERβ pathway, but at least in part, through suppression of NF-κB activity, which protects from atherosclerosis triggered by monocyte adherence.

Differential resolution of inflammation and recovery after renal ischemia–reperfusion injury in Brown Norway compared with Sprague Dawley rats

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.

TAp63 Regulates VLA-4 Expression and Chronic Lymphocytic Leukemia Cell Migration to the Bone Marrow in a CD74-Dependent Manner

The hallmark of chronic lymphocytic leukemia (CLL) is the relentless accumulation of mature lymphocytes, mostly due to their decreased apoptosis. CD74 was recently shown to serve as a survival receptor on CLL cells. In this study, we show that stimulation of CD74 with its natural ligand, migration inhibitory factor, initiates a signaling cascade that results in upregulation of TAp63, which directly regulates CLL survival. In addition, TAp63 expression elevates the expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the bone marrow (BM). Thus, CD74 and its target genes TAp63 and VLA-4 facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that rescues them from apoptosis. These results could form the basis of novel therapeutic strategies aimed at blocking homing of CLL cells in their return to the BM and attenuating their survival.

Abstract 2346: Contribution of VLA-4 to ovarian cancer metastasis and platinum resistance

Abstract Ovarian cancer is the fourth leading cause of cancer-related death among women. The relatively poor prognosis for ovarian cancer (5-year survival of 30-40%) reflects 1) the advanced stage of disease at diagnosis and 2) the frequent development of recurrent disease refractory to front line therapy. Ovarian cancers metastasize by attaching to and invading through the mesothelium, a single cell layer of mesothelial cells lining the peritoneal cavity. The presence of invasive peritoneal metastases is associated with a poor prognosis for ovarian cancer (5 yr survival <25%). We identified the integrin VLA-4 and its ligand VCAM-1 as regulators of mesothelial invasion. VLA-4 expression correlates with the ability of ovarian cancer cells to invade mesothelial monolayers. Additionally, VLA-4 is expressed on primary ovarian cancer cells obtained from patient tumors or ascites. The contribution of VLA-4 to ovarian cancer peritoneal metastasis was evaluated using a mouse xenograft model and function-blocking antibodies directed against VLA-4. Mice were injected intraperitoneally with platinum-resistant human ovarian cancer cells; one week later the mice were randomized into four groups and treated with 1) anti-VLA-4 antibodies, 2) carboplatin, 3) anti-VLA-4 and carboplatin, or 4) control IgG. Treatment with either anti-VLA-4 therapy or carboplatin alone had no effect on tumor burden compared to the IgG control group. However, the combined treatment of anti-VLA-4 and carboplatin significantly reduced tumor burden compared to the control or either treatment alone. This study supports a role for the active involvement of VLA-4 expressed by tumor cells in the regulation of ovarian cancer chemoresponsiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2346.

Differential integrin expression by T lymphocytes: Potential role in DMD muscle damage

The expression and function of integrin-type extracellular matrix receptors, VLA-4 and VLA-5, and laminin receptor VLA-6 on the surface of CD3 +CD4 + and CD3 +CD8 + defined T cell populations was evaluated in the blood of Duchenne muscular dystrophy (DMD) patients and healthy individuals. Both the number of CD4 + and CD8 + T cell subsets expressing VLA-4 or VLA-5 and the fibronectin-driven T cell migration was significantly higher in DMD patients. These data indicate that interactions of VLA-4 and/or VLA-5 with fibronectin may drive T lymphocytes to specific niches within muscle, contributing to tissue damage and fibrosis in DMD patients.

