Urokinase plasminogen activator amino-terminal peptides inhibit development of the rat ventral prostate.

The plasma membrane urokinase plasminogen activator receptor (uPAR) localizes and enhances activation of pro-uPA. Active uPA, in turn, promotes increased degradation of the extracellular matrix (ECM) by activation of plasminogen. uPAR binds to ECM molecules and integrins, which can affect cellular adhesion, signal transduction, and gene regulation. The current study examines the expression and function of uPAR in developing rat ventral prostates (VPs). We report that newborn VPs express uPAR mRNA and protein. In addition, the function of uPAR-bound uPA during in vitro prostatic development was studied by adding recombinant peptide competitive inhibitors of uPA-uPAR binding. Newborn VP explants were cultured in serum-free media for one week with 10(-8) M testosterone plus chimeric peptides containing a human immunoglobulin G Fc domain and either human uPA amino acids 1-138 (hu-uPA 1-138) as a control or mouse uPA amino acids 1-138 (mo-uPA 1-138) or 1-48 (mo-uPA 1-48). Hu-uPA 1-138-treated VPs underwent normal ductal branching morphogenesis and tissue differentiation. In contrast, VPs treated with mo-uPA 1-138 or mo-uPA 1-48 displayed a dose-dependent perturbation of ductal branching. Differentiation of both epithelial and mesenchymal tissues was also impaired. Mo-uPA 1-48-treated VPs contained significantly more apoptotic cells. These observations suggest that disruption of uPA binding to uPAR results in a retardation of the development of newborn VPs.

Inhibition of prostate cancer neovascularization and growth by urokinase-plasminogen activator receptor blockade.

Binding of the serine protease urokinase (u-PA) to its receptor on tumor cell surfaces facilitates proteolysis and tumor invasion. We undertook this study to determine whether the role of u-PA in prostate cancer induced angiogenesis and secondary tumor growth by developing a homologous, immunocompetent in vivo model in which the tumors cells secrete an inhibitor of the murine u-PA receptor. A mutant recombinant murine u-PA that retains receptor binding but not proteolytic activity was made by PCR mutagenesis. Mutant u-PA and a reporter gene pRK luciferase were transfected and stably expressed in the highly metastatic rat Dunning MAT-LyLu prostate cancer cell line. Several clones expressing mutant u-PA and luciferase were identified by Western blotting, plasminogen zymography, and reverse transcription-PCR. One of these clones, 5C4, was injected s.c. into Copenhagen rats. Compared to animals injected with clones expressing pRK luciferase alone, tumors in animals injected with 5C4 cells were significantly smaller. Moreover, there were fewer lung micrometastases in the 5C4 animals. Primary tumor angiogenesis was measured by microvessel quantification of tissue stained with antibodies against von Willebrand factor. Mean microvessel density in 5C4 tumors was 4.3-fold lower than that in animals with tumors derived from the control tumor cell line (P < 0.0001). Significant inhibition of tumor growth was also observed for two additional MAT-LyLu cell lines expressing mutant u-PA. These findings suggest that cell surface u-PA contributes to prostate cancer growth by enhancing angiogenesis.

The role of type IV collagenases in rat bladder development and obstruction.

The role of type IV collagenases during rat bladder development and in response to partial bladder outlet obstruction was evaluated. Gelatinase gel zymography was performed on developing rat bladders (gestation d 16 and 19, at birth, 5, 10, 15, 20, 30, and 75 d postnatally), after partial obstruction of the bladder outlet in young adults and after separation of the epithelium from the mesenchyme in young adults. Bladder function was assessed by cystometry in obstructed animals. During development, the 72-kD type-IV collagenase [matrix metalloproteinase (MMP)-2, both latent and activated] was maximally expressed in the fetal period and decreased with age; whereas the 92-kD gelatinase (MMP-9) was not expressed in developing or adult bladders. MMP-2 was localized to the bladder mesenchyme and was undetectable in isolated epithelium. In 46 obstructed rats, there was an 8-fold increase in bladder volume and weight along with smooth muscle hypertrophy (mean smooth muscle cell diameter 7.09 +/- 0.11 microns versus 4.65 +/- 0.05 microns in normal animals, p < 0.001). Obstructed rats had increased quantities of latent and activated MMP-2 and MMP-9 compared with sham-operated and normal controls. These findings suggest that expression and activation of type IV collagenases (MMP-2 and 9) are developmentally regulated and play a role in bladder remodeling during developmental morphogenesis and after partial outlet obstruction.

