Eosinophil cationic protein stimulates migration of human lung fibroblasts in vitro.

Asthma is characterized by eosinophilic inflammation and remodelling of the airways. Eosinophil cationic protein (ECP) is a protein released by activated eosinophils and the hypothesis that ECP contributes to the development of structural changes in the airways of asthmatics has been posed. Fibroblast recruitment is an important step in the remodelling process, and we therefore put the question whether ECP stimulates migration of human lung fibroblasts. Human peripheral eosinophils isolated from buffycoats from healthy individuals were cultured and conditioned media (CM) were collected. Native ECP was extracted from human peripheral eosinophils by gel filtration, ion-exchange and chelating chromatography. The ability of eosinophil CM and ECP to stimulate fibroblast migration was determined using the 48-well Boyden chamber. ECP concentrations in CM were assayed by ECP-CAP-FEIA. Both CM and ECP significantly stimulated fibroblast migration (48.4+/-cells/field versus 33+/-2 and 36+/-6 versus 25+/-4; P<0.001 and 0.05 respectively) in a time- and concentration-dependent manner. Adding neutralizing ECP antibodies attenuated fibroblast migration induced by both ECP as well as CM. ECP stimulates migration of human lung fibroblasts, suggesting a potential mechanism for eosinophils in the fibrotic response. This may be an important mechanism by which ECP promotes remodelling of extracellular matrix leading to airway fibrosis in asthmatics.

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro.

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.

Malignant effusions are sources of fibronectin and other promigratory and proinvasive components.

Metastatic dissemination is the primary cause of death in ovarian cancer (OvCa) patients, and dissemination to pleural and peritoneal effusions is a common clinical event. Effusion samples were collected from 15 OvCa patients. Twenty-six samples were collected prospectively, two were archival, and eight were taken from patients with other malignancies. Twenty-nine samples were from malignant ascites, and seven specimens were pleural fluids. In addition, six ascites and two pleural fluids from noncancer patients were studied as effusion controls. Effusion supernatants were tested for migration-stimulation activity, using A2058 human melanoma cells as the index responder cell. Malignant samples induced a 400-1200% increase in migration. Sixty percent of the migration was inhibited by incubation of the malignant fluid with antifibronectin (FN) antibody, in contrast to 75% inhibition of control fluid-stimulated migration (P = 0.017). Gelatin zymography and Western blot analyses showed that latent and activated MMP-2 and MMP-9 collagenases, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were present in all malignant fluids. Serial samples were taken from several patients, and a trend for correlation between MMPs and clinical behavior of the tumors was shown. Free TIMP-2 correlated with CA-125 levels in two patients for whom serial samples were available. The demonstration of promigratory and proinvasive activity in malignant effusions is consistent with their association with other metastatic disease in OvCa patients and their function as a haven for metastatic cells.

Regulation of matrix metalloprotease activity in malignant mesothelioma cell lines by growth factors.

BACKGROUND: The extracellular matrix metalloproteases (MMPs) produced by tumour and stromal cells are believed to play a key role in tumour cell invasion and metastasis. Malignant mesothelioma is a highly aggressive tumour. Previous studies have shown that malignant mesothelioma cells express MMP-1, -2, -3, -7 and -9. However, the regulation of MMP expression in malignant mesothelioma cells has not been thoroughly investigated. METHODS: The effects of a number of growth factors on the secretion of MMP-2, -3 and -9 in malignant mesothelioma cells were studied by substrate zymography. The expression of relevant growth factor receptors in malignant mesothelioma cells was also examined using RT-PCR. RESULTS: The exposure of malignant mesothelioma cells to different growth factors including epidermal growth factor (EGF), transforming growth factor-alpha, amphiregulin, heparin binding EGF, beta-cellulin (BTC), stem cell factor, insulin-like growth factors I and II, acidic and basic fibroblast growth factors, and hepatocyte growth factor increased secretion of MMP-9 and/or MMP-3. EGF receptor ligands BTC and EGF had the most potent effect on MMP-9 and MMP-3 production, respectively. Production of MMP-2 was not affected by any growth factors used in this study. We have also demonstrated mRNA expression of different growth factor receptors in malignant mesothelioma cells, and found that the tyrosine kinase inhibitor genistein inhibited the increased production of MMPs resulting from stimulation with different growth factors. CONCLUSION: This study shows, for the first time, the broad extent of regulation of MMPs by various growth factors in malignant mesothelioma cells.