1151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN)

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN) Tomoyuki Kato, Mayu Yagi, Atsushi Yamagishi, Noriyuki Hosoya, Toshihiko Sakurai, Hayato Nishida, Sei Naito, Masaaki Tsukigi, Hisashi Kawazoe, Akinori Muto, Akira Nagaoka, and Yoshihiko Tomita Tomoyuki KatoTomoyuki Kato More articles by this author , Mayu YagiMayu Yagi More articles by this author , Atsushi YamagishiAtsushi Yamagishi More articles by this author , Noriyuki HosoyaNoriyuki Hosoya More articles by this author , Toshihiko SakuraiToshihiko Sakurai More articles by this author , Hayato NishidaHayato Nishida More articles by this author , Sei NaitoSei Naito More articles by this author , Masaaki TsukigiMasaaki Tsukigi More articles by this author , Hisashi KawazoeHisashi Kawazoe More articles by this author , Akinori MutoAkinori Muto More articles by this author , Akira NagaokaAkira Nagaoka More articles by this author , and Yoshihiko TomitaYoshihiko Tomita More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.650AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical instillation of Bacillus Calmette-Guerin (BCG) is an effective treatment for superficial high grade bladder cancer. Although both immunological and direct effects on tumor cells have been proposed, the complete mechanisms for its effect are not fully understood. Presence of fibronectin has been shown to be necessary for binding and internalization of BCG into tumor cells to exert direct effect on them. We have previously demonstrated the correlation between high expression of fibronectin receptor VLA5 (alpha 5 beta 1 integrin) and high malignant potential of urothelial cancer (UC). Here, we assessed the direct antitumor effects of BCG for UC cell lines which beard various amount of VLA5 on the cell surface. METHODS The human UC cell lines (T24, HT1376 and RT4) and Tokyo 172 BCG strain (Immunobladder, Japan BCG Laboratory), BCG cell wall skeleton (BCG-CWS SMP105, Dainippon Sumitomo Pharma) were used in this study. Viability of cell lines was evaluated with crystal violet stainig assay. Fluorescence of apoptotic nuclei stained with Propidium Iodide (PI) was measured with a FACScan. DNA synthesis was evaluated with BrDU incorporation assay. RESULTS First, expression of VLA5 on the surface of cell lines was checked. T24 and HT1376, high grade UC, beard large amount of VLA5 on the cell surface and RT4, a low grade UC, had traces of VLA5. Treatment with BCG resulted in dose dependent decrease of relative viability in T24 (p<0.0001) and HT1376 (p<0.0001). This effect was partially reversed by coculturing cells with VLA5 blocking antibody. In RT4, viability was not altered by treatment with BCG. Sub-G1 fraction of PI stained T24 increased from less than 1% (untreated) to more than 4% (1 mg/ml of BCG) after exposure to BCG and it suppressed BrDU incorporation (p=0.004) in T24 cells. Autoclaved killed BCG also demonstrated cell growth retardation effect in T24 (p<0.0001) and HT1376 (p<0.0001). BCG-CWS SMP105 also delayed T24 and HT1376 cell growth (p<0.0001). CONCLUSIONS These findings suggest that BCG exerts direct antitumor effect on UC cells via VLA5 with induction of apoptosis and inhibition of DNA synthesis. Moreover, killed BCG and BCG cell wall skeleton were able to exert similar effect making this approach attractive for clinical application where potentially serious adverse effect of viable BCG can be avoided while maintaining antitumor effect using killed BCG or cell extracts. Yamagata, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445-e446 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoyuki Kato More articles by this author Mayu Yagi More articles by this author Atsushi Yamagishi More articles by this author Noriyuki Hosoya More articles by this author Toshihiko Sakurai More articles by this author Hayato Nishida More articles by this author Sei Naito More articles by this author Masaaki Tsukigi More articles by this author Hisashi Kawazoe More articles by this author Akinori Muto More articles by this author Akira Nagaoka More articles by this author Yoshihiko Tomita More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chlamydia pneumoniaeinfection suppressesStaphylococcusenterotoxin B-induced proliferation associated with down-expression of CD25 in lymphocytes

