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Biomedical subjects

R F Todd

Publications and source records attributed to R F Todd.

At least 19 recordsLinked to original sources
PubMed2000

Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3 proximity on neutrophils.

Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3.

Adult↗
PubMed2000

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Antibodies, Monoclonal↗
PubMed1999

Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes.

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.

Benzoquinones↗
PubMed1998

Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes.

Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10-100 microg/ml)/Mn2+ (50 microM) for 2 h were examined by electrophoretic mobility shift assay. NF-kappa B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-kappa B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-kappa B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn2+ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-kappa B.

Adjuvants, Immunologic↗
PubMed1998

Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins.

The mechanisms by which Ebola virus evades detection and infects cells to cause hemorrhagic fever have not been defined, though its glycoprotein, synthesized in either a secreted or transmembrane form, is likely involved. Here the secreted glycoprotein was found to interact with neutrophils through CD16b, the neutrophil-specific form of the Fc gamma receptor III, whereas the transmembrane glycoprotein was found to interact with endothelial cells but not neutrophils. A murine retroviral vector pseudotyped with the transmembrane glycoprotein preferentially infected endothelial cells. Thus, the secreted glycoprotein inhibits early neutrophil activation, which likely affects the host response to infection, whereas binding of the transmembrane glycoprotein to endothelial cells may contribute to the hemorrhagic symptoms of this disease.

Cell Line↗
PubMed1998

A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells.

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50- to 65-kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA-dependent, plasmin-mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated-suPAR (B-suPAR) bound in a specific fashion to THP-1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B-suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B-suPAR to THP-1 cells was enhanced four- to sevenfold by 24-h exposure of cells to PMA or by co-incubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B-suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by co-incubation with uPA. B-suPAR biding to PMA-differentiated THP-1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA-differentiated THP-1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B-suPAR. Whereas the binding of suPAR did not measurably affect cell-associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane-associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin-sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand.

Biotin↗
PubMed1998

A microscopic study of Fc gamma RIII-mediated respiratory burst in neutrophils.

We investigated the relationship between the respiratory burst in neutrophils and the membrane distribution of the IgG receptor, Fc gamma RIII. Fc gamma RIII receptors were labeled with a fluoresceinated antibody that does not block binding of immune complexes. The respiratory burst was detected using covalently bound rosamine stain previously described for flow cytometric applications. This method allows visualization of intracellular oxidant production in fixed cells using attenuated illumination with a laser. Strong cytosolic oxidation of rosamine was observed only in those cells that displayed prominent receptor endocytosis upon interaction with insoluble immune complexes. Soluble immune complexes or insoluble complexes in the presence of cytochalasin B did not stimulate endocytosis of Fc gamma RIII and induced no rosamine oxidation. Extracellular superoxide production measured by the cytochrome c test did not correlate with intracellular rosamine oxidation: it was maximal in cytochalasin-treated cells and did not require any visible receptor rearrangement. Our results demonstrate the utility of the rosamine stain as an intracellular marker of the oxidative burst, support the role of Fc gamma RIII in neutrophil activation and emphasize the compartmental regulation of the oxidative burst.

Antigen-Antibody Complex↗
PubMed1998

Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells.

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34+ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples: in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 x 10(3) ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.

Acute Disease↗
PubMed1998

Aberrant integrin (CR4; alpha(x)beta2; CD11c/CD18) oscillations on neutrophils in a mild form of pyoderma gangrenosum.

