Alterations in surfactant neutral lipid composition during the development of bleomycin-induced pulmonary fibrosis.

The purpose of this investigation was to correlate changes in the neutral lipids of pulmonary surfactant with previously observed changes in surfactant phospholipids and lung compliance in the rat model of bleomycin-induced pulmonary fibrosis. Bronchoalveolar lavage fluid (BAL) obtained at 0, 1, 3, 7, 14, 30 and 120 days after transtracheal instillation of bleomycin was used as a source of surfactant lipids. The mean concentration of neutral lipids in normal BAL was 439 nmol/lung and was composed of 55% cholesterol (CHO), 27% cholesterol ester (CE) and 19% free fatty acids (FFA). CHO was elevated at 1 day, reaching a maximum 4-fold increase in concentration at 14 days before subsiding to normal at 120 days. In contrast to CHO, CE and FFA were significantly reduced at 1 day after bleomycin with FFA below detectable levels. However, both these species were twice normal levels at 3-30 days before returning to normal at 120 days. The fatty acid composition of CE did not change; however, unsaturated fatty acids were significantly increased in FFA between 3 and 120 days. The data indicate that there are significant alterations in the neutral lipid composition of pulmonary surfactant at various stages of bleomycin induced lung injury. The significance of these changes are not fully understood; however, the possibility exists that an abnormal surfactant results which in turn affects lung function.

Role of Ca2+ and Mg2+ in neutrophil hexose transport.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids.

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.

Fluorescent markers for studying phagosome-lysosome fusion.

Lysosomotropic fluorescent aminoacridines such as acridine orange and quinacrine have achieved prominence as markers for studying lysosome-phagosomes fusion, especially in macrophages. Experiments described demonstrate that because the aminoacridines traverse biological membranes with facility, they diffuse throughout the system, and ultimately accumulate intra- or extracellularly where they are most efficiently bound. Their presence or absence in phagosomes is therefore not unequivocally indicative of fusion or nonfusion. Alternative fluorescent lysosomal markers are described, and systems defined for which the aminoacridines may probably be used with confidence.

Oxidation of glucosamine by human polymorphonuclear leukocytes.

When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced 14CO2 from [1-14C]glucosamine at a rate 10-25% of that produced from glucose under the same conditions. The production of CO2 from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.

Oxidation of 2-deoxyglucose by human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) can metabolize [1-14C]2-deoxyglucose to 14CO2 when stimulated by either phorbol myristate acetate (PMA) or opsonized zymosan. Oxidation of 2-deoxyglucose is about 10-120% as efficient as that of glucose in normal PMNL; it does not occur in defective cells obtained from patients with chronic granulomatous disease. The increased oxidation of [1-14C]2-deoxyglucose induced by PMA is not sufficient to explain the inhibition of transport induced by that compound; conversely increased transport of 2-deoxyglucose induced by zymosan-activated serum does not result in a significant increase in oxidation of the hexose. Oxidation of [1-14C]2-deoxyglucose appears to be mediated by the hexose monophosphate shunt as indicated by the following (1) oxidation of [1-14C]2-deoxyglucose in intermediate in activity between that of [1-14C]glucose and [6-14C]glucose; (2) the reaction is insensitive to cyanide or azide; and (3) shunt enzymes measured in a cell-free extract from human PMNL can react with 2-deoxyglucose compounds with approximately 10% the efficiency shown towards the corresponding glucose derivatives.

Humoral immunity to Streptococcus pneumoniae induced by a pneumococcal ribosomal protein fraction.

Isolation of a protective subfraction of ribosomes from Streptococcus pneumoniae has been achieved, and the immune response it induces has been investigated. Mice immunized with pneumococcal ribosomes or purified protein extracted from the ribosomal preparation (2-CE protein) exhibit similar survival rates upon challenge by virulent S. pneumoniae. In contrast, recipients of purified ribosomal ribonucleic acid were never protected against pneumococcal challenge. Serum from mice immunized with pneumococcal ribosomes or 2-CE protein passively immunized syngeneic recipients against pneumococcal challenge, whereas spleen cells from the same donors were unable to transfer immunity. Passive immunization with antiribosome serum could be abrogated by absorption with whole ribosomes, 2-CE protein, or various serotypes of S. pneumoniae (capsular types 2, 3, 6, and 14). Antiribosome serum significantly enhanced clearance of S. pneumoniae from mouse blood in vivo and in vitro. This required phagocytic cells, since antiribosome serum alone, with or without complement, supported growth of S. pneumoniae to an extent comparable to normal serum. The data suggest that the primary immunogen of pneumococcal ribosomes resides in the protein fraction. Further, the immunity induced by the protein fraction is mediated by antibody that appears to function as an opsonin for S. pneumoniae.

Role of extracellular calcium and neutrophil degranulation responses to 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine.

The rabbit polymorphonuclear neutrophil degranulation response to 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine depends on extracellular calcium. In the absence of this bivalent cation, neutrophil suspensions pretreated with cytochalasin B responded to the lipid by releasing minimal amounts of lysozyme and beta-glucuronidase. Incremental increases in extracellular calcium over a range of 20-200 microM led to increasing amounts of lipid-stimulated enzyme release. In contrast, extracellular magnesium neither supported nor enhanced the degranulation responses. Verapamil (25-200 microgram/ml), a calcium channel blocker, inhibited degranulation. Neutrophil suspensions exposed to the phosphocholine stimulus rapidly took up radiolabeled extracellular calcium. The kinetics of this calcium uptake were similar to the kinetics of enzyme release, and the amount of calcium taken up correlated closely with the amount of released lysozyme and beta-glucuronidase. Finally, in a dosage which blocked degranulation, verapamil inhibited calcium uptake. Thus, the rapid association of extracellular calcium with the neutrophil may mediate, at least in part, the degranulating actions of the phosphocholine stimulus.

