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C Cabañas

Publications and source records attributed to C Cabañas.

At least 37 records · Page 2Linked to original sources
PubMed1997

Prevention of cytokine-induced changes in leukocyte adhesion receptors by nonsteroidal antiinflammatory drugs from the oxicam family.

OBJECTIVE: To explore the effect of the nonsteroidal antiinflammatory drugs (NSAIDs) piroxicam and meloxicam on quantitative and qualitative changes in leukocyte adhesion receptors induced by cytokines and other activation stimuli. METHODS: The expression of CD11b and L-selectin during neutrophil activation with tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), FMLP, phorbol myristate acetate (PMA), and calcium ionophore A23187 was assessed by flow cytometry. Enzyme-linked immunosorbent assays were used to quantitate soluble L-selectin shed after neutrophil stimulation. Enzyme release was measured to determine neutrophil degranulation by proinflammatory stimuli. Changes in affinity state of beta 1 and beta 2 integrins after neutrophil and T lymphocyte stimulation were assessed, by flow cytometry, using the monoclonal antibodies (MAb) HUTS-21 (anti-beta 1) and CBRM1/5 (anti-CD11b), which recognize activation-dependent epitopes on these two integrins. RESULTS: Pretreatment of neutrophils with either NSAID prevented the changes in L-selectin and CD11b expression induced by TNF alpha, GM-CSF, and FMLP, but not those induced by PMA or A23187. Furthermore, piroxicam significantly decreased the amount of L-selectin shed by cytokine-treated neutrophils, whereas it did not exert this effect on PMA- or A23187-treated neutrophils. Piroxicam also decreased the release of gelatinase and lysozyme induced by TNF alpha, but not by PMA. Interestingly, piroxicam prevented the conformational changes that beta 2 integrins underwent upon activation of neutrophils: the appearance of the activation epitope of CD11b, detected by the CBRM1/5 MAb, was blocked by piroxicam in TNF alpha-treated neutrophils. Moreover, in chemokine-treated T lymphocytes, the expression of activation epitopes on beta 1 integrins was also diminished by piroxicam. In contrast, this NSAID did not affect the beta 1 integrin conformational changes induced by PMA or Mn++. CONCLUSION: Our results indicate that members of the oxicam family are able to interfere with events of neutrophil function, such as their degranulation and cytokine-mediated activation changes in adhesion molecules, both in neutrophils and in lymphocytes. Such effects may significantly contribute to the antiinflammatory activity of these drugs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Functional relevance during lymphocyte migration and cellular localization of activated beta1 integrins.

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the beta1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for beta1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common beta1 subunit of VLA proteins. Engagement of beta1 integrins through this LIBS epitope inhibited T lymphoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized beta1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-beta1 activating antibodies.

Actin Cytoskeleton↗
PubMed1996

Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common beta 1 chain.

The very late activation antigens (VLA) or beta 1 integrins mediate cell attachment to different extracellular matrix proteins and intercellular adhesions. The ligand binding activity of these adhesion receptors is not constitutive and can be regulated by temperature, presence of extracellular divalent cations, stimulatory monoclonal antibodies (mAbs), and cellular activation. We have generated three novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes on the common beta 1 subunit (CD29) of VLA integrins whose expression correlates with the ligand binding activity of these heterodimeric glycoproteins. This correlation has been demonstrated for several integrin heterodimers in different cell systems using a variety of extracellular and intracellular stimuli for integrin activation. Thus, the presence of micromolar concentrations of extracellular Mn2+, preincubation with the activating anti-beta 1 mAb TS2/16, and cell treatment with phorbol esters or calcium ionophores, induced the expression of the HUTS beta 1 epitopes on T lymphoblasts. Using a panel of human-mouse beta 1 chimeric molecules, we have mapped these epitopes to the 355-425 sequence of the beta 1 polypeptide. This segment represents therefore a novel regulatory region of beta 1 that is exposed upon integrin activation. Interestingly, binding of HUTS mAbs to partially activated VLA integrins results in maximal activation of these adhesion receptors and enhancement of cell adhesion to beta 1 integrin ligands collagen, laminin, and fibronectin.

Antibodies, Monoclonal↗
PubMed1996

Regulation of integrin function.

Cells communicate with their environment through several kinds of cell surface receptor. One of the most important families of cell adhesion receptors are the integrins, which include receptors that mediate cell-cell as well as cell-extracellular matrix interactions. A distinctive feature of integrins is their variable adhesive competence that is reversibly modified depending on the state of cell differentiation and/or activation or in response to environmental signals. The acquisition of adhesive function by integrins may be a consequence of conformational changes in these receptors that result in an increased ligand binding affinity. In addition, cells can control integrin-mediated adhesion through other mechanisms, including receptor clustering and association to cytoskeleton, phenomena that regulate the avidity of integrins for ligand molecules without altering their monovalent affinity. These phenomena have collectively been designated as 'post-receptor occupancy events'. These two interesting aspects of the regulation of integrin function are reviewed.