Decreased prostate cancer cell migration by inhibition of the insulin-like growth factor II/Mannose-6-Phosphate receptor.

The 270-kDa insulin-like growth factor II (IGF-II)/cation-independent mannose-6-phosphate receptor (MPR) is a multifunctional receptor protein. Endocytoses and intracellular transport of soluble enzymes bearing mannose6-phosphate (M-6-P) residues to lysosomes is mediated by the IGF-II/MPR. We examined human prostate cancer cells for IGF-II/MPR expression to determine whether this receptor mediates cell migration. PC3 human prostate cancer cells were studied for intracellular IGF-II/MPR by immunoblotting. PC3 cell surface IGF-II/MPR expression was assessed by flow cytometric analysis. Cell motility was quantitated by a scratch migration assay, and IGF-II/MPR blockade was achieved using M-6-P or affinity-purified rabbit anti-bovine cation-independent IGF-II/MPR immunoglobulin. IGF-II/MPR is expressed in the cytoplasm and on the surface of PC3 prostate cancer cells. The mean number of PC3 cells migrating per high powered field in medium containing polyclonal anti-IGF-II/MPR immunoglobulin or M-6-P decreased significantly (5 ± 4 cells and 34 ± 5 cells, respectively) compared with control medium containing mouse immunoglobulin G (70 ± 12 cells) or mannose-1-phosphate (67 ± 7 cells). This decreased PC3 cell migration following cell surface IGF-II/MPR blockade suggests that the IGF-II/MPR may play an important role in prostate cancer cell motility.

Upregulation of the 72-kDa type IV collagenase in epithelial and stromal cells during rat tracheal gland morphogenesis.

Submucosal glands secrete most of the mucus that lubricates the tracheal surface and protects it from irritants and infection. These glands develop postnatally in the rat, permitting convenient study of the mechanisms controlling this process. One such mechanism involves degradation of the supportive connective tissue matrix at the front of the growing glands. We recently showed that tracheal gland cell invasion of collagen gels in vitro is dependent on secretion of a 72-kDa type IV collagenase. In the present study, we show that the activity of this enzyme (also referred to as matrix metalloproteinase-2 or gelatinase A) is elevated at the time of gland development in vivo. That this increase is at least partly mediated at the level of steady-state mRNA was indicated by semiquantitative PCR analysis of gland-enriched, microdissected tissue samples. Immunohistochemistry revealed that the enzyme was present at the interface between the glands and extracellular matrix. In situ hybridization revealed that the cognate mRNA was present in epithelial cells of glands undergoing morphogenesis (particularly Postnatal Day 7) but not in those of adult glands or the surface epithelium. At all ages, stromal cells below the surface epithelium were labeled; labeling intensity was highest at the time and location of gland morphogenesis. These findings suggest that the 72-kDa type IV collagenase is developmentally regulated in gland and stromal cells at the level of steady-state mRNA and plays a role in the degradation of extracellular matrix during tracheobronchial gland morphogenesis.

Development of male urogenital epithelia elicited by soluble mesenchymal factors.