Expression and activity of matrix metalloproteases in human malignant mesothelioma cell lines.

The extracellular matrix metalloproteases (MMPs) secreted by various human tumor cells play a crucial role in tumor cell invasion and metastasis, but their expression in malignant mesothelioma (MM) cells has not been examined. In this study, we have investigated the spectrum of MMPs and tissue inhibitors of metalloproteases (TIMPs) produced by 8 MM cell lines. Using RT-PCR, we found that all investigated MM cell lines expressed genes encoding mRNA for MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and TIMPs 1, 2 and 3. We also found that 6/8 MM cell lines expressed MMP-7 (matrilysin) and 3/8 MM cell lines expressed MMP-10 (stromelysin-2). MMP-11 (stromelysin-3) was not detected in any of the MM cell lines. Production of MMP-2 and MMP-9 was confirmed using gelatin zymography. In addition, all MM cell lines secreted a 66 kDa metalloprotease, while 3/8 MM cell lines secreted 46, 48, 51 and 63 kDa metalloproteases which specifically degraded the extracellular matrix components fibronectin, vitronectin and laminin. The 66 kDa protease was identified as MMP-3 by Western blot. Our results reveal a broad spectrum of MMPs and TIMPs produced by MM cells and indicate that different substrate specificities of MMPs may play a role in MM cell invasion.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

Epidermal growth factor receptor ligands are chemoattractants for normal human mesothelial cells.

Signalling through epidermal growth factor (EGF) receptor leads to several cellular responses including cell division and cell migration. Since EGF receptors are expressed on normal mesothelial cells, this study investigated whether EGF receptor ligands act as chemoattractants on these cells. The study used Boyden chambers fitted with filters coated with the adhesive matrix proteins fibronectin, laminin, collagen type IV and the nonmatrix adhesive molecule poly-L-lysine, for the migration studies. Normal mesothelial cells migrated to EGF receptor ligands such as EGF, transforming growth factor (TGF)-alpha and heparin-binding epidermal growth factor (HB-EGF) at concentrations ranging 0.024-100 ng x mL(-1) (with a peak stimulation at 6.25 ng x mL(-1)), if matrix proteins were present as adhesive substrates. This migration was integrin-dependent, since the same cells failed to migrate in the absence of extracellular matrix molecules or when the Boyden chamber assay was performed in the presence of anti-beta1 integrin monoclonal antibodies. These findings describe for the first time epidermal growth factor receptor ligands acting as chemoattractants on normal mesothelial cells, and that signalling through epidermal growth factor receptors leading to mesothelial cell migration also requires the activation of integrins.

Spectrum of extracellular matrix degrading enzymes in normal and malignant T lymphocytes.

Human T cells produce and release fibronectin degrading neutral serine proteases with a molecular weight of 50 kD, 70-80 kD (doublet) and 95 kD and have a cell associated 400 kD fibronectin degrading enzyme. In addition, human T cells produce proteases with m.w. 50, 70-80 kD and 400 kD which degrade laminin. CD 4+ T lymphocytes from a non-malignant cloned human T cell line produce a 92 kD gelatinase (MMP 9) and malignant T cell lines release, in addition to the 92 kD gelatinase, low amounts of a 72 kD gelatinase (MMP 2). Purification of the enzymatic activities using benzamidine sepharose yields a 50 kD and a 70 kD band of which the 50 kD band has fibronectin degrading capacity. The purified enzymes do not react with monoclonal antibodies to various previously characterized proteolytic enzymes present in T cells. T lymphocytes from a non-malignant cloned human T cell line produce high amounts of the 50 and 70-80 kD proteases directly after stimulation with anti-CD 3 monoclonal antibodies whereafter the production of these enzymes declines with time. The expression of the 400 kD fibronectin-degrading protease is downregulated by crosslinking of alpha 4 beta 1-integrin receptors on T cells using monoclonal antibodies. Thus, T lymphocytes produce several matrix degrading enzymes with multiple substrate specificities. The expression of these enzymes is controlled partly by lymphocyte activation signals or by direct signalling via beta 1-integrins.

Growth-factor-dependent migration of human lung-cancer cells.

