Reduced clearance of proteins labeled with diisopropylfluorophosphate in portacaval-shunted rats

Portacaval shunting is a model for hepatic encephalopathy that causes chronic hyperammonemia, disruption of metabolic, signaling, and neurotransmitter systems, and progressive morphological changes. Exposure of cultured cells to ammonia raises intralysosomal pH and inhibits proteolysis, and the present study tested the hypothesis that proteolytic capacity is diminished in portacaval-shunted rats. Proteins were labeled in vivo with tracer doses of diisopropylfluorophosphate (DFP) and clearance of label was assayed. This approach labeled proteins independent of protein synthesis, which is reported to be altered in shunted rats, and avoided complications arising from re-utilization of labeled amino acids that causes underestimation of degradation rate. Characterization of DFP labeling showed that protein labeling was fast, about 50 % of the label was released during a 24 h interval, labeling by DFP metabolites was negligible, inhibition of brain acetylcholinesterase was not detectable, and labeling by [³H]- and [¹⁴C]DFP was equivalent. To assay degradative capacity, proteins were first labeled with [³H]DFP, followed by labeling with [¹⁴C]DFP that was given 24 or 72 h later. The ³H/¹⁴C ratio in each animal was used as a relative measure of removal of ³H-labeled proteins. ³H/¹⁴C ratios were generally significantly higher in portacaval-shunted rats than in controls, consistent with reduced proteolytic capacity. Assays of amino acid incorporation into brain protein generally replicated literature reports, supporting the conclusion that protein synthesis unlikely to be markedly inhibited and amino acid recycling influences calculated protein synthesis rates in shunted rats. Therapeutic strategies to reduce ammonia level would help normalize lysosomal functions and protein and lipid turnover.     

Protein biosynthetic activity of polymorphonuclear leukocytes in inflammatory arthropathies. Increased synthesis and release of fibronectin

We have investigated protein synthesis and release by polymorphonuclear leukocytes (PMN) to compare the protein biosynthetic activity of peripheral blood PMN and inflammatory synovial fluid (SF) PMN from patients with inflammatory arthropathies. We analyzed and compared the protein profiles produced by these cells, using patient matched peripheral blood and SF PMN as well as peripheral blood PMN from normals. Twenty-five patients with either rheumatoid arthritis, psoriatic arthritis or gout were studied. Fluorographs of SDS-polyacrylamide slab gels, performed using cell supernatants from metabolically labelled cells, revealed an increased release of de novo synthesized proteins by inflammatory SF PMN compared to peripheral blood PMN. Under reducing conditions, 4 clearly distinguishable high molecular mass products were observed (Mr 230,000, 185,000, 170,000 and 95,000). Two of the protein bands were found to be gelatin binding (Mr 230,000 and Mr 95,000). By Western blot, the Mr 230,000 protein was found to be fibronectin and the Mr 95,000 protein was shown to be identical to a recently described gelatinase. Thus, the activation of PMN in inflammation is accompanied by an increased release of a number of de novo synthesized proteins, including fibronectin. Our studies directly pertain to the in vivo inflammatory process since the PMN were not activated artificially in vitro.     

Induction of FcγR-III (CD16) expression on neutrophils affected by paroxysmal nocturnal haemoglobinuria by administration of granulocyte colony-stimulating factor

Summary. The inducibility of glycosyl-phosphatidylinositol (GPI)-anchored proteins on affected paroxysmal nocturnal haemoglobinuria (PNH) neutrophils (PMN) after both in vitro and in vivo stimulation was investigated. FcγR-III (CD16), decay-accelerating factor (DAF/CD55)and 20 kD homologous restriction factor (HRF20/CD59) were demonstrated to be concurrently deficient on unstimulated defective PNH PMN. Upon in vitro stimulation with either N -formyl-methionyl-leucyl-phenylalanine (fMLP), zymosan-activated serum (ZAS), or recombinant human granulocyte colony-stimulation factor (G-CSF), neither CD16 nor CD55 expression was induced on defective PNH PMN. G-CSF was administered to two patients with PNH when their conditions were complicated by bacterial infections, or to prevent infections associated with the extraction of teeth or cataract surgery. CD16 expression was induced on the defective PNH PMN in both cases during the administration of G-CSF, but the expression of CD55 and CD59 was not. CD16, induced on the defective PNH PMN during the administration of G-CSF, was phosphatidylinositol-specific phospholipase C (PIPLC)-sensitive, implying that it had GPI-linkage to the membranes. The patients treated with G-CSF recovered from infection or evaded infection. These observations suggest that a deficiency of GPI-anchored proteins is not always seen in defective PNH blood cells, at least under certain stimulation conditions.     

Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart

OBJECTIVE: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins. METHODS: Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n = 10) or without IPC (n = 8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. RESULTS: IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6 +/- 2.8 to 3.4 +/- 0.9 (P < 0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm2 myocardium) in the ischemic zone from 243 +/- 19 to 118 +/- 19 (P < 0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r = 0.8, P < 0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204 +/- 57% in the control group to 76 +/- 7% (P < 0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group. CONCLUSION: These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax.     

An in vitro model for polymorphonuclear-leukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic membranes

Alteration of the extracellular matrix by inflammatory cells is believed to be important in both lung injury and the subsequent restoration of lung architecture. Here we describe the results of the interaction between an acellular human amnionic membrane model and stimulated human polymorphonuclear neutrophils (PMN) in vitro. Polymorphonuclear neutrophil suspensions were placed on one surface of the amnion, and either the chemotactic peptide FMLP or the cell membrane activator phorbol myristate acetate (PMA) was placed on the opposite side of the amnion. Stroma and basement membrane sides of the amnion were separately exposed to the PMN. The PMN suspension was removed and centrifuged, and the supernatant was assayed for superoxide anion (O2-.) and for elastase activity. Injury to the acellular amnion was evaluated by transmission electron microscopy and by measurement of fibronectin (FN) released from the membrane matrix. Although both stimulants cause a concentration-dependent release of O2-., only PMA stimulated elastase release. These effects were similar when either the stroma or the basement membrane side was exposed to PMN. PMA-stimulated cells and supernatants from PMA-stimulated cells caused solubilization of membrane at different incubation times. Electron microscopy confirmed the disruption of the basement membrane of the amnion by PMA-stimulated PMN. Oxidant scavengers (SOD and catalase) did not prevent matrix degradation, and elastase inhibition by a specific chloromethylketone inhibitor diminished FN release on both sides of the amnion by activated PMN supernatants, but only on the basement membrane side by intact PMN. We conclude that in this model, elastase rather than oxygen radicals solubilizes FN from the matrix.     

Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-binding protein shedding: relationship to GH receptor down-regulation

GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splicing of a common GHR precursor messenger RNA to encode secreted GHBP (the source of the vast majority of GHBP in rodents); and 2) proteolysis of the cell-associated GHR with shedding of GHBP (a mechanism operative in rabbits and humans). We previously observed that phorbol ester (PMA)-induced activation of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolysis and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA-induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding were also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chinese hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibition of either PKC or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. Thus, different pathways leading to metalloprotease-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth factor (PDGF) and serum. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteolysis, which was inhibited by IC3. Interestingly, PMA-, PDGF-, and serum-induced GHR proteolysis was associated with substantial decreases in GH-induced activation of Janus kinase-2, which were also prevented by IC3. These findings suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.     

Degradation of human articular cartilage by neutrophils in synovial fluid

Objective. To determine whether surface-adherent immunoglobulins are capable of mediating synovial fluid (SF) neutrophil degradation of proteoglycan and collagen in intact, normal human articular cartilage, and to define the respective roles of neutrophil serine proteases and metalloproteases in degrading these cartilage constituents. Methods. Pellet explants of normal human articular cartilage pretreated with bovine serum albumin (BSA) or IgG were incubated with polymorphonuclear cells suspended in SF (PMN-SF), or with supernatants derived from neutrophils stimulated with surface-associated IgG. Proteoglycan degradation was measured by assaying release of ³⁵S-proteoglycan fragments from cartilage explants prelabeled with ³⁵S-sulfate. Collagen degradation was measured by assaying hydroxyproline content in the PMN-SF preparations or neutrophil supernatants following their incubation with unlabeled explants. Results. Significant release of both ³⁵S fragments and hydroxyproline was noted following incubation of PMN-SF with IgG-treated pellets, compared with pellets treated with BSA. IgG preparations derived from pooled normal serum or rheumatoid arthritis SF were equally efficacious in mediating PMN degradation of cartilage collagens. Explant release of ³⁵S fragments during incubation with PMN supernatant was completely inhibited when serine proteases were inactivated by diisopropyl fluorophosphate (DFP); however, release of ³⁵S fragments was enhanced when metalloprotease activity was present in the supernatant. Release of hydroxyproline during incubation of explants with PMN supernatant was comparable in the presence of DFP or EDTA, but was markedly enhanced when both serine and metalloprotease activity were present in the supernatant. Conclusion. Neutrophils in SF are capable of degrading both proteoglycans and collagens in intact human articular cartilage. Degradation of these cartilage constituents is facilitated by immunoglobulins adherent to the cartilage surface and by the synergistic action of PMN serine and metalloproteases released during activation of neutrophils with surface-associated immunoglobulin.     