Androgen-dependent development of male secondary sexual glands is mediated by paracrine mesenchymal-epithelial interactions that regulate a complex array of biological events such as epithelial morphogenesis, growth, and cytodifferentiation. It is not known whether the action of mesenchyme on epithelial development in the male genital tract requires cell-cell contact or whether soluble, diffusible mediators are involved. To examine paracrine effects of urogenital sinus mesenchyme (UGM) on epithelial development, conditioned media (CM) from embryonic mouse UGM of normal (wild-type) and androgen-insensitive Tfm (testicular feminization) mice were tested for growth and morphogenetic effects on heterotypic tissue recombinants composed of rat or mouse bladder mesenchyme plus neonatal mouse seminal vesicle epithelium (BLM+SVE) or rat or mouse bladder mesenchyme plus neonatal mouse bulbourethral gland epithelium (BLM+BUG-E). Addition of a concentrate of CM from wild-type UGM grown in the presence of dihydrotestosterone (DHT) induced epithelial growth and complex epithelial morphogenesis in BLM+SVE recombinants, whereas CM from DHT-treated Tfm UGM or a saline control were without effect. CM from wild-type UGM elicited similar trophic effects in BLM+BUG-E recombinants, but in addition induced precocious mucous epithelial differentiation in BLM+BUG-E recombinants. These results suggest that the normally androgen-dependent epithelial growth and branching morphogenesis in developing male urogenital organs is elicited by soluble mesenchymal factors. Two-dimensional gel electrophoresis of proteins synthesized and secreted by wild-type UGM revealed several androgen-dependent proteins with molecular weights of approximately 30 kDa that are absent in CM of Tfm UGM either in the presence or absence of DHT.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered metabolic and adhesive properties and increased tumorigenesis associated with increased expression of transforming growth factor beta 1.

Transforming growth factor-beta (TGF-beta) is a potent mediator of cell proliferation and extracellular matrix formation, depending on the cell type and the physiological conditions. TGF-beta is usually secreted in a "latent" complex that needs activation before it can exert its effects. Several observations correlate increased expression of TGF-beta 1 with tumorigenesis. To evaluate the physiological relevance of increased TGF-beta 1 synthesis in tumor cells we established cell clones overexpressing TGF-beta 1 and observed the resulting physiological changes in TGF-beta overproducing cells in vitro and in vivo. As a model system we used the human E1A-transformed 293 tumor cells, which are insensitive to the direct growth modulatory effects of TGF-beta. The selection of this cell line allows an assessment of physiological alterations independent of TGF-beta induced proliferative changes. The use of two TGF-beta 1 expression vectors containing either the natural or a modified TGF-beta 1 precursor cDNA permitted the establishment of separate 293 cell lines overexpressing latent or active TGF-beta. Comparison of the resulting changes in glycolytic rate, adhesiveness and integrin and plasminogen activator expression established that, in vitro, both types of clones behaved similarly, indicating that expression of latent TGF-beta induces autocrine changes in the tumor cells and thus suggesting that some level of cell-associated activation occurs. TGF-beta overexpression resulted in an increased metabolic rate due to enhanced glycolysis, a property long associated with tumor cells. This increased glycolysis was not associated with altered proliferation. Cells overexpressing TGF-beta also displayed enhanced fibronectin mRNA and plasminogen activator synthesis and increased adhesiveness in vitro. They showed enhanced survival when plated sparsely on plastic in the absence of serum, and attached more readily to laminin. In addition, synthesis of several beta 1 integrins, in particular the alpha 1/beta 1, alpha 2/beta 1, and alpha 3/beta 1, all of which recognize laminin, were enhanced. Finally, cells overexpressing active TGF-beta, but not latent TGF-beta, also showed increased tumorigenicity in nude mice. Thus, an increase in endogenous TGF-beta synthesis confers several proliferation-independent phenotypic changes which may be of significance for the survival of the tumor cell inoculum or its subsequent growth, and for tumor formation and development. In the case of cells expressing active TGF-beta, the release of active TGF-beta into the vicinity of the tumor cells may also result in a more hospitable environment for tumor growth.

Invasion by cultured human follicular thyroid cancer correlates with increased beta 1 integrins and production of proteases.

