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• W1844711036 abstract "Allograft inflammatory factor (AIF)-1 is a cytoplasmic, calcium-binding protein whose expression in transplanted human hearts correlates with rejection and development of coronary artery vasculopathy (CAV). AIF-1 is constitutively expressed in monocytes/macrophages, but its expression in human lymphocytes has not been described. After immunohistochemical analysis of human coronary arteries with CAV, we identified AIF-1 expression in CD3-positive lymphocytes. AIF-1 was differentially expressed in peripheral blood mononuclear cells and in the T-lymphoblastoid MOLT-4 cell line exposed to various cytokines, suggesting a role for AIF-1 in T-lymphocyte activation. To determine AIF-1 function, MOLT-4 cells were stably transduced by AIF-1 retrovirus. Overexpression of AIF-1 in these cells led to a 238% increase in cell number compared to empty vector controls. AIF-1 polymerized nonmuscle actin and MOLT-4 cells overexpressing AIF-1 migrated 95% more rapidly than empty vector controls. Primary human vascular smooth muscle cells cultured in conditioned media from AIF-1-transduced MOLT-4 cells proliferated 99% more rapidly than vascular smooth muscle cells cultured in conditioned media from empty vector-transduced MOLT-4 cells. These data indicate that AIF-1 is expressed in activated T lymphocytes, that its expression enhances activation of lymphocytes, and that AIF-1 expression in activated lymphocytes may have important ramifications for activation of adjacent arterial vascular smooth muscle cells and development of CAV. Allograft inflammatory factor (AIF)-1 is a cytoplasmic, calcium-binding protein whose expression in transplanted human hearts correlates with rejection and development of coronary artery vasculopathy (CAV). AIF-1 is constitutively expressed in monocytes/macrophages, but its expression in human lymphocytes has not been described. After immunohistochemical analysis of human coronary arteries with CAV, we identified AIF-1 expression in CD3-positive lymphocytes. AIF-1 was differentially expressed in peripheral blood mononuclear cells and in the T-lymphoblastoid MOLT-4 cell line exposed to various cytokines, suggesting a role for AIF-1 in T-lymphocyte activation. To determine AIF-1 function, MOLT-4 cells were stably transduced by AIF-1 retrovirus. Overexpression of AIF-1 in these cells led to a 238% increase in cell number compared to empty vector controls. AIF-1 polymerized nonmuscle actin and MOLT-4 cells overexpressing AIF-1 migrated 95% more rapidly than empty vector controls. Primary human vascular smooth muscle cells cultured in conditioned media from AIF-1-transduced MOLT-4 cells proliferated 99% more rapidly than vascular smooth muscle cells cultured in conditioned media from empty vector-transduced MOLT-4 cells. These data indicate that AIF-1 is expressed in activated T lymphocytes, that its expression enhances activation of lymphocytes, and that AIF-1 expression in activated lymphocytes may have important ramifications for activation of adjacent arterial vascular smooth muscle cells and development of CAV. Although progress in immunosuppressive therapy has been successful in reduction of graft rejection, the vascular narrowing indicative of graft vascular disease remains the major complication that limits long-term survival of solid organ transplantation.1Weis M von Scheidt W Cardiac allograft vasculopathy.Circulation. 1997; 96: 2069-2077Crossref PubMed Scopus (401) Google Scholar In the case of cardiac allografts, this disease is characterized by a diffuse, concentric intimal hyperplasia extending throughout the coronary arterial tree, veins, and capillaries. Consequently, interventions useful for patients with conventional coronary artery disease are not applicable to the majority of patients with coronary artery vasculopathy (CAV) because of its extensive nature. The pathogenesis of CAV is believed to involve a chronic immune response of the recipient to the donor vasculature in which activated recipient immune cells damage the endothelium, resulting in the production of