Cell Adhesion Molecules↗
PubMed1996

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Animals↗
PubMed1994

Functional regulation of the human integrin VLA-1 (CD49a/CD29) by divalent cations and stimulatory beta 1 antibodies.

We have investigated the regulation by divalent cations Mg2+, Ca2+ and Mn2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg2+ or microM levels of Mn2+. In contrast, Ca2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg(2+)-supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin beta 1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-alpha 1 specific monoclonal antibody, 5E8D9.

Antibodies, Monoclonal↗
PubMed1993

Ligand intercellular adhesion molecule 1 has a necessary role in activation of integrin lymphocyte function-associated molecule 1.

The signaling that causes the leukocyte integrin lymphocyte function-associated molecule (LFA-1) to bind firmly to its ligand intercellular adhesion molecule 1 (ICAM-1) is transduced indirectly through other T-cell receptors and is termed inside-out signaling. We show here that the high-affinity state of LFA-1 is characterized by expression of the LFA-1 epitope detected by monoclonal antibody 24. This epitope is expressed not in response to the initial agonist-mediated signal but when LFA-1 binds to ICAM-1, indicating that ligand binding induces an alteration in LFA-1. As would be predicted, the monoclonal antibody 24 epitope is confined to the LFA-1, which is located at the site of contact between T cells and ICAM-1-expressing transfectants. When a fixation protocol for "freezing" receptors is used, only T cells that are fixed after prior exposure to ICAM-1 bind firmly to ICAM-1 a second time. This suggests that, in addition to the inside-out signaling, a previously unrecognized requirement for full activation of the leukocyte integrin LFA-1 is the initial interaction with its ligand ICAM-1. Thus, activation of LFA-1 is in part achieved by an induced fit imposed from without by interaction with ligand.

Animals↗
PubMed1992

Camptothecin induces differentiation and stimulates the expression of differentiation-related genes in U-937 human promonocytic leukemia cells.

We have studied the effect of the DNA topoisomerase I inhibitor camptothecin on growth, differentiation, and gene expression in U-937 human promonocytic leukemia cells. At a concentration of 20 nM, camptothecin caused significant DNA strand breakage and decreased the growth activity by accumulating cells preferentially at the G2 phase of the cycle. The growth arrest occurred concomitantly with an increase in cell size. Under those conditions, camptothecin induced differentiation, as demonstrated by (a) the capacity of the cells to generate reactive oxygen species, (b) the increase in the surface expression of the leukocyte integrins CD11b/CD18 and CD11c/CD18, (c) the increase in the cellular content of the intermediate filament protein vimentin, and (d) the decrease in the surface expression of the transferrin receptor. Camptothecin also induced the expression of differentiation markers in other human myeloid cells, namely, the promonocytic THP-1 and the myelomonocytic HL-60 cell lines. Northern blot assays revealed that camptothecin stimulated the expression of CD11b, CD11c, and vimentin at the mRNA level. Moreover, the drug increased the transcription rate of the vimentin gene, as shown by "run-on" transcription assays.

Camptothecin↗
PubMed1992

Regulated expression on human macrophages of endoglin, an Arg-Gly-Asp-containing surface antigen.

Endoglin is an endothelial homodimeric membrane antigen containing the tripeptide arginine-glycine-aspartic acid (RGD), which is a recognition motif for adhesion receptors of the integrin family. We have investigated the expression of endoglin by monocyte/macrophage cells from different tissue compartments and at different stages of cell differentiation. Although endoglin is absent from peripheral blood monocytes, it is expressed by in vitro differentiated monocytes as determined by flow cytometry using the endoglin-specific monoclonal antibody 44G4 and 8E11. Furthermore, Northern blot analyses revealed a correlation between the presence of endoglin mRNA and the surface expression of the antigen by in vitro differentiated monocytes. Immunostaining of frozen tissue sections with the 8E11 monoclonal antibody demonstrated the presence of endoglin not only in the endothelium of all the tissues studied, but also on the interstitial macrophages present in the red pulp of the spleen. Using as a model of macrophage differentiation monocytic cell lines treated with phorbol esters, we found that the reactivity of the 8E11 monoclonal antibody is greatly increased on U-937 and HL-60 cells during their PMA-induced differentiation. These findings clearly demonstrate for the first time the regulated expression of the putative adhesion molecule endoglin by macrophages.

Antibodies, Monoclonal↗
PubMed1992

Modulation by dexamethasone of insulin binding and insulin receptor mRNA levels in U-937 human promonocytic cells.

