Rat Polymorphonuclear Neutrophils Research Articles

The role of the complement system in the neutrophil functions stimulatedin vitroby an alkali-insoluble cell wall fraction ofParacoccidioides brasiliensis

We investigated the capacity of an alkali-insoluble cell wall polysaccharide fraction (F1) of Paracoccidioides brasiliensis to induce rat polymorphonuclear neutrophil (PMN) migratory and chemiluminescence (CL) responses. Normal rat serum pre-incubated with F1 induced a chemotactic neutrophil response which was fully abolished by heat-inactivation. The participation of the alternative complement pathway was more effective than that of the classical pathway since depletion of factor B by heating at 50 degrees C reduced PMN migration, whereas blockade of the classical pathway with EGTA left the migratory response practically unchanged. Opsonized serum F1 induced a significant release of oxygen radicals from PMN as measured by CL. The complement system was also found to be involved in this activity since serum inactivation at 56 degrees C altered the CL response. In addition to complement-derived fragments, other serum opsonins, probably cross-reacting antibodies, were required for optimal interaction between PMN and opsonized particles. These results contribute to the understanding of the role of fungal components and of the complement system in the inflammatory response observed in paracoccidioidomycosis.

Polyphosphoinositide metabolism in polymorphonuclear cells from healthy and thermally injured rats: effect of the immunomodulator RU 41740.

Burn trauma is associated with alterations of various components of host defenses, including impaired neutrophil functions. In an animal model of experimental thermal injury, we studied if the modifications of cellular reactivity result from alterations in signalling systems by comparing polyphosphoinositide breakdown, particularly the production of inositol phosphates (IP, IP2 IP3), in healthy and burned rat polymorphonuclear neutrophil leukocytes (PMNs). Neutrophil activators such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and serum-opsonized zymosan increased in vitro production of inositol phosphates in PMNs from healthy rats. The immunomodulator RU 41740 had no effect by itself, but decreased the stimulating effect of fMLP and zymosan. In PMNs from burned rats, the stimulating effects of fMLP and zymosan were decreased, while RU 41740 stimulated inositol phosphate generation. In vivo treatment with RU 41740 inhibited the activation of phosphoinositide metabolism by fMLP or zymosan in healthy rat PMNs. Similar treatment of burned rats after injury restored the stimulating effect of fMLP and zymosan on inositol phosphate accumulation in PMNs. Thus, RU 41740 can modulate fMLP and zymosan receptor-mediated signal transduction, inducing an attenuation of the phosphatidylinositol hydrolysis response. After burn injury, when the activating effects of fMLP and zymosan are inhibited, RU 41740 can, on the contrary, stimulate phospholipase C-mediated polyphosphoinositide turnover and the formation of intracellular messengers such as IP3. These data show that RU 41740 has different effects on polyphosphoinositide metabolism in rat PMNs, according to the physiological and pathological state of the animals. Interestingly, it has a beneficial action on the post-burn decrease in PMN reactivity.

Biochemical characteristics of alveolar macrophagespecific peroxidase activities in the rat

The biochemical characteristics of endogenous macrophage peroxidases (Po), and their relationship to myeloperoxidase (MPO), have heretofore been poorly understood and were examined in the current study. Rat alveolar macrophages (AM) were homogenized and fractionated by differential centrifugation into lysosomal and microsomal fractions. The Po activities in both fractions were separated using HPLC gel-filtration and two main activities were detected. One, in the lysosomal fraction, had a relative molecular mass ( M r) of 58,000, while the other, associated with the microsomal fraction corresponded to M r 74,000. By comparison MPO from rat polymorphonuclear neutrophils (PMN) had M r 140,000. The 58- and 74-kDa Po activities also differed from MPO with respect to their apparent K m for H 2O 2 and optimum pH of activity. Using o-dianisidine as a substrate, the K m for H 2O 2 of the 58- and 74-kDa Po species was 0.4 and 0.19 m m, respectively, compared to 0.011 m m for MPO. Using monochlorodimedon, the corresponding values were 0.22 and 0.195 m m for the 58- and 74-kDa activities and 0.026 m m for MPO. With either substrate, MPO exhibited optimum activity at pH 5.4, compared to 5.2 for the 58-kDa activity and 4.8 for the 74-kDa species. Thus, rat AM contain two endogenous Po activities with biochemical characteristics distinct from those of MPO. Our findings suggest that these activities represent novel peroxidases that may play an important role in the oxidative metabolism of AM.

