Two-chamber Assay Research Articles

Senescent Human Pancreatic Stellate Cells Secrete CXCR2 Agonist CXCLs to Promote Proliferation and Migration of Human Pancreatic Cancer AsPC-1 and MIAPaCa-2 Cell Lines.

Interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) play an important role in the progression of pancreatic cancer. Recent studies have shown that cellular senescence and senescence-associated secretory phenotype factors play roles in the progression of cancer. This study aimed to clarify the effects of senescence-induced PSCs on pancreatic cancer cells. Senescence was induced in primary-cultured human PSCs (hPSCs) through treatment with hydrogen peroxide or gemcitabine. Microarray and Gene Ontology analyses showed the alterations in genes and pathways related to cellular senescence and senescence-associated secretory phenotype factors, including the upregulation of C-X-C motif chemokine ligand (CXCL)-1, CXCL2, and CXCL3 through the induction of senescence in hPSCs. Conditioned media of senescent hPSCs increased the proliferation—as found in an assessment with a BrdU incorporation assay—and migration—as found in an assessment with wound-healing and two-chamber assays—of pancreatic cancer AsPC-1 and MIAPaca-2 cell lines. SB225002, a selective CXCR2 antagonist, and SCH-527123, a CXCR1/CXCR2 antagonist, attenuated the effects of conditioned media of senescent hPSCs on the proliferation and migration of pancreatic cancer cells. These results suggest a role of CXCLs as senescence-associated secretory phenotype factors in the interaction between senescent hPSCs and pancreatic cancer cells. Senescent PSCs might be novel therapeutic targets for pancreatic cancer.

δ-Catenin Promotes Bevacizumab-Induced Glioma Invasion.

The combination of bevacizumab with temozolomide and radiotherapy was shown to prolong progression-free survival in newly diagnosed patients with glioblastoma, and this emphasizes the potential of bevacizumab as a glioma treatment. However, although bevacizumab effectively inhibits angiogenesis, it has also been reported to induce invasive proliferation. This study examined gene expression in glioma cells to investigate the mechanisms of bevacizumab-induced invasion. We made a human glioma U87ΔEGFR cell xenograft model by stereotactically injecting these cells into the brain of animals. We administered bevacizumab intraperitoneally three times per week. At 18 days after tumor implantation, the brains were removed for histopathology and mRNA was extracted. In vivo, bevacizumab treatment increased glioma cell invasion. qRT-PCR array analysis revealed upregulation of δ-catenin (CTNND2) and several other factors. In vitro, bevacizumab treatment upregulated δ-catenin expression. A low concentration of bevacizumab was not cytotoxic, but tumor cell motility was increased in scratch wound assays and two-chamber assays. Overexpression of δ-catenin increased the tumor invasion in vitro and in vivo However, δ-catenin knockdown decreased glioma cell invasiveness. The depth of tumor invasion in the U87ΔEGFR cells expressing δ-catenin was significantly increased compared with empty vector-transfected cells. The increase in invasive capacity induced by bevacizumab therapy was associated with upregulation of δ-catenin expression in invasive tumor cells. This finding suggests that δ-catenin is related to tumor invasion and migration.

Schizandrin A exerts anti-tumor effects on A375 cells by down-regulating H19.

Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.