cytokines that elicit activation and proliferation of medial vascular smooth muscle cells (VSMCs).2Barnhart G Pascoe E Mills S Accelerated coronary arteriosclerosis in cardiac transplant recipients.Transplant Rev. 1987; 1: 31-46Abstract Full Text PDF Scopus (46) Google Scholar The activation of VSMCs is responsible for most of the obliterative arterial intimal thickening present in solid organ allografts as well as in CAV.3Libby P Salomon R Payne D Schoen FJ Pober JS Functions of vascular wall cells related to development of transplantation-associated coronary arteriosclerosis.Transplant Proc. 1989; 21: 3677-3684PubMed Google Scholar, 4Ventura HO Mehra MR Smart FW Cardiac allograft vasculopathy: current concepts.Am Heart J. 1995; 129: 791-798Abstract Full Text PDF PubMed Scopus (106) Google ScholarThe important role of T lymphocytes in promotion of CAV, even in the absence of rejection, has been well established. Activated T lymphocytes, in addition to macrophages and VSMCs, make up much of the cellular content of the neointima in CAV vessels.5Dong C Redenbach D Wood S Battistini B Wilson JE McManus BM The pathogenesis of cardiac allograft vasculopathy.Curr Opin Cardiol. 1996; 11: 183-190Crossref PubMed Scopus (35) Google Scholar Products of activated T cells, granzyme A and perforin, have been identified as markers of rejection and compromised cardiac function in transplanted human hearts.6Alpert S Lewis NP Ross H Fowler M Valantine HA The relationship of granzyme A and perforin expression to cardiac allograft rejection and dysfunction.Transplantation. 1995; 60: 1478-1485Crossref PubMed Scopus (28) Google Scholar In animals, it has been shown that increasing the frequency of donor-reactive T cells did not mediate acute rejection but did increase the rate and severity of transplant vasculopathy.7He C Schenk S Zhang Q Valujskikh A Bayer J Fairchild RL Heeger PS Effects of T cell frequency and graft size on transplant outcome in mice.J Immunol. 2004; 172: 240-247PubMed Google Scholar Several studies have demonstrated that T-helper cells play an essential function in development and severity of neointimal thickening associated with allograft vasculopathy,8Yun JJ Fischbein MP Whiting D Irie Y Fishbein MC Burdick MD Belperio J Strieter RM Laks H Berliner JA Ardehali A The role of MIG/CXCL9 in cardiac allograft vasculopathy.Am J Pathol. 2002; 161: 1307-1313Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Yamada A Laufer TM Gerth AJ Chase CM Colvin RB Russell PS Sayegh MH Auchincloss Jr, H Further analysis of the T-cell subsets and pathways of murine cardiac allograft rejection.Am J Transplant. 2003; 3: 23-27Crossref PubMed Scopus (43) Google Scholar, 10Szeto WY Krasinskas AM Kreisel D Krupnick AS Popma SH Rosengard BR Depletion of recipient CD4+ but not CD8+ T lymphocytes prevents the development of cardiac allograft vasculopathy.Transplantation. 2002; 73: 1116-1122Crossref PubMed Scopus (37) Google Scholar suggesting a fundamental role in activation of vascular cells. Consequently, identification and characterization of proteins that link vascular/immune communication may elucidate the molecular mechanisms of many vascular diseases.Allograft inflammatory factor-1 (AIF-1) is a 143-amino acid, cytoplasmic, evolutionarily conserved, calcium-binding protein. AIF-1 is constitutively expressed in macrophages and glial cells, and has been implicated in the inflammatory process of several cell types. Data from several groups in diverse systems advocate an important role for AIF-1 in inflammatory processes ranging from expression in infiltrating macrophages in rat cardiac allografts,11Utans U Arceci R Yamashita Y Russell ME Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection.J Clin Invest. 1995; 95: 2954-2962Crossref PubMed Scopus (237) Google Scholar in microglial injury and activation,12Schluesener HJ Seid K Meyermann R Effects of autoantigen and dexamethasone treatment on expression of endothelial-monocyte activating polypeptide II and allograft-inflammatory factor-1 by activated macrophages and microglial cells in lesions of experimental autoimmune encephalomyelitis, neuritis, and uveitis.Acta Neuropathol. 