Chamber Chemotaxis Assay Research Articles

The Cytoplasmic Domain of Tissue Factor Inhibits Migration and Enhances Adhesion of Human Breast Cancer Cells.

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that when complexed with its cofactor, factor VIIa (FVIIa), initiates blood coagulation. Apart from hemostasis, TF has been shown to have roles in cellular signaling, development, inflammation, metastasis and angiogenesis. We showed previously that both the cytoplasmic and extracellular domains of TF are required for the full metastatic effect of TF. Recently, we showed that TF-FVIIa-FXa complex induces cellular signaling in human breast cancer cells and is associated with enhanced cell migration and prevention of apoptosis. However, the role of the cytoplasmic domain of TF in tumor cell function is not fully known. In the present study, the role of the cytoplasmic domain of TF in cell migration and adhesion was investigated using the Adr-MCF-7 cell line, a multidrug resistant subline of the human breast cancer cell line, MCF-7. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of PAR1 and PAR2. Adr-MCF-7 cells were retrovirally transfected with either a cDNA construct encoding a FLAG epitope tag fused to the transmembrane and cytoplasmic domains of TF (known as FLAG-TFCD) or vector (LXSN) alone as a control, and stable, polyclonal cell lines selected using G418. Expression of the FLAG-TFCD construct was verified by RT-PCR, Western blot analysis and flow cytometry. To test the effect of overexpression of the FLAG-TFCD construct on cell motility a modified Boyden chamber chemotaxis assay was used. The control LXSN cell line had a nearly 9 fold increase in cell migration [33.5± 3.2 cells/hpf (mean± SEM)]using the combination of rFVIIa (10 nM) and FX (150 nM) as the chemoattractant compared with 0.1% bovine serum albumin (BSA) [3.9± 1.2 cells/hpf]. In contrast, the FLAG-TFCD cell line had no increase in migration of using the combination of rFVIIa and FX [6.6± 0.57 cells/hpf] compared with BSA [5.4± 0.67 cells/hpf]. We then examined the ability of the transfected cell lines to adhere to type IV collagen. The number of adherent cells for the transfected cell line, FLAG-TFCD, was nearly 3 fold higher than that for the LXSN line using a colorimetric MTS assay (0.295± 0.041 vs 0.119± 0.011). Moreover, treatment of the FLAG-TFCD cells with the combination of rFVIIa and FX increased the adhesion by nearly 2 fold compared with untreated FLAG-TFCD cells (0.561±0.055 vs 0.296±0.044). In summary, overexpression of TF cytoplasmic domain leads to inhibition of tumor cell migration and enhancement of cell adhesion and potentially acts as a dominant negative in these cellular processes. These data suggest that a function of the cytoplasmic domain of TF in metastasis is to regulate tumor cell migration and adhesion.

Chemotaxis towards hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding

The accumulation of the extracellular matrix glycosaminoglycan hyaluronan by tumours and tumour-associated stroma promotes cancer cell invasion and metastasis. Using the Dunn chamber chemotaxis assay, we demonstrate for the first time that high molecular mass hyaluronan acts as a soluble chemoattractant promoting the directional migration of MDA-MB-468 and MDA-MB-231 breast cancer cells. Moreover, chemotaxis towards hyaluronan, but not foetal bovine serum, can be abrogated following treatment of the cells with siRNA oligonucleotides to downregulate CD44 expression. These data indicate that CD44 is the principal receptor mediating this response and that CD44 expression is not a general requirement for cell migration and gradient sensing, rather it elicits a ligand-specific response. However, expression of CD44 alone is not sufficient to drive chemotaxis towards hyaluronan, as NIH-3T3 fibroblasts were unable to respond to a hyaluronan gradient even when transfected with high levels of human CD44. For NIH-3T3 cells to bind exogenous hyaluronan, it was necessary to both increase the level of receptor expression and remove a hyaluronan pericellular matrix. Together, these studies reveal a direct mechanism for promoting cell invasion into the hyaluronan-rich matrix and predict that in the complex multicellular environment in vivo, multiple mechanisms exist to regulate the ability of a cell to respond to a chemotactic hyaluronan gradient.