Metabolic fate of platelet-activating factor in neutrophils.

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-acetyl-GPC) incubated with rabbit polymorphonuclear leukocytes was metabolized to 1-O-alkyl-2-acyl-GPC containing long chain groups at the 2 position. Within 5 min, 60% of the total label added to the cell suspension was found in the 1-O-[3H]alkyl-2-acyl-GPC. At earlier time points, 1-O-[3H]alkyl-2-lyso-GPC was present. No metabolites were detected that would indicate the ether bond had been cleaved or the polar head group had been altered by the cells. At subaggregating and subdegranulating concentrations of labeled 1-O-[3H]alkyl-2-acyl-GPC, the same pattern of product formation was observed. Furthermore, when 1-O-[3H]alkyl-2-lyso-GPC was added to the cells, it was taken up and acylated in the same manner as that from 1-O-alkyl-2-acetyl-GPC. The nature of the long chain acyl residues incorporated into the 2 position was then examined by argentation chromatography and high performance liquid chromatography. Argentation chromatography of 1-O-[3H]alkyl-2-acyl-3-acetylglycerols obtained from 1-O-[3H]alkyl-2-acyl-GPC after acetolysis indicated that mono-, di-, and tetraenoic fatty acids were the predominant molecular species being incorporated into the 2 position of the molecule (18, 55, and 18%, respectively). Furthermore, high performance liquid chromatographic analysis using synthetic 1-O-alkyl-2-acyl-GPC standards indicated that three fatty acids, linoleic, arachidonic, and oleic (50, 15, and 12%, respectively), were the major chains being incorporated into the 1-O-hexadecyl-linked species. Analysis of 1-O-[3H]alkyl-2-acyl-GPC derived from exogenously added 1-O-[3H]alkyl-2-lyso-GPC revealed the same distribution of acyl groups linked to the 2 position. The findings are consistent with a pathway in which two enzymatic activities are responsible for the metabolism of exogenous platelet-activating factor in the rabbit neutrophils: one that hydrolyzes the acetyl residue (acetylhydrolase) and another that transfers a fatty acyl chain to the 1-O-alkyl-2-lyso-GPC formed (possibly acyl-CoA:1-O-alkyl-2-lyso-GPC acyltransferase). These events appear to play an important role in inactivating this potentially lethal phospholipid.

1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine: a novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187.

Rabbit peritoneal neutrophils incorporated [14C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[14C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the sn-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-O-alkyl-2-lyso-GPC is the metabolic precursor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.

Selective acylation of lyso platelet activating factor by arachidonate in human neutrophils.

Human polymorphonuclear leukocytes (PMN) incubated with 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet activating factor) inactivated the compound by removing the acetyl group and replacing it with a long chain acyl residue. The nature of the acyl group added at the 2-position of the 1-O-[3H]alkyl-2-acyl-GPC formed was examined by argentation chromatography and by reverse phase high performance liquid chromatography. A striking selectivity for arachidonate was observed in the acylation reaction. The major labeled component of the starting material was the 1-O-hexadecyl-linked species; high performance liquid chromatography analysis revealed that 75 to 80% of this component was acylated by arachidonate. Similarly, based on argentation thin layer chromatography, approximately 80% of the total starting material was acylated by tetraenoic acyl residues. The incorporation of 1-O-[3H]alkyl-2-lyso-GPC into 1-O-alkyl-2-acyl-GPC by the PMN was compared; no difference in the acylation pattern was observed with the 2-acetyl and 2-lyso precursors. Thus, activation of the PMN does not appear to be required to elicit the selectivity for arachidonate. When labeled 1-palmitoyl-2-lyso-GPC was compared in the system under the same conditions, it was also preferentially acylated by arachidonate; thus, it is not clear at this time whether or not the selectivity for arachidonate is physiologically limited to platelet activating factor. Our findings suggest a close relationship exists between the metabolism of platelet activating factor and arachidonate in human PMN.

Correlation of changes in pulmonary surfactant phospholipids with compliance in bleomycin-induced pulmonary fibrosis in the rat.

Qualitative and quantitative analysis was performed on phospholipids isolated from bronchoalveolar lavage fluid during the development of pulmonary fibrosis in the rat. A single transtracheal injection of 2.0 units of bleomycin was administered to rats to induce lung injury. Animals were killed at 0, 3, 7, 14, 30, and 120 days after bleomycin treatment. Total lipid phosphorus in BAL from animals given bleomycin increased from 1.6 mumol/lung in normal animals to 3.2 mumol/lung at 14 and 30 days. The increase in phospholipids was primarily in phosphatidylcholine with minor increases in phosphatidylinositol and phosphatidylethanolamine. These quantitative changes were accompanied by qualitative changes that included an increase in the percentage of total phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylinositol. In contrast, the percentage of phosphatidylglycerol was significantly reduced. Plasma phospholipid analysis indicated that these alterations were not due to plasma contamination. The functional significance of the phospholipid changes was assessed by comparing air- and saline-filled compliance measurements at similar times after bleomycin. Abnormal compliance measurements were observed at 3, 7, and 14 days after bleomycin. At 3 and 7 days the predominant compliance defect was at the air-liquid interface; however, at 14 days, when phospholipids were significantly elevated, the defect was primarily due to tissue components.(ABSTRACT TRUNCATED AT 250 WORDS)