IL-6/STAT3 Plays a Regulatory Role in the Interaction Between Pancreatic Stellate Cells and Cancer Cells.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis, a characteristic feature of pancreatic cancer. Although it is still controversial, previous studies have suggested that PSCs promote the progression of pancreatic cancer by regulating the cell functions of cancer cells. PSCs produce large amounts of IL-6, which promotes the accumulation of myeloid-derived suppressor cells via a signal transducers and activator of transcription 3 (STAT3)-dependent mechanism. But the role of IL-6/STAT3 pathway in the interaction between PSCs and pancreatic cancer cells remains largely unknown. To clarify the role of IL-6/STAT3 in the interaction between PSCs and cancer cells. Human pancreatic cancer cells (Panc-1 and SUIT-2 cells) were treated with conditioned medium of immortalized human PSCs (PSC-CM). The effects of PSC-CM and IL-6 neutralization on the mRNA expression profiles were examined using Agilent's microarray. Activation of STAT3 was assessed by Western blotting using an anti-phospho-specific antibody. Cellular migration was examined by a two-chamber assay. The expression of markers related to epithelial-mesenchymal transition (EMT) was assessed by real-time reverse transcription PCR. PSC-CM induced the activation of STAT3 in pancreatic cancer cells. Neutralization of IL-6 suppressed the PSC-CM-induced upregulation of genes including complement factor B, lipocalin, and chemokine (C-C motif) ligand 20. Inhibition of IL-6/STAT3 pathway by anti-IL-6 antibody or a STAT3 inhibitor (NSC74859) inhibited the PSC-CM-induced migration and the expression of EMT-related markers (Snail and cadherin-2) in pancreatic cancer cells. IL-6/STAT3 pathway regulates the PSC-induced EMT and alterations in gene expression in pancreatic cancer cells.

The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins.

The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinson's disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.

CD73 expression in RPE cells is associated with the suppression of conventional CD4 cell proliferation

CD73 is intensively involved in the regulation of immune responses through the conversion of pro-inflammatory ATP to immunosuppressive adenosine. Herein, we clarified whether cells in the retina express CD73 and participate in the regulation of inflammatory eye diseases such as experimental autoimmune uveitis (EAU). First, immunofluorescence staining was performed to compare the distribution of CD73+ cells in the retinas of EAU-induced and normal B10RIII mice. The results revealed that a layer of cells in the normal retina that was consistent with the location of retinal pigment epithelial (RPE) cells strongly expressing CD73, and the expression was markedly reduced in the presence of EAU. Thereafter, EAU was also induced in C57BL/6 mice by active immunization or adoptive transfer. CD73 expression in isolated RPE cells was assessed by real-time RT-PCR and western blotting, and the catalytic abilities of the cells to convert AMP to adenosine were determined using HPLC analyses. Compared to the normal control, significantly decreased CD73 expression and AMP catalytic ability were found in the RPE cells isolated from inflamed eyes. CD73 expression and activity were also studied in cultured RPE cells treated with different stimuli, such as Toll-like receptor ligands and cytokines. Highly varied functional CD73 expression was observed in RPE cells through cytokines or Toll-like receptor agonist treatments. Finally, whether RPE cells could regulate the immune response, particularly the proliferation of CD4 cells, through surface-expressed CD73 was determined using a two-chamber assay. The robust inhibition of conventional T-cell proliferation was uniquely observed when CD73+ RPE cells in the upper chamber were in the presence of AMP. To further confirm the function of CD73 in RPE cells, Cd73−/− RPE cells were isolated, and CD73-rescued control cells were constructed. CD73+Cd73−/− RPE, not Cd73−/− RPE, significantly suppressed interacted CD4 cells proliferation and cytokine production. Taken together, these data suggest that naive RPE cells suppressed the immune response through their high expression of CD73. The expression of CD73 in RPE cells could be regulated through many factors, and down-regulated CD73 expression attenuated the suppressive effect of RPE on the proliferation of conventional CD4 cells.

α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma

Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, α-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with α-parvin expression. Twenty-one (37.5 %) ILCs stained positive for α-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). α-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. α-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that α-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.

