Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Trypanosoma cruzi infection modulates intrathymic contents of extracellular matrix ligands and receptors and alters thymocyte migration.

Several T cell abnormalities have been described in the course of acute Trypanosoma cruzi infection in mice, including severe effects on the thymus. In the present study, looking at the expression of extracellular matrix ligands in the thymus, we observed that deposits of fibronectin and laminin increased progressively during the course of infection, reaching a maximum at the peak of parasitemia and thymic atrophy. Concomitantly, membrane expression of fibronectin and laminin receptors (VLA-4, VLA-5 and VLA-6) was also enhanced on thymocyte subsets of infected mice. These results correlated with changes in intrathymic thymocyte migration ability during the acute phase of infection, when a higher fibronectin-dependent transmigratory activity of CD4(+)CD8(+) thymocytes was observed. Strikingly, we detected higher frequency of immature and high VLA-expressing CD4(+)CD8(+) T cells in the peripheral lymphoid organs of infected mice at the peak of parasitemia. These cells seemed to be thymus dependent, since significantly lower amounts of them were found in thymectomized mice, and some of them carry "prohibited" Vbeta segments of the TCR. Our data suggest an imbalance in the intrathymic cell trafficking following acute T. cruzi infection, likely due to dysregulated extracellular matrix-dependent interactions.

The potential role of integrin receptor subunits in the formation of local recurrence and distant metastasis by mouse breast cancer cells.

BACKGROUND: The mechanisms by which surgical injury fosters tumor growth are examined. METHODS: TA3Ha mouse breast tumor line and its subline (TA3AD) differing in their metastatic abilities as tested by two models were used. In model a, TA3Ha/TA3AD tumors were grown in the mammary fat pads of mice and then surgically removed with a curative intent. In model b, TA3Ha/TA3AD cells were injected intravenously into mice subjected to liver or spleen wedge resection. Frequency of tumor formation at various sites was assessed. Expression of integrin, immunoglobulin, and proteoglycan cell adhesion receptors on TA3Ha and TA3AD cells was examined by flow cytometry. The roles of these receptors in metastasis were examined by blocking them by selected ligands and/or antibodies. RESULTS: Frequencies of local recurrence and axillary metastasis after surgical resection, were 43% (32/74), and 37% (27/74) with TA3Ha tumors and 4% (1/29) at both sites with TA3AD tumors. Tumors at surgically injured spleen and the liver were seen in 75% (141/189) and 45% (107/240) of the mice with TA3Ha cells and in 8% (3/38) and 10% (4/42) of the mice with TA3AD cells. alpha 5 and CD44 receptors were expressed by TA3Ha cells but not by TA3AD cells. Other receptors examined were similarly expressed by both cell lines. Blocking of alpha 5 receptor by fibronectin reduced tumor implantation in a dose-dependent manner. CONCLUSIONS: The data suggest a correlation among the ability to implant at surgically injured sites, to form local recurrence, and to express the fibronectin receptor subunit.

Expression of laminin 5, fibronectin, and epithelium-associated integrins in recurrent aphthous ulcers.

Recurrent aphthous ulceration (RAU) is characterized by an ulcerated lesion that persists longer than traumatic ulcers of similar size. This delayed healing phase of the lesion was investigated for extracellular matrix components and matrix receptors (integrins). The hypothesis tested was that aphthous ulcers may lack key extracellular matrix components, or their receptors, that are necessary for the migration of marginal keratinocytes from the ulcer edge. We immunocytochemically stained biopsy specimens of RAUs and non-involved mucosal specimens from HIV+ and non-infected individuals to investigate the presence and distribution of molecules reported to be associated with reepithelialization of mucosal and cutaneous wounds. Fibronectin, laminin type 5 (kalinin), and integrin subunits beta 1, beta 4, alpha 6, and alpha v were consistently found at the margins of RAU, as they are in traumatic ulcers. The alpha 5 and beta 6 subunits were not always present. We also found alpha v in the intact stratified squamous epithelium adjacent to ulcers. Immunohistochemical stains showed distruption in the deposition of laminin 5 and an apparent lack of fibronectin at the edges of some ulcers. Although these tissue results do not determine which integrin subunits are paired with each other, they do show some alterations in their expression in RAU. Absence of one or more of these molecules at the migrating front may contribute to delayed epithelial regeneration. It is likely that the absence or inappropriate expression of keratinocyte integrins or their extracellular matrix receptors occurs after the causative factors (currently unknown) of the lesion are gone. The reason for the altered expression of these molecules may be related to the secretory products (including lymphokines and proteinases) of the lymphocytic infiltrate.