1999; 97: 119-126Crossref PubMed Scopus (60) Google Scholar and in the allograft response of phylogenetically distant species such as carp and marine sponges.13Fukiki K Shin DH Nakao M Yano T Molecular cloning of carp CC chemokine CXC, chemokine receptors, allograft inflammatory factor-1, and natural killer cell enhancing factor by use of suppression subtractive hybridization.Immunogenetics. 1999; 49: 909-914Crossref PubMed Scopus (120) Google Scholar, 14Kruse M Steffen R Batel R Muler I Muller W Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto-and allograft response in marine sponges.J Cell Sci. 1999; 112: 4305-4313Crossref PubMed Google Scholar In humans, the AIF-1 gene maps to a the major histocompatibility complex class III region on chromosome 6p21.3, which is known for clusters of genes involved in the inflammatory response. Although constitutively expressed in lymphoid tissue, we have determined that AIF-1 is not expressed in VSMCs, but can be induced by injury and inflammatory cytokines.15Autieri MV Mu A Carbone C Expression of allograft inflammatory factor-1 (AIF1) is a marker of activated human VSMC and arterial injury.Arterioscler Thromb Vasc Biol. 2000; 20: 1737-1744Crossref PubMed Scopus (77) Google Scholar Overexpression of AIF-1in VSMCs leads to increased proliferation and migration, and expression of AIF-1 in cardiac allografts is associated with the severity of CAV.16Autieri MV Carbone CM Over expression of allograft inflammatory factor-1 promotes proliferation of vascular smooth muscle cells by cell cycle deregulation.Arterioscler Thromb Vasc Biol. 2001; 21: 1421-1426Crossref PubMed Scopus (45) Google Scholar, 17Autieri MV Kelemen SE Wendt KW AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.Circ Res. 2003; 92: 1107-1114Crossref PubMed Scopus (61) Google Scholar, 18Autieri MV Kelemen SE Thomas BA Feler ED Goldman BI Eisen HJ Allograft inflammatory factor-1 (AIF-1) expression correlates with cardiac rejection and development of cardiac allograft vasculopathy.Circulation. 2002; 106: 2218-2223Crossref PubMed Scopus (58) Google ScholarThe function of AIF-1 in T lymphocytes has not been reported, nor have the functional consequences of lymphocyte expression on local VSMC pathophysiology been explored. In this study we determine that AIF-1 is expressed in infiltrating T lymphocytes in human coronary arteries with CAV as well as in cytokine-activated cultured lymphocytes. The purpose of this study is to determine the functional significance of AIF-1 expression in lymphocytes, and determine how this expression might impact development of CAV. In this article, we describe AIF-1 expression in CD3-positive lymphocytes in human coronary arteries with CAV, and characterize the effects of AIF-1 overexpression on T-lymphocyte migration and proliferation. We also determine the effects of AIF-1-activated lymphocytes on human VSMC function.Materials and MethodsImmunohistochemistryCoronary arteries from transplanted hearts with CAV were used in this study. All tissue procurement protocols were approved by the Institutional Review Board for Human Studies at Temple University. Tissue sections were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 μm, deparaffinized in xylene, and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 1.5% hydrogen peroxide in methanol for 15 minutes. After blocking with normal serum, sections were incubated with primary antibodies for 1 hour at room temperature. Antibody for CD3 (T-lymphocyte marker) was used at a concentration of 2 μg/ml. AIF-1 antibody, which has been previously described, was used at 1.0 μg/ml.18Autieri MV Kelemen SE Thomas BA Feler ED Goldman BI Eisen HJ Allograft inflammatory factor-1 (AIF-1) expression correlates with cardiac rejection and development of cardiac allograft vasculopathy.Circulation. 