PRL-3 Regulates Myeloma Cell Survival, Proliferation, Adhesion and Migration

AbstractAbstract 1321 Introduction:Multiple myeloma (MM) is a neoplastic monoclonal proliferation of bone marrow plasma cells. Despite advances in treatment in recent years, MM is still a fatal disease. Phosphatase of regenerating liver-3 (PRL-3) is a protein expressed in primary MM cells and MM cell lines, but not in normal plasma cells. A recent study showed that siRNA against PRL-3 suppresses MM-cell migration (Fagerli et al, 2008) and another study has identified PRL-3 as a marker gene for a subgroup of patients with MM (Broyl et al, 2010). Methods:The human myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226 were used in this study. Apoptosis was measured by Annexin V-FITC binding by flow cytometry, proliferation was measured by methyl-3H-thymidine incorporation, migration against a stromal cell derived factor-1α (SDF-1α) gradient was studied in a Transwell two-chamber assay and adhesion was measured as percentage adhered cells to fibronectin after stimulation with the pro-adhesive cytokines hepatocyte growth factor (HGF), insulin like growth factor-1 (IGF-1) and SDF-1α. RT-PCR and Western blotting were used to measure expression of pro- and anti-apoptotic proteins. Western blotting was used to map intracellular signaling pathways. INA-6 cells stably expressing exogenous PRL-3 were generated using retroviral transduction. The small molecular inhibitor PRL-3 Inhibitor I was used for PRL-3 inhibition. Results:PRL-3 inhibitor I reduced survival of the myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226. IC50 was between 15 and 50 μM depending on cell line. Treatment with the inhibitor down-regulated the anti-apoptotic protein Mcl-1 and led to activation of the intrinsic apoptotic pathway. Inhibition of PRL-3 reduced interleukin (IL)-6-induced phosphorylation of STAT-3. Treatment with 10 μM of PRL-3 inhibitor I also significantly reduced migration against a SDF-1α gradient and HGF-, IGF-1- and SDF-1α -mediated adhesion of the cell line INA-6. By retroviral transduction we made PRL-3-overexpressing INA-6 cells. INA-6 cells are dependent on IL-6 to grow, but overexpression of PRL-3 led to a major increase in cell proliferation even in the absence of IL-6 as well as increased survival at suboptimal concentrations of IL-6. Conclusion:PRL-3 is expressed in MM cells but not in normal plasma cells and can represent a cancer-specific target. Overexpression of PRL-3 in the IL-6-dependent human myeloma cell line INA-6 renders the cells less dependent of IL-6 and increases survival and proliferation. On the other hand, the use of an inhibitor against PRL-3 significantly decreases survival of five MM-cell lines and reduces adhesion and migration of the cell line INA-6. IL-6-STAT3 signaling is important in MM cell survival and proliferation and we here show that PRL-3 possibly influences cell survival as a positive regulator of this signaling pathway. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

Aspirin and Heparin Effect on Basal and Antiphospholipid Antibody Modulation of Trophoblast Function

Low molecular weight (LMW) heparin, with or without aspirin (acetylsalicylic acid [ASA]), is used to prevent complications in antiphospholipid syndrome in pregnancy. Our objective was to elucidate the actions of low-dose LMW heparin and ASA on basal and antiphospholipid antibody-induced modulation of trophoblast function. The human first-trimester trophoblast cell line (HTR-8) was treated with or without antiphospholipid antibody in the presence of no medication, low-dose LMW heparin, low-dose ASA, or combination therapy. Interleukin (IL)-6, IL-8, IL-1β, growth-regulated oncogene-α, vascular endothelial growth factor (VEGF), placental growth factor, soluble FMS-like tyrosine kinase-1, and soluble endoglin were measured in the supernatant. Cell migration was performed using a two-chamber assay. Low molecular weight heparin improved basal trophoblast migration and induced potent increases in growth-regulated oncogene-α and soluble FMS-like tyrosine kinase-1. Aspirin did not affect basal function. Combined therapy promoted migration but did not reverse the LMW heparin-induced soluble FMS-like tyrosine kinase-1 effect. Antiphospholipid antibody increased IL-8, IL-1β, growth-regulated oncogene-alpha, VEGF, placental growth factor, and soluble endoglin secretion, while decreasing cell migration and IL-6 and soluble FMS-like tyrosine kinase-1 secretion. The antiphospholipid antibody-induced cytokine changes were best reversed with LMW heparin, with partial reversal of IL-8 and IL-1β upregulation. The antiphospholipid antibody-induced angiogenic changes were worsened by LMW heparin, with increased soluble FMS-like tyrosine kinase-1 secretion. The therapies did not reverse antiphospholipid antibody-induced decrease in migration. In the absence of antiphospholipid antibodies, LMW heparin induces potentially detrimental proinflammatory and antiangiogenic profile in the trophoblast. In the presence of antiphospholipid antibodies, single-agent LMW heparin may be the optimal therapy to counter trophoblast inflammation, but also induces an antiangiogenic response. These findings may explain the inability of current therapies to consistently prevent adverse outcomes.

