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R F Todd

Publications and source records attributed to R F Todd.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

LPS induces CD14 association with complement receptor type 3, which is reversed by neutrophil adhesion.

CD14, a glycosylphosphatidyl inositol (GPI)-linked membrane protein, is a key membrane binding site for LPS (endotoxin). Although CD14 lacks transmembrane and cytoplasmic sequences, it activates CR3-mediated leukocyte adhesion and cytokine release. Since CR3 has been shown to interact with other GPI-linked membrane proteins, we tested the hypothesis that CD14 can physically associate with CR3. Using qualitative and quantitative resonance energy transfer microscopy, we show that LPS in the presence of serum or LPS binding protein triggers formation of CD14-CR3 complexes. Kinetic studies show that CD14-CR3 complexes dissociate as neutrophils attach to substrates. We speculate that LPS-charged CD14 enhances CR3-mediated adhesion by directly binding to CR3.

Adult↗
PubMed1996

Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (alpha M beta 2' CD11b/CD18) during neutrophil polarization.

Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.

Adult↗
PubMed1996

Integrins as promiscuous signal transduction devices.

Recent studies suggest physical and functional interactions of glycosylphosphatidylinositol (GPI)-linked proteins (CD14, CD16b and CD87) with leukocyte beta 2 integrins. As discussed in this article, it now appears that beta 2 integrins relay proinflammatory information from GPI-anchored membrane receptors to the cytoplasm via exodomain interactions.

Animals↗
PubMed1995

Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels.

Hematopoietic stem cell gene therapy holds the promise of being able to treat a variety of inherited and acquired diseases of the hematopoietic stem cell. However, to date, genetic modification of the human hematopoietic stem cell has been relatively inefficient. Here, we report the results of using a bioreactor system to expand hematopoietic cells after a brief retrovirus infection using a high titer, replication defective virus encoding for murine CD18. The retrovirus transduced culture continued to produce genetically modified hematopoietic progenitors for up to 6 weeks, the duration of the culture period. Up to one-third of the long-term culture initiating cell (LTC-IC) are genetically modified by the culture conditions. Murine CD18 can be expressed on the cell surface of up to 20% of the mature cells generated by the culture system, suggesting that clinically significant levels of gene transfer may be occurring. These results demonstrate the feasibility of using continuous perfusion bioreactors as a method of efficiently modifying human hematopoietic stem cells.

Animals↗
PubMed1995

Enzyme-linked immunoabsorbent assay detection of a soluble form of urokinase plasminogen activator receptor in vivo.

The receptor for urokinase plasminogen activator (uPA-R, CD87) is a glycosylphosphatidylinositol (GPI)-anchored 50 to 65 kD glycoprotein that, by regulating membrane-associated plasmin activity, may facilitate the invasion of inflammatory and malignant cells. Certain other GPI-anchored glycoproteins are shed from the cell membrane and exist as soluble products in vitro and in vivo. To determine if uPA-R undergoes a similar phenomenon, we have developed a sensitive enzyme-linked immunoabsorbent assay (ELISA) (using a rabbit antiserum as both capture and detection reagents) to measure the quantity of soluble uPA-R (suPA-R) in tissue culture supernatants and biologic fluids. Using this ELISA, we have detected suPA-R in the culture supernatants of U-937 cells and human monocytes stimulated in vitro by certain soluble inflammatory mediators (Sitrin et al, Blood 84:1268, 1994; Mizukami et al., Clin Res 42:115A, 1994). To determine if suPA-R exists in vivo, we have screened the plasma of 20 normal volunteers (mean +/- SD, 3 +/- 3 ng/mL; median, 2 ng/mL; range, 1 to 11 ng/mL [serum values slightly higher]); the plasma of 13 ICU patients with clinical sepsis syndrome (mean +/- SD, 30 +/- 11 ng/mL; median, 11 ng/mL; range, 4 to 221 ng/mL); and the extravascular fluids (pleural, pericardial, and peritoneal) of 84 individuals with presumed inflammatory or malignant conditions (mean +/- SD, 21 +/- 39 ng/mL; median, 10 ng/mL; range, 2 to 253 ng/mL). Among the latter specimens, most were inflammatory exudates (only six were malignant by positive cytology) with the highest quantities of suPA-R associated with neutrophilic exudates. The solubility of suPA-R contained within these fluids was confirmed by reanalysis after ultracentrifugation to remove particulate material. When tested in a uPA ligand capture ELISA, representative specimens of extravascular body fluids and sepsis plasma contained suPA-R capable of binding uPA ligand (generally representing a small fraction of the immunoreactive material). We conclude from these data that suPA-R is immunologically detectable in vitro and in vivo with high concentrations of receptor found under conditions of inflammatory stimulation. The possibility of suPA-R's biologic activity is suggested by its partial retention of ligand binding capacity.