Human lung tumors express different types of growth-factor receptors and corresponding ligands that might modulate several biological functions such as proliferation, differentiation, adhesion, and chemotaxis. In the present study, we have investigated the expression of different growth-factor receptors and their ligands in 5 established human lung-cancer cell lines. Using RT-PCR, we found that IGF-II/mannose-6-phosphate (M6P), c-met, EGF and c-kit receptors are expressed in 5/5 human lung-cancer cell lines. In order to investigate the biological function of these receptors, we performed Boyden-chamber assays using various growth factors as chemo-attractants. Human non-small-cell-lung-cancer cells (non-SCLC) migrated to recombinant human (rh)IGF I and IGF II at concentrations ranging from 1 to 1000 ng/ml, to HGF at 10 to 100 ng/ml, to EGF at 1 to 100 ng/ml and SCF at 1 to 50 ng/ml. In addition, we performed Boyden-chamber assays using U-1810-, U-1752- and Wart-derived serum-free conditioned medium as chemo-attractants. Serum-free conditioned medium stimulated migration of producer cells in a dose-dependent manner. The autocrine motility stimulating effect of U-1810-derived serum-free conditioned medium could be inhibited by 50% in the presence of neutralizing ahIGF-II antibodies in the assay, suggesting a possible autocrine motility loop in vitro.

A tumor derived motility factor that stimulates cell migration on extracellular matrix.

A factor that stimulates migration of lung carcinoma cells on biological substrata was purified from the human lung adenocarcinoma cell line WART. A partially purified autocrine motility factor-like substance, termed haptotaxin, was added to the lower compartment of Boyden chambers and the filters were coated on the upper, lower or both sides with different concentrations of the extracellular matrix (ECM) components fibronectin, laminin or collagen type IV. These adhesive proteins coated on the lower surface of the filter promoted the migration (haptotaxis) of lung carcinoma cells. This effect was greatly enhanced by the addition of haptotaxin. In contrast, ECM components (including gelatin) coated on the upper surface or on both filter surfaces did not stimulate tumor cell migration. However, the addition of haptotaxin also timulated cell migration under these conditions. Haptotaxin did not stimulate migration on filters coated with bovine serum albumin or on uncoated filters. Haptotaxin could not be absorbed by fibronectin, laminin, collagen type IV or gelatin, and soluble ECM components did not affect the locomotor effect of haptotaxin. Substrata coated with fibronectin, laminin and collagen type IV induced adhesion and spreading of lung carcinoma cells in a dose dependent fashion. Haptotaxin potentiated adhesion and spreading of tumor cells on these substrata but did not in itself mediate adhesion and spreading of the cells. Anti-VLA 2 antibodies inhibited migration to haptotaxin on gelatin and laminin coated filters but did not affect haptotaxin-induced migration on fibronectin or collagen type IV substrata. Anti-VLA-5 monoclonal antibodies inhibited haptotaxin-induced migration on fibronectin coated filters but not such migration on filters coated with other ECM molecules showing that tumor cells must interact specifically with ECM components in order to migrate to haptotaxin.

Production of a motility factor by a newly established lung adenocarcinoma cell line.

We have established and characterised a cell line, designated WART, from a patient with primary adenocarcinoma of the lung. This cell line grows with a doubling time of approximately 15 hours, forms colonies in soft agarose, is tumorigenic in athymic nude mice, and has a complex karyotype with both structural and numerical abnormalities. WART serum free conditioned medium (SFCM) contains a factor which stimulates motile behavior of WART cells. This factor with an apparent molecular weight of 67 kDa induced in an autocrine fashion prominent pseudopodia, and chemotactic and chemokinetic responses. Heparin affinity chromatography, ion exchange and molecular sieve chromatography accompanied by SDS-PAGE analysis showed that the motility inducing activity was associated with a major band with molecular weight 67 kDa. The motility inducing activity of the 67 kDa protein was not sensitive to reduction with either dithiotreitol or mercaptoethanol which distinguishes it from A-2058 melanoma autocrine motility factor (AMF)/autotaxin, HT-1080 fibrosarcoma AMF and scatter factor which lose their biological activity upon reduction. This 67 kDa motility inducing factor did not augment DNA synthesis indicating that its locomotor activity is independent of mechanisms regulating cell growth. Pertusis toxin inhibited the motile response induced by the 67 kDa protein indicating a signal transduction pathway involving G proteins. Due to its production of the motility stimulating protein the cell line could facilitate studies of invasion and metastasis of human lung tumors.

Hepatocyte growth factor/scatter factor stimulates chemotaxis and growth of malignant mesothelioma cells through c-met receptor.

