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Biomedical subjects

S D Wright

Publications and source records attributed to S D Wright.

At least 145 records · Page 8Linked to original sources
PubMed1986

Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites.

Preincubation of resident peritoneal macrophages with 10-100 ng/ml LPS for 60 min resulted in the cells becoming primed for enhanced (three-to eightfold higher) arachidonic acid (20:4) secretion in response to a variety of triggers. The half-maximal concentration of LPS required for priming was 10 ng/ml irrespective of whether the trigger was particulate (examples: zymosan or immune complexes) or soluble (such as PMA or A23187). Similarly, the time required for half-maximal priming of macrophages was 20 min irrespective of which trigger was used. The primed state persisted for at least 30 h. LPS-priming of macrophages also affected the kinetics of 20:4 metabolite secretion. The lag phase characteristically observed when 20:4 secretion is triggered was reduced in LPS-primed cells. Furthermore, LPS-primed cells secreted 20:4 metabolites when challenged with latex beads, while unprimed cells did not. These data suggest that stimuli such as zymosan, which elicit 20:4 secretion in macrophages, promote two signals, a priming signal and a triggering signal. LPS is capable of establishing the priming signal but not the triggering signal, while latex promotes the triggering signal but is unable to prime the cells for 20:4 release. LPS did not effect the profile of 20:4 metabolites secreted in response to any of the triggers, nor did it effect the profile of products synthesized from exogenously added 20:4, suggesting that it did not regulate the 20:4 cascade at the level of either the cyclooxygenase or lipoxygenase pathways. Macrophages respond to LPS without the intervention of T lymphocytes, since the macrophages from nude mice could be primed for enhanced 20:4 secretion.

Animals↗
PubMed1986

Interferon-gamma depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin.

Cultivation of human monocytes with recombinant IFN-gamma causes a 5-10-fold depression in their binding of EC3b or EC3bi. This effect is observed within 18 h and is expressed for 5 d in the presence of 100 U/ml IFN-gamma. The capacity of IFN-gamma-treated phagocytes to bind EC3b and EC3bi is fully restored if the phagocytes are allowed to spread for 45 min on surfaces coated with Fn. IFN-gamma-treated cells express normal levels of cell surface C3b and C3bi receptors as measured with monoclonal anti-receptor antibodies, and spreading on Fn does not alter receptor number. We conclude that cultivation with IFN-gamma causes a change in the nature of these receptors that prevents them from interacting with ligand. Immunoelectron microscopy shows that C3bi receptors are expressed on the apical surface of the IFN-gamma-treated MO and that these receptors exhibit normal capacity to migrate in the plane of the membrane. Thus, the nature of the change caused by IFN-gamma is not related to changes in receptor number, location, or mobility. While spreading of IFN-gamma-treated cells on Fn enables C3 receptors to bind ligand, it does not enable them to promote phagocytosis. Treatment of cells with PMA alone does not affect binding or phagocytosis, but treatment of cells with both Fn and PMA enables cells to phagocytose EC3b and EC3bi. These data indicate that the binding and signaling activities of C3 receptors are separately regulated. Fn enables receptors to bind ligand and PMA enables them to signal phagocytosis.

Antigens, Surface↗
PubMed1986

Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes.

Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases to levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for by alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity (deactivation). In contrast with its effects on C3 receptors, PMA causes only a reduction in the capacity of Fc receptors to bind and ingest IgG-coated erythrocytes. Deactivation of Fc receptors also appears to involve a qualitative alteration in receptors, because the binding affinity of soluble immune complexes is sharply reduced upon stimulation of PMN with PMA. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, we loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc receptors, suggesting that the deactivation is the result of a dephosphorylation event.

Animals↗
PubMed1986

Fc receptors on monocytes cause OKT3-treated lymphocytes to internalize T3 and to secrete IL-2.

Monocytes cause OKT3-treated T cells to secrete IL-2 and to lose cell surface T3. We have studied the fate of the "lost" T3. Immunofluorescence microscopy on permeabilized cells showed that monocytes induce T cells to internalize T3. Furthermore, experiments with radioiodinated T cells showed that the internalized T3 was not degraded and exhibited an unaltered polypeptide composition for at least 16 hr. The role of Fc receptors in inducing internalization was also investigated. Fc receptors were depleted from monocytes by allowing the phagocytes to spread on immune complexes. Such depleted monocytes exhibit a fourfold reduction in their ability to promote both internalization of T3 and production of IL-2. A comparable reduction is seen if F(ab')2 fragments of OKT3 were employed in place of intact IgG. Furthermore, monocyte Fc receptors that have been blocked by heat-aggregated human IgG also show much reduced capability for induction of OKT3-mediated T-cell proliferation. We therefore conclude that Fc receptors bind to the Fc domain of OKT3 and thereby cause OKT3-treated T cells to internalize T3 and become activated.