Chlamydia pneumoniae (Chlamydophila pneumoniae) infects lymphocytes and modulates their immune functions; this is critical in the development of chronic inflammatory diseases associated with this pathogen. Therefore, to clarify this immune modulation due to C. pneumoniae infection, the effect of this infection on the proliferation of human peripheral blood lymphocytes was examined. Lymphocytes were proliferated by stimulation with Staphylococcus aureus enterotoxin B, and the cell number was increased up to 3 times the unstimulated lymphocyte number. Further, induction of CD25 expression was observed in 55.8% of lymphocytes. Infection with C. pneumoniae suppressed the proliferation of almost half the lymphocytes induced by stimulation with S. aureus enterotoxin B, and CD25 induction was inhibited in 64.7% of lymphocytes. Inhibition of CD25 expression was observed in both infected and uninfected lymphocytes in culture. However, the expression of VLA4 was not affected by C. pneumoniae infection. Furthermore, inhibition was observed only by infection with viable C. pneumoniae and not by the heat-killed bacteria. These results suggest that C. pneumoniae affects lymphocyte function by inhibiting proliferation and CD25 expression in response to immunological stimulation, possibly via humoral mediator(s).

A Unique Population of Tissue-Memory CD4+ T cells in the Airways after Influenza Infection that is Dependent on the Integrin VLA-1 (45.8)

Abstract During the immune response to influenza infection, activated T cells are distributed to both lymphoid and extralymphoid tissues, including the infected airways where direct recognition of viral antigen-bearing cells takes place. The collagen binding α1β1 integrin VLA-1 is essential for the development of memory CD8+ T cells in the airways but while expressed by some CD4+ T cells, its significance has not been demonstrated. We investigated the role of VLA-1 on virus-specific CD4+ T cells during and after primary or secondary influenza infection of mice. The proportion of CD4+ cells expressing CD49a (α1 integrin) was low in all tissues sampled during primary infection, but increased in the airways after viral clearance. Furthermore, during the first 24hr of a secondary influenza challenge, the majority of IFN-γ secreting effector CD4+ T cells from the airways were in the CD49a+ population. Airway CD49a+ CD4+ cells also expressed reduced markers of apoptosis compared to CD49a- cells, and fewer memory or effector CD4+ cells could be recovered from airways of α1-/- mice, though lymphoid tissues appeared unaffected. These data suggest VLA-1 expression defines a population of tissue-memory CD4+ T cells that act as rapid effectors upon re-infection, and VLA-1 expression is integral to their accumulation in the airways.

Identification of a Unique Population of Tissue-Memory CD4+ T Cells in the Airways after Influenza Infection That Is Dependent on the Integrin VLA-1

During the immune response to influenza infection, activated T cells are distributed to both lymphoid and extralymphoid tissues, including the infected airways where direct recognition of viral Ag-bearing cells takes place. The collagen-binding alpha(1)beta(1) integrin VLA-1 is essential for the development of memory CD8(+) T cells in the airways, and although expressed by some CD4(+) T cells, its significance has not been demonstrated. We investigated the role of VLA-1 on virus-specific CD4(+) T cells during and after primary or secondary influenza infection of mice. The proportion of CD4(+) cells expressing CD49a (alpha(1) integrin) was low in all tissues sampled during primary infection but increased in the airways after viral clearance. Furthermore, during the first 24 h of a secondary influenza challenge, the majority of IFN-gamma-secreting effector CD4(+) T cells from the airways was in the CD49a(+) population. Airway CD49a(+)CD4(+) cells also expressed reduced markers of apoptosis compared with CD49a(-) cells, and fewer memory or effector CD4(+) cells could be recovered from airways of alpha(1)(-/-) mice, although lymphoid tissues appeared unaffected. These data suggest VLA-1 expression defines a population of tissue memory CD4(+) T cells that act as rapid effectors upon reinfection, and VLA-1 expression is integral to their accumulation in the airways.