We have previously shown that the beta2 integrins CR3 and CR4 physically and functionally interact with urokinase receptors (uPAR) on neutrophil plasma membranes in an oscillatory fashion. In this study we have analyzed neutrophils from patient SC, a 34 y old African American female, with aberrant skin window results and recurrent perianal abscesses and pretibial lesions diagnosed as pyoderma gangrenosum. Although untreated migrating normal neutrophils exhibited 20 s sinusoidal oscillations in CR4-uPAR proximity, neutrophils from SC demonstrated a faster oscillation (10 s) in the form of a flyback sawtooth wave. This waveform mimicked that observed for normal neutrophils treated with subsaturating doses of the kinase inhibitors staurosporine, genistein, and erbstatin. As beta2 integrins are regulated by phosphorylation, we tested the hypothesis that the aberrant CR4-uPAR proximity oscillations seen in SC's neutrophils are due to defective kinase activity that might be balanced by a decrease in phosphatase activity. When SC's cells are exposed to subsaturating concentrations of the phosphatase inhibitor pervanadate, this caused the CR4-uPAR oscillations to become sinusoidal in shape with a 20 s period, as seen in normal migrating neutrophils. Although SC's neutrophils were deficient in spontaneous and N-formyl-methionyl-leucyl-phenylalanine-induced polarization, 0.5 microM pervanadate returned cell polarization to nearly normal levels, thus paralleling the acquisition of normal receptor interactions. Inasmuch as SC's cellular phenotype is mimicked by kinase inhibitors and corrected by phosphatase inhibitors, we suggest that a mutation(s) affecting the kinetics of intracellular signaling enzymes, but not blocking the pathway per se, may be responsible for this clinical state.

Adult↗
PubMed1998

Aberrant neutrophil trafficking and metabolic oscillations in severe pyoderma gangrenosum.

Having previously associated metabolic oscillations with cell locomotion, we hypothesized that patients with abnormalities in neutrophil trafficking may display aberrant intracellular oscillations. A pyoderma gangrenosum patient exhibiting aberrant leukocyte trafficking in vivo and skin ulceration without infection was identified. This patient's neutrophils constitutively overexpressed and clustered the leukocyte integrins CR3 and CR4 and failed to display appropriate integrin-to-GPI receptor interactions. Increased levels of tyrosine phosphorylation were observed. NAD(P)H oscillations, which are sinusoidal in normals, were chaotic with multiple frequency components in this patient's neutrophils. Normal cell shape and sinusoidal NAD(P)H oscillations were restored by providing a pulsed electric field to drive metabolic oscillations and by temperature reduction. N-acetyl-D-glucosamine disrupted CR3 clusters and sinusoidal NAD(P)H oscillations returned. Anecdotal reports suggest that local hypothermia is clinically useful for this patient. These data define the first metabolic oscillation-associated disease and suggest that pyoderma gangrenosum can be classified as a dynamical disease at the cellular level.

Acetylglucosamine↗
PubMed1997

Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components.

We have shown previously that the urokinase-type plasminogen activator receptor (uPAR) physically associates with beta2 integrins on human leukocyte membranes. We now report that uPAR associates with certain members of the beta1 and beta3 integrin families expressed by a nonhematopoietic fibrosarcoma cell line (HT1080) when adherent to certain extracellular matrix molecules. Flow cytometry studies indicated that HT1080 cells expressed uPAR and beta1 and beta3 integrins. Double staining immunofluorescence was used to label uPAR and beta1 and beta3 integrins. The staining patterns of uPAR and beta1 integrins were strikingly similar when attached to fibronectin, laminin, or vitronectin but not polylysine-coated substrates. Resonance energy transfer (RET) between uPAR and beta1 integrins was observed, especially at focal adhesion plaques; this indicates that these molecules are within about 7 nm of each other on these cell membranes. uPAR and beta3 integrin coclustering and RET were also observed on tumor cells adherent to vitronectin but not to fibronectin, laminin, or polylysine-coated surfaces. Because N-acetyl-D-glucosamine was found previously to inhibit beta2 integrin-uPAR association, we tested the effect of saccharides on the beta1-uPAR and beta3-uPAR colocalization and RET. Colocalization and RET between uPAR and beta1 or beta3 integrins were effectively inhibited by N-acetyl-D-glucosamine on extracellular matrix-coated surfaces. To better define which members of beta1 and beta3 integrin families associate with uPAR, we studied the association of several alpha subunits with uPAR on tumor cells. We found that: (a) alpha5 colocalizes with uPAR on cells attached to fibronectin-coated surfaces; (b) alpha5 and alpha(v) colocalize with uPAR on cells adherent to vitronectin; and (c) alpha3 and alpha6 associate with uPAR on cells attached to laminin. In further support of physical associations between integrins and uPAR on tumor cells, uPAR was found to coimmunoprecipitate with beta1 integrins in Brij-58 lysates of HT1080 cells (as detected by anti-uPAR Western blotting of material isolated from an anti-beta1 integrin immunoaffinity column). Thus, uPAR may laterally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for tumor cell migration and invasion.

