Chemotactic and mitogenic components of the alveolar macrophage response to particles ad neutrophil chemoattractant.

The pulmonary response to instilled particulates involves initial efflux of polymorphonuclear leukocytes (PMNs) and increased production of alveolar macrophages (AMs). The relationships of the cell migration and division components of the AM production to the initial PMN response after intrapulmonary carbon or latex administration are examined. Supernatants of lung lavages taken during early PMN migration to the alveoli promote sequential migration of PMNs, migration of monocytes, and division of pulmonary interstitial cells in normal mice. Supernatants taken during the phase of increased cell division in the lung produce no such effects, implying tht factors responsible for chemotaxis and mitosis are generated rapidly and are short-lived. A possible source is the interaction of AMs with particles, since supernatants of such incubations induce an inflammatory response in vivo. Similarly, when a synthetic chemoattractant is used, efflux of PMNs is followed by macrophages arising from migration of monocytes and from division of interstitial cells. The results suggest that particulate instillation in the lung stimulates a standard inflammatory response in which rapid generation of a factor(s) chemotactic for PMNs also attracts mononuclear cells to the alveoli. The initial efflux of cells may be explained by migration from the blood, but continued demand or replacement requires mitotic activity of precursors. For the alveolar macrophages, this includes division of cells in the pulmonary interstitium.     

Characteristics of alveolar macrophages in experimental septic lung.

We investigated the pathogenesis of lung injury in sepsis (septic adult respiratory distress syndrome) by focusing on the functional changes of alveolar macrophages (AMs). Sepsis was induced in male WK rats by cecal ligation and puncture. Histological examination of the lungs from this experimental model revealed edematous change at 24 h after the surgery. The protein and endotoxin concentrations in the bronchoalveolar lavage fluid (BALF) increased with time after the surgery. The time course studies of AM function after surgery indicated that AMs from septic rats were activated by endotoxins. Specifically, this was suggested by the finding that AM adherence to and spreading on a plastic dish had increased. On stimulation, these AMs enhanced generation of superoxide anions and increased release of lysosomal enzymes, such as beta-glucuronidase. On the other hand, AMs in sepsis generated much smaller amounts of arachidonate lipoxygenase metabolites, such as leukotriene B4 (LTB4) and 12- and 5-hydroxyeicosatetraenoic acids (HETEs), on stimulation than did AMs from sham rats or untreated rats. However, the concentrations of immunoreactive LTC4 in the BALF of septic rats seemed to be higher than in untreated rats. It is suggested that the AMs of septic rats released lipoxygenase metabolites in alveoli and that these AMs could not be stimulated in vitro. These functional changes in the AMs of septic rats progressed along with the sepsis. These results implicate AMs in the development and progression of septic lung injury by releasing superoxide anions, beta-glucuronidase, and arachidonate metabolites. Furthermore, we speculate that reduced production of LTB4 by septic AMs may increase host susceptibility to severe pulmonary infection during septic ARDS.     

Inhibition of interferon gamma-induced macrophage microbicidal activity against Leishmania major by liposomes: inhibition is dependent upon composition of phospholipid headgroups and fatty acids

Multilamellar liposomes of phosphatidylcholine and phosphatidylserine at a 7:3 molar ratio significantly inhibited activation of murine resident peritoneal macrophages by recombinant murine interferon-gamma for cytotoxicity against amastigotes of the protozoan parasite Leishmania major; other macrophage effector functions, such as particle phagocytosis or tumoricidal activity, were unaffected. This inhibition was not due to direct toxic effects of liposomes against parasite or macrophage, was fully reversible, and was directed at one or more early events in macrophage-LK interactions which ultimately induce microbicidal activity. Liposomes containing some natural phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidic acid or diphosphatidyl glycerol), but not phosphatidylcholine, phosphatidylglycerol, or several synthetic saturated phospholipids, prevented the induction of macrophage microbicidal activity. Inhibition by liposomes of various composition was not related to the efficiency with which these vesicles were ingested by macrophages. Inhibitory activity was directly influenced by changes in the phospholipid head group, as well as by the number of unsaturated bonds in phospholipid fatty acids: for a given phospholipid in liposomes, inhibition was directly related to the number of unsaturated bonds among the fatty acids. These data support a role for phospholipids in postbinding regulation of macrophage activation and add to our understanding of how liposome delivery systems can be designed to avoid potential microbicidal suppressive effects.     