LrrA, a novel leucine-rich repeat protein involved in cytoskeleton remodeling, is required for multicellular morphogenesis in Dictyostelium discoideum

Cell sorting by differential cell adhesion and movement is a fundamental process in multicellular morphogenesis. We have identified a Dictyostelium discoideum gene encoding a novel protein, LrrA, which composes almost entirely leucine-rich repeats (LRRs) including a putative leucine zipper motif. Transcription of lrrA appeared to be developmentally regulated with robust expression during vegetative growth and early development. lrrA null cells generated by homologous recombination aggregated to form loose mounds, but subsequent morphogenesis was blocked without formation of the apical tip. The cells adhered poorly to a substratum and did not form tight cell–cell agglomerates in suspension; in addition, they were unable to polarize and exhibit chemotactic movement in the submerged aggregation and Dunn chamber chemotaxis assays. Fluorescence-conjugated phalloidin staining revealed that both vegetative and aggregation competent lrrA − cells contained numerous F-actin-enriched microspikes around the periphery of cells. Quantitative analysis of the fluorescence-stained F-actin showed that lrrA − cells exhibited a dramatically increase in F-actin as compared to the wild-type cells. When developed together with wild-type cells, lrrA − cells were unable to move to the apical tip and sorted preferentially to the rear and lower cup regions. These results indicate that LrrA involves in cytoskeleton remodeling, which is needed for normal chemotactic aggregation and efficient cell sorting during multicellular morphogenesis, particularly in the formation of apical tip.

INTERLEUKIN 8 DIMERIZATION AS A MECHANISM FOR REGULATION OF NEUTROPHIL ADHERENCE-DEPENDENT OXIDANT PRODUCTION

Interleukin 8 (IL-8), a member of the CXC subfamily of chemoattractant cytokines, induces a range of functional responses in human neutrophils via its interactions with two high-affinity cell-surface receptors, CXCR1 and CXCR2. Like other CXC chemokines, IL-8 forms homodimers at physiologic concentrations. Monomers and dimers bind to CXC receptors with high affinity and induce various functions. Binding to glycosaminoglycans decreases the dimerization constant, enhancing surface-bound dimer formation. However, a specific role for IL-8 dimerization has not been identified. We explored the hypothesis that certain neutrophil responses to IL-8 were induced primarily by the IL-8 dimers. To this end, two dimerization-deficient IL-8 mutant proteins, M3 and M4, were used in various functional assays. In contrast to native IL-8, these proteins existed primarily as monomers at micromolar concentrations. The mutants retained high-affinity binding to both CXC receptors and potently induced neutrophil calcium flux, chemotaxis, and elastase release. In contrast to native IL-8, neither mutant inhibited tumor necrosis factor alpha-induced oxidant production. Additionally, M4 was less effective than native IL-8 at desensitizing neutrophil migration. These data suggest that although IL-8 dimers or monomers are sufficient for several neutrophil functions, dimers may participate in suppression of specific surface-dependent neutrophil responses.

Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration

Cigarette smoke, specifically the nicotine contained within, has been shown to cause ultrastructural changes in vascular endothelium resulting in the development of atherosclerosis. Our study examines the effects of nicotine on vascular smooth muscle cell (VSMC) migration and attempts to eludicidate the cellular mechanisms governing those effects. Bovine aortic VSMC were cultured in 10% fetal bovine serum (FBS) growth media and exposed to 10 −8 nicotine for varying periods of time. Boyden chamber chemotaxis assays and a scrape injury model using confluent cells were used to assess cell motility. Activation of the mitogen-activated protein kinases (MAPK), p38 and p44/42, was assessed using Western blotting methods. Nicotine, itself, did not cause significant VSMC migration. However, augmented migration was seen in nicotine-treated VSMCs (16.6 ± 3-fold) and media (17.0 ± 4-fold) with 10% FBS as chemoattractant. Inhibitors of p38 and p44/42 diminished this migration by 48.5 ± 6% and 29.4 ± 2%, respectively. Immunoblotting verified p38 and p44/42 activation with nicotine and inhibition with inhibitors of p38 and p44/42. Nicotine-treated endothelial cell (EC) conditioned media (CM) was shown to increase migration 20.3 ± l.l-fold. This chemotactic effect was diminished both with heat treatment and serial dilution. In conclusion, nicotine enhances the chemotactiveness of VSMC. This migration is mediated via the MAPKs p38 and p44/42. Nicotine causes EC production of a chemoattractant molecule that enhances VSMC migration.

Thioredoxin-1 ameliorates myosin-induced autoimmune myocarditis by suppressing chemokine expressions and leukocyte chemotaxis in mice.

Cardiac myosin-induced myocarditis is an experimental autoimmune myocarditis (EAM) model used to investigate autoimmunological mechanisms in inflammatory heart diseases and resembles fulminant myocarditis in humans. We investigated the therapeutic role of thioredoxin-1 (TRX-1), a redox-regulatory protein with antioxidant and antiinflammatory effects, in murine EAM. EAM was generated in 5-week-old male BALB/c mice by immunization with porcine cardiac myosin at days 0 and 7. Recombinant human TRX-1 (rhTRX-1), C32S/C35S mutant rhTRX-1, or saline was administered intraperitoneally every second day from day 0 to 20. In addition, rabbit anti-mouse TRX-1 serum or normal rabbit serum was administered intraperitoneally on days -1, 2, and 6. Animals were euthanized on day 21. Histological analysis of the heart showed that TRX-1 significantly reduced the severity of EAM, whereas mutant TRX-1 failed to have such an effect, and anti-TRX-1 antibody enhanced the disease markedly. Immunohistochemical analysis showed that TRX-1 significantly suppressed cardiac macrophage inflammatory protein (MIP)-1alpha, MIP-2, and 8-hydroxydeoxyguanosine expression and macrophage infiltration into the heart in EAM. Although serum levels of MIP-1alpha were not suppressed by TRX-1 until day 21, both an in vitro chemotaxis chamber assay and an in vivo air pouch model showed that TRX-1 significantly suppressed MIP-1alpha- or MIP-2-induced leukocyte chemotaxis. However, real-time reverse transcription-polymerase chain reaction showed that TRX-1 failed to decrease chemokine receptor expression increased in the bone marrow cells of EAM mice. TRX-1 attenuates EAM by suppressing chemokine expressions and leukocyte chemotaxis in mice.

Cytokines modulate cilomilast response in lung fibroblasts

Fibroblasts, as a major source of extracellular interstitial connective tissue matrix, play an important role in wound healing and the development of fibrosis. The phosphodiesterase (PDE) 4 inhibitor cilomilast inhibits fibroblast chemotaxis and fibroblast-mediated gel contraction. Using the Boyden blindwell chamber chemotaxis assay and the type I collagen gel contraction model, this study investigated whether specific cytokines modulate cilomilast's inhibitory effect through regulation of endogenous PGE2 production. Human recombinant IL-1β stimulated PGE2 production and shifted the cilomilast concentration–dependence curve to the left in both assay systems, indicating increased sensitivity to cilomilast. In contrast, human recombinant IL-4 inhibited PGE2 production and shifted the cilomilast concentration–dependence curve to the right in both systems. In summary, the inhibitory effect of cilomilast on fibroblast migration and collagen gel contraction is modulated by IL-1β and IL-4 through regulation of PGE2 production.