Antibodies, Monoclonal↗
PubMed1997

A sensitive flow cytometric method for measuring the oxidative burst.

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sensitivity of this assay in detecting the response to chemotactic peptide N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) was comparable to that observed with the cytochrome c oxidation test. We compared the new method to previously described methods using intracellular fluorescent stains. Our method showed the lowest nonspecific oxidation and the best sensitivity to FMLP-induced activation. It was equally or slightly less efficient than the green fluorescent stain dihydrorhodamine 123 in detection of neutrophil activation by immune complexes and PMA but much more sensitive than red fluorescent stains hydroethidine and dihydrotetramethylrosamine. It can be successfully used in kinetic experiments and yields a fluorescence signal that remains stable for at least 1.5 h after formaldehyde fixation with antioxidant added.

Flow Cytometry↗
PubMed1997

Proximity oscillations of complement type 4 (alphaX beta2) and urokinase receptors on migrating neutrophils.

Migrating neutrophils utilize beta2 integrins for substrate attachment and urokinase receptors (uPAR) to focus pericellular proteolysis. Our studies show that CR3 associates with uPAR on resting cells, whereas uPAR associates with CR4 at lamellipodia of migrating cells. Using resonance energy transfer (RET) microscopy, we show that the molecular proximity between CR4 and uPAR oscillates on migrating cells, thus suggesting that CR4 molecules periodically bind/release uPAR. Cell contact with fibrinogen, endothelial cells, chemotactic factors and indomethacin, and treatment with sub-optimal doses of signal transduction inhibitors, affect the oscillations' period, amplitude, and/or waveform. The oscillations were indistinguishable in period and 180 degrees out-of-phase with cytosolic NAD(P)H autofluorescence oscillations. Thus, CR4 and CR3 identify a neutrophil's axis of migration and CR4 may restrain uPAR at lamellipodia. Oscillations in signal transduction and energy metabolism may coordinate cell adherence, local proteolysis, oxidant release, actin assembly, and cell extension.

Adult↗
PubMed1997

Beta 2 (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors.

Fig. 1 depicts our current thinking about the ways in which Mo1 and p150,95 form cis interactions with other leukocyte receptors. With respect to the associations of Mo1 with Fc gamma RIIIB and uPAR, the inhibitory effect of saccharides such as NADG suggests a lectin-carbohydrate interaction that may involve the recognition of Mo1's beta-glucan site for N-linked carbohydrates4 that are expressed by both Fc gamma RIIIB and uPAR. This hypothesis is supported by the results of Stockl et al., who showed that the binding of C-terminal-specific mAb VIM12 to Mo1, which enhances the phospholipase C-mediated release of Fc gamma RIIIB, was inhibited by NADG. However, unlike the sample lectin-carbohydrate interaction that appears to govern the association between Mo1 and Fc gamma RIIIB, effective Mo1-dependent uPAR signaling also depends on the binding of intact uPA to uPAR (the receptor-binding ATF of uPA proving insufficient to prime neutrophils for an enhanced burst response to FMLP). We speculate that ATF (residues 6-135) binds to uPAR while the carboxyl terminal fragment (residues 136-411), which includes a glycosylation site at residue 144, binds to the lectinlike site of Mo1, thus fostering the linkage between the two receptors. In support of this model is the fact that exposure of neutrophils to ATF reduced the degree of molecular proximity between Mo1 and uPAR (the latter probably occupied by endogenous intact uPA) and increased the molecular association between Mo1 and Fc gamma RIIIB (both as detected by quantitative RET). This hypothesis is analogous to the concept proposed by Nykjaer et al in which plasminogen activator inhibitor-1 initially binds to uPA to form a complex that secondarily binds to the alpha 2 macroglobulin receptor, leading to internalization of the complex. Whereas the contribution of intact uPA to the interaction between Mo1 and uPAR remains speculative (based on the indirect data available), no such ambiguity exists for the role of the LPS/LBP ligand in regulating the association between Mo1 and CD14. In this circumstance, no physical linkage exists between the two receptors without the ligand complex. This observation is consistent with the previously described affinity of the beta 2 integrins for LPS, leading to the notion that the LPS portion of the LPS/LPB complex binds to Mo1, serving to link it with LPS/LBP bound to CD14. The observed reversibility of the interactions between the integrin glycoproteins and uPAR or CD14 illustrates the fact that these associations can be highly dynamic and tied to cellular processes that include directed motility (Mo1-uPAR), adherence to substrates (Mo1-CD14), and energy metabolism (p150,95-uPAR). We speculate that the GPI-anchored receptor proteins serve as rapidly diffusible, expendable "scouts" for the beta 2 integrins, which serve to expand their ligand binding repertoire in a cis-acting fashion.