Expression of CXCR4, VLA-1, LFA-3 and transducer of ERB in G-CSF-mobilised progenitor cells in acute myocardial infarction

G-CSF induced mobilisation of progenitor cells is a multistep processes involving chemokines, growth factors, matrix-degrading enzymes, and cell adhesive interactions mediated by specific receptors on haematopoietic cells. This study's aim was to investigate progenitor cells mobilised during myocardial infarction after treatment with granulocyte-stimulating factor (G-CSF). In the randomised, double-blind, placebo-controlled REVIVAL-2 study, 114 patients with acute myocardial infarction were included. Five days after successful percutaneous coronary intervention patients received either 10 microg/kg G-CSF (n=56) or placebo (n=58) subcutaneously for five days. Venous blood samples were analysed on day(s) 1, 3, 5 and 7 after therapy, and progenitor cell mobilisation and surface expression of VLA-4, LFA-1 and CXCR-4 was measured on circulating progenitor cells using flow cytometry. G-CSF induced a significant increase in circulating progenitor cells (72 +/- 20 cells/microl vs. 4.5 +/- 0.8 cells/microl, p<0.05). Surface expression of LFA-1, VLA-4 and CXCR4 on progenitor cells was decreased by 44%, 49% and 60% after G-CSF as compared to placebo (p<0.05). In accordance, mRNA expression of CXCR4 was reduced. Moreover, anti-proliferative transducer of ERB (TOB) mRNA was decreased, suggesting an increased proliferative potential of the mobilised progenitor cells. Decreased expression of adhesion and chemokine receptors on G-CSF mobilised progenitor cells in acute myocardial infarction may alter the homing capacity of circulating cells to the myocardium.

T cell activation profiles in different granulomatous interstitial lung diseases – a role for CD8+CD28null cells?

Lymphocytes play a crucial role in lung inflammation. Different interstitial lung diseases may show distinct lymphocyte activation profiles. The aim of this study was to examine the expression of a variety of activation markers on T lymphocyte subsets from blood and bronchoalveolar lavage fluid (BALF) of patients with different granulomatous interstitial lung diseases and healthy controls. Bronchoalveolar lavage cells and blood cells from 23 sarcoidosis patients, seven patients with hypersensitivity pneumonitis and 24 healthy controls were analysed. Lymphocyte activation status was determined by flow cytometry. Lymphocytes were stained with antibodies against CD3, CD4, CD8, CD25, CD28, CD69, very late antigen-1 (VLA)-1, VLA-4 and human leucocyte antigen D-related (HLA-DR). In general, CD28, CD69 and VLA-1 expression on BALF CD4+ lymphocytes and HLA-DR expression on BALF CD8+ lymphocytes was different in patients with hypersensitivity pneumonitis and sarcoidosis patients with parenchymal involvement. This BALF lymphocyte phenotype correlated with carbon monoxide diffusing lung capacity (Dlco) values across interstitial lung diseases (ILD) (r2 = 0.48, P = 0.0002). In sarcoidosis patients, CD8+CD28(null) blood lymphocytes correlated with lower Dlco values (r = -0.66, P = 0.004), chronic BALF lymphocyte activation phenotype (r2 = 0.65, P < 0.0001), radiographic staging (stage I versus stage II and higher, P = 0.006) and with the need for corticosteroid treatment (P = 0.001). Higher expression of CD69, VLA-1 and HLA-DR and lower expression of CD28 on BALF lymphocytes suggests prolonged stimulation and chronic lymphocyte activation in patients with ILD. In sarcoidosis, blood CD8+CD28(null) cells might be a new biomarker for disease severity but needs further investigation.

Additive effects of combination treatment with anti-inflammatory and neuroprotective agents in experimental autoimmune encephalomyelitis

We studied the effects of combination treatment with an anti-inflammatory agent, interferon (IFN)-β, and a putative neuroprotective agent, an estrogen receptor (ER)-β ligand, during EAE. Combination treatment significantly attenuated EAE disease severity, preserved axonal densities in spinal cord, and reduced CNS inflammation. Combining ERβ treatment with IFNβ reduced IL-17, while it abrogated IFNβ-mediated increases in Th1 and Th2 cytokines from splenocytes. Additionally, combination treatment reduced VLA-4 expression on CD4 + T cells, while it abrogated IFNβ-mediated decreases in MMP-9. Our data demonstrate that combination treatments can result in complex effects that could not have been predicted based on monotherapy data alone.