Natural hemozoin stimulates syncytiotrophoblast to secrete chemokines and recruit peripheral blood mononuclear cells

BackgroundPlacental malaria is associated with local accumulation of parasitized erythrocytes, deposition of the parasite hemoglobin metabolite, hemozoin, and accumulation of mononuclear cells in the intervillous space. Fetal syncytiotrophoblast cells in contact with maternal blood are known to respond immunologically to cytoadherent Plasmodium falciparum-infected erythrocytes, but their responsiveness to hemozoin, a potent pro-inflammatory stimulator of monocytes, macrophages and dendritic cells, is not known. MethodsThe biochemical and immunological changes induced in primary syncytiotrophoblast by natural hemozoin was assessed. Changes in syncytiotrophoblast mitogen-activated protein kinase activation was assessed by immunoblotting and secreted cytokine and chemokine proteins were assayed by ELISA. Chemotaxis of peripheral blood mononuclear cells was assessed using a two-chamber assay system and flow cytometry was used to assess the activation of primary monocytes by hemozoin-stimulated syncytiotrophoblast conditioned medium. ResultsHemozoin stimulation induced ERK1/2 phosphorylation. Treated cells secreted CXCL8, CCL3, CCL4, and tumor necrosis factor and released soluble intercellular adhesion molecule-1. Furthermore, the dependence of the hemozoin responses on ERK1/2 stimulation was confirmed by inhibition of chemokine release in syncytiotrophoblast treated with an ERK pathway inhibitor. Hemozoin-stimulated cells elicited the specific migration of PBMCs, and conditioned medium from the cells induced the upregulation of intercellular adhesion molecule-1 on primary monocytes. ConclusionsThese findings confirm an immunostimulatory role for hemozoin and expand the cell types known to be responsive to hemozoin to include fetal syncytiotrophoblast. The results provide further evidence that syncytiotrophoblast cells can influence the local maternal immune response to placental malaria.

Antiphospholipid antibodies limit trophoblast migration by reducing IL-6 production and STAT3 activity

PROBLEM: Women with antiphospholipid antibodies (aPL) are at risk of recurrent miscarriage and pre-eclampsia. aPL target the placenta by binding to beta(2)-glycoprotein I (beta(2) GPI) expressed by the trophoblast. The objective of this study was to evaluate if and how aPL affect first trimester trophoblast migration. METHOD OF STUDY: First trimester trophoblast cells were treated with anti-beta(2) GPI monoclonal antibodies. Migration was determined using a two-chamber assay. Interleukin (IL)-6 production was evaluated by RT-PCR and enzyme-linked immunosorbent assay, and signal transducer and activator of transcription 3 (STAT3) activation was assessed by western blot. RESULTS: Trophoblast cells constitutively secreted IL-6 in a time-dependent manner and this directly correlated with STAT3 phosphorylation. In the presence of anti-beta(2) GPI Abs, trophoblast IL-6 mRNA levels and secretion was downregulated in a Toll-like receptor 4/MyD88-independent manner and this correlated with a reduction in phosphorylated STAT3 levels. In addition, the anti-beta(2) GPI Abs reduced the migratory potential of trophoblast. Heparin was able to reverse aPL-dependent inhibition of trophoblast IL-6 secretion and migration. CONCLUSION: This study demonstrates that aPL limit trophoblast cell migration by downregulating trophoblast IL-6 secretion and STAT3 activity. As heparin was unable to prevent these effects, our findings may explain why women with antiphospholipid syndrome, treated with heparin, remain at risk of developing obstetrical syndromes, associated with impaired deep placentation, such as pre-eclampsia.