Treatment with 5 x 10(-6) M dexamethasone stimulated insulin binding in human promonocytic U-937 cells. When curvilinear Scatchard plots were examined according to the one-site model, only changes in affinity, but not in receptor numbers, were observed. However, when the two-site model was applied, an increase in both the affinity and the number of the high affinity-low capacity sites was observed, with maximum values at 15 h. By contrast, the low affinity-high capacity sites did not undergo significant alterations. Northern blot assays revealed two insulin receptor-related mRNAs of approximately 11 and 7 kb in size. Dexamethasone increased the levels of these RNAs, following similar kinetics to those of high affinity receptor expression. This suggests that the 11 and 7 kb species carry information for high affinity insulin receptors, and that in U-937 cells the expression of this receptor subclass is primarily regulated at the mRNA level.

Cell Line↗
PubMed1992

Divalent cation regulation of the function of the leukocyte integrin LFA-1.

The integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells serves as a useful model for analysis of leukocyte integrin functional activity. We have assessed the role of divalent cations Mg2+, Ca2+, and Mn2+ in LFA-1 binding to ligand intercellular adhesion molecule-1 (ICAM-1) and induction of the divalent cation-dependent epitope recognized by mAb 24. Manganese strongly promoted both expression of the 24 epitope and T cell binding to ICAM-1 via LFA-1, suggesting that Mn2+ is able to directly alter the conformation of LFA-1 in a manner that favors ligand binding. Since Mn2+ also promotes functional activity of other integrins, parallels in mechanism of ligand binding may span the integrin family. In contrast, induction of 24 epitope expression by Mg2+ required removal of Ca2+ from T cell LFA-1 with EGTA. Furthermore, binding of mAb 24 to T cell LFA-1 in the presence of either Mn2+ or Mg2+ was found to be specifically inhibited by Ca2+, suggestive of a negative regulatory role for Ca2+ in the control of leukocyte integrin function. Analysis of T cell binding to ICAM-1 via LFA-1 in the presence of Mg2+ or Mn2+, confirmed that Ca2+ exerted inhibitory effects upon LFA-1 function. The implication of our findings is that Ca2+ bound with relatively high affinity to LFA-1 may serve to maintain an inactive state. Thus induction of function and 24 epitope expression may occur as a result of displacement of Ca2+ from leukocyte integrins or alternatively, such activators may be able to impose the required conformational change in the presence of bound Ca2+.

Animals↗
PubMed1992

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function.

The leukocyte integrins (CD11/CD18 or beta 2-type integrins) are expressed exclusively on leukocytes and participate in many adhesion-dependent functions (Arnaout, M.A. 1990. Blood. 75:1037-1050; Springer, T. A. 1990. Nature. (Lond.) 346:425-434; Dustin, M. L., and T. S. Springer. 1991. Annu. Rev. Immunol. 9:27-66). The avidity of leukocyte integrin binding to their ligands or counter-receptors is dependent upon response to intracellular signals (Wright, S. D., and B. C. Meyer. 1986. J. Immunol. 136:1759-1764; Dustin, M. A., and T. S. Springer. 1989. Nature (Lond.). 341:619-624). We have investigated the effects of a novel mAb (mAb 24) which defines a leukocyte integrin alpha subunit epitope that is Mg(2+)-dependent and may be used as a "reporter" of the activation state of these receptors (Dransfield, I., and N. Hogg. 1989. EMBO (Eur. Mol. Biol. Organ) J. 8:3759-3765; Dransfield, I., A.-M. Buckle, and N. Hogg. 1990. Immunol. Rev. 114:29-44; Dransfield, I., C. Cabañas, A. Craig, and N. Hogg. 1992. J. Cell Biol.) Data is presented to show that this mAb inhibits monocyte-dependent, antigen-specific T cell proliferation and IL-2-activated natural killer cell assays which are both dependent on lymphocyte function-associated antigen-1 (LFA-1), and complement receptor type 3 (CR3)-mediated neutrophil chemotaxis to f-Met-Leu-Phe. This inhibitory effect is not caused by the prevention of receptor/ligand binding because LFA-1/ICAM-1, LFA-1/ICAM-2,3 and CR3/iC3b interactions are, under activating conditions, promoted rather than blocked by mAb 24. As it does not interfere with mitogen-stimulated T cell proliferation, it is unlikely that mAb 24 transduces a "negative" or antiproliferative signal to the T cells to which it is bound. Using a model system of transient activation of LFA-1, we have found that mAb 24 prevents "deadhesion" of receptor/ligand pairs, possibly locking leukocyte integrins in an "active" conformation. It is speculated that inhibition of leukocyte integrin function by this mAb reflects the necessity for dynamic leukocyte integrin/ligand interactions.

Antibodies, Monoclonal↗
PubMed1992

Leukocyte integrin activation.