2002; 106: 2218-2223Crossref PubMed Scopus (58) Google Scholar Sections were then incubated with biotinylated secondary antibody (1:200) followed by avidin-biotin-peroxidase complex in the Vectastain Elite kit (both from Vector Laboratories, Burlingame, CA). The reaction product was visualized using 3,3′-diaminobenzidine (Vector Laboratories) as the chromogenic substrate, producing a reddish-brown stain. The sections were counterstained with hematoxylin.Cells and CulturePrimary human coronary VSMCs were obtained as cryopreserved secondary culture from Cascade Corp. (Portland, OR) and subcultured as described.17Autieri MV Kelemen SE Wendt KW AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.Circ Res. 2003; 92: 1107-1114Crossref PubMed Scopus (61) Google Scholar Cells from passage 3 to 6 were used in the described studies. All cytokines and phytohemagglutinin A (PHA) were purchased from Sigma (St. Louis, MO). MOLT-4 cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI medium supplemented with 10% fetal calf serum. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy human donors by venipuncture, isolated by Ficoll-Hypaque gradient centrifugation, and cultured as for MOLT-4 cells.Retroviral Stable Transduction and Proliferation AssayAIF-1 retrovirus (AIF-1RV) was constructed using a kit from Clontech (La Jolla, CA) according to the manufacturer's instructions as previously described.17Autieri MV Kelemen SE Wendt KW AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.Circ Res. 2003; 92: 1107-1114Crossref PubMed Scopus (61) Google Scholar Briefly, the protein-coding region of the AIF-1 cDNA was inserted into the retroviral packaging vector pLXSN containing the gentimycin resistance gene. For some samples, cDNA coding for the 9-amino acid hemagglutinin antigen (HA) tag was inserted 3′ to the AIF-1 sequence. This was transfected into an ectotropic 293-derived packaging cell line.19Yee JK Friedmann T Burns JC Generation of high-titer pseudotyped retroviral vectors with very broad host range.Methods Cell Biol. 1994; 43: 99-112Crossref PubMed Scopus (392) Google Scholar Media was then collected from these cells and used to infect an amphitropic second packaging cell line, PT-67, according to the manufacturer's instructions. This supernatant containing recombinant high-titer virus was then used to infect MOLT-4 cells in two 4-hour exposures of viral supernatant in the presence of 8 μg/ml of Polybrene (∼40 to 50% stable transduction was achieved). Stably transduced G418-resistant cells were pooled from each transduction rather than individual clones to avoid the effects of clonal variation. For proliferation assays, equal numbers of stable transfectants were seeded onto 12-well plates at a density of 7500 cells/ml. Medium was changed on the 4th day, and after 1, 4, and 7 days, viable, trypan blue-excluding cells were counted using a standard hemocytometer. Statistical analyses from at least three independent experiments were performed by paired sample t-test.Migration and ChemotaxisFor analysis of chemotaxis, 6.5-mm diameter transwell Boyden chamber plates (Costar, Cambridge, MA) with 8-μm polycarbonate membrane pore size were seeded with stably transduced MOLT-4 cells (50,000 cells per membrane) in 600 μl of medium containing 0.5% fetal calf serum as described.20Grabski R Dewit J De Braekeleer J Malicka-Blaskiewicz M De Baetselier P Verschueren H Inhibition of T-cell invasion across cultured fibroblast monolayers by phenothiazine-related calmodulin inhibitors: impairment of lymphocyte motility by trifluoperazine and chlorpromazine, and alteration of the monolayer by pimozide.Biochem Pharmacol. 2001; 61: 1313-1317Crossref PubMed Scopus (10) Google Scholar Sixty μl of fetal calf serum was placed in the lower chamber and cells were incubated for the indicated times at 37°C, at which time cells in the lower chamber were recovered and counted in a hemocytometer. Experiments were performed in triplicate from three independently derived stably transduced groups of MOLT-4 cells.Western BlottingMOLT-4 cell extracts were prepared and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and protein transfer were performed as described.2Barnhart G Pascoe E Mills S Accelerated coronary arteriosclerosis in cardiac transplant recipients.Transplant Rev. 1987; 1: 31-46Abstract Full Text PDF Scopus (46) Google Scholar Membranes were incubated