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.

Hepatocyte growth factor promotes migration of human myeloma cells

Multiple myeloma is characterized by the accumulation and dissemination of malignant plasma cells in the bone marrow. Cell migration is thought to be important for these events. We studied migration in a Transwell two-chamber assay and tested the motogenic effect of various cytokines. In addition to insulin-like growth factor-1 and stromal cell-derived growth factor-1alpha, previously known as chemoattractants for myeloma cells, we identified hepatocyte growth factor as a potent attractant for myeloma cells. Hepatocyte growth factor-mediated migration was dependent on phosphatidylinositol-3-kinase, involved the MAPK/Erk signaling cascade and VLA-4 integrins, but did not involve Akt, mTOR or G proteins.

Immunologic activation of human syncytiotrophoblast by Plasmodium falciparum

BackgroundMalaria during pregnancy is characterized by the sequestration of malaria-infected red blood cells (iRBC) in the intervillous spaces of the placenta, often accompanied by the infiltration of maternal mononuclear cells, causing substantial maternal and foetal/infant morbidity. The iRBC bind to receptors expressed by the syncytiotrophoblast (ST). How ST responds to this interaction remains poorly understood. Because it is known that ST is immunoactive and can respond to infectious agents, the consequences of this ST-iRBC interaction should be investigated.MethodsAn in vitro system was used to assess the biochemical and immunological changes induced in ST by ST-adherent iRBCs. Changes in ST mitogen-activated protein kinase (MAPK) activation were assessed by immunoblotting and mRNA expression levels of selected cytokine and chemokines in primary ST bound by iRBC were determined using real-time, reverse transcription PCR. In addition, secreted cytokine and chemokine proteins were assayed by standard ELISA, and chemotaxis of PBMC was assessed using a two-chamber assay system.ResultsFollowing iRBC/ST interaction, ST C-Jun N-terminal kinase 1 (JNK1) was activated and modest increases in the mRNA expression of TGF-β and IL-8/CXCL8 were observed. In addition, this interaction increased secretion of MIF and MIP-1α/CCL3 by ST and induced migration of PBMC towards iRBC-stimulated ST.ConclusionResults from this study provide the first evidence that ST participates in shaping the local immunological milieu and in the recruitment of maternal immune cells to the maternal blood space during placental malaria infection.

Disruption of Endothelial Microfilaments Selectively Reduces the Transendothelial Migration of Monocytes

The transendothelial migration of leukocytes (diapedesis) is a central event in inflammatory and immunological processes. Although leukocyte–endothelium interactions occurring during diapedesis have been investigated intensively, little is known about the actual transmigration and the molecular mechanisms involved. Toward this end we analyzed whether the endothelial cytoskeleton plays a direct role during the transendothelial migration of monocytes. Filter-grown monolayers of human microvascular endothelial cells (HMEC-1) were treated with cytoskeleton stabilizing or destabilizing drugs and the effect of this treatment on the transmigration of peripheral blood monocytes was analyzed in a two-chamber assay. Our results show that taxol-induced stabilization of microtubules causes a reduction of leukocyte transmigration through HMEC-1, while the opposite effect is induced by the destabilization of microtubules with colchicine or nocodazol. Disruption of microfilaments with cytochalasin B or latrunculin A, on the other hand, significantly reduces the transendothelial migration although monocyte adhesion and endothelial permeability for macromolecules are slightly increased. An active participation of the endothelial microfilament system with a direct role of unconventional, calmodulin-regulated myosins is suggested by the finding that monocyte transmigration is decreased upon treatment of the endothelial cells with the Ca2+/CaM antagonist triflouperazine.