Parallel inhibition of neutrophil arachidonic acid metabolism and lysosomal enzyme secretion by nordihydroguaiaretic acid

Arachidonic acid at concentrations from 0.2 to 2.0 × 10?⁶M induces the secretion of lysosomal enzymes from cytochalasin B-treated rabbit neutrophils. These concentrations of arachidonic acid are metabolized primarily to hydroxyeicosatetraenic acids rather than to cyclooxygenase products. A good correlation is observed between the extent of arachidonic acid metabolism and the secretion of lysosomal enzymes. Nordihydroguaiaretic acid (1?10μM) inhibits both lysosomal enzyme secretion and the production of lipoxygenase products by neutrophils.     

Inhibition of chemotactic factor-induced neutrophil responsiveness by arachidonic acid

Arachidonic acid when added simultaneously with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) inhibits the ability of the latter to initiate several but not all of its effects on rabbit peritoneal neutrophils. Stimulated neutrophil aggregation, calcium uptake, and increases in the steady state level of exchangeable calcium are all inhibited by 1-10 microM arachidonic acid. The binding of f-Met-Leu-Phe and the parameters of intracellular calcium redistribution (calcium efflux and changes in the steady state level of exchangeable calcium in the absence of extracellular calcium) and of stimulated sodium uptake are, on the other hand, unaffected by the same concentrations of arachidonic acid. Arachidonic acid, the saturated analog of arachidonic acid, was found not to inhibit f-Met-Leu-Phe-stimulated aggregation and calcium uptake. Arachidonic acid, therefore, in addition to its well-described agonist properties, also possesses antagonist activities toward rabbit neutrophils. These results add a new level of complexity to the study of the role of arachidonic acid in cell activation.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.     

Stimulation of lysosomal enzyme secretion by growth factors

Levels of extracellular lysosomal enzymes are relatively high in tumors and especially so at their periphery. By degrading the intercellular matrix, these and other nonlysosomal enzymes could facilitate invasion and metastasis by tumor cells. Using a rapid assay, we have shown that cells transformed by a variety of agents can be stimulated in culture by several growth factors to secrete lysosomal enzymes. These factors have little or no stimulatory activity on their nontransformed counterparts. The basal rate of secretion of N-acetylglucosaminidase (NAGA) and the efficiency of the stimulus are greater in transformed cells in log phase of growth. These observations suggest that altered or increased responsiveness to paracrine and autocrine growth factors not only may be responsible for the persistent division of malignant cells but also may promote their invasiveness.     

Mechanisms of lysosomal enzyme secretion by human monocytes

Secretion of lysosomal enzymes by human monocytes in response to various stimuli and the effect of conditioned media from lymphocytes and neutrophils was studied. Monocytes were found to release β-glucosaminidase in response to NH₄Cl and to particles (zymosan, opsonised zymosan, asbestos and latex), but do not respond to some soluble stimuli like formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, cytochalasin B, concanavalin A and $\text{N-}\text{acetylmuramyl-}\text{l}\text{-alanyl-}\text{d}\text{-isoglutamine}$. Neutrophil conditioned medium or neutrophil components did not have any effect on secretion. When treated with lymphokines the cells are more responsive, especially to zymosan. Even through there are similarities in the secretory activities of mouse macrophages and human monocytes, there are several differences both in the quantity of the response and in the mechanisms involved.     

Processing, transport, and secretion of the lysosomal enzyme acid phosphatase in Dictyostelium discoideum