with a 1:2000 dilution of AIF-1 primary antibody, and a 1:2000 dilution of secondary antibody. Equal protein concentrations of cell extracts were determined by Bradford assay and equal loading on gels were verified by Ponceau S staining of the membrane. Reactive proteins were visualized using enhanced chemiluminescence.Actin Interaction and PolymerizationThe AIF-1 protein-coding region was cloned into the pGEX vector and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein according to the manufacturer's protocol (Pharmacia, Inc., Uppsala, Sweden). The affinity pull-down and actin polymerization assays were performed as described.15Autieri MV Mu A Carbone C Expression of allograft inflammatory factor-1 (AIF1) is a marker of activated human VSMC and arterial injury.Arterioscler Thromb Vasc Biol. 2000; 20: 1737-1744Crossref PubMed Scopus (77) Google Scholar Briefly, extracts from MOLT-4 cells were first precleared with GST Sepharose beads, and then incubated with GST Sepharose beads (lane 3), or with AIF-1/GST Sepharose beads (lane 4). Pellets were washed and interacting proteins separated by SDS-PAGE and identified by Western blot with anti-actin antibody. In vitro nonmuscle actin polymerization was determined by differential centrifugation in a kit purchased from Cytoskeleton, Inc. (Denver, CO), according to the manufacturer's instructions. Briefly, 23 μmol/L of AIF-1 and nonmuscle actin were incubated together for 30 minutes in F-actin buffer containing ATP and Ca2+, then centrifuged at 8000 × g, the speed at which only cross-linked actin will pellet. Protein in the pellets and supernatants were analyzed by SDS-PAGE, Coomassie staining, and densitometry using NIH Image software.ResultsAIF-1 Is Expressed in CD3-Positive LymphocytesSeveral different cell types are involved in the pathogenesis and progression of CAV.1Weis M von Scheidt W Cardiac allograft vasculopathy.Circulation. 1997; 96: 2069-2077Crossref PubMed Scopus (401) Google Scholar Because AIF-1 expression has been linked to the development and severity of CAV, it was important to determine which cell populations expressed this protein. AIF-1 expression in activated VSMCs and monocytes has been previously reported. In this study, immunohistochemical analysis of human coronary arteries with CAV determined AIF-1 expression in CD3-positive lymphocytes (Figure 1A). Expression of AIF-1 in human lymphocytes has not been described, and subsequent experiments were designed to characterize this expression and its functional implications in T-lymphocyte pathophysiological processes.AIF-1 Is Differentially Expressed by Cytokine StimulationDifferential cytokine induction of AIF-1 has been described in monocytes and VSMCs.11Utans U Arceci R Yamashita Y Russell ME Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection.J Clin Invest. 1995; 95: 2954-2962Crossref PubMed Scopus (237) Google Scholar, 15Autieri MV Mu A Carbone C Expression of allograft inflammatory factor-1 (AIF1) is a marker of activated human VSMC and arterial injury.Arterioscler Thromb Vasc Biol. 2000; 20: 1737-1744Crossref PubMed Scopus (77) Google Scholar To determine whether AIF-1 expression could be modulated by cytokines in lymphocytes, we challenged normal, human PBMCs and the human T-lymphoblastoid cell line MOLT-4 to various cytokines, and examined AIF-1 expression by Western blot. Several similarities in sensitivity to stimuli and degree of AIF-1 expression by these stimuli were noted between these cells. Figure 2 shows a constitutive expression of AIF-1 in both unstimulated PMBCs and MOLT-4 cells. T-lymphocyte-conditioned medium and PHA elicited the strongest induction of AIF-1 expression, with mean increases of 4.4- and 4.8-fold for conditioned medium, and 3.8- and 4.1-fold for PHA, in PBMCs and MOLT-4 cells, respectively. It is interesting to note that AIF-1 expression paralleled proliferation of these cells, as both of these stimuli induced proliferating cell nuclear antigen to the greatest degree. Although both of these cell populations express AIF-1 and displayed a similar response to stimuli, MOLT-4 cells were used as a surrogate