A Nycodenz gradient method for the purification of neutrophils from the peripheral blood of rats

A Nycodenz gradient technique is described which permits the separation of functionally active polymorphonuclear neutrophils (PMN) from the peripheral blood of rats. PMN are obtained at greater than 90% purity and fractionate at a peak density of 1.0919 g/ml. The method is suitable for isolating PMN when the circulating PMN count is low (<20%) as on normal rats and when the count is high (>30%), as a result of inducing inflammation in the subcutaneous air pouch of rats by the intra-pouch injection of peptone. The chemotactic responsiveness of rat PMN was found to be markedly less than that of human PMN when the formyl peptides were used as chemoattractants but not when zymosan-activated serum was the chemoattractant. Blood PMN from normal rats and from peptone-treated rats showed no significant difference in their response to ZAS indicating that priming of their activity by the induction of long term (8 day) inflammation was not a feature of these experiments. However, air pouch-derived PMN displayed a highly significant reduction in activity compared with their isologous blood-PMN. The Nycodenz method offers an alternative to the Percoll separation method for rat blood and will be useful when comparative studies of elicited and non-elicited PMN are required since the latter are obtained in sufficiently high yield and purity for microassays on their function to be performed.

Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.

Purification and antimicrobial properties of three defensins from rat neutrophils

Three cysteine-rich cationic peptides, designated RatNP-1, RatNP-3, and RatNP-4, were purified from an acid extract of rat polymorphonuclear neutrophils, sequenced, and tested for antimicrobial activity. The peptides ranged from 29 to 32 amino acids in length (Mr, 3,252 to 3,825), and each contained all eight invariantly conserved "framework" residues that are characteristic of defensins. Each of the peptides killed Escherichia coli ML-35, Acinetobacter calcoaceticus HON-1, Staphylococcus aureus 502A, and Candida albicans 820 in vitro. RatNP-1, the most cationic rat defensin, was also the most potent. With this report, a total of 13 distinct defensins have been characterized in the polymorphonuclear leukocytes of four mammalian species. The existence of the defensin system in rats should facilitate investigations of the in vivo role of defensins in experimental infections.

Fluorophore-labeled ether lipids: Substrates for enzymes of the platelet-activating factor cycle in peritoneal polymorphonuclear leukocytes

Cell-free preparations of ionophore-stimulated peritoneal rat polymorphonuclear neutrophils (PMNs) incubated with 1-( N-dansyl-11-amino-1-undecyl)- sn-glycerol-3-phosphorylcholine (dansyllyso-PAF) converted this fluorescent lyso ether lipid into two different classes of products. In the absence of acetyl-CoA 1-( N-dansyl-11-amino-1-undecyl)-2-long chain acyl- sn-glycerol-3-phosphorylcholine (dansylalkyl-2-acyl-GPC) was the only identified new fluorescent phospholipid. In the presence of acetyl-CoA an additional new product, 1-( N-dansyl-11-amino-1-undecyl)-2-acetyl- sn-glycerol-3-phosphorylcholine (dansyl-PAF), was formed. The formation of dansyl-PAF in PMN homogenates was only transient with a maximum after about 4 min. When PMN homogenates were incubated with dansyl-PAF the formation of dansyllyso-PAF was observed prior to the formation of dansyl-2-acyl-GPC. Thus, our data indicate that enzymatically formed dansyl-PAF is completely remodeled into dansylalkyl-2-acyl-GPC by the sequential action of PAF acetylhydrolase and CoA-independent transacylase. These results demonstrate that peritoneal rat PMNs contain lyso-PAF acetyltransferase, PAF acetylhydrolase, and CoA-independent transacylase and that fluorophore-labeled ether lipids provide an easy means to assay enzymes which catalyze important enzymatic reactions involved in the biosynthesis and remodeling of platelet-activating factor.

Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor.

Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.

Effect of rat polymorphonuclear leukocyte granule components on the growth and survival of Pseudomonas aeruginosa and Salmonella typhimurium

Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate buffer (pH 4.0), dialyzed against phosphate-buffered saline (pH 7.0), and tested for bactericidal activity against selected target bacteria. Salmonella typhimurium LT-2 and a series of progressively rough lipopolysaccharide outer membrane mutants derived from it were used to monitor antimicrobial activity. Although an antimicrobial potential was present in rat granule contents for S. typhimurium, the growth of Pseudomonas aeruginosa PAO-1 in antimicrobial assay mixtures containing rat granule contents was substantially enhanced over control values. The growth enhancement property of the granule protein was heat resistant and promoted increased oxygen consumption by whole cells.

Bactericidal activity of granule contents from rat polymorphonuclear leukocytes.

Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate (pH 4), dialyzed against phosphate-buffered saline (pH 7), and tested for bactericidal activity. Bactericidal assays consisted of mixing rat granule extract with 1 x 10(3) to 3 x 10(3) bacterial cells per ml at 37 degrees C for 1 h in a medium suited for bacterial growth. The granule extract demonstrated a distinctive dose-dependent bactericidal activity against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT-2, independent of added hydrogen peroxide or other active oxygen derivatives. The rough bacterial mutants showed an ordered increase in sensitivity to the rat lysosomal extracts inversely related to the length of their lipopolysaccharide carbohydrate side chains. Fractionation of the rat polymorphonuclear neutrophil granule extract with Sephadex G-100 column chromatography revealed an elution profile containing three major areas (peaks) of protein. Polyacrylamide gel electrophoresis and examination of enzymatic activity showed that these peaks contained myeloperoxidase (peak A), neutral protease (peak B), and lysozyme (peak C) activities. Also observed in peak C were cationic protein species whose cathodal electrophoretic migration was faster than that for lysozyme. Only peak C exhibited a bactericidal activity against the rough mutants of S. typhimurium LT-2 similar to that obtained for the unfractionated granule extract, with susceptibility of the bacterial mutants increasing with a progressive loss of carbohydrate residues in the lipopolysaccharide of the cell wall. The bactericidal activity of the peak C protein fraction was dose dependent. Boiling the unfractionated granule extract or peak C for 30 min had little affect on their antimicrobial activity when reacted against a deep-rough lipopolysaccharide mutant. However, trypsin pretreatment of these fractions significantly reduced their antimicrobial activity for the same mutant chemotype.

Effects of bradykinin and some of its fragments on smooth muscles and chemotaxis

Bradykinin (BK) and two of its C-terminal fragments, namely H-Phe-Ser-Pro-Phe-Arg-OH and H-Ser-Pro-Phe-Arg-OH were found to be potent inhibitors of the chemotaxis of rat polymorphonuclear neutrophils (PMN). The activity of the three peptides was significantly enhanced by SQ 14225, a rather specific inhibitor of kininase II. A shorter C-terminal sequence of BK (Phe-Arg-OH) was inactive. The whole peptide (BK) exerted potent actions on blood pressure and on isolated organs, e.g. the rabbit mesentric vein or the guinea pig ileum, but none of its fragments showed similar effects. The present findings suggest that rat PMN possess membrane receptors which cannot be considered identical to B 1- and B 2-receptors, previously characterized on isolated smooth muscles.

Influence of hydrocortisone on uptake and elimination of 32P-labelled E. coli by rat polymorphonuclear neutrophils (PMN) in vitro.

Hydrocortisone was tested for inhibition of uptake and of elimination of E. coli by monolayers of rat peritoneal PMN. 32P was used as label of the bacteria and their degradation products eliminated from the phagocytes. The cellular uptake was reduced by hydrocortisone (1-2 mg per ml) both in the presence and absence of serum, while the elimination of bacterial label was reduced by 0.5 mg per ml and higher concentrations of the drug.

Effects of divalent cations on adhesiveness of rat polymorphonuclear neutrophils in vitro.

AbstractCell preparations rich in polymorphonuclear neutrophils (PMN) were obtained from peritoneal exudates of rats without the use of any anticoagulant. The adhesiveness of these PMN to glass bead columns coated with rat serum were studied quantitatively using suspending solutions free of added serum protein. A dependence of the PMN adhesiveness upon divalent cations was demonstrated. Added singly Mg2+, Ni2+, Zn2+, Co2+, Mn2+, Cu2+, or Cd2+ were found to be effective whereas Ca2+, Sr2+ and Ba2+ were ineffective. A possible auxilliary role for Ca2+ when added with Mg2+ is suggested by the data. The ineffectiveness of the ions Ca2+, Sr2+ and Ba2+ was shown by use of an ion electrode not to be due to the unavailability of the ionized species. Procedures are described for obtaining highly reproducible results with the Orion Divalent Cation Electrode. The ineffectiveness of the ions Ca2+, Sr2+ and Ba2+ were also shown not to be due to action as general protoplasmic poisons. The effective ions are distinguished from the ineffective ones by characteristic ranges of ionic radii, coordination number, second ionization potentials, electronegativities and affinity constants. Removal of components of complement from the cells by washing in 0.05 M EDTA, and heating all serum used for 30 minutes at 56°C had no significant effect on the adhesiveness of the PMN. A role for complement, therefore appears largely excluded.