Antibodies, Monoclonal↗
PubMed1986

Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i.

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In contrast, a rise in [Ca++]i of macrophages was measured during phagocytosis occurring in a serum-free saline of pH 7.85, and as a consequence of incubation with quin2 A/M. We estimate that had a change in [Ca++]i occurred during phagocytosis, aequorin would have detected a rise from 0.1 to 1.0 microM taking place in as little as 2% of the macrophage's cytoplasmic volume. We therefore suggest that either Ca++ is not involved as a cytoplasmic signal for phagocytosis or that increases in [Ca++]i during phagocytosis are confined to such small regions of cytoplasm as to be below the limits of detection by our cellular averaging method. Our data emphasizes, moreover, the need for well-defined, nonperturbing conditions in such measurements of [Ca++]i.

Aequorin↗
PubMed1986

Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

Animals↗
PubMed1985

Fibronectin receptor of human macrophages recognizes the sequence Arg-Gly-Asp-Ser.

When cultured human monocytes (MO) were spread on fibronectin (Fn)-coated surfaces, C3 receptors on the MO exhibited markedly enhanced capacity to promote phagocytosis. The activation of C3 receptors by Fn was mediated by a receptor that recognizes a sequence, Arg-Gly-Asp-Ser (RGDS), present in the cell-binding domain of Fn. Soluble, RGDS-containing peptides inhibited the activation of C3 receptors caused by surface-bound Fn, and surface-bound, RGDS-containing peptides themselves caused activation of the C3 receptors of attached MO. Although soluble, RGDS-containing peptides bound to Fn receptors, such monovalent ligation was insufficient to activate C3 receptors.

Amino Acid Sequence↗
PubMed1985

Ligated complement receptors do not activate the arachidonic acid cascade in resident peritoneal macrophages.

Receptors for IgG stimulate the release of approximately 20% of cellular arachidonic acid (20:4) from murine resident peritoneal macrophages. In contrast, C3 receptors do not trigger the secretion of any 20:4 in excess of that released constitutively from the cells. Since the ability of C3 receptors to promote phagocytosis is regulated, we compared resting macrophages, whose C3 receptors do not promote phagocytosis of C3-coated particles, and lymphokine-treated cells, whose receptors do promote ingestion. Despite their ability to promote phagocytosis, the C3 receptor of lymphokine-treated macrophages remain unable to initiate release of 20:4. We speculate that the intracellular signals that initiate phagocytosis are distinct from those that initiate release of 20:4.

Animals↗
PubMed1985

OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2.

OKB7, an IgG2 mouse monoclonal antibody, binds to the C3d receptor (CR2) of human B lymphocytes in or near the active site of the receptor. OKB7 precipitates the same molecule as monoclonal antibodies B2 and HB-5 which have previously been reported to react with the C3d receptor at epitopes distant from the active site. The epitope recognized by OKB7 is distinct from those bound by B2 and HB-5 as shown in competitive binding experiments and is near the complement binding site since OKB7 blocks EAC3d rosetting at concentrations as low as 1 microgram/ml in the absence of cross-linking antibodies.

Animals↗
PubMed1984

Structure and modulation of Fc and complement receptors.

Recent experiments have revealed the structure of some phagocytosis-promoting receptors. The C3b receptor is a single-chain membrane glycoprotein of Mr 205,000, while the C3bi receptor is composed of two surface glycoprotein chains, of Mr 180,000 and 100,000. Fc receptors all appear to be single-chain glycoproteins of approximately Mr 50,000. Despite this structural similarity, Fc receptors display a broad range of heterogeneity with respect to ligand specificity. One type of Fc receptor (Fc gamma 2b/gamma 1R) appears to function as a ligand-dependent ion channel; the ion flux initiated by the ligation of this receptor may represent the proximal signal sent by this Fc receptor. The second signal sent by other Fc receptors and by the C3 receptors is uncharacterized, except for the observation that the second signal generated by C3 receptors is distinct from that of Fc gamma 2b/gamma 1R.

Animals↗
PubMed1984

Communication between receptors for different ligands on a single cell: ligation of fibronectin receptors induces a reversible alteration in the function of complement receptors on cultured human monocytes.

