The strength of integrin binding between neutrophils and endothelial cells

The firm adhesion of activated polymorphonuclear neutrophils to endothelial cells in blood vessels is achieved through binding of the integrin intercellular adhesion molecule. To contribute to the better understanding of this adhesion step, our investigation is aimed at the relationship between integrin expression and the strength of neutrophil binding to endothelial cells. Flow cytometry and 3D scanning microscopy are used to study integrin expression and distribution, respectively. It is found that CD11b/CD18 integrin expression is localized in clusters distributed irregularly over the neutrophil surface. After cell activation, the cluster distribution polarizes, increasing the local CD11b/CD18 density concurrently with nearly doubled integrin expression. The neutrophil adhesion efficiency is measured in a flow chamber coated successively by various substrates, including endothelial cells in an activated state. Analysis of the flow dependence of the number of attached cells reveals the prevailing number of neutrophils with stronger binding to the endothelium when both cells are in the activated state in comparison with non-activated cells.     

Flow injection analysis of binding reaction between fluorescent lectin and cells

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.     

L-dopa binding sites in rodent melanoma cells.

Rapid, saturable, specific and stereoselective binding of L-dopa to crude membranes and purified nuclei from rodent amelanotic melanoma cells is reported. Cross-linking of [3H]dopa to melanoma cell surface emphasized proteins of approx. 55, 30, 25 and less than 20 kDa. It is suggested that these binding sites may regulate melanocyte activity.     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

Psoralen activity and binding sites in melanotic and amelanotic human melanoma cells

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.     

Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells

Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Adrenocorticotropin binding activity of B16/C3 melanoma cells

The binding of adrenocorticotropic hormone (ACTH) to B16/C3 murine melanoma cells was found to be specific and saturable. The binding capacity of the cells changed as a function of the age of the culture. Scatchard analysis revealed one class of high-affinity ACTH binding sites. The specificity of ACTH binding to the cells was tested by displacement experiments with human leukocyte interferon (alpha-IFN) and alpha-melanocyte stimulating hormone (alpha-MSH) as the competitors. Structure-activity relationship of ACTH, alpha-MSH and alpha-IFN was discussed.     

Kinetics of oxygen consumption by phagocytosing human neutrophils

Oxygen consumption by phagocytosing human neutrophils commences after a lag of ～ 25 secs after particle uptake, reaches a maximal rate of ～ 35 nmols/10⁷ cells/min and remains linear for ～ 60 secs. A strict temporal and stoichiometric relationship exists between particle uptake and oxygen consumption. For each particle taken up, 0.2 fmols of oxygen is consumed in a very brief and self limiting process.     

Kinetics of superoxide production by stimulated neutrophils.

Oxygen-derived active species and superoxide radical in particular are generated and excreted upon granulocyte activation and are instrumental in host defense against bacterial and fungal infections. Associated with the activation of neutrophils is an apparent transitory oxy-radical production. Evidence from independent methods has previously suggested that radical production peaks shortly following neutrophil stimulation and decays within minutes. However, since neutrophil function in the body might reasonably be expected to last beyond the few minutes following stimulation, cessation of the production of oxy-radicals is unexpected. In an attempt to reconcile this discrepancy, the formation kinetics of superoxide by stimulated human neutrophils was reinvestigated by three independent methods: electron spin resonance, chemiluminescence, and ferricytochrome c reduction. The present results demonstrate that under appropriate experimental conditions stimulated neutrophils have the capacity to produce superoxide for several hours. The reasons for the previously reported "apparent" ephemeral nature of oxy-radical formation are discussed.     

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory responses in human neutrophils

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN₃), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H₂O₂-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[³H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (V_ist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects ofthe ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the bioogical activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.     

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.     

Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells

Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.     

Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.     

