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Biomedical subjects

A F Lopez

Publications and source records attributed to A F Lopez.

At least 73 records · Page 4Linked to original sources
PubMed1991

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.

Binding, Competitive↗
PubMed1991

Inhibition of human monocyte adhesion by interleukin-4.

The adhesion of monocytes to vascular surfaces is central to inflammation and atherogenesis; however, very little is known about regulatory factors that can prevent these processes. Here we report the inhibition of human monocyte adhesion to human endothelial layers and plastic by interleukin-4 (IL-4), a T-cell-derived glycoprotein with pleiotropic activities. The inhibitory effects of IL-4 were seen with basal and cytokine-stimulated monocyte adhesion, were apparent at low concentration, and were abolished by inactivating IL-4. No direct toxic effect of IL-4 on monocytes was detected. Inhibition of adhesion was accompanied by small increases in monocyte surface expression of the leukocyte-functional antigen group of adhesion structures, suggesting that absolute levels of expression may be less important than the functional status of such molecules in the regulation of monocyte adhesion. In addition, inhibition by IL-4 of cytokine-stimulated monocyte adhesion was not associated with changes in the surface expression of cytokine receptors. These results suggest a role for IL-4 in the regulation of monocyte adhesion, and may provide for a common mechanism for the inhibitory effects of IL-4 on monocyte function.

Antigens, CD↗
PubMed1991

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.

Anions↗
PubMed1991

Human interleukin-3 mRNA accumulation is controlled at both the transcriptional and posttranscriptional level.

Interleukin-3 (IL-3) is a hematopoietic growth factor that regulates the differentiation of multilineage and committed progenitor cells and the functions of some mature blood cells. The expression of human IL-3 appears to be restricted to stimulated T lymphocytes. We have investigated the kinetics and mechanisms involved in the induction of IL-3 expression in the human T lymphocytic tumor cell line Jurkat. We show that accumulation of IL-3 mRNA is controlled at both the transcriptional and posttranscriptional level. Transcription of the IL-3 gene in these cells appears to be constitutive but no IL-3 mRNA was detected in unstimulated cells, indicating that in resting cells IL-3 mRNA is highly unstable. Treatment with phytohemagglutinin (PHA) induced a small and transient increase in the IL-3 gene transcription rate and led to the production of detectable levels of IL-3 mRNA and protein. Optimal induction of IL-3 expression required a second stimulus. Costimulation of Jurkat cells with both phorbol myristate acetate and PHA caused both a transient increase in IL-3 gene transcription, which is dependent on new protein synthesis, and also a transient increase in mRNA stability.

Cell Line↗
PubMed1991

Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.

Cells, Cultured↗
PubMed1990

IL-3 and granulocyte-macrophage colony-stimulating factor stimulate two distinct phases of adhesion in human monocytes.

IL-3 and granulocyte-macrophage CSF are hemopoietic growth factors involved in monocytopoiesis and functional stimulation of circulating blood monocytes. We demonstrate that both cytokines enhance the adhesion of purified human monocytes to cultured human umbilical vein endothelial cells and to plastic surfaces. The stimulation seen was biphasic: an early phase detectable by 10 min, and a late phase seen after 9 h of in vitro culture. IL-3- and granulocyte-macrophage-CSF-stimulated adhesion was seen at concentrations as low as 6 pM, with maximal monocyte adhesion of up to 60% seen at concentrations of 60 pM and above. Both phases of stimulated adhesion were partially inhibited by a monoclonal antibody to CD18, the common beta-chain of the leukocyte functional Ag family of adhesion molecules, but not by an antibody to CD11b, the alpha-chain of MAC-1. However, a difference in the mechanism by which the early and late phases of stimulated adhesion arise could be shown by the use of cycloheximide as an inhibitor of protein synthesis. Although the late phase was totally dependent on de novo protein synthesis, early phase adhesion was not inhibited by cycloheximide, suggesting receptor redistribution or conformational change as the mechanism mediating enhanced adhesion at this time. These findings may be relevant to the pathogenesis of inflammatory disease and may have implications for the clinical use of these cytokines.

Antibodies, Monoclonal↗
PubMed1990

Identification of a novel integrin beta subunit expressed on cultured monocytes (macrophages). Evidence that one alpha subunit can associate with multiple beta subunits.