Enhanced superoxide generation and the decreased superoxide scavenging activity of peripheral blood leukocytes in Beh?et's disease--effects of colchicine.

Enhanced superoxide radical (O2-) generation by polymorphonuclear cells (PMN) has been suggested to mediate tissue damage associated with Beh?et's disease (BD). Because superoxide dismutase (SOD) provides protection from O2- we investigated whether the superoxide scavenging activity (SSA) of PMN, mononuclear cells (MNC) or plasma has a correlation to the amount of O2- generated by PMN in BD. We also studied the effects of colchicine, a potent inhibitor of phagocytosis, on the SSA of PMN. In BD, O2- release by both non-stimulated PMN and those stimulated with opsonized zymosan (OZ), or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), was enhanced. The SSA of PMN, but not MNC and plasma, was significantly lower in BD patients as compared with healthy controls. The SSA of PMN showed a strong negative correlation with their O2- release. The SSA of PMN was lower in the BD patients with elevated erythrocyte sedimentation rates and C-reactive protein levels than in those with normal test results. In addition, we found that colchicine treatment increased the SSA of PMN toward normal levels in BD patients. In vitro studies demonstrated that the SSA in PMN obtained from healthy adults was decreased after stimulation with either OZ or PMA. Colchicine could prevent the decrease in the SSA when PMN were stimulated with OZ, but not when stimulated with PMA. Our results suggest that the enhanced O2- release by PMN in vivo may be responsible for the decreased SSA of PMN in BD and the PMN might be able to release more O2- in tissues. Colchicine may increase the SSA of PMN toward normal levels by blocking phagocytosis.     

IDENTIFICATION OF COMPONENTS OF (NA+ + K+)-ADENOSINE TRIPHOSPHATASE BY DOUBLE ISOTOPIC LABELING AND ELECTROPHORESIS

A microsomal adenosine triphosphatase (ATPase) that requires both sodium and potassium ions is thought to be identical with, or an integral part of, the active cation transport system located in cell membranes. Attempts to isolate and purify (Na(+) + K(+))-ATPase have met with limited success because solubilization of microsomal protein causes partial, if not complete, loss of enzymatic activity. We now report the isolation from rat kidney microsomes of proteins which, though enzymatically inactive, could still be identified as components of the (Na(+) + K(+))-ATPase system.Phosphoproteins known to be intermediates in the hydrolysis of ATP by (Na(+) + K(+))-ATPase were prepared by incubating rat kidney microsomes with gamma-labeled ATP(33) in the presence of sodium or with P(32)-orthophosphate in the presence of ouabain. After the P(32)- and P(33)-labeled microsomes had been dissolved in phenol-acetic acid-urea, the resultant solutions were mixed and subjected to polyacrylamide gel electrophoresis. The radioactivity from both phosphorus isotopes was found almost exclusively in one of the resultant 21 protein bands. In contrast, the radioactive protein from DFP(32)-labeled microsomes moved slightly faster than the radioactive protein from microsomes labeled with P(33)-orthophosphate in the presence of ouabain. DFP inhibits (Na(+) + K(+))-ATPase by reacting with a nucleophilic site at or near the active site. These results suggest that while a single protein component of (Na(+) + K(+))-ATPase accepts the terminal phosphate from ATP, the final splitting of this phosphoprotein intermediate may be catalyzed by nucleophilic sites on a second protein.     

The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.

The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.     

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.     