Formation of tissue factor–factor VIIa–factor Xa complex promotes cellular signaling and migration of human breast cancer cells

Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor (F)VIIa. Recently, TF has been shown to promote cellular signaling, tumor growth, angiogenesis, and metastasis. In the present study, we examined the pathway by which TF-FVIIa complex induces cellular signaling in human breast cancer cells using the Adr-MCF-7 cell line. This cell line has high endogenous TF expression as measured by flow cytometry and expression of protease-activated receptors 1 and 2 (PAR1 and PAR2) as determined by reverse transcriptase-polymerase chain reaction analysis. Both PAR1 and PAR2 are functionally active as determined by induction of p44/42 mitogen-activated protein kinase (MAPK) phosphorylation using specific agonist peptides. We found that MAPK phosphorylation in this cell line was strongly induced by the combination of FVIIa and factor (F)X, but not by FVIIa alone at a concentration of FVIIa that approaches physiological levels. Induction of MAPK phosphorylation involved the formation of TF-FVIIa-FXa complex and occurred by a pathway that did not require thrombin formation, indicating a critical role for FXa generation. In addition, induction of MAPK phosphorylation was found to be independent of PAR1 activation. We then examined whether TF-FVIIa complex formation could promote tumor cell migration using a modified Boyden chamber chemotaxis assay. The combination of FVIIa and FX, but not FVIIa alone, strongly induced migration of tumor cells by a pathway that probably involves PAR2, but not PAR1 activation. MAPK phosphorylation was found to be required for the induction of cell migration by the combination of FVIIa and FX. These data suggest that TF-FVIIa-mediated signaling in human breast cancer cells occurs most efficiently by formation of the TF-FVIIa-FXa complex. One of the physiological consequences of this signaling pathway is enhanced cell migration that is probably mediated by PAR2, but not PAR1 activation.

Allurin, a 21 kD sperm chemoattractant, is rapidly released from the outermost jelly layer of theXenopus egg by diffusion and medium convection

Allurin, a 21 kD protein from Xenopus laevis egg jelly, has been demonstrated to attract sperm by video microscopy and by quantitative chemotaxis chamber assays. Here, we use immunocytochemistry to demonstrate that this sperm chemoattractant is located in the outermost layer of egg jelly (J3) and is rapidly released into the surrounding medium. SDS-PAGE analysis and Western blotting confirm the appearance of allurin in the medium within 1.5 min and separation of proteins in the medium by anion exchange FPLC, shows that nearly half of the allurin released over a 12-hr period is discharged in the first 5 min. The kinetics of allurin release from J3 and its appearance in the medium were quantitatively accounted for, by computer simulation of mathematical diffusion and convection models. Comparison of simulation data to quantitative measurements of allurin appearance in the medium suggests that allurin, although larger than most chemoattractants, is effectively dispersed by a combination of diffusion and medium mixing at the jelly surface during spawning. Our model further predicts that the innermost jelly layer, J1, is less permeable to allurin than the other layers, allowing it to act as a "reflector" to speed up allurin discharge.

Matrix metalloproteinase regulation of sphingosine-1-phosphate-induced angiogenic properties of bone marrow stromal cells