Depletion of alveolar macrophages by treatment with 2-chloroadenosine aerosol.

Alveolar macrophages (AMs) are localized in the alveoli and alveolar ducts of the lung and are the only macrophages living in an aerobic environment. Recent studies have demonstrated that AMs play a central role in lung diseases, such as pneumonia and acute respiratory distress syndrome. It has become important to find a simple, effective way to eliminate AMs in order to investigate the function of AMs in vivo. 2-Chloroadenosine (2-CA), a purine analog, is reported to be selectively cytotoxic to cultured macrophages, and we hypothesized that it would deplete the number of AMs in the bronchoalveolar lavage fluid (BALF) of mice without any effect on neutrophil or lymphocyte counts. After mice had inhaled 1 mM aerosolized 2-CA for 2 h, AMs were found to be significantly depleted at 0 h [(4.42 +/- 0.16) x 10(4)/ml], 24 h [(4.17 +/- 0.89) x 10(4)/ml], 48 h [(3.17 +/- 0.21) x 10(4)/ml], and 72 h [(5.00 +/- 0.64) x 10(4)/ml] compared with concentrations in untreated controls [(12.1 +/- 0.21) x 10(4)/ml]. Neutrophil and lymphocyte counts in BALF did not change and histological changes in the lung were not observed after 2-CA treatment. The lung wet-to-dry weight ratio did not change at 0, 24, and 48 h after 2-CA aerosol application. The 2-CA aerosol had no effect on lung vascular permeability, as assessed by the intravenous administration of Evans blue, or on other phagocytes, as assessed by Kupffer cell counts. Our study demonstrates the efficacy of 2-CA in reducing AM numbers in vivo.     

Syk and paxillin are differentially phosphorylated following adhesion to the plastic substrate in rat alveolar macrophages.

Adhesion is associated with tyrosine phosphorylation in many types of cells. Although macrophages are known to adhere and phagocytose foreign particles, the signal transduction pathway of macrophages in response to adhesion to the foreign substrate has not been fully investigated. In the present study we investigated tyrosine-phosphorylated proteins and phosphorylation of paxillin in alveolar macrophages (AMs) following adhesion to a plastic substrate. Adhesion to a plastic dish resulted in tyrosine phosphorylation of a 68 000 MW protein, which was shown, by immunoprecipitation and immunoblotting in the present study, to be a rat Syk kinase. Treatment with erbstatin reduced both tyrosine phosphorylation of Syk and adherence of AMs, while treatment with cytochalasin B inhibited spreading of AMs but did not inhibit tyrosine phosphorylation of Syk. These results suggest that tyrosine phosphorylation of Syk plays an important role in adhesion of AMs to the plastic substrate, but not in AM spreading. Paxillin is known to be tyrosine phosphorylated following adhesion to the extracellular matrix in many types of cells. However, paxillin appeared to be serine/threonine phosphorylated rather than tyrosine phosphorylated following adhesion of AMs to the plastic substrate. Treatment with A23187 (a calcium ionophore), but not phorbol 12-myristate 13-acetate (PMA; a protein kinase C stimulator), induced tyrosine phosphorylation of Syk in non-adherent AMs. Treatment with either A23187 or PMA caused electromobility changes of paxillin that were mainly a result of serine/threonine phosphorylation. These results suggest that adhesion to the plastic substrate leads to two differently regulated events in AMs: tyrosine phosphorylation of Syk and serine/threonine phosphorylation of paxillin, both of which are probably mediated by an increase in intracellular calcium.     