Receptors for the third component of complement (C3) on cultured human monocytes (MO) bind ligand-coated particles but do not initiate phagocytosis. The function of these receptors, however, is altered dramatically after MO attach to surfaces coated with fibronectin (FN) or after MO are exposed to phorbol esters. FN and phorbol esters "activate" C3 receptors such that they promote vigorous phagocytosis. Here we show that activation of C3 receptors requires the continuous presence of FN or phorbol esters and is rapidly reversible when these stimuli are removed. Activation does not change the number or distribution of C3 receptors on the surface of MO. We conclude that the function of C3 receptors is regulated by reversible reactions that are initiated by ligation of a different class of receptors on the surface of the same cell.

Cell Communication↗
PubMed1984

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.

Antibodies, Monoclonal↗
PubMed1983

Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes.

We have measured the release of H2O2 from granulocytes, monocytes, and macrophages during spreading on ligand-coated culture surfaces. While IgG-coated surfaces stimulate vigorous release of H2O2, neither C3b- nor C3bi-coated surfaces promoted appreciable release of H2O2 despite full ligation of C3b and C3bi receptors. We also measured release of H2O2 from cultured monocytes spreading on surfaces coated with both fibronectin and C3. Under such circumstances, the C3 receptors elicit a strong phagocytic response, but no H2O2 release was recorded. We conclude that the C3b and C3bi receptors of monocytes and granulocytes do not signal the generation of toxic oxygen intermediates from these cells.

Cells, Cultured↗
PubMed1983

Fibronectin and serum amyloid P component stimulate C3b- and C3bi-mediated phagocytosis in cultured human monocytes.

Fibronectin (FN) and serum amyloid P component (SAP) markedly enhance phagocytosis mediated by the C3b and C3bi receptors of cultured human monocytes but not of granulocytes. (The C3b and C3bi receptors of granulocytes can be activated by treatment of these phagocytes with PMA.) Activation of monocyte C3 receptors by FN is developmentally regulated: Freshly explanted monocytes respond to FN with a small increase in C3 receptor-mediated phagocytosis while monocytes matured in culture exhibit a much greater response. The mechanism of action of FN on C3 receptors of cultured monocytes is unique in two respects. First, while substrate-bound FN or SAP activate monocyte C3 receptors, soluble FN does not. Second, stimulation of the basal surface of monocyte plasma membranes by substrate-bound FN activates C3b and C3bi receptors on the apical surface of the plasma membrane, i.e., at sites remote from the segments of membrane in contact with the FN or SAP.

Amyloid↗
PubMed1983

Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM10, all directed against the C3bi receptor of human monocytes and macrophages (M phi). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on M phi adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from M phi lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM10 binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from 125I-surface-labeled M phi. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human M phi is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.

Animals↗
PubMed1982

Tumor-promoting phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes.

Monocytes were isolated in high yield (approximately 80%) and purity (greater than 90%) by Percoll gradient centrifugation and incubated in Teflon culture vessels. Using this culture method, we routinely recovered 80% of the cells originally placed into culture. Studies of the C3b and C3b' receptors of these monocytes showed that the function of both receptors could be dramatically altered by treating the cells with tumor-promoting phorbol esters. Both C3b and C3b' receptors of human monocytes efficiently mediate attachment of erythrocytes coated with the corresponding ligands, but do not promote their ingestion. However, monocytes treated with phorbol myristate acetate (PMA) or phorbol didecanoate ingest C3b- and C3b'-coated erythrocytes. Phorbol esters that are inactive as tumor promoters do not stimulate C3 receptor-mediated phagocytosis. The ability of monocytes to respond to PMA by activation of C3 receptors is developmentally regulated. Freshly isolated monocytes do not take up C3b- or C3b'-coated erythrocytes in response to PMA, but after 3 d of culture they show strong PMA-stimulated uptake. The stimulatory effect of PMA on monocyte C3b and C3b' receptor function occurs within minutes, is stable for hours, is cycloheximide insensitive, and can be inhibited with colchicine. Several lines of evidence indicates that phagocytosis of C3b or C3b'-coated erythrocytes is specifically mediated by the monocytes' C3b and C3b' receptors. First, erythrocytes attached to monocytes with concanavalin A are not ingested when the monocytes are treated with PMA. Second, monocytes plated on IgG-bearing substrates lose Fc receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b- or C3b'-coated erythrocytes after PMA treatment. Third, PMA-treated monocytes plated on C3b-coated surfaces lose C3b receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b'-coated erythrocytes. Conversely, PMA-treated monocytes plated on C3b'-coated surfaces show reduced C3b' receptors activity on their nonadherent surfaces but retain the capacity to ingest C3b-coated erythrocytes.

Cations↗