to further characterize the role of AIF-1 expression in T-lymphocyte activation because they are a homogenous cell population and responses to stimuli would not vary from experiment to experiment, and more importantly, because they are immortalized, which facilitates stable transduction for long-term functional studies.Figure 2Expression of AIF-1 protein in activated human lymphocytes. Human PBMCs and the human T-myeloblast cell line, MOLT-4, were serum-starved for 48 hours, and protein from 1) serum-starved, and cells treated for 40 hours with 2) 15% fetal calf serum; 3) T-cell conditioned media; 4) PHA; 5) transforming growth factor-β; and 6) tumor necrosis factor-α. Extracts from these cells were subjected to Western blot with anti-AIF-1, proliferating cell nuclear antigen, CD3, and GAPDH antibody. Bands were quantitated by densitometry, and normalized to GAPDH. Blot shown is representative of three performed from different groups of PMBC and MOLT-4 cells. Bar graph indicates percent expression above unstimulated cells for three groups of experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AIF-1 Expression Enhances T-Lymphocyte ProliferationTo characterize the functional effects of AIF-1 expression in lymphocytes, we overexpressed AIF-1 by retroviral (RV) gene transduction in MOLT-4 cells and examined various parameters of T-lymphocyte activation including proliferation and migration. MOLT-4 cells were transduced with AIF-1 RV containing the protein-coding region of AIF-1 cDNA (AIF-1-RV) tagged with the hemagglutinin epitope (HA) or with vector alone (empty RV), and stable transductants were selected with antibiotic and pooled. Constitutive and retroviral delivered AIF-1 protein expression was verified by Western blot (Figure 3A). To examine effects on proliferation, equal numbers of stable transductants were seeded into 12-well plates, and after 1, 4, and 7 days, viable, trypan blue-excluding cells were trypsinized and counted. Figure 3B shows that MOLT-4 cells that constitutively overexpress AIF-1 demonstrate a mean 238% increase in proliferation compared with control cells (4.3 × 105 versus 1.2 × 105, for AIF-1 and empty vector, respectively) (P < 0.001). Table 1 demonstrates the population doubling times for four separate experiments that were calculated and overall, AIF-1-expressing MOLT-4 cells have a 41% decrease in doubling time (47.8 ± 4.3 hours for empty vector, and 28.2 ± 0.8 hours for AIF-1, respectively). These differences are not due to an increase in viability of AIF-1-expressing cells because both control and AIF-1-overexpressing cells had less than 5% trypan blue-positive cells. Together, these results indicate that increased expression of AIF-1 leads to increased proliferation of MOLT-4 cells.Figure 3AIF-1 mediated proliferation of MOLT-4 cells. A: Stable expression of AIF-1 protein in MOLT-4 cells. The human T-myeloblast cell line, MOLT-4, was transduced with retrovirus containing AIF-1 tagged with the hemagglutinin (HA) or empty vector retrovirus, and selected with antibiotic. Resistant populations were pooled and subjected to Western blot with anti-AIF-1 to determine endogenously expressed, or HA tag antibody to identify virally transduced AIF-1. CD3 antibody was used as a loading control. Blot shown is representative of three performed from different groups of stably transduced MOLT-4 cells. B: Overexpression of AIF-1 results in enhanced MOLT-4 proliferation. Equal numbers of pooled MOLT-4 cells stably transduced with AIF-1 retrovirus or empty vector were seeded into 12-well plates and grown in growth medium. After 1, 4, and 7 days, viable trypan blue-excluding cells were harvested and counted in triplicate. Viability was greater than 95% for both empty vector and AIF-1-transduced cells. Numbers on the y-axis indicate cells per well. Values are means of three independent transfections with similar results (P < 0.001). Error bars are SEM from three independent transfections each performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Calculated Doubling Times of Stably Transduced MOLT-4 CellsExperiment1234Mean±SDEmpty