Hepatocyte growth factor (HGF) and its receptor c-met are present in several human tissues but their expression in mesothelial cells has not been examined. In this study, we have investigated the expression of HGF and c-met in normal human mesothelial cells and 11 human malignant mesothelioma cell lines. Using RT-PCR and Western blotting we found that HGF is produced by 3/11 mesothelioma cell lines whereas c-met is expressed in 11/11 mesothelioma cell lines. In addition, c-met expression was also found in 6/6 cell samples obtained from pleural fluids of patients with mesothelioma. In contrast, neither normal cultured mesothelial cells nor mesothelial cells obtained directly from patients without mesothelioma expressed HGF nor c-met. We have also analysed the biological function of HGF and c-met in mesothelioma cell lines. Recombinant human (rh) HGF stimulated both directional (chemotactic) and random (chemokinetic) motility in all mesothelioma cell lines tested. Furthermore, mesothelioma serum free conditioned medium containing HGF stimulated mesothelioma cell migration. This effect could be blocked in the presence of neutralizing anti-HGF monoclonal antibodies (MAbs) in the assay. Addition of HGF to mesothelioma cells cultured on collagen type IV was associated with induction of bipolar shape and protrusion of prominent pseudopodia. We have also found that rhHGF was mitogenic for mesothelioma cells. Our findings suggest that expression of HGF/c-met is involved not only in mesothelioma progression but also in its growth and migration and that c-met expression found in mesothelioma cells taken directly from patients may be of diagnostic importance.

Integrin dependent migration of lung cancer cells to extracellular matrix components.

Since tumour progression is dependent on the ability of malignant cells to interact with the extracellular matrix (ECM), we have investigated the significance of beta1 and beta3 integrins for migration of lung cancer cells to components of the ECM. In an in vitro hapto- and chemotactic assay system, five cell lines representing the major types of lung cancer were examined: adenocarcinoma (WART); squamous cell carcinoma (U-1752); small cell lung cancer (SCLC) (U-1906, 054 A) and large cell lung cancer (LCLC) (U-1810). Flow cytometric analyses were performed to characterize their integrin expression. U-1906, 054 A, WART and U-1752 all expressed beta1 integrins whereas U-1810 did not. However, U-1810 and U-1752 expressed beta3 integrins. All cell lines except U-1810 and U-1752 showed hapto- and chemotactic motility to fibronectin, laminin and type IV collagen and this motility was beta1 integrin-dependent except in the case of U-1810. However, the hapto- and chemotactic responses differed markedly between the separate cell lines and there was no distinct pattern to separate non-small cell lung cancer (NSCLC) from SCLC. No or very little migration was seen in control experiments with bovine serum albumin (BSA) or serum-free medium alone, indicating that the migration of the lung cancer cells require adhesion molecules, soluble or substratum bound. We have found the involvement of beta1 integrins in lung cancer cell migration in vitro towards fibronectin, laminin and type IV collagen except in the case of U-1810. The U-1810 cell line clearly differed from the rest of the cell lines by lacking expression of beta1 integrins.

Platelet-derived growth factor (PDGF) BB acts as a chemoattractant for human malignant mesothelioma cells via PDGF receptor beta-integrin alpha3beta1 interaction.

Platelet-derived growth factor BB (PDGF BB) and the PDGF receptor beta are expressed on mesothelioma cells, but their biological function has not yet been defined. In the present study we used Boyden chambers fitted with filters coated with the adhesive matrix proteins fibronectin, laminin, collagen type IV or the nonmatrix adhesive molecule poly-L-lysine (PLL). Mesothelioma cells migrated towards PDGF BB at concentrations ranging from 0.78 to 12.5 ng/ml if matrix proteins were present as adhesive substrates. This migration was integrin dependent since the same cells failed to migrate if the adhesive interactions necessary for migration were provided by molecules other than integrins. Migration of mesothelioma cells on fibronectin, laminin or collagen-type IV in response to PDGF BB was inhibited if the cells were pretreated with blocking antibodies to alpha3beta1 integrin. These findings describe for the first time PDGF BB as a chemoattractant for malignant mesothelioma cells and that collaboration between PDGF receptor beta and integrin alpha3beta1 is necessary for the motile response of these cells to PDGF BB.

Differential motile response of human malignant mesothelioma cells to fibronectin, laminin and collagen type IV: the role of beta1 integrins.