Reduced expression of platelet surface glycoprotein receptor IIb/IIIa at hyperthermic temperatures.

BACKGROUND: Hyperthermic temperatures exist from the heat dissipation of the implantable energy source of an artificial heart. This procedure as well as therapies for cancer and thermal injuries pose a new medical problem. Among many reported effects of heat on biologic systems, platelet functions such as maximal aggregation and adhesion are known to be reduced. Using flow cytometry, we have studied platelet dysfunction at elevated temperatures and have gained a mechanistic comprehension of the loss of platelet function. EXPERIMENTAL DESIGN: Platelet rich plasma was incubated at differing temperatures for 1 hour. Immediately after, the platelets were stained using mAb against glycoprotein IIb/IIIa (GPIIb-IIIa) (CD41a) and other platelet surface glycoproteins (GP) involved in aggregation and adhesion. Relative fluorescence intensity was measured using single-labeled, laser flow cytometry to determine changes in GP surface expression. In addition, scanning electron microscopy was used to evaluate morphologic changes. RESULTS: Hyperthermic temperatures between 40 and 44 degrees C significantly lowered the mAb cell surface binding in vitro of GP that participate in aggregation and adhesion. The most dramatic temperature-dependent loss of mAb binding was demonstrated by anti-GPIIb-IIIa, the mAb against the fibrinogen receptor. mAb binding to this receptor at 44 degrees C was decreased to 6.2% of a base-line fluorescence intensity of 654 (arbitrary units). The ADP-induced aggregation of platelets incubated at the same temperature also decreased to 2.1% of maximum aggregation. Other mAb, such as those against the von Willebrand factor receptor (GPIb) (CD42b), the thrombospondin receptor (GPIV) (CD36), and GPIIIa (CD61), also showed statistically significant reduction of mAb binding but to a lesser degree. Finally, scanning electron microscopy as well as side-scatter density plots from flow cytometry revealed that platelets became more spherical after incubation at 44 degrees C. CONCLUSIONS: The significant reduction in mAb binding correlates with functional impairment exhibited during hyperthermic incubation. Our results support the loss of binding ability of surface GP that are involved in aggregation and adhesion as a mechanism of platelet dysfunction upon heating. GPIIb-IIIa appeared the most susceptible to heat and the principal agent in thermal induced loss of platelet function. Significant morphologic changes at 44 degrees C, the critical temperature at which ADP-induced aggregation ceases, may contribute as well.

Coordinated expression of the vitronectin receptor and the urokinase-type plasminogen activator receptor in metastatic melanoma cells.

Integrin alpha v beta 3 is a marker of progression in malignant melanoma. Previously we reported that human melanoma cells derived from regional lymph node metastases had increased alpha v beta 3-mediated adhesion to lymph node vitronectin. In the present study, the expression and function of alpha v beta 3 were further investigated with emphasis on the functional relationship between alpha v beta 3 and the urokinase-type plasminogen activator system of proteolysis. We found that metastases-derived melanoma MeWo LNI 6I (6I) and MIM/8 LNI cells had a markedly increased expression of alpha v mRNA transcripts relative to the parent lines which was reflected in significantly elevated levels of the alpha v beta 3 heterodimers on the cell surface. These cells also expressed elevated levels of urokinase plasminogen activator receptor (uPAR) mRNA and had higher levels of surface bound urokinase plasminogen activator as detected by immunolabeling. To determine whether the expression of uPAR and alpha v were linked, alpha v synthesis in the metastatic melanoma cells was suppressed using alpha v antisense phosphorothioate oligonucleotides. This resulted in a marked decrease in detectable alpha v mRNA and protein and a corresponding substratum-specific reduction in cell adhesion to vitronectin. When uPAR expression in these cells was subsequently analyzed, we found a reduction of approximately 50% in uPAR mRNA levels. On the other hand, ligation of the alpha v beta 3 receptor on the melanoma cells by immobilized antibody resulted in a twofold increase in uPAR mRNA. The results suggest that the expression of uPAR in metastatic melanoma cells is linked to the expression and function of the vitronectin receptor.