CD11 Antigens↗
PubMed1997

Ectodomain interactions of leukocyte integrins and pro-inflammatory GPI-linked membrane proteins.

Although glycosylphosphatidyl-inositol (GPI) linked membrane proteins do not possess transmembrane or cytosolic sequences they elicit transmembrane signals. Using microscopic fluorescence imaging and resonance energy transfer (RET) techniques we have shown that certain pro-inflammatory GPI-linked membrane proteins can interact with leukocyte beta 2 integrins (complement receptor type 3 (CR3) and 4 (CR4) and the leukocyte function-associated antigen-1 (LFA-1)). For example, physical associations between CR3 and Fc gamma RIIIB, CR3 and urokinase receptors, and CR3 and CD14 (lipopolysaccharide receptor) have been found. Although Fc gamma RIIIB appears to be constitutively associated with CR3, urokinase receptors and CD14 associations with CR3 are influenced by their ligation status and cell function (e.g. adherence and locomotion). CR3-to-urokinase receptor interactions have been confirmed by immunoprecipitation techniques. Immunoprecipitation of CR3 from Brij-58 lysates after biotinylation of neutrophil membranes revealed proteins of M(r) = 40,000, 50,000, 74,000 and 120,000, in addition to bands corresponding to the integrin alpha and beta chains. Cell functions such as transmembrane signaling and superoxide release/priming have been linked to these interactions. Importantly, reagents that affect the lectin-like site of CR3, such as N-acetyl-D-glucosamine, alpha-methyl-D-mannoside and beta-glucan alter these interactions and, in parallel, leukocyte functions. Thus, the interactions of GPI-linked proteins and integrins can be highly dynamic events linked to cell activities. Our studies suggest that it may be possible to develop new drugs directed at the lectin-like site of beta 2 integrins that block GPI-linked protein-to-integrin coupling thereby controlling inflammatory cell processes including cell adherence, locomotion and activation. Such drugs may be useful in clinical conditions such as ischemia-reperfusion injury, sepsis, arthritis and others.

CD18 Antigens↗
PubMed1996

CR3 (alphaM beta2; CD11b/CD18) restores IgG-dependent phagocytosis in transfectants expressing a phagocytosis-defective Fc gammaRIIA (CD32) tail-minus mutant.

Previous studies have suggested that complement receptors cooperate with Fc receptors to mediate Ab-dependent effector functions. In the present study, we tested the capacity of complement receptor type 3 (CR3; alphaM beta2; CD11b/CD18) to participate in Fc gammaRIIA (CD32)-mediated phagocytosis. To test this hypothesis, we transfected a phagocytosis-defective tail-minus mutant of Fc gammaRIIA into fibroblasts that did or did not express CR3. As controls, cells exposed to the transfection protocol but not expressing either CR3 or Fc gammaRIIA constructs were also selected. Expression of cell surface receptors was confirmed by both flow cytometry and fluorescence microscopy. Cells were tested for their ability to bind and internalize IgG-opsonized erythrocytes and for surface proximity of CR3 and Fc gammaRIIA using resonance energy transfer techniques. Resonance energy transfer studies showed a physical proximity between CR3 and Fc gammaRIIA on cell surfaces. Cells expressing only the tail-minus mutant of Fc gammaRIIA were unable to internalize erythrocytes but showed significant binding ability. In contrast, cells expressing both mutant Fc gammaRIIA and CR3 internalized opsonized erythrocytes. These results show that CR3 can complement the phagocytic function of defective Fc gammaRIIA. These data suggest that CR3 is capable of transducing a phagocytic signal generated by Fc gammaRIIA recognition events that mediates internalization of IgG-opsonized targets.

3T3 Cells↗
PubMed1996

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes.

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

Antibodies↗