Adult↗
PubMed1995

Interaction of Fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

3T3 Cells↗
PubMed1995

Human urokinase-type plasminogen activator primes neutrophils for superoxide anion release. Possible roles of complement receptor type 3 and calcium.

Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.

3T3 Cells↗
PubMed1995

Compartmentalized roles for leukocytic adhesion molecules in lung inflammatory injury.

By using the model of acute injury caused by intrapulmonary deposition of IgG immune complexes, blocking mAb to CD11a, CD11b, L-selectin, and intercellular adhesion molecule-1 (ICAM-1) were administered either i.v. or intratracheally (i.t.). The effects of these interventions were assessed according to lung injury, lung content of myeloperoxidase (MPO), TNF-alpha, and cellular content in bronchoalveolar lavage (BAL) fluids, and up-regulation of pulmonary vascular ICAM-1. In animals treated i.v. with Abs to CD11a, L-selectin, or ICAM-1 lung injury was significantly attenuated in parallel with reduced lung content of MPO. Under similar conditions, treatment with anti-CD11b had no effect. However, when the same mAb were administered i.t., anti-CD11a and anti-L-selectin were without protective effects, whereas i.t. administered anti-CD11b and anti-ICAM-1 were each highly protective. The protective effects of anti-CD11b were related to profound reductions in BAL levels of TNF-alpha, pulmonary vascular up-regulation of ICAM-1, and lung content of MPO. The protective effects of i.t.-administered anti-ICAM-1 were not associated with reduced BAL levels of TNF-alpha. Protective effects of mAb were also reflected in reductions of retrievable neutrophils in BAL fluids. mAb to rat CD11b and CD18 but not to rat CD11a suppressed in vitro production of TNF-alpha by immune complex-stimulated rat alveolar macrophages. The mAb did not reduce NO2-/NO3- generation in stimulated macrophages but all mAb (except anti-ICAM-1) reduced O2- responses in macrophages. These data suggest a compartmentalized role for adhesion molecules in lung inflammatory injury after intraalveolar deposition of IgG immune complexes, with CD11a, L-selectin, and ICAM-1 being important in the vascular compartment for neutrophil recruitment, whereas in the alveolar compartment CD11b and ICAM-1 (but not CD11a and L-selectin) seem to play key roles.

Alveolitis, Extrinsic Allergic↗
PubMed1995

Function of the urokinase receptor (CD87) in neutrophil chemotaxis.

During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase-type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN chemotaxis, PMNs were pretreated with an anti-CD87 monoclonal antibody (mAb), a neutralizing anti-uPA mAb, or uPA. PMN chemotaxis was profoundly suppressed by the anti-CD87 mAb but was unaffected by anti-uPA mAb or uPA. The role CD87 plays in chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADG, D-mannose, and mannoside) inhibit PMN chemotaxis. We conclude that CD87 plays a crucial role in PMN chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.

Antibodies, Monoclonal↗
PubMed1995

Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.

Animals↗
PubMed1995

Cell surface-bound urokinase-type plasminogen activator facilitates infiltration of freshly isolated granulocytes into fibrin matrix.

Human cell lines of myelo/monocytic origin express the cellular receptor for urokinase-type plasminogen activator (uPA-R). The receptor localizes urokinase-type plasminogen activator (uPA) to the surface of the cell, where it can convert plasminogen to the active serine proteinase plasmin. Plasmin may subsequently account for proteolysis of pericellular proteins. We demonstrated the expression of the uPA-R by freshly isolated neutrophilic granulocytes by using a specific mAb. In freshly isolated granulocytes we detected only a weak occupation of the uPA-R; further uPA binding by granulocytes was saturable and proceeded in a dose-dependent manner. Receptor-bound uPA retained its enzymatic activity. Saturation of isolated granulocytes with exogenous uPA enhanced cellular infiltration into a fibrin matrix in vitro. uPA-dependent infiltration was inhibited by an anti-catalytic monoclonal anti-uPA antibody. The findings show that circulating neutrophilic granulocytes express the cell surface uPA-R and suggest that surface-binding of uPA may facilitate the infiltration of granulocytes into a fibrin clot, a process that might add to thrombolysis in vivo.

Cell Movement↗