Covalent binding of growth hormone to surface receptors on rat adipocytes

GH specifically binds to receptors on the surface of adipocytes and produces a variety of biological effects in these cells. To gain insight into the nature of the GH receptors, [125I] human GH ([125I]hGH) was cross-linked to surface binding sites on intact rat adipocytes using the bifunctional reagent disuccinimidyl suberate. Plasma membranes were isolated, and after solubilization with sodium dodecyl sulfate (SDS), the proteins were subjected to electrophoresis on 5% or 7.5% polyacrylamide gel. Autoradiography of the 7.5% gels revealed three iodinated bands corresponding to apparent molecular weights of 56, 130, and more than 240 kilodaltons. The more than 240-kilodalton band contained approximately as much 125I as the 130-kilodalton species and about twice as much as the 56-kilodalton species. When run on the more porous 5% gel, the more than 240-kilodalton band resolved into two bands, corresponding to apparent molecular weights of 240 and 310 kilodaltons. Excess unlabeled human or bovine GH, but not ovine PRL, competed with [125I]hGH for binding and prevented the formation of all of the labeled bands. Treatment of the membranes and extracted proteins with dithiothreitol resulted in the generation of additional 130-kilodalton material at the expense of both the 310- and 240-kilodalton species, but failed to alter the amount of 125I that migrated with the 56-kilodalton species. The same pattern of labeling was seen regardless of whether protease inhibitors were present during isolation of membrane proteins or when membrane proteins were isolated under conditions that favored proteolysis, suggesting that the 56-kilodalton species is not a degradative product of the higher molecular weight species. When [125I]hGH was cross-linked to adipocytes in which total binding was decreased by hypophysectomy or starvation of the donor rats or by treatment of the cells with cycloheximide, there was a proportionate diminution in labeling of all species. It thus appears that the GH receptor contains a 130-kilodalton subunit, a portion of which is in disulfide linkage with higher molecular weight complexes and, in addition, contains a 56-kilodalton species. It cannot be determined from these studies if the various labeled protein complexes are components of a single or multiple classes of GH receptors in the adipocyte membrane.     

Direct effects of neutrophil oxidants on elastase-induced extracellular matrix proteolysis

Oxidant species produced by human polymorphonuclear leukocytes (PMN) inactivate alpha-1-protease inhibitor and thus may indirectly enhance neutrophil elastase-induced proteolysis. It is unclear, however, if PMN-derived oxidants directly enhance proteolysis of extracellular matrix by neutrophil elastase. Matrix was produced by neonatal rat aortic smooth muscle cells and pulse-labeled with 3H-lysine to allow identification of the collagen-specific amino acid, hydroxylysine (3H-HL), and the elastin specific amino acid, desmosine (3H-DES). The smooth muscle cells were lysed, and the remaining matrix was used as a culture surface and a proteolytic substrate for intact PMN and purified neutrophil elastase. Proteolysis of collagen and elastin were quantified by chromatographic separation of the marker amino acids 3H-HL and 3H-DES, which were released into the supernatant or remained in the matrix after a 3-h incubation at 37 degrees C. The peptide, formyl-methionine-leucine-phenylalanine (FMLP), produced more rapid release of myeloperoxidase than did phorbol myristate acetate (PMA), which produced more release of O2- and H2O2 than did FMLP. The percent release of total matrix 3H-DES in the presence of PMN + FMLP was 2.45 +/- 0.19% (mean +/- SE, n = 6) and with PMN + PMA it was 1.32 +/- 0.1% (n = 6, p less than 0.01). The release of matrix 3H-HL did not differ. Neutrophil cytoplasts, which produced O-2 and H2O2 but lacked azurophilic granules, did not significantly enhance either elastin or collagen degradation by purified neutrophil elastase (NE).(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant prolylcarboxypeptidase activates plasma prekallikrein

The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.     

Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium

Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking postfixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere with the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of dissected human umbilical vein tissue. In either experimental model system, both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites. VE-cadherin moved away to different ends of the transmigration site, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 minutes. The relative positions of some junction proteins might rearrange to form a new interendothelial contour after PMNs had transmigrated through multicellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, whereas PECAM-1-containing junctions were opened during PMN transendothelial migration.     

Ligand-induced association between the T-cell antigen receptor and two glycoproteins.