High Expression of the Very Late Antigen (VLA)-4 (CD49d) Integrin Predicts for Reduced Risk of Relapse and Better Outcome in Pediatric Acute Myeloid Leukemia (AML): A Report From the Children's Oncology Group.

Abstract 1592▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-618 BackgroundPrevious studies highlighted the importance of the cell adhesion molecule, VLA-4, for chemoresistance and minimal residual disease (MRD) in AML, suggesting promise as therapeutic target. By comparison, the prognostic role of VLA-4 in AML remains controversial with retrospective studies implying either adverse or favorable prognosis. Therefore, we prospectively evaluated VLA-4 expression in participants of a recent Children's Oncology Group (COG) AML pilot protocol. MethodsCOG-AAML03P1 enrolled 340 newly diagnosed children (aged 1 month - 21 years) with de novo non-acute promyelocytic AML, excluding those with Down syndrome, and tested the feasibility of combining gemtuzumab ozogamicin (GO) with intensive induction chemotherapy followed by GO-containing intensification therapy or matched related donor stem cell transplantation; 216 patients submitted diagnostic marrow specimens for flow cytometric determination of VLA-4 expression that was then correlated with patient demographics, laboratory characteristics, and clinical outcome. Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa; n=73), high risk (-5/5q-, monosomy 7, or FLT3/ITD with high allelic ratio; n=25), or standard risk (all other patients with cytogenetic/molecular data; n=101); 17 patients had insufficient data for risk classification. ResultsAmong the 216 diagnostic specimens, the mean fluorescence intensity (MFI) of VLA-4 expression varied over 35-fold from a baseline of 30 to 1110 (median, 219.5). Patients with high VLA-expression (>median MFI) were younger (p<0.001), had a lower prevalence of FLT3/ITD (p=0.002). Presence of extramedullary disease (EMD, chloroma or CNS involvement) was significantly higher in patients with high VLA-4 expression (16% vs. 5%, p=0.013), where 17/22 (77%) of patients with EMD had high VLA-4 expression. We initially inquired whether VLA-4 expression as a continuous variable correlated with disease outcome. We demonstrated that over the range of VLA-4 expression levels, every 10-unit increase in VLA-4 MFI corresponded to a 2% decrease in relapse risk (RR; p=0.023) and 2% increase in disease-free survival (DFS) from end of induction I (p=0.015). We subsequently divided the study patients into two cohorts based on median MFI and determined the clinical outcome per median VLA-4 MFI for the entire cohort as well as in specific risk groups (i.e., high risk, low risk, and standard risk). In the evaluation of the entire cohort, patients with high VLA-4 expression had a significantly superior DFS (67% vs 48%, p=0.023) and lower RR (24% vs 44%, p=0.011) compared to those with lower VLA-4 expression. In subgroup analyses, high VLA-4 expression was associated with significantly superior DFS (69% vs 34%, p=0.011) and lower RR (26% vs 61%, p=0.009) in patients with standard-risk AML but not in patients with high or low risk disease. In multivariate Cox regression analyses that included age, cytogenetics, and molecular prognostic factors, low VLA-4 expression remained significantly associated with elevated RR (hazard ratio for low VLA-4: 2.25, p=0.011). Finally, we determined the impact of MRD in the context of VLA-4 expression. The prevalence of MRD was similar for patients with low or high VLA-4 expression (29% vs. 25%, p=0.70). In patients with low VLA-4 expression, those with MRD had a RR of 75% compared to that of 35% for the MRD negative cohort (p=0.005). Corresponding DFS was 19% and 54% for those with and without MRD (p=0.018), a difference mainly attributable to increased RR. In patients with high VLA-4 expression, those with MRD had a RR of 31% vs. 23% for the MRD-negative patients (p=0.28) with a corresponding DFS of 46% and 75% for the MRD positive and negative patients (p=0.014), a difference mainly explained by higher treatment-related mortality in MRD positive patients. ConclusionThis study demonstrates significant heterogeneity of VLA-4 expression in pediatric AML and its association with EMD and clinical outcome. This study further demonstrates that VLA-4 expression is an independent prognostic factor for clinical outcome and can identify the risk status in patients with no identifiable cytogenetic or molecular risk factors. DisclosuresNo relevant conflicts of interest to declare.