Differential sensitivity of thoracic malignant tumors to adenovirus-mediated drug sensitization gene therapy.

Malignant mesothelioma may prove to be an attractive candidate for somatic gene therapy with replication-deficient recombinant adenovirus transfer of a toxic, or drug sensitization gene. Transfer of the herpes simplex thymidine kinase type I gene (HSVtk), followed by exposure to the acyclic nucleoside drug ganciclovir, has been shown to be an effective tumor cell killing system. To study generalized applicability, we tested a number of thoracic malignant cell lines for their sensitivity to gancyclovir after infection with an adenoviral vector containing the HSVtk gene (Ad.RSVtk). Using the concentration of gancyclovir required to kill 50% of the cells (IC50) as a measure of sensitivity, we detected variable sensitivity among cell lines, with mesothelioma most sensitive (IC50 = 0.075 to 2.8 mumol/L gancyclovir), and non-small-cell carcinoma lines having an intermediate sensitivity (IC50 = 1.5 to 100 mumol/L). In contrast, an ovarian carcinoma line was extremely resistant (IC50 > 2000 mumol/L). To study the possible mechanisms for these differences, we studied cell lines with regard to their ability to be infected with an adenoviral vector containing a marker gene (Ad.CMVlacZ) and expression of the vitronectin receptor alpha v (an integrin cell adhesion molecule shown to be required for adenovirus internalization after initial binding). We found that the degree of lacZ transduction correlated with HSVtk sensitivity, whereas vitronectin receptor expression did not, suggesting that differences in initial viral binding ability, rather than internalization, may explain the sensitivity differences seen in vitro.

Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones.

Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix.

CD36 induction on human monocytes upon adhesion to tumor necrosis factor-activated endothelial cells.

Cell adhesion between circulating monocytes and the endothelium is a critical component of vascular thromboregulation and atherogenesis. The biochemical and genetic consequences of adhesion are poorly understood. We have found that monocyte surface expression of CD36, an integral membrane receptor for thrombospondin, collagen, and oxidized low density lipoprotein, increased dramatically upon adhesion to tumor necrosis factor-activated human umbilical vein endothelial cells (HUVEC). Expression was assessed by indirect immunofluorescence microscopy and immunoblotting using monoclonal antibodies to CD36. Steady-state CD36 mRNA levels, detected by RNase protection assay, also showed a similar pattern of up-regulation. To verify the adhesion dependence of the observed phenomenon, monocytes were co-cultured with tumor necrosis factor-activated HUVEC in a transwell apparatus that physically separated monocytes from the endothelial cells. Under these conditions, no increase in CD36 expression was detected, demonstrating that the enhanced monocyte CD36 expression observed is not due to soluble factors released by HUVEC. To characterize the specific adhesion molecules involved in the process, co-culture assays were performed on murine L cells transfected with either human E-selectin or intercellular adhesion molecule-1 cDNAs. A dramatic increase in CD36 mRNA was seen upon monocyte adhesion to E-selectin-transfected L cells compared with adhesion to intercellular adhesion molecule-1 or control transfectants. Furthermore, monoclonal antibodies to E-selectin inhibited the adhesion-dependent up-regulation of CD36 mRNA induced by transfected L cells or cytokine-activated endothelial cells. These findings demonstrate adhesion-dependent gene regulation of monocyte CD36 and suggest the possible involvement of E-selectin in initiating this process.

Expression of vitronectin receptor on human NK cells and its role in protein phosphorylation, cytokine production, and cell proliferation.