A recognized model of tumor invasion requires cells to adhere to epithelial basement membrane and extracellular matrix components triggering release of proteases thus allowing cancer cells to invade the substrate. This adhesion is mediated by beta 1 integrins, a family of receptors to substrates such as collagen, laminin, and fibronectin. In order to study tumor invasion in follicular thyroid cancer (FTC), we used cell lines derived from a single patient's FTC primary tumor (FTC-133), neck lymph node metastases (FTC-236), and lung metastases (FTC-238). In vitro invasion as determined by the ability of the tumor cells to penetrate Matrigel was assessed by scanning electron microscopy. FTC-133 did not invade, FTC-236 was moderately invasive, and FTC-238 was highly invasive. Immunoprecipation with a monoclonal antibody to beta 1 integrin subunits and SDS-PAGE showed increased synthesis and flow cytometry showed increased expression of this subunit in FTC-236 and FTC-238 compared to FTC-133. Proteolytic activity was assessed by gelatin zymography. FTC-238 cell extract and conditioned media exhibited a more complex array of proteases consistent with activated type I collagenase and stromelysin compared to the less invasive clones, however 72 and 92 kd gelatinases consistent with type IV collagenases were present in the conditioned media from all three lines. In conclusion, in vitro invasion parallels in vivo metastasis by the source cells in the FTC-133/236/238 cell-lines. The ability to invade basement membrane preparation correlates with increased synthesis and expression of beta 1 integrins and activation of tumor proteases.

Defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis.

Juvenile periodontitis (JP) is a disease characterized by severe gingival infections. PMN from some JP patients exhibit abnormal chemotactic responsiveness when challenged with the synthetic formyl peptide, FMLP. While investigating PMN function in JP, we found a patient in whom abnormal PMN chemotactic responses to FMLP were associated with a defective population of PMN formyl peptide receptor(s) (FPR). JP PMN failed to respond chemotactically when challenged with FMLP, but exhibited normal chemotactic responses upon exposure to purified human C5a. Furthermore, JP PMN were capable of degranulating and generating superoxide anion radicals as well as normal PMN upon exposure to FMLP. Binding studies demonstrated that JP PMN had a diminution in the number of high-affinity FPR. Studies in which FPR was radiolabeled by chemical cross-linking demonstrated that JP PMN FPR exhibited the same molecular weight and N-linked glycosylation as normal PMN FPR. JP PMN FPR, however, was more resistant to papain cleavage than normal PMN FPR. Autoradiograms obtained from 2D-PAGE of normal and JP PMN FPR demonstrated decreased amounts of FPR isoforms in JP PMN.

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils.

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4.

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.

Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.

Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.

Removal of human polymorphonuclear leukocyte surface sialic acid inhibits reexpression (or recycling) of formyl peptide receptors. A possible explanation for its effect on formyl peptide-induced polymorphonuclear leukocyte chemotaxis.

Removal of surface sialic acid specifically inhibits human polymorphonuclear leukocyte (PMN) chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neuraminidase-treated (NT)-PMN bound and internalized [3H]FMLP (used as receptor marker) as well as normal PMN. NT-PMN, however, retained more [3H]FMLP-associated radioactivity than normal PMN. Subcellular fractionation studies demonstrated that NT-PMN retained more sedimentable (100,000 X G for 180 min) [3H]FMLP-associated radioactivity within light Golgi-containing fractions than normal PMN. Furthermore, NT-PMN exhibited a defect in their ability to reexpress (or recycle) a population of FMLP receptors. Abnormal receptor recycling was associated with inhibition of FMLP-induced PMN chemotaxis. Thus, it appears that recycling of formyl peptide receptors may be necessary for optimal PMN chemotactic responses to FMLP. We postulate that removal of PMN surface sialic acid inhibits FMLP-induced PMN chemotaxis by blocking the reexpression (or recycling) of a population of formyl peptide receptors, perhaps by preventing trafficking of desialated receptors through a light Golgi pathway.