Role of Stromal Microenvironment in Non-Pharmacological Resistance of CML to Imatinib through Lyn/CXCR4 Interactions.

Abstract 4248In patients with chronic-phase chronic myeloid leukemia (CML), imatinib resistance is of increasing importance. We have recently reported that the constitutively activated Bcr-Abl tyrosine kinase in CML suppresses CXCL12/CXCR4-mediated migration of CML cells to the bone marrow (BM) stroma. This finding can explain the characteristic leukocytosis in CML. In turn, tyrosine kinase inhibitor imatinib inhibits Bcr-Abl, enhances migration of CML cells towards CXCL12-producing BM stromal cells which in turn promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). In this study, we further investigated the molecular mechanisms of imatinib-induced CML cell migration and adherence to the bone marrow-derived stromal cells (MSC). Src-related kinase Lyn regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase and is known to interact with CXCL12/CXCR4 signaling. Lyn frequently localizes in lipid raft fractions, which act as signal transduction platforms for a variety of intracellular processes. Therefore, we investigated the effects of imatinib on the localization of activated Lyn in the lipid raft structures of KBM-5 CML cells under co-culture conditions with CXCL12-secreting MSC or recombinant CXCL12. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that CXCR4 and phosphorylated CXCR4 localized in the higher-density detergent-soluble non-raft cell surface regions in KBM5 cells in the presence and absence of imatinib, with or without MSC, which suggests that CXCR4 does not directly associate with lipid rafts. In contrast, Lyn was present both in the low-density raft and in the high-density non-raft fractions, which contained CXCR4. We have further demonstrated co-localization of CXCR4 with Lyn, and their direct interaction was confirmed by co-immunoprecipitation. Notably, the active form of phosphorylated p-LynTyr396 clustered in lipid rafts, while inactive p-LynTyr507 in non-raft fractions. In suspension KBM-5 cultures imatinib depleted both, p-LynTyr396 and p-LynTyr507. In contrast, under MSC co-culture conditions imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396 in lipid rafts, and p-LynTyr396 further accumulated in non-raft fractions, likely associating with CXCR4. Knock-down of Lyn by siRNA, Src inhibitor treatment or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12, indicating the critical role of p-LynTyr396 in cell migration. Since the a4b1 integrin VLA-4 represents a cooperative molecular pathway guiding BM homing in addition to CXCL12/CXCR4, we next investigated the localization and expression of VLA-4 in KBM5 cells. Imatinib decreased VLA-4 protein expression both in lipid raft and non-raft fractions without affecting VLA-4 gene expression levels as determined by quantitative RT-PCR. Interestingly, VLA-4 reduction by imatinib or lipid raft destruction by MbCD did not affect the ability to adhere to fibronectin.In conclusion, these findings demonstrate that under conditions mimicking BM microenvironment imatinib restores CXCL12-dependent migration through interactions between CXCR4 and active p-Lyn Tyr396 in non-raft microdomains of CML cells and that p-Lyn Tyr396 localized in lipid rafts is contributing to the CML cell migration. We propose that while CXCR4 is segregated from lipid raft fractions, imatinib through Bcr-Abl kinase inhibition induces the compartmental changes of multivalent Lyn complex between lipid raft and non-raft fractions, restoring the interactions between Lyn and CXCR4 and stimulating cell migration.Our findings indicate that leukemic BM microenvironment may be involved in imatinib resistance in a subset of CML patients through activation of Lyn kinase, consistent with reported higher clinical activity of Bcr-Abl/Src inhibitor dasatinib in patients with imatinib-resistant CML. We propose that BM stroma cells produce abundant CXCL12 and attract migrating cells through adhesive interactions with the extracellular matrix, which may in turn facilitate lodging into BM niches of imatinib-exposed CML cells and promote non-pharmacological resistance to this agent. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Adhesion Receptor Expression by Acute Myeloid Leukemia (AML) Bone Marrow Derived or Circulating Blasts and AML Stem Cells: The Key to Overcoming Chemoresistance.