The vitronectin receptor (VnR) is one member of a subset of cell adhesion receptors within the integrin supergene family which shares the beta 3 subunit (IIIa). We show here that the VnR is absent from the surface of monocytes freshly isolated from blood but is expressed on these cells after a period of in vitro culture. Such cultured monocytes (macrophages) from a patient with type I Glanzmann's thrombasthenia, however, failed to express the VnR. Instead, immunoprecipitation with a monoclonal antibody directed to the VnR alpha chain (alpha v) revealed a novel integrin comprising alpha v associated noncovalently with a 100-kDa beta subunit (beta 3b), immunologically unrelated to the VnR beta subunit (beta 3a). This same novel integrin complex was also identified on 10-day-old macrophages from healthy donors, but on these cells, the beta 3b subunit was co-expressed with the classical VnR complex of alpha v beta 3a. The novel beta 3b subunit was not identified by monoclonal or polyclonal antibodies to IIIa (beta 3a) nor by a monoclonal antibody to the classical VnR complex. The beta 3b subunit could be distinguished from beta 3a by its relatively greater migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction, by its distinct isoelectric point upon two-dimensional gel electrophoresis, and by one-dimensional peptide mapping. Neither platelets nor B lymphoblasts from this patient with Glanzmann's thrombasthenia expressed any VnR on their surface, whereas control cells from a normal donor expressed the classical VnR but not the beta 3b subunit. The two beta chains, and hence also the combined receptor complexes, appeared to be differentially regulated. These findings provide the first example of an integrin alpha chain complexed with more than a single beta chain in the same cell. Furthermore, the differential regulation of expression of the different beta subunits that associate with the VnR alpha chain on cultured monocytes suggests a role for the novel receptor complex during monocyte/macrophage differentiation.

Adult↗
PubMed1990

Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity.

The human T cell-derived cytokines interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 were examined for their ability to bind specifically to human basophils and to regulate their function. Scatchard analysis of equilibrium binding studies showed that IL-3 and GM-CSF, bound to basophils with apparent dissociation constants (KD) = 8 x 10(-11) M and 3.9 x 10(-11) M, respectively. Specificity studies under conditions that prevent receptor internalization showed that the binding of IL-3, GM-CSF, and IL-5 was not inhibited by tumor necrosis factor (TNF)-alpha, IL-1 beta, interferon (IFN)-gamma, or G-CSF. However, receptors for IL-3, GM-CSF, and IL-5 interacted with each other on the basophil membrane, showing a unique spectrum of cross-reactivity, with IL-3 competing for GM-CSF and IL-5 binding, whereas GM-CSF and IL-5 showed little or no competition for IL-3 binding. In order to relate the binding properties of these cytokines to function, they were tested for their ability to influence basophil histamine release in an IgE/anti-IgE-dependent system. We found a hierarchy in the stimulation of basophil with the order of potency being IL-3 greater than GM-CSF greater than IL-5. In addition, IL-3 stimulated larger amounts of histamine release than GM-CSF or IL-5. The observation that IL-3 interacts with receptors for GM-CSF and IL-5 may have a bearing on its stronger functional effects and suggests a major role for IL-3 in the pathogenesis of hypersensitivity syndromes.

Basophils↗
PubMed1990

Specific binding of human interleukin-3 and granulocyte-macrophage colony-stimulating factor to human basophils.

The human T cell-derived cytokines interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined for their ability to bind to human basophils. Basophils were obtained from the peripheral blood of a patient with chronic myeloid leukemia undergoing basophilic differentiation after purification on a density gradient of metrizamide. Binding studies with 125I-labeled IL-3 and 125I-labeled GM-CSF demonstrated that basophils express a single class of high-affinity receptors for each of these molecules. Saturation binding curves with 125I-labeled IL-3 revealed that IL-3 bound specifically to basophils, and analysis according to the method of Scatchard revealed that basophils express 800 to 900 receptors per cell with an apparent dissociation constant of 2.6 x 10(-11) mol/L. Saturation-binding curves with 125I-labeled GM-CSF revealed that basophils express 100 to 200 receptors per cell with an apparent dissociation constant of 4 x 10(-11) mol/L. The demonstration of high-affinity receptors for IL-3 and GM-CSF on human basophils suggests a role for these cytokines in the regulation of basophil function.

Basophils↗
PubMed1990

Human tumour necrosis factor-alpha (TNF-alpha) directly stimulates arachidonic acid release in human neutrophils.

The ability of tumour necrosis factor-alpha (TNF-alpha) to directly stimulate phospholipid turnover from human neutrophils was studied. Stimulation with recombinant human (rH) TNF-alpha induced the release of significant amounts of radioactivity from [3H]arachidonic acid-labelled neutrophils. This stimulation was equipotent to that induced by the bacterial tripeptide formyl-methionyl-leucylphenylalanine (FMLP). The time of maximum stimulated release varied between donors, with the most common maximal stimulation being 45 min. Dose-response experiments indicated that 100-1000 U/ml rH TNF-alpha were required for the maximum stimulatory effect. High-performance liquid chromatography analysis of the supernatants revealed that the radioactivity was associated with arachidonic acid, but not with its metabolites, indicating that TNF-alpha stimulates the release of arachidonic acid from cellular phospholipids but does not stimulate its metabolism. A comparison of TNF-alpha with other cytokines indicated that stimulation of arachidonic acid release paralleled the 'priming' of neutrophils for enhanced superoxide production, raising the possibility that phospholipid turnover and priming of neutrophils are causally related.