vector49.743.643.954.147.8 ± 4.3AIF-127.028.828.029.128.2 ± 0.8Equal numbers of pooled, stably transduced MOLT-4 were seeded into 12-well plates. Media was changed on the 4th day, and after 1, 4, and 7 days, cells were trypsinized and counted using a standard hemocytometer. Doubling times were calculated as described in Materials and Methods. Open table in a new tab AIF-1 Interacts with and Polymerizes Nonmuscle ActinIn human VSMCs, we previously found AIF-1 to interact with and polymerize F-actin, and we hypothesized that AIF-1 would have the same effects in lymphocytes.17Autieri MV Kelemen SE Wendt KW AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.Circ Res. 2003; 92: 1107-1114Crossref PubMed Scopus (61) Google Scholar Protein extracts from MOLT-4 cells were precleared with GST protein, then incubated with an affinity resin consisting of a recombinant AIF-1-GST fusion protein coupled to glutathione Sepharose beads or GST Sepharose beads as a negative control. The beads were washed, interacting proteins were separated by SDS-PAGE, and actin interaction identified by Western blot (Figure 4A). To verify this interaction and determine the effect of AIF-1 on actin dynamics, purified nonmuscle cell F-actin was incubated with recombinant AIF-1 protein, and after incubation, F-actin was pelleted at low speed (8000 × g), which allows pelleting only of heavy, cross-linked F-actin. Controls for this experiment included α-actinin, a known actin crosslinking protein, and bovine serum albumin, which does not interact with or crosslink actin.21Dubreuil RR Structure and evolution of the actin crosslinking proteins.BioEssays. 1991; 13: 219-226Crossref PubMed Scopus (66) Google Scholar This experiment demonstrates that the majority of actin was recovered in the pellet, along with a portion of interacting AIF-1 (Figure 4B). Overall, these data indicate that AIF-1 binds to and has a polymerizing effect on nonmuscle actin.Figure 4AIF-1 interacts with and polymerizes nonmuscle actin. A: Extracts from MOLT-4 cells were incubated overnight with recombinant AIF-1/GST fusion protein. Beads were washed, boiled, and interacting proteins were separated by SDS-PAGE. Actin was identified by Western blotting with anti-pan actin antibody. B: AIF-1 polymerization of nonmuscle actin. Nonmuscle F-actin was incubated with 23 μg of recombinant AIF-1 protein; bovine serum albumin (BSA), which does not bind actin; and α-actinin, an actin polymerizing protein (included in the kit and were used as negative and positive controls, respectively) for 30 minutes, and then centrifuged at 8000 × g. Supernatant (s) and pellets (p) were collected and proteins separated by SDS-PAGE and identified by Coomassie blue staining. Presence of actin in the pellet indicates polymerization.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AIF-1 Expression Enhances Lymphocyte MigrationBecause cytoskeletal rearrangement and actin polymerization are involved in cell motility, we examined the possibility that migration of MOLT-4 cells would be altered by AIF-1 overexpression. Figure 5 demonstrates that AIF-1-overexpressing MOLT-4 cells migrate 381% and 315% more rapidly at 4 and 10 hours, respectively, than do empty vector control cells (P < 0.05 and 0.01). This is consistent with previous studies showing VSMCs that overexpress AIF-1 migrate more rapidly in response to platelet-derived growth factor.17Autieri MV Kelemen SE Wendt KW AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.Circ Res. 2003; 92: 1107-1114Crossref PubMed Scopus (61) Google ScholarFigure 5AIF-1 expression enhances lymphocyte chemotaxis. Stably transduced MOLT-4 cells were seeded onto Boyden chamber membranes and exposed to 10% fetal calf serum for the times indicated. MOLT-4 cells that traversed the membrane into the lower chamber were collected and counted. Values are means from experiments performed in triplicate from three independent, re" @default.
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• W1844711036 title "Expression of Allograft Inflammatory Factor-1 in T Lymphocytes" @default.
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