Kinetics of desolvation-mediated protein-protein binding

The role of desolvation in protein binding kinetics is investigated using Brownian dynamics simulations in complexes in which the electrostatic interactions are relatively weak. We find that partial desolvation, modeled by a short-range atomic contact potential, is not only a major contributor to the binding free energy but also substantially increases the diffusion-limited rate for complexes in which long-range electrostatics is weak. This rate enhancement is mostly due to weakly specific pathways leading to a low free-energy attractor, i.e., a precursor state before docking. For alpha-chymotrypsin and human leukocyte elastase, both interacting with turkey ovomucoid third domain, we find that the forward rate constant associated with a collision within a solid angle phi around their corresponding attractor approaches 10(7) and 10(6) M(-1)s(-1), respectively, in the limit phi approximately 2 degrees. Because these estimates agree well with experiments, we conclude that the final bound conformation must be preceded by a small set of well-defined diffusion-accessible precursor states. The inclusion of the otherwise repulsive desolvation interaction also explains the lack of aggregation in proteins by restricting nonspecific association times to approximately 4 ns. Under the same reaction conditions but without short range forces, the association rate would be only approximately 10(3) M(-1)s(-1). Although desolvation increases these rates by three orders of magnitude, desolvation-mediated association is still at least 100-fold slower than the electrostatically assisted binding in complexes such as barnase and barstar.     

MSH binding in bomirski amelanotic hamster melanoma cells is stimulated by L-tyrosine

Bomirski Ab amelanotic melanoma cells have recently been shown to undergo striking phenotypic changes when precursors of the melanogenic pathway, L-tyrosine and L-dopa, are added to the culture medium. The changes include increased tyrosinase activity andde novo synthesis of melanosomes and melanin. L-tyrosine and L-dopa appeared to elicit these responses through separate but overlapping regulatory pathways. Here we show an additional effect of L-tyrosine: stimulation of MSH binding capacity. Cells cultured for 24–48 hours in the presence of 200 M L-tyrosine display a 3–4 fold increase in their ability to bind¹²⁵l--MSH. L-dopa did not stimulate MSH binding under the same conditions. In control experiments neither L-tyrosine nor L-dopa had any effect on insulin binding. The amelanotic cells respond to MSH with increased dendrite formation, increased tyrosinase activity without melanin production, and decreased growth rate.     

Comparative antiproliferative and receptor binding activities of interferons alpha and beta on lymphoblastoid and melanoma cells

The relative antiproliferative and receptor binding characteristics of the hitherto little-characterized interferon alpha 4a on cells of lymphoid and epithelial origin are compared with two other type I interferons, alpha 2 a and beta. Using the lymphoblastoid cell line, Daudi, interferons alpha 4 a and alpha 2 b had similar antiproliferative activity, and were about 10-fold more active than IFN beta. By contrast, using the melanoma cell line Sk-Mel-28, IFN beta was the most active, whereas IFN alpha 2b and IFN alpha 4a were respectively 60-fold and greater than 1000-fold less active than on Daudi cells. Receptor binding did not correlate with antiproliferative sensitivities, but confirmed a shared receptor component for these three interferons. These results indicate that the antiproliferative activities of three type I IFNs differs markedly on different cell types and that this is unlikely to be due to receptor binding, but more likely a post receptor binding event.     

Thrombospondin binding by human squamous carcinoma and melanoma cells: relationship to biological activity

Human squamous carcinoma cells attach and spread on thrombospondin (TSP)-coated culture dishes but exhibit significant variability among individual cell lines in their degree of responsiveness. Using a highly responsive squamous carcinoma line and a cell line which is much less responsive (as well as a human melanoma cell line which does not respond at all in the adhesion assay), we have examined binding of exogenous radiolabeled TSP. The cells which were the most responsive to TSP in the adhesion assay bound the greatest amount of radiolabeled ligand. Binding was time- and dose-dependent, saturable, inhibitable with excess unlabeled TSP, reversible, and specific. The less-responsive squamous carcinoma cells bound only 25-30% of the amount of TSP bound by the highly responsive cells while the nonresponsive melanoma cells bound less than 10% of the amount bound by the highly responsive squamous carcinoma cells. Our previous studies (J. Varani et al. (1986) Exp. Cell Res. 167, 376) have shown that the highly responsive squamous carcinoma cells also synthesized the greatest amount of TSP as indicated by biosynthetic labeling studies. The less-responsive squamous carcinoma cells were intermediate in synthetic activity and no synthetic activity was seen with the melanoma cells. These findings suggest that the amount of ligand bound may determine the degree of biological responsiveness and that endogenously synthesized TSP may be the source of that ligand.