Arachidonic Acids↗
PubMed1990

Heterogeneity of recombinant granulocyte-macrophage colony-stimulating factor-mediated enhancement of neutrophil adherence to endothelium.

Human recombinant (r) and chemically synthesized granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to enhance the attachment of neutrophils to monolayers of human umbilical vein endothelial cells by direct action upon the neutrophil. Using synthetic peptides of GM-CSF with truncated amino and carboxy termini, a region between amino acids 14 and 24 was found to be essential for neutrophil attachment. In analysis of the response of neutrophils from individual donors, a heterogeneity in their capacity to respond to GM-CSF by increased adherence was observed. The level of response to GM-CSF did not depend on receptor number. However, a positive correlation (r = 0.58) was found between the ability to respond to GM-CSF and the level of response to tumor necrosis factor--suggesting a link between the responses of neutrophils to these two cytokines. The stimulation of neutrophil adhesiveness to endothelial cells by rGM-CSF and the heterogeneity in donor response may have important implications for the clinical administration of GM-CSF.

Adult↗
PubMed1989

Synthesis and expression of the gene encoding human interleukin-3.

To perform structure-function studies of human interleukin-3 (hIL-3) we have synthesized a cDNA encompassing the complete coding region of 484 bp. The strategy we employed involved construction of the cDNA in four sections. Each fragment contained six to ten oligodeoxyribonucleotides. Unique restriction sites were engineered to flank the natural sequence for cloning. Naturally occurring restriction sites were placed internally to these, to allow ligation of the four fragments. The gene was cloned into a modified pJL4 vector and expressed in COS cells. Biological assays of supernatants collected from these cells, for both mature cell function and proliferative activity, showed that synthetic hIL-3 had the same activity as that previously determined for recombinant hIL-3.

Animals↗
PubMed1989

Recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor show common biological effects and binding characteristics on human monocytes.

Two human hemopoietic growth factors involved in monocytopoiesis, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied for their ability to stimulate blood monocytes and to bind to the monocyte membrane. Both cytokines maintained monocyte/macrophage numbers during long-term culture and increased cell size as compared with controls. Effects on cell numbers were present at low cytokine concentrations (6 to 20 pmol/L), whereas enhanced 3H-thymidine incorporation was observed only at higher concentrations (greater than or equal to 60 pmol/L). Autoradiographic studies showed only 1% to 3% of stimulated monocytes with nuclear grains. These results suggest that the primary mechanism for IL-3 and GM-CSF-induced maintenance of monocyte/macrophage numbers in humans is through an effect on cell survival. Surface receptors for both IL-3 and GM-CSF were studied by using 125I-labeled recombinant human (rh) cytokines and performing Scatchard analyses. Both cytokines showed curvilinear Scatchard plots, and computer analyses favored a two-site binding model. High-affinity binding data for 125I rhIL-3 (Kd 7.7 to 38.2 pmol/L; receptor number/cell 95 to 580) and for 125I rhGM-CSF (Kd 4.7 to 38.9 pmol/L; receptor number/cell 8 to 67) show similar binding affinities for the two cytokines but a lower receptor number/cell for 125I rhGM-CSF. Low-affinity binding characteristics for 125I rhIL-3 (Kd 513 to 939 pmol/L; receptor number/cell 179 to 5,274) and for 125I rhGM-CSF (Kd 576 to 1,120 pmol/L; receptor number/cell 130 to 657) show a similar pattern for the two cytokines. Specificity of 125I rhIL-3 and 125I rhGM-CSF binding to monocytes was established by the ability of the homologous cytokine to inhibit binding and the inability of a range of other cytokines to compete at 100-fold excess molar concentration. It is important, however, that binding of 125I rhIL-3 was partially inhibited by rhGM-CSF and that rhIL-3 partially inhibited binding of 125I rhGM-CSF to the monocyte membrane under conditions shown to prevent receptor internalization. The degree of inhibition varied between 25% and 80% in different experiments, and quantitative inhibition experiments showed that 1,000-fold excess concentrations of competitor failed to inhibit binding of the heterologous ligand completely. These results demonstrate that human IL-3 and GM-CSF have similar effects on growth and survival of human monocytes in vitro and suggest that these and other common biological effects may be mediated either through a common receptor or through distinct receptors associated on the monocyte membrane.