Bone marrow-derived stromal cells (MSC) are able to acquire histological and immunophenotypic characteristics consistent with endothelial cells (EC). In this study we examined the effect of sphingosine-1-phosphate (S1P), a platelet-derived bioactive lysophospholipid that is believed to specifically stimulate EC migration and tube formation, on the angiogenic properties of MSC. MSC were isolated from murine bone marrow and cultured in the presence of diverse angiogenic growth factors. Using a chemotaxis chamber and Matrigel tubulogenesis assay, we measured the extent of MSC migration and capillary-like structure formation. Western blots and zymography were used to assess the levels and activation states of soluble and membrane-bound matrix metalloproteinase (MMP). We found that S1P strongly induced MSC migration and in vitro capillary-like structure formation. Ilomastat, a broad-spectrum MMP inhibitor, antagonized several angiogenic and S1P-mediated events in MSC. These included 1) the inhibition of S1P-induced tube formation, 2) the inhibition of concanavalin-A (Con-A)-mediated proMMP-2 activation, and 3) the inhibition of S1P- and Con-A-induced caspase-3 activity. Moreover, S1P induced membrane type-1 (MT1)-MMP mRNA and protein expression, but paradoxically antagonized its cell surface proteolytic processing. In addition, anti-angiogenic agents such as Ilomastat, Neovastat, and green tea polyphenol epigallocatechin-3-gallate antagonized the S1P-induced migration of MSC as well as that of transfected COS-7 cells overexpressing the recombinant receptor for S1P, EDG-1. Collectively, our results indicate a crucial role for S1P/EDG-1-mediated angiogenic and survival events in the regulation of microvascular network remodeling by MSC, and may provide a new molecular link between hemostasis and angiogenesis processes.

Specific inhibition of human skin fibroblast chemotaxis to platelet-derived growth factor A-chain homodimer by transforming growth factor-beta1.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) have been suggested to play important roles in wound healing. We investigated the effect of TGF-beta1 on the mitogenic and chemotactic activities of PDGF A-chain homodimer (PDGF-AA) and B-chain homodimer (PDGF-BB) in primary cultures of human skin fibroblasts. TGF-beta1 inhibited the growth-promoting activity of both PDGFs. Proliferative responses to basic fibroblast growth factor and epidermal growth factor were also restricted by TGF-beta1. A Boyden chamber chemotaxis assay revealed that the chemotactic migration to PDGF-AA was inhibited by TGF-beta1 pretreatment, but in contrast, the response to PDGF-BB was not affected by the same treatment. Western blot analysis showed that TGF-beta1 downregulated PDGF alpha-receptors, but not beta-receptors, indicating that the isoform-specific inhibition of chemotaxis is related to differential effects of TGF-beta1 on PDGF receptor expression. The present findings suggest that TGF-beta1 may act antagonistically towards PDGFs in humans under certain conditions, and this antagonistic nature of TGF-beta1 must be considered when it is applied to human wounds as a therapeutic agent.

Exploitation of interleukin-8-induced neutrophil chemotaxis by the agent of human granulocytic ehrlichiosis.

The agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium with a tropism for neutrophils; however, the mechanisms of bacterial dissemination are not yet understood. Interleukin-8 (IL-8) is a chemokine that induces neutrophil migration to sites of infection for host defense against pathogens. We now show that HGE bacteria, and the HGE-44 protein, induce IL-8 secretion in a promyelocytic (HL-60) cell line that has been differentiated along the neutrophil lineage with retinoic acid and in neutrophils. Infected HL-60 cells also demonstrate upregulation of CXCR2, an IL-8 receptor, but not CXCR1. Human neutrophils migrate towards Ehrlichia sp.-infected cells in a chemotaxis chamber assay, and this movement can be blocked with antibodies to IL-8. Finally, immunocompetent and severe combined immunodeficient mice administered CXCR2 antisera, and CXCR2(-/-) mice that lack the human IL-8 receptor homologue, are much less susceptible to granulocytic ehrlichiosis than are control animals. These results demonstrate that HGE bacteria induce IL-8 production by host cells and, paradoxically, appear to exploit this chemokine to enhance infection.

Signal transduction pathways in directed migration of human monocytes induced by human growth hormone in vitro

The human growth hormone (GH) was shown to modulate leukocyte functions such as stimulating directed migration of human monocytes in vitro. Dimerisation of GH-receptors leads to the activation of various signalling mechanisms. As transduction of GH signals to monocytes is unknown, we investigated GH signalling mechanisms in monocyte migration using a modified Boyden chamber chemotaxis assay. Inhibition of tyrosyl phosphorylation of GH receptor-associated tyrosine kinase by tyrphostin-23 or staurosporine blocked GH-stimulated monocyte migration down to random levels. Furthermore, pre-incubation with effective concentrations of 4B-phorbol-12-myristate-13-acetate (PMA), staurosporine and bisindolylmaleimide I, inhibitors of protein kinase C, significantly decreased GH-induced migration, suggesting that PKC is involved in the signalling cascade. Additionally, phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) activation seems to be required. This study revealed signalling pathways in monocyte movement toward GH in vitro.