Chemokine receptor 2-mediated accumulation of fungicidal exudate macrophages in mice that clear cryptococcal lung infection

Clearance of pulmonary infection with the fungal pathogen Cryptococcus neoformans is associated with the accumulation and activation of lung macrophages. However, the phenotype of these macrophages and the mechanisms contributing to their accumulation are not well-defined. In this study, we used an established murine model of cryptococcal lung infection and flow cytometric analysis to identify alveolar macrophages (AMs) and the recently described exudate macrophages (ExMs). Exudate macrophages are distinguished from AMs by their strong expression of CD11b and major histocompatibility complex class II and modest expression of costimulatory molecules. Exudate macrophages substantially outnumber AMs during the effector phase of the immune response; and accumulation of ExMs, but not AMs, was chemokine receptor 2 (CCR2) dependent and attributable to the recruitment and subsequent differentiation of Ly-6C(high) monocytes originating from the bone marrow and possibly the spleen. Peak ExM accumulation in wild-type (CCR2(+/+)) mice coincided with maximal lung expression of mRNA for inducible nitric oxide synthase and correlated with the known onset of cryptococcal clearance in this strain of mice. Exudate macrophages purified from infected lungs displayed a classically activated effector phenotype characterized by cryptococcal-enhanced production of inducible nitric oxide synthase and tumor necrosis factor α. Cryptococcal killing by bone marrow-derived ExMs was CCR2 independent and superior to that of AMs. We conclude that clearance of cryptococcal lung infection requires the CCR2-mediated massive accumulation of fungicidal ExMs derived from circulating Ly-6C(high) monocytes.     

Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity.

In order to manipulate the physical properties of the macrophages membrane, methods were developed which potentiated the incorporation of exogenously supplied fatty acids into membrane lipids. Chromatograms of macrophages which were grown in the presence of a variety of fatty acids demonstrated that exogenously supplied unsaturated fatty acids (palmitoleic, oleic, elaidic, linoleic, linolenic and arachidonic acids) were readily incorporated into the cells and selectively altered the fatty acyl composition of macrophage phospholipids. Up to 38% of the total cellular phospholipids were found to be derived from the exogenously added fatty acid supplements. The incorporation of the different fatty acids into cellular phospholipids had striking effects on cellular phagocytic activity. These effects were found to correlate with the degree of unsaturation, and the cis- or trans-double bond configuration. Thus, macrophage phagocytic ingestion rates of 125I-labelled Shigella flexneri were found to alter by more than 2-fold after the cells were cultivated in the presence of cis unsaturated fatty acids.     

Arachidonic acid metabolism is altered in sarcoid alveolar macrophages

Macrophages produce various arachidonic acid (AA) metabolites which may either enhance or suppress inflammatory processes. We investigated AA metabolite production by alveolar macrophages (AMs) from 11 patients with pulmonary sarcoidosis and 9 normal volunteers. We assessed the production of both cyclooxygenase products (prostaglandin (PG) E2, thromboxane B2 (TXB2), PGF2 alpha, and 6-keto-PGF1 alpha) and lipoxygenase products (leukotrienes (LT) and hydroxyeicosatetraenoic acids (HETEs] in AM cultures. We found that sarcoid AMs produced less PGE2, TXB2, 6-keto-PGF1 alpha, and HETEs in both the unstimulated and the calcium ionophore-stimulated states compared with normal AMs. Sarcoid AMs also produced less PGF2 alpha and LTs in the unstimulated state after 1 hr of incubation, but following calcium ionophore stimulation, these differences did not achieve statistical significance. We conclude that sarcoid AMs have a reduced capacity to produce AA metabolites compared with that of normal AMs.     