In this paper, we provide evidence that the vitronectin receptor (VNR) alpha v beta 3 is expressed on human NK cells. The presence of this VNR on freshly purified NK cells was demonstrated by flow cytometry analysis, as well as biochemically, after 125I-labeled surface lactoperoxidase labeling and immunoprecipitation. mAbs LM142 and LM609 specific for alpha v and alpha v beta 3, respectively, precipitated a heterodimer of alpha- and beta-chains with approximate molecular masses of 155 and 110 kDa under nonreducing conditions. Under reducing conditions, there was an apparent decrease in the molecular mass of the alpha-chain, which is likely to result from the release of a protein of 20 to 30 kDa linked by internal disulfide bond to the alpha v-chain. Integrin alpha v beta 3 expressed on NK cells became functional, i.e., was able to bind its ligand, vitronectin (VN), only after cellular activation or when costimulation with an additional signal was provided. Thus, NK cells adhered to plastic-immobilized VN only after IL-2 activation, and RGD-containing synthetic peptides or mAbs specific for alpha v beta 3 complex inhibited this binding. To assess the role of the VNR in signal transduction, anti-beta 3 mAb was used to cluster the VNR on NK cells and, thereby, mimic the process that occurs during formation of adhesive contacts. Cross-linking of VNR on fresh NK cells stimulated phosphorylation on tyrosine residues of several intracellular proteins. The major increase in tyrosine phosphorylation was observed in proteins of approximate molecular masses of 75 and 120 kDa. Therefore, signal transduction by the VNR on NK cells induced activation of intracellular protein kinases. Ligand engagement of the VNR on NK cells also costimulated cytokine production and proliferation of NK cells. Binding of NK cells to plastic-immobilized VN served as a costimulus with either anti-Fc gamma RIII or IL-2 to produce IFN-gamma, TNF-alpha, and cell proliferation. Our findings suggest that occupancy and subsequent clustering of VNRs play a role in the activation and function of human NK cells.

The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix.

The human ovarian surface epithelium (OSE), or ovarian mesothelium, is functionally complex as seen by its capacity to proliferate, migrate, and contribute to ovulation and ovulatory repair in response to cyclic hormonal and environmental changes. We wished to determine whether this phenotypic versatility is reflected in cell-extracellular matrix interactions in primary and low-passage culture. Comparisons of cultures maintained on different substrata revealed that these cells form cohesive monolayers on plastic, while fibrin clots enhance cell dispersion, and thus may provide a migratory cue. The cells invaded Matrigel as multicellular aggregates, while collagen gels mediated a morphologic epithelial-mesenchymal conversion. On plastic, the cells produced extracellular matrix components characteristic of epithelial basement membrane (laminin and collagen type IV), as well as stroma (collagen types I and III). In addition, ovarian surface epithelial cells secreted serine proteases and matrix metalloproteinases. The levels of chymotrypsin- and elastase-like proteases were dictated by the substratum: low levels were secreted by cells grown on plastic, intermediate levels on collagen gels and fibrin clots, and most protease was produced on Matrigel. The rate of cell proliferation varied with the substrata and was inversely related to protease secretion. Integrin expression was greatest on plastic and least on collagen gels where integrins were downregulated with time. alpha 6/beta 4 was absent from all cells while varying levels of alpha 2, alpha 3, alpha 5, beta 1, and vitronectin receptor were detected depending on the culture substratum employed. In low-passage cultures of human ovarian surface epithelial cells, then, cell shape, growth, protease production, and integrin expression are modulated by the extracellular matrix. The cells, in turn, alter extracellular matrix by synthesis, lysis, and physical remodeling, and express both stromal and epithelial characteristics. The broad repertoire of these functions may be related to their mesodermal origin, and may reflect the expression of a dual, epithelio-mesenchymal phenotype by relatively immature, uncommitted cells. The results demonstrate the great complexity and versatility of these interactions which render OSE cells capable of participating in numerous physiological and pathological processes.

Integrin alpha v beta 3 differentially regulates adhesive and phagocytic functions of the fibronectin receptor alpha 5 beta 1.