Certain stages of the immune response require interaction of leukocytes with each other and with non-hematopoietic cells. One of the systems implicated in these interactions involves an integrin, LFA-1 (Lymphocyte Function Antigen-1), expressed by all leukocytes at their cell surface, and a molecule belonging to the immunoglobulin superfamily, ICAM-1 (Intercellular Adhesion Molecule-1). The avidity of LFA-1 for ICAM-1 is transient. It is modulated both by regulation of ICAM-1 expression and by activation of LFA-1 molecules constitutively expressed on leukocyte membranes. This activation, which induces a conformational change in the molecule, depends on the presence of divalent cations, notably Mg++. This has been demonstrated by using a specific monoclonal antibody, MAb 24. In addition to being a ligand for LFA-1, ICAM-1 is sometimes used as a cell receptor by pathogens such as Plasmodium falciparum, the causative organism of malaria. Very careful study of the binding site of this pathogen using specific antibodies, mutagenesis studies and the construction of a three-dimensional model of the molecule suggests some interesting therapeutic possibilities for the treatment of malaria.

CD3 Complex↗
PubMed1991

Lymphocyte-fibroblast adhesion. A useful model for analysis of the interaction of the leucocyte integrin LFA-1 with ICAM-1.

The adhesion of human T lymphoblasts to ICAM-1-expressing normal dermal fibroblasts has been assessed as a sensitive model system for the analysis of the interaction of the leucocyte integrin LFA-1 with its counter-receptor ICAM-1. Using this model system, the effects of factors known to regulate the activity of LFA-1 have been quantitated: temperature; concentration of divalent cations; and exposure to phorbol esters. We show here that under the appropriate assay conditions, this model system represents a useful and simple alternative to the detection of leucocyte binding to purified ICAM-1 and also has the additional advantage of permitting more sensitive quantification than is possible using the homotypic adhesion assay.

Antibodies, Monoclonal↗
PubMed1991

Induction of LFA-1-mediated homotypic adhesions in promonocytic U-937 cells occurs independently of cell differentiation.

The differentiation of monocytes into macrophages occurs along with a marked increase in LFA-1-dependent intercellular adhesions. Similarly, the phorbol ester-induced differentiation of U-937 promonocytic cells into macrophage-like cells is morphologically characterized by an important increase in LFA-1/ICAM-1-dependent intercellular homotypic adhesions. Since an important functional role in activation of human T cells has been demonstrated for LFA-1-dependent adherence, we have analyzed whether the induction of LFA-1-dependent intercellular adhesion of human monocytic cells is necessarily accompanied by differentiation of these cells. We found that treatment of the promonocytic U-937 cells with the anti-LFA-1 mAb NKI-L16 induces formation of intercellular clusters, but does not induce cell differentiation as determined by several differentiation markers. These markers include the arrest of cell proliferation, production of reactive oxygen species, changes in the cell surface expression of differentiation-associated antigens such as the transferrin receptor, CD11b and CD11c and changes in the levels of several specific gene transcripts such as CD18 antigen, c-myc, ornithine decarboxylase and vimentin. These findings suggest that LFA-1-dependent adhesion and differentiation of monocytic cells are independent processes.

Antibodies, Monoclonal↗
PubMed1991

Effects of poliovirus replication on undifferentiated and differentiated monocytic U937 cells: comparative studies with human macrophages.

Poliovirus infection of either undifferentiated or differentiated U937 cells produced a decrease in the percentage of cells positive for the surface expression of the CD4, CD11c, CD14, or 8E11 antigens. The number of 4F2 surface molecules per cell increased in infected normal U937 cells, but was unaffected in differentiated cells. The level of O2- production in infected differentiated U937 cells was approximately 50% of that found when not infected. Finally, poliovirus RNA levels and infectious particle production were similar in either cell type.

Antigens, CD↗
PubMed1991

Differentiation of human promonocytic leukemia U-937 cells with DNA topoisomerase II inhibitors: induction of vimentin gene expression.

The administration of the DNA topoisomerase II inhibitors 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (10(-7) M), VP-16 (2 x 10(-7) M), or novobiocin (1.5 x 10(-4) M) reduces the growth activity of human promonocytic leukemia U-937 cells, by arresting them preferentially at the G2 (m-AMSA and VP-16) or at the G1 and G2 (novobiocin) phases of the cell cycle. Under these conditions, m-AMSA and VP-16 induce the differentiation of the cells efficiently, as proved both by an increase in the production of reactive oxygen species and by the activation of the surface expression of CD11b and CD11c, two differentiation-specific antigens. Novobiocin also induces the expression of those differentiation markers, but to a lesser extent. Analyses by Northern blot indicate that the topoisomerase II inhibitors reduce the levels of c-myc and beta-actin mRNA and increase the levels of vimentin mRNA. The expression of vimentin is also stimulated at the protein level, as indicated by immunofluorescence assays. This represents one of the few known instances in which topoisomerase inhibitors stimulate gene expression in eukaryotic cells.

Amsacrine↗