Different cell surface and phagocytic properties in mononuclear phagocytes from blood and alveoli. A comparative study of blood monocytes and alveolar macrophages from human nonsmokers using flow cytofluorometry

Flow cytofluorometry was used to compare blood monocytes (BMs) and alveolar macrophages (AMs) from the same nonsmoking subject (n = 13). Autofluorescence was quantified, cell surface markers (HLA-DR, CR3) were detected by monoclonal antibodies, and phagocytic ability was determined using C3b-coated yeast particles. AMs expressed more HLA-DR (p less than 0.001) and CR3 (p less than 0.01) on their surfaces than did BMs. The phagocytic capacity was enhanced in AMs compared to BMs (p less than 0.001) and the cells showed an increased autofluorescence (p less than 0.001) in the alveoli compared to blood. The findings suggest that the mononuclear phagocyte is activated when it migrates from blood to alveoli in order to adapt to the milieu in the alveolar space.     

Proliferation of alveolar macrophages in hyperoxia

The effect of oxygen on the proliferative response of alveolar macrophages (AMs) was investigated. Alveolar macrophages cultured in hyperoxic atmosphere (95% O2 + 5% CO2) for 18 hours showed increased incorporation of [3H]-thymidine and proliferation in contrast to those cultured in a control atmosphere (95% air + 5% CO2). The proliferating cell was shown to be a macrophage by morphology, esterase staining, and phagocytic ability. The results suggest an oxygen-induced proliferation of AMs that may play a critical role in AM influx into the alveoli particularly at times of hyperoxia, eg, the neonatal period.     

Differential recognition of structural details of bacterial lipopeptides by toll-like receptors

The question which detailed structures of bacterial modulins determine their relative biological activity and respective host cell receptors was examined with synthetic variants of mycoplasmal lipopeptides as model compounds, as well as recombinant outer surface protein A (OspA) of Borrelia burgdorferi and lipoteichoic acid. Mouse fibroblasts bearing genetic deletions of various toll-like receptors (TLR) were the indicator cells to study receptor requirements, primary macrophages served to measure dose response. The following results were obtained: (i) the TLR system discriminates between modulins with three and those with two long-chain fatty acids in their lipid moiety, in that lipopeptides with three fatty acids were recognized by TLR2, whereas those with two long-chain fatty acids and lipoteichoic acid required the additional cooperation with TLR6; (ii) substitution of the free N terminus of mycoplasmal lipopeptides with an acetyl or palmitoyl group decreased the specific activity; (iii) removal of one or both ester-bound fatty acids lowered the specific activity by five orders of magnitude or deleted biological activity; (iv) oxidation of the thioether group lowered the specific activity by at least four orders of magnitude. The implications of these findings for physiological inactivation of lipopeptides and host-bacteria interactions in general are discussed.     

Relationship of oxidant-mediated cytotoxicity to phospholipid metabolism in endothelial cells

Exposure to oxidants permeabilizes cell membranes and liberates unesterified fatty acids (UFA) in a variety of cell types, including endothelial cells. Products of phospholipase activity, particularly UFA and lysophosphatides, possess potent detergent-like properties, and we postulated that oxidant injury might be mediated by the accumulation of these toxic phospholipase products. Several radiolabels were incorporated into defined positions in the phospholipids of cultured, confluent bovine pulmonary endothelial cells (BPAEC). The release of radiolabeled fatty acids and the accumulation of cell-associated phospholipase products were measured and compared to a standard cytotoxicity assay (51Cr release) in response to an oxidant stress, in this case 0.1 to 10 mM hydrogen peroxide (H2O2). H2O2 caused time- and dose-dependent 51Cr release as well as liberation of saturated ([14C]stearic acid) and unsaturated ([3H]arachidonic acid) fatty acids and the accumulation of phospholipase A2 and C products. The ability of BPAEC to incorporate UFA into complex phospholipids was shown to be severely impaired in the presence of H2O2. Further studies showed that H2O2 caused depletion of BPAEC adenosine triphosphate (ATP) content to undetectable levels, and that the depletion of cellular ATP by iodoacetic acid induced substantial release of [3H]arachidonic acid but not [14C]stearic acid from BPAEC. This finding suggests that release of UFA in response to an oxidant stress may be due in part to a defect in ATP-dependent reacylation pathways and need not reflect any increase in phospholipase activities. Also unsaturated fatty acids were found to be toxic to BPAEC upon adding them to supernatants of cultured monolayers.     