Meclofenamate sodium monohydrate inhibits chemotactic factor-induced human polymorphonuclear leukocyte function. A possible explanation for its antiinflammatory effect.

Meclofenamate sodium monohydrate (MSM), a potent nonsteroidal antiinflammatory agent, specifically inhibits chemotactic factor-induced human polymorphonuclear leukocyte functions: chemotaxis, degranulation, and generation of superoxide anion radicals. These effects of MSM were found to be dependent upon the concentrations of drug not bound to albumin (free drug), and were caused by its ability to interfere at both a receptor and post-receptor (i.e., a step distal to mobilization of polymorphonuclear leukocyte intracellular Ca2+) level. These unique actions of MSM may provide a partial explanation for its potent antiinflammatory effect.

Human neutrophils contain subpopulations of specific granules exhibiting different sensitivities to changes in cytosolic free calcium.

We examined the role of mobilization of intracellular calcium in the ability of human neutrophils to discharge specific granule constituents upon stimulation with the synthetic chemotactic factor, N-formyl-met-leu-phe. Extracellular calcium was not required for optimal secretion of the specific granule markers lactoferrin and vitamin B12-binding protein. Depletion and chelation of intracellular calcium, as well as reconstitution experiments, however, revealed different calcium requirements for stimulated secretion of these markers. N-formyl-met-leu-phe-induced secretion of vitamin B12-binding protein required half-maximal change in intracellular calcium of greater than 20 nM, while lactoferrin requirements were approximately 140 nM. Thus, it appears that cytosolic free calcium modulates fusion of subpopulations of specific granules which with the neutrophil plasma membrane.

Preparation and characterization of a derivative of wheat germ agglutinin which specifically inhibits polymorphonuclear leukocyte chemotaxis to the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine.

A nonagglutinating derivative of wheat germ agglutinin (WGA), prepared by treating the native lectin with cyanogen bromide and formic acid and purified by affinity chromatography on an N-acetyl-D-glucosamine column, inhibited human polymorphonuclear leukocyte (PMN) chemotaxis to the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The WGA derivative (WGA-D) did not influence either the ability of PMN to migrate randomly or their chemotactic response to the complement-derived peptide C5a. Similarly, WGA-D had no effect on either FMLP-induced PMN polarization or other FMLP-induced PMN functions (i.e., selective discharge of lysosomal enzymes from cytochalasin B-treated cells, generation of superoxide anion). The inhibition of FMLP-induced PMN chemotaxis by WGA-D could not be reversed by washing the cells, or by incubating lectin-treated PMN at 37 degrees C for 20 min. The inhibitory effect of WGA-D was mediated by its specific binding to N-acetyl-D-glucosamine residues on the cell surface. WGA-D did not alter the specific binding of [3H]-FMLP to its receptor(s) on the PMN membrane. The data presented here suggest that WGA-D inhibits FMLP-induced PMN chemotaxis at a step distal to stimulus recognition.

A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors.

We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.

Removal or oxidation of surface membrane sialic acid inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis.

Polymorphonuclear leukocyte (PMN) surface membrane glycoproteins are probably involved in the phenomenon of stimulus-response coupling. Consequently, we examined the effects of either removal or oxidation of surface membrane-associated sialic acid residues on some responses of human PMN to chemotactic factors. Treatment of human PMN with either neuraminidase or sodium metaperiodate did not affect the ability of these cells to migrate randomly, but did inhibit their ability to respond chemotactically to the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Treated PMN responded normally, however, to the complement-derived peptide C5a, and to the lipoxygenase product leukotriene B4. Enzymatic removal or oxidation of membrane sialic acid residues did not affect either FMLP-induced PMN degranulation or FMLP-induced generation by PMN of superoxide anion radicals. Removal of sialic acid did not significantly alter specific binding of [3H]FMLP to its receptor(s) on the PMN membrane. These findings indicate that sialic acid residues on the PMN surface membrane play an important role in modulating PMN responses to FMLP.