Beta1 integrins are widely expressed in human tissues but their presence and function on malignant mesothelioma cells have not been examined. In this study, we have investigated the expression and function of beta1 integrins in 7 human malignant mesothelioma cell lines. Immunofluorescence staining and FACS analysis showed similar expression of beta1 integrins with strongest expression of alpha3beta1 in all investigated mesothelioma cell lines. Using the Boyden chamber assay, we found that mesothelioma cell lines migrated to soluble (chemotaxis) and substrate-bound (haptotaxis) fibronectin, laminin and collagen type IV. In order to investigate the biological function of integrins in mesothelioma cells, we pre-incubated the cells with blocking anti-integrin monoclonal antibodies (MAbs) prior to the adhesion and migration assays. Anti-beta1 antibodies inhibited cell adhesion, chemotaxis and haptotaxis in all cell lines. Generally, anti-alpha2 integrin antibodies inhibited cell adhesion, chemotactic and haptotactic migration to collagen type IV, whereas antibodies to the alpha5 and alpha6 subunits inhibited cell adhesion and migration to fibronectin and laminin, respectively. Preincubation of mesothelioma cells with anti-alpha3 antibodies inhibited the migration to either collagen type IV, laminin or fibronectin in all cell lines. Interestingly, in 3 cell lines anti-alpha3 antibodies inhibited cell migration to laminin and collagen type IV without affecting the ability of the cells to adhere to these proteins. Furthermore, in 2 cell lines, antibodies to the alpha3 chain inhibited chemotaxis but not haptotaxis to collagen type IV, indicating the presence of distinct signalling pathways.

Triggering of motile behavior in T lymphocytes via cross-linking of alpha 4 beta 1 and alpha L beta 2.

The mechanisms by which T lymphocytes are transformed from passively transported cells during circulation in the vascular system to actively migrating cells during extravasation are unknown. Therefore, the possibility that lymphocyte receptors are capable of inducing motility was investigated using a modified Boyden chamber assay. Cross-linking of alphaL beta2 and alpha4 beta1 on human T lymphocytes (T cell line and peripheral blood T cells) with immobilized mAbs induced motile behavior on fibronectin, laminin, collagen type IV, and poly-L-lysine. This induction of T cell migration was very potent and in most cases more efficient than pretreatment of the cells with phorbol esters. In contrast, control Abs to several other integrin- and non-integrin molecules present on T lymphocytes did not induce T cell migration. Anti-CD3 Abs themselves did not trigger motile behavior. However, anti-CD3 promoted T cell migration in the Boyden chamber system if present simultaneously with 40-kDa alpha4 beta1 binding fibronectin fragments or alphaL beta2 binding intercellular adhesion molecule-1/hIgG1Fc fusion proteins on the upper side of the filter. Abs to other surface components on T cells did not trigger motility when presented together with the 40-kDa fibronectin fragments or the intercellular adhesion molecule-1/hIgG1Fc fusion proteins. The induction of motile behavior could be blocked if the T cells were pretreated with Genistein and Calphostin C, indicating the involvement of a protein tyrosine kinase and protein kinase C-dependent signaling pathway in triggering of T cell motility via integrins. These results indicate that alphaL beta2 and alpha4 beta1 on T lymphocytes can selectively trigger motile behavior when cross-linked by their endothelial or extracellular matrix ligands. Furthermore, these data indicate that cross-linking of CD3 facilitates ligand binding and subsequent triggering of a motile phenotype by alphaL beta2 and alpha4 beta1.

T lymphocyte migration: the influence of interactions via adhesion molecules, the T cell receptor, and cytokines.

Although lymphocytes have been studied extensively with respect to a number of motile aspects the understanding of directed lymphocyte motility and its regulation has increased relatively slowly. T lymphocyte migration/translocation in vivo and in vitro are critically dependent on the avidity of adhesive lymphocyte receptors for endothelial cell ligands and extracellular matrix (ECM) components and on the capacity of the lymphocytes to undergo a motile response. Lymphocytes are rendered motile by adhesion to endothelial cells and ECM components. Thus, T lymphocytes exhibit chemotactic and haptotactic migration to the ECM components fibronectin, laminin, and collagen type IV. This directed migration is mediated by beta 1-integrins and separate T-lymphocyte lines have a functional specialization using either alpha 4 beta 1 or alpha 5 beta 1 during chemo- and haptotaxis to ECM components, although the same cell line may use both integrins for adhesion. Noteworthy, signals triggering T cell migration to ECM components seem to be delivered preferentially via alpha 4 beta 1 or alpha L beta 2. The T cell antigen receptor cannot by itself trigger T lymphocyte migration to fibronectin, laminin, or collagen type IV but does so in collaboration with signals via alpha 4 beta 1. It follows that the migration-triggering signals can be separated from the integrin interactions with matrix components that mediate the chemo- and haptotactic migration per se. This suggests that T cell recruitment to inflammatory sites is induced by antigen receptor signals and beta 1- and beta 2-integrin signals in synergy. Cytokines with chemokinetic properties may collaborate with lymphocyte counterreceptors on endothelial cells and with ECM components in control of the lymphocyte migratory pathways and specifically attract lymphocyte subsets to different compartments. T lymphocytes are endowed with multiple enzymes, classified as serine proteinases or metalloproteinases, which can degrade extracellular matrix components. These enzymes may play an important role for the capacity of T cells to migrate and infiltrate tissues.