Binding, Competitive↗
PubMed1989

Carbohydrate-specific monoclonal antibodies bind to human granulocytes and stimulate antibody-dependent cellular cytotoxicity.

Mouse monoclonal antibodies which specifically recognize human granulocytes are used to study the classification, differentiation, and function of these cells. Mouse monoclonal antibody WEM-G1 specifically binds to human neutrophils and eosinophils. It also affects granulocyte function by stimulating granulocyte-mediated antibody-dependent cellular cytotoxicity. Biochemical studies presented here show that WEM-G1 recognizes the sugar sequence 3-fucosyllactosamine, Gal beta 1-4[Fuc alpha 1-3]GlcNAc. This sequence is present in granulocyte glycolipids and in glycoproteins of average approximate Mr 165,000 and 105,000. WEM-G1 is thus similar to other monoclonal antibodies that recognize this sequence on granulocytes and various other cells. Some of these 3-fucosyllactosamine-specific antibodies affect several other granulocyte functions. Knowledge of the biochemical structure of the WEM-G1 antigen suggested testing granulocyte function with other monoclonal antibodies of similar specificity. Antibodies recognizing both the identical oligosaccharide structure and a related sequence, Gal beta 1-4GlcNAc-R, were also found to stimulate granulocyte-mediated antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1989

Human interleukin 4 regulates the phenotype of lymphocytes generated during mixed lymphocyte culture and inhibits the IL-2-induced development of LAK function in normal and leukaemic cells.

This study examined the immunoregulatory role of recombinant interleukin 4 (IL-4), also known as B-cell stimulating factor 1, on the generation of cytotoxic effector cells from normal and leukaemic human blood mononuclear cells. When tested on cells from normal individuals, the addition of IL-4 to mixed lymphocyte cultures led to a dose-dependent proliferation of T-helper cells (CD3, 4 positive) with a concomitant decrease in phenotypic and functional cytotoxic T cells and natural killer (NK) cells. IL-4 also inhibited the interleukin-2 (IL-2)-induced generation of lymphokine-activated killer (LAK) activity when added at the beginning of mixed lymphocyte culture. When tested on mature leukaemic NK cells, IL-4 also inhibited the ability of IL-2 to induce LAK function using a short-term culture system. These results show that IL-4 acts on both normal and leukaemic cells and suggests that it acts at more than one level during the development of LAK function.

Antigens, Differentiation↗
PubMed1989

Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony-stimulating factor to human eosinophils.

125I-labeled recombinant human interleukin 3 (IL-3) bound, at 4 degrees C, to a single class of high-affinity receptors on human eosinophils with an apparent dissociation constant (Kd) of 470 pM, but it did not bind to human neutrophils. 125I-labeled recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) also bound to a single class of high-affinity receptors on eosinophils with an apparent Kd of 44 pM and on neutrophils with an apparent Kd of 70 pM. These binding characteristics were consistent with the biological activities of IL-3 and GM-CSF on eosinophils and with the lack of stimulation of neutrophil function by IL-3. Specificity studies under conditions shown to prevent receptor internalization showed that the binding of 125I-labeled IL-3 to eosinophils was partially inhibited by GM-CSF but not by other cytokines. Reciprocal experiments with 125I-labeled GM-CSF showed that IL-3 but not other cytokines partially inhibited binding to eosinophils. In contrast, the binding of 125I-labeled GM-CSF to neutrophils was not inhibited by IL-3 or other cytokines tested. Quantitative inhibition binding experiments on eosinophils showed that the reciprocal inhibition between IL-3 and GM-CSF was not complete up to a concentration of heterologous ligand of 100 nM. These results show that (i) IL-3 binds to eosinophils but not neutrophils and (ii) IL-3 and GM-CSF specifically interact on the surface of eosinophils, providing a possible mechanism for the overlapping activities of IL-3 and GM-CSF on these cells.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1989

Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase the adherence of purified peripheral blood monocytes to plastic surfaces and to monolayers of human umbilical vein endothelial cells. With plastic surfaces as a model 9-hr culture with GM-CSF was necessary for enhancement, and maximum levels were obtained after 24-hr stimulation. GM-CSF-stimulated adherence must require new RNA and protein synthesis because actinomycin D and cycloheximide abolished existing adherence and prevented further monocyte attachment. Interestingly, shorter incubations (1-2 hr) with cycloheximide increased adherence, suggesting a labile inhibitor. Formaldehyde fixation of monocytes but not of human vein endothelial cells abolished adherence, indicating the need for actively metabolizing monocytes. Thus, a hemopoietic growth factor, responsible for the proliferation and differentiation of monocytes, can also alter their adhesive characteristics. These observations may have important implications in pathological situations and in the in vivo use of GM-CSF.

Biological Factors↗