Abstract 2658Poster Board II-634Adhesion of AML blasts within the bone marrow microenvironment confers chemotherapy resistance. CXCR4 plays a primary role in the retention of stem cells within the marrow, and is associated with poor prognosis in AML (Rombouts et al Blood 104:550, 2004, Konoplev et al Cancer 109:1152, 2007; Spoo et al Blood 109:786, 2007). A CXCR4 inhibitor enhanced chemotherapy cytotoxicity for human AML cells on stroma (Zeng et al Mol Cancer Ther 5:3113, 2006). VLA4 expression is also correlated with prognosis in AML, and our group and others have shown that disruption of VLA4 enhances chemotherapy cytotoxicity (Matsunaga et al Nat Med 9:1158, 2003; Becker et al Blood 113:866, 2009). However, given the number of potential ligands and receptors involved in the interaction of leukemia cells within the bone marrow stroma, there are likely other important binding sites. Fundamental to determining mechanisms to overcome drug resistance will be identification of all critical interactions responsible for retention and/or adhesion to the bone marrow stroma. We therefore sought to fully characterize all potential adhesive interactions utilized by AML blasts. We also examined the leukemia stem cell population, the small number of leukemia cells that are able to sustain and perpetuate the leukemia. We have now defined the adhesion receptor expression pattern (18 receptors) for populations of AML blasts, including circulating vs. marrow, and putative stem cell populations as defined by surface markers. Bone marrow and peripheral blood samples have been obtained at presentation for a total of 38 patients with AML. We performed flow cytometry analysis, gating on sequential populations of cells by forward scatter/side scatter, CD45 vs. side scatter, CD34+, or CD34+/CD38-/CD123+ populations. Expression levels were measured by percent of positive cells (%EXPR) and mean fluorescence intensity (MFI). Our analysis of adhesion receptor expression by AML blasts revealed that some receptors were expressed by 85%–92% of blasts, including CD11a, CD29, CD44, CD49d, CD49e, CD31; some were expressed by <5%, including CD49a, CD49c, CD106; and others exhibited moderate expression 18–62%, such as L-selectin, ICAM-1, CXCR4, CD49f. For a given patient, there was no significant correlation between CXCR4 % EXPR or MFI and expression of other receptors, such as CD49d, CD49f, or CD29. However, there was significant positive correlation (by Spearman rank order correlation) between %EXPR on marrow vs. circulating blasts from the same patient for CD29 [p=.01], CD11a (LFA-1) [p=.004], CXCR4 [p=.03], CD31 (PECAM-1) [p=.03], L-selectin [p=.007], CD44 [p=.04], while others were different, including CD49d [p=.41], CD49e [p=.08], and CD29f [p=.17] (all two-sided p values). When comparing the whole CD34+ population to the CD34+CD38-CD123+ population, there was no significant difference in MFI for the receptors CD29, CD44, or CD49f, but the MFI of CD49d and CD49e was significantly lower on the more primitive (stem) cells [p=0.003 for each ]. When we examined the CD34+/CD38-/CD123+ population isolated by sequential CD38 depletion then CD34 selection, and gated on CD123+ cells, we found uniform high percent expression >90% for CD49d, CD49f, CD44, and the percent expression of CD49f was also lower for the primitive cells in some patients. Thus, there are several adhesion receptors expressed at high level that may contribute to biological behavior and chemotherapy resistance. Overcoming chemoresistance in leukemia stem cells may require targeting of multiple receptors in order to achieve a clinical response. Disclosures:Becker:Leukemia and Lymphoma Society: Research Funding.