Glycosaminoglycans from two human malignant mesothelioma cell lines: determination, distribution, and effect of platelet-derived growth factor on their synthesis.

The synthesis and distribution of glycosaminoglycans (GAGs) were studied in two human malignant mesothelioma cell lines: one with fibroblast-like morphology and the other with epithelial differentiation. Analyses using highly sensitive high-pressure liquid chromatography techniques and agarose gel electrophoresis showed that these cells produce not only hyaluronan (HA) but also galactosaminoglycans (GalAGs, chondroitin sulfate and (or) dermatan sulfate) and heparan sulfate (HS). In both cell lines most of the HA (87-90%) and GalAGs (57-66%) are secreted into the extracellular matrix. Although HS is mainly bound to the cell surface in fibroblast-differentiated cells (75%), in epithelial type cells only 40% occurs in the cell-associated fraction. The amounts of secreted GAGs are 6- to 8-fold higher in epithelial than in fibroblast-like mesothelioma cultures. In cells with the fibroblast phenotype, the beta-homodimer of platelet-derived growth factor (PDGF) in a concentration of 1.5 ng/mL stimulates HA and GalAG synthesis 5-fold and that of HS 10-fold, whereas higher concentrations suppress this stimulatory effect. The stimulatory effect, observed at low concentrations of this growth factor, was completely blocked by the addition of antibodies against this factor. In epithelially differentiated cells, the production of all GAGs was suppressed after addition of this factor, even at low concentrations. We therefore suggest that mesothelioma cells can produce GAGs, the synthesis of which is dependent on the presence and concentration of PDGF beta-homodimer. The differences between the two cell lines regarding the effect of this growth factor on GAG synthesis indicates that the regulation of this synthesis is complex, other factors also being important.

Functional specialization of fibronectin-binding beta 1-integrins in T lymphocyte migration.

We have investigated the role of alpha 4 beta 1 and alpha 5 beta 1 integrins in adhesion and migration of T lymphocytes to extracellular matrix proteins. Fibronectin, collagen type IV, and laminin promoted haptotactic and chemotactic migration of lymphoid T cell lines and 12-O-tetradecanoylphorbol 13-acetate-stimulated blood lymphocytes, as determined using a modified Boyden chamber system. Adhesion studies of the T cell lines indicated involvement of both alpha 4 beta 1 and alpha 5 beta 1 integrins in the binding to fibronectin. In contrast, migration assays demonstrated that haptotactic and chemotactic migration to fibronectin in most cases was mediated by only one of the beta 1 integrins. FACS analysis demonstrated comparable amounts of alpha 4 beta 1 and alpha 5 beta 1 on the various cell lines, indicating that utilization of the integrins for migration is not determined by their expression on the cells. Haptotactic migration toward a 120-kDa fibronectin fragment containing the RGD sequence, confirmed the selectivity of the different beta 1 integrins in directing migration. Thus, T cells using alpha 5 beta 1 for haptotaxis against fibronectin were migrating against the 120 kDa fragment whereas T cells using alpha 4 beta 1 were not. These results indicate that the response of T cells to haptotactic and chemotactic signals usually is mediated selectively via alpha 4 beta 1 or alpha 5 beta 1 although binding of fibronectin to the cells is not restricted to only one of the integrins. Cholera toxin and 8-Br-cAMP but not pertussis toxin inhibited migration of T cell lines to fibronectin. Adhesion of these cells to fibronectin was not influenced by any of the toxins. Thus, both in their integrin utilization and in their signaling pathways, adhesion and migration show substantial differences in T cells.