Extracellular ATP or ADP Induce Chemotaxis of Cultured Microglia through Gi/o-Coupled P2Y Receptors

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

The C-terminal domain of thrombospondin-1 induces vascular smooth muscle cell chemotaxis

Objective: Thrombospondin-1 (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-1 has six domains of repeating homologous amino acid sequences: N-terminal, procollagen homology, type 1 repeat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The purpose of this experiment was to determine which domains of TSP-1 induce VSMC chemotaxis. Methods: A modified Boyden Chamber chemotaxis assay was used to assess VSMC migration. Serum-free medium, TSP-1, or each of the fusion proteins (10 and 20 μg/mL) synthesized for the different domains were placed in the bottom wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top wells. After 4 hours at 37°C, migrated VSMCs were recorded as cells per five fields (400×) and analyzed with the paired t test. To verify the fusion protein data, we performed chemotaxis assays with antibodies to each of the domains (25 μg/mL) combined with TSP-1 (20 μg/mL) in the bottom wells and VSMCs in the top wells. Results: The COOH domain significantly stimulated VSMC chemotaxis (P = <.001). To a lesser extent, the N-terminal and T3 domains also induced chemotaxis (P <.05). However, only the anti-COOH antibody (C6.7) and the anti–integrin-associated protein portion of COOH antibody (D4.6) significantly inhibited TSP-1–induced VSMC chemotaxis (by 85% and 92%, respectively). Conclusions: These results implicate the COOH domain as the portion of the TSP-1 molecule primarily responsible for VSMC chemotaxis. This experiment suggests that future strategies in the prevention of VSMC migration, an initial step in the development of vascular lesions, may involve selective inhibition of the COOH domain of TSP-1. (J Vasc Surg 2001;33:595-600.)

Effects of fibronectin cleaved by neuropsin on cell adhesion and migration.

Neuropsin is a serine protease cloned from the mouse hippocampus. Since neuropsin is a secreted protein which effectively cleaves fibronectin, it may affect cell adhesion or cell migration by modulating the content and/or chemical characteriscs of fibronectin in extracellular matrix (ECM). In adhesion assays, alpha5B2 cells expressing integrin alpha5beta1 bound less effectively to fibronectin teated with neuropsin than intact fibronectin. In Boyden chamber chemotaxis assays, the fibronectin-induced migration of alpha5B2 cells was not affected by neuropsin treatment. These findings suggest that neuropsin regulates the local microenvironment by modulating the interaction between cells and fibronectin in ECM.

Focal Adhesion Kinase Facilitates Platelet-derived Growth Factor-BB-stimulated ERK2 Activation Required for Chemotaxis Migration of Vascular Smooth Muscle Cells

The focal adhesion (FAK) non-receptor protein-tyrosine kinase (PTK) links both extracellular matrix/integrin and growth factor stimulation to intracellular signals promoting cell migration. Here we show that both transient and stable overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) inhibits serum and platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (SMC) migration in wound healing and in vitro Boyden Chamber chemotaxis assays, respectively. Expression of FRNK, but not a point mutant of FRNK (FRNK L1034S), disrupted the formation of a complex containing both FAK and the activated PDGF-beta receptor and resulted in reduced tyrosine phosphorylation of endogenous FAK at the Tyr-397 binding site for Src family PTKs. As demonstrated using FAK-deficient and FAK-reconstituted fibroblasts, FAK positively contributed to PDGF-BB-stimulated ERK2/MAP kinase activity, and in SMCs, ERK2/MAP kinase activity was required for PDGF-BB-stimulated chemotaxis. Stable expression of FRNK but not FRNK L1034S expression in SMCs lowered the extent and duration of stimulated ERK2/MAP kinase activation at low but not at high PDGF-BB concentrations. Importantly, stable expression of FRNK in SMCs did not affect SMC morphology or proliferation in culture. Because the increased migration of vascular SMCs in response to extracellular matrix proteins and growth factors contributes to neointima formation, our results show that FAK inhibition by FRNK expression may provide a novel approach to regulate abnormal vascular SMC migration in vivo.