We have identified two cell surface glycoproteins of 34 and 38 kDa (gp34 and gp38) that associate with the T-cell antigen receptor (TCR). The coimmunoprecipitation of these proteins with the TCR is increased by treatment with monoclonal antibodies (mAbs) directed against the TCR prior to cell solubilization and immunoprecipitation. Treatment of T cells with mAbs directed against other cell surface molecules, CD2 or HLA, does not induce the association of these proteins with the TCR. The coimmunoprecipitation of gp34 and gp38 with the TCR requires solubilization in the presence of an alkylating agent, suggesting that subunit alkylation stabilizes the interaction. J.CaM1 and J.CaM2 are signal-transduction mutant cell lines derived from Jurkat. These cell lines fail to activate the inositol phospholipid second messenger pathway in response to anti-TCR mAbs. Treatment with mAb C305 (anti-TCR) induces the association of gp34 and gp38 with the TCR in J.CaM2 cells but not in J.CaM1. J.CaM1 modulates the TCR normally in response to anti-TCR antibody treatment. This observation suggests that gp34 and gp38 are involved in the signal-transduction pathway of the TCR complex rather than receptor internalization. Furthermore, since these proteins do associate with the TCR of J.CaM2, the induced association with the TCR is not a consequence of signal transduction.     

Transmembrane signaling in human polymorphonuclear neutrophils: 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid modulates receptor agonist-triggered cell activation.

15(S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) exerted a time- and concentration-dependent inhibition of superoxide anion (O2-) production and exocytosis of both azurophil and specific granule constituents from human polymorphonuclear neutrophils (PMN) stimulated with the receptor-specific agonists, N-formylmethionylleucylphenylalanine (FMLP), platelet-activating factor, and leukotriene B4, but not that elicited by phorbol 12-myristate 13-acetate. 15-HETE did not alter the binding of FMLP to its specific receptors on PMN but, rather, appeared to interfere with a subsequent process in signal transduction. Receptor-coupled production of inositol 1,4,5-trisphosphate (InsP3) and increases in cytosolic free calcium elicited with FMLP, platelet-activating factor, and leukotriene B4 were suppressed by 15-HETE. 15-HETE did not, however, inhibit the mobilization of 45Ca from intracellular stores elicited by the addition of InsP3 to permeabilized PMN. 15-HETE suppressed O2- production and increases in intracellular [Ca2+] induced when cell-surface receptors were bypassed and the PMN were activated directly by the guanine nucleotide-binding protein (G protein) activators aluminum fluoride (AlF4-) and mastoparan. 15-HETE, however, did not perturb all G protein functions because cAMP production in FMLP-activated PMN was essentially unaffected by 15-HETE. These data support the proposition that 15-HETE modulates receptor-triggered activation of PMN either by uncoupling G protein stimulation of phospholipase C or by directly inhibiting phospholipase C, thus inhibiting the InsP3-dependent rise in intracellular [Ca2+] that is prerequisite for PMN responsiveness to receptor agonists.     

Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by- products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.     

Mechanisms of adhesion among cells from neural tissues of the chick embryo.

In order to analyze the molecular mechanisms of cell adhesion during development, proteins on the surface of chick embryonic neural cells were compared with proteins released after placing these cells in culture. One of the components released into culture, F1 (molecular weight, Mr 140,000), was derived by proteolytic cleavage of a cell surface precursor with a molecular weight of at least 240,000. Another protein, F2, recovered from culture as a dimer (Mr 1110,000), appeared to be a product of limited proteolytic cleavage of F1. Cells in retinal tissue possessed a surface protein of Mr 150,000 that also appeared to be derived by limited proteolytic cleavage of the cell surface precursor. Antibodies to F2 interacted with determinants on the cell surface protein of Mr 150,000, and specifically prevented homologous and heterologous binding among dissociated retinal and brain cells. In contrast, antibodies to F1 failed to prevent cell-cell adhesion and did not crossreact with F2. These data suggest that the cell surface protein of Mr 150,000 generated by limited proteolysis is involved in adhesion of both retinal and brain cells. Cell-cell binding of both retinal and brain cells varied as a function of developmental age and brain cells acquired their binding properties at an earlier time than retinal cells. Similar results were obtained in experiments on the binding of retinal and brain cells of different ages to nylon fibres coated with antibodies to F2. The results of the molecular and cellular experiments are incorporated in a model for cell adhesion invoking both proteolytic activation and modulation of cell surface ligands.