To explain the different secretion kinetics of lysosomal enzymes in Dictyostelium discoideum, previous investigators have hypothesized the existence of a heterogeneous population of lysosomes containing either the enzyme acid phosphatase or other hydrolase enzymes. This proposal predicts that at least two targeting mechanisms exist for lysosomal enzymes in this organism. To begin to investigate this possibility, the transport, processing, and targeting of acid phosphatase was studied by using a combination of radiolabel pulse-chase procedures, subcellular fractionations, and indirect immunofluorescence microscopy. Acid phosphatase was initially synthesized in axenically growing cells as a 56-kDa precursor polypeptide that was proteolytically processed after 20 min to a 55-kDa mature protein. This enzyme was rapidly transported from the endoplasmic reticulum to Golgi complex (halftime of 3 min) as measured by the acquisition of resistance to the enzyme endoglycosidase H. Furthermore, Percoll gradient fractionations indicated that radiolabeled forms of acid phosphatase reached dense lysosomal vesicles at about the same time as final processing was occurring. Proper sorting of acid phosphatase in D. discoideum apparently was not critically dependent on low intravacuolar pH since the addition of ammonium chloride did not stimulate the missorting and secretion of acid phosphatase. These results are very similar to previous observations concerning other Dictyostelium lysosomal enzymes. Consistent with the existence of a heterogeneus population of lysosomes, the percentage of radiolabeled acid phosphatase secreted 4 h into a chase period was 15-fold lower as compared with another lysosomal enzyme, beta-glucosidase. However, acid phosphatase, alpha-mannosidase, and beta-glucosidase were all predominantly colocalized as determined by indirect immunofluorescence, which for the first time demonstrates the homogeneous nature of the lysosomal system in D. discoideum. Taken together these results suggest that the processing and transport of acid phosphatase may be similar in nature to the glycosidases. However, the different kinetics of secretion of acid phosphatase versus the colocalized glycosidase enzymes suggests that an undefined mechanism operates to distinguish these classes of enzymes at a step after localization to lysosomes but prior to secretion.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

Inhibition of lysosomal enzyme liberation by indomethacin in vivo

The volume of the peritoneal exudate induced in the rat by iota carrageenan is reduced by subcutaneous administration of indomethacin while the concentrations of three lysosomial enzymes in the exudate are slightly increased or not modified. Thus, the total enzymatic activities of the exudate are reduced by indomethacin. The leucocyte accumulation remains unchanged in the indomethacin treated rats. During the development of the peritoneal exudate, the circulating plasma displays a high degree of lysosomial enzymes activity which is suppressed by indomethacin at the dose of 4 mg/kg.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7-34 and 9-50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7-37, 30-72 and 17-72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.     

Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor.

Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.     

Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migration in vivo and in vitro. The in vivo chemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS-PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migration in vitro as well as in vivo. This was not modified by dexamethasone pretreatment.     

Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs

Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while diminishing its action against other cellular targets. Thus, a set of NDGA analogs (7a-7h) was prepared and examined for inhibitory potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs was based upon a cursory Topliss approach in which one of NDGA's aromatic rings was modified with various substituents. Structural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may serve as a new lead in developing novel therapeutic agents for breast cancer.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Inhibition of receptor-mediated uptake of a lysosomal enzyme into fibroblasts by chloroquine, procaine and ammonia

Cultured human skin fibroblasts take up α- -iduronidase by receptor-mediated pinocytosis. Certain lysosomotropic amines such as chloroquine, ammonia and procaine inhibit this process, without affecting the fluid endocytosis of dextran. In contrast to the competitive inhibition by mannose 6-phosphate, the inhibition by amines is non-competitive and is therefore presumed not to affect binding of the enzyme to receptors. The dose response curves are very steep, and equations that best fit the data use a power of inhibitor concentration (i² for procaine, i⁴ for chloroquine), indicating interaction of several amine molecules at the inhibitory site(s). The inhibition is reversed by removal of the amine from the medium and does not result from accelerated efflux of endocytosed enzyme. We suggest that the amines interfere with delivery of receptor-bound enzyme to lysosomes.     

Purification and characterization of a neutrophil chemotactic factor from Dirofilaria immitis

A neutrophil chemotactic factor (NCF-Di) was purified from a crude extract of Dirofilaria immitis adult worm by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. NCF-Di showed a single protein band by both polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. The molecular weight of NCF-Di was estimated to be 17,000 by gel filtration on Sephadex G-150, and 14,000 by SDS-PAGE. NCF-Di was an acidic protein with isoelectric point of 4.5. NCF-Di was absorbed neither to lentil lectin-Sepharose nor to concanavalin A-Sepharose. The chemotactic activity of NCF-Di was heat labile (56 C, 1 hr), but was resistant to periodate oxidation. These results suggest that NCF-Di is a simple peptide which has few or no sugar chains. These physicochemical properties of NCF-Di were compared to previously reported parasite-derived chemoattractants or purified allergen of D. immitis.