The plasma protein fibronectin is an important opsonin in wound repair and host defense. To better understand the process of fibronectin-mediated phagocytosis, we have transfected K562 cells, which endogenously express alpha 5 beta 1, with alpha v beta 3. In these transfectants, antibodies to alpha v beta 3 block phagocytosis of fibronectin-opsonized beads completely, even though half the ingestion occurs through endogenous alpha 5 beta 1 receptors. alpha 5 beta 1-mediated adhesion to fibronectin-coated surfaces is unaffected by alpha v beta 3 ligation. Neither alpha v beta 5 nor alpha M beta 2 ligation affects alpha 5 beta 1 phagocytic function in transfectants expressing these receptors. Pharmacologic data suggest that alpha v beta 3 ligation suppresses the phagocytic competence of high affinity alpha 5 beta 1 receptors through a signal transduction pathway, perhaps involving protein kinase C. In addition to its significance for phagocytosis, alpha v beta 3 regulation of alpha 5 beta 1 function may be significant for its roles in cell migration, metastasis, and angiogenesis.

The expression of osteoclast markers on foreign body giant cells.

The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (M phi Ag), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50-200 mM and expressed VNR and a restricted range of M phi Ag. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express M phi Ag such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.

The integrin alpha v gene: identification and characterization of the promoter region.

We isolated a 15.5 kilobase pair DNA fragment that contains the 5' end of the human vitronectin receptor alpha subunit (alpha v) gene. The nucleotide sequence of the 5' flanking region, first exon and part of the first intron of the alpha v gene was determined. The sequence showed that the 5' end of the alpha v gene lies within a CpG island. The transcriptional initiation site was mapped 169 base pairs upstream of the alpha v translational initiation site. The 5' flanking region of the alpha v gene does not contain TATA or InR transcriptional control elements but does contain four Sp1 binding sites, two Ets binding sites and one GATA binding site. The identified alpha v gene 5' flanking region directed the expression of human growth hormone in transfected HeLa cells. Successive deletions of the 5' flanking region demonstrated a 222 bp region that exerts a strong positive effect on alpha v promoter activity.

Requirement of vascular integrin alpha v beta 3 for angiogenesis.

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.

Emergence of alpha 5 beta 1 fibronectin- and alpha v beta 3 vitronectin-receptor expression in melanocytic tumour progression.

Cell adhesion is crucial in the process of tumour progression. As integrins are important receptor molecules involved in cell adhesion, we studied the distribution of the alpha 1-6, alpha v, alpha IIb, beta 1, beta 3, and beta 4 integrin subunits in tissue sections of common naevocellular naevi (n = 22), dysplastic naevi (16), thin (24) and thick primary cutaneous melanomas (28), and melanoma metastases (25). We found correlated expression of alpha 1/alpha 2, of alpha 4/alpha 5/beta 3, and of alpha 6/beta 4. Decrease of alpha 6 and beta 4, and increase of alpha 4 and alpha v were found to be correlated with melanoma progression. Furthermore, expression of alpha 5 and beta 3 was detected only in primary melanoma and melanoma metastasis. Our findings indicate that during melanoma progression alterations in integrin expression occur, the most striking being emergence of alpha 5 beta 1 fibronectin and alpha v beta 3 vitonectin receptor.

Regulation of vitronectin receptor expression by retinoic acid on human melanoma cells.

The integrin family of adhesion receptors is likely to be important for tumor cell invasion and dissemination. We have studied the effects of the differentiating agents retinoic acid on integrin expression by the human melanoma cell line MeWo. Our results show that this agent inhibits cellular proliferation, increases melanin content and induces morphological changes in MeWo cells. Functionally, these alterations are associated with an enhanced adhesion to matrix protein vitronectin and higher levels of expression of vitronectin receptor on the cell surface. This is accompanied by increased levels of alpha v integrin mRNA. Although the mechanism by which retinoic acid regulates the expression of vitronectin receptor in MeWo cells needs further examination, this system may represent a good model for understanding the role of this receptor in melanoma progression, as well the molecular basis for retinoic acid therapy in these tumors.

Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression.

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-1 alpha (IL-1 alpha) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-1 alpha, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.