Chicken Soup Inhibits Neutrophil Chemotaxis In Vitro

Chicken soup has long been regarded as a remedy for symptomatic upper respiratory tract infections. As it is likely that the clinical similarity of the diverse infectious processes that can result in "colds" is due to a shared inflammatory response, an effect of chicken soup in mitigating inflammation could account for its attested benefits. To evaluate this, a traditional chicken soup was tested for its ability to inhibit neutrophil migration using the standard Boyden blindwell chemotaxis chamber assay with zymosan-activated serum and fMet-Leu-Phe as chemoattractants. Chicken soup significantly inhibited neutrophil migration and did so in a concentration-dependent manner. The activity was present in a nonparticulate component of the chicken soup. All of the vegetables present in the soup and the chicken individually had inhibitory activity, although only the chicken lacked cytotoxic activity. Interestingly, the complete soup also lacked cytotoxic activity. Commercial soups varied greatly in their inhibitory activity. The present study, therefore, suggests that chicken soup may contain a number of substances with beneficial medicinal activity. A mild anti-inflammatory effect could be one mechanism by which the soup could result in the mitigation of symptomatic upper respiratory tract infections.

Influence of synthetic polymers on neutrophil migration in three-dimensional collagen gels.

In vitro studies of cell migration within three-dimensional polymeric materials are essential for understanding cell behavior and for developing new biomedical materials. Human neutrophil motility was examined in hydrated collagen gels containing various synthetic polymers. Physical mixtures of collagen and certain water-soluble polymers formed stable gels that were good substrates for cell migration. Addition of either polyethylene glycol (PEG) or the pluronictrade mark copolymer F68 did not change the morphological or mechanical properties of collagen gels, as determined by SEM and oscillatory rheometry; however, addition of either polymer significantly inhibited cell motility in both a modified 96-well chemotaxis chamber assay and a direct visual assay. Although the mechanism for this observed polymer inhibition of neutrophil migration is not yet clear, these results suggest that PEG and F68, two widely used biomedical polymers that are considered to be relatively "inert," may cause significant inhibition of cell motility.

Inducer-specific bidirectional regulation of endothelial interleukin-8 production by thalidomide

Interleukin-8 (IL-8) is a potent neutrophil chemotaxin, which can also be produced by endothelial cells to facilitate leukocyte emigration. The aim of this study was to determine the effects of the anti-inflammatory drug thalidomide (THD) on chemotaxin release from endothelial cells. Human umbilical vein endothelial cells (HUVEC) were stimulated with tumor necrosis factor alpha (TNFα) or endotoxin (LPS) in the presence or absence of various concentrations of THD. Endothelium-derived interleukin-8 (eIL-8) in supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) and biological activity of the harvested eIL-8 was tested in Boyden chamber chemotaxis assays on PMNL. THD itself had no effect on eIL-8 release. Upon stimulation with TNFα or LPS, HUVEC produced increased amounts of eIL-8 and THD affected this process in a bidirectional manner, with augmentation of TNFα- and inhibition of LPS-effects. Functionality of eIL-8 was confirmed in chemotaxis experiments and by inhibition of chemotactic effects of supernatants with anti-human IL-8 monoclonal antibodies. Results explain and emphasize immunomodulatory properties of THD in cytokine- and endotoxin-induced inflammation and regulation of transendothelial migration.