Correlation Between Modification of Membrane Phospholipids and Some Biological Activity of Lymphocytes, Neutrophils and Macrophages

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. the results are as follows.

a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested.

b) the phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids.

c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.

d) the cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested.     

A comparative study of the lipids of plasma membranes of normal cells and those infected and transformed by rous sarcoma virus

Comparative studies of the phospholipids of Rous sarcoma virus (RSV)-transformed chicken embryo cells, uninfected normal cells, and cells infected but not transformed by the transformation-defective mutant of RSV td105, were done. Phospholipids of whole cells as well as those of isolated plasma membranes were analyzed by thin-layer and gas-liquid chromatography. The plasma membrane preparations used were significantly enriched in 5′-nucleotidase enzyme activity over that of whole cell homogenates. Our results indicated that transformation by RSV and infection by td105 do not significantly alter the molar ratios of the various phospholipids compared to uninfected normal cells. However, comparison of the fatty acyl chains of total phospholipid as well as isolated specific phospholipids revealed that the phospholipids of transformed cells have higher percentages of unsaturated fatty acyl chains than those of uninfected normal cells; phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SP) have higher percentages of unsaturated fatty acyl chains, while phosphatidylcholine (PC) and phosphatidylserine (PS) remained unchanged. The percentages of unsaturated fatty acyl chains of PE and PI of td 105-infected cells increased, while that of PC and PS decreased resulting in no overall change. These results are consistent with the concept that altered fluidity of membranes of transformed cells is due to the selection of phospholipids with specific fatty acyl chains by new or modified proteins incorporated into the membranes of transformed and/or virus-producing cells.     

Evidence for protein-mediated fatty acid efflux by adipocytes

Aim: The hormonally controlled mobilization and release of fatty acids from adipocytes into the circulation is an important physiological process required for energy homeostasis. While uptake of fatty acids by adipocytes has been suggested to be predominantly protein‐mediated, it is unclear whether the efflux of fatty acids also requires membrane proteins. Methods: We used fluorescent fatty acid efflux assays and colorimetric assays for free fatty acids and glycerol to identify inhibitors with effects on fatty acid efflux, but not lipolysis, in 3T3‐L1 adipocytes. We assessed the effect of these inhibitors on a fibroblast‐based cell line expressing fatty acid transport protein 1, hormone‐sensitive lipase and perilipin, which presumably lacks adipocyte‐specific proteins for fatty acid efflux. Results: We identified 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS) as an inhibitor of fatty acid efflux that did not impair lipolysis or the cellular exit of glycerol but lead to an accumulation of intracellular fatty acids. In contrast, fatty acid efflux by the reconstituted cellular model for fatty acid efflux was responsive to lipolytic stimuli, but insensitive to DIDS inhibition. Conclusion: We propose that adipocytes specifically express an as yet unidentified DIDS‐sensitive protein that enhances the efflux of fatty acids and therefore may lead to novel treatment approaches for obesity‐related disorders characterized by abnormal lipid fluxes and ectopic triglyceride accumulation.     

Kinetics of tumour necrosis factor and prostaglandin production by murine resident peritoneal macrophages as affected by dietary n-3 polyunsaturated fatty acids.

Cell-associated and secreted tumour necrosis factor (TNF), prostaglandin (PG) E2, and 6-keto PGF1 alpha were monitored at various times following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide (LPS). Macrophages were obtained from mice maintained on diets containing 1.5 wt% n-3 polyunsaturated fatty acids (PUFA)+ 1.5 wt% n-6 fatty acids; 1.5 wt% n-6 fatty acids; or 3 wt% n-6 fatty acids, for 4 weeks. Cell-associated TNF increased transiently in the resident peritoneal macrophages from mice consuming all diets and decreased after TNF secretion had reached maximum and plateaued. Macrophages from mice consuming the n-3 PUFA contained more cell-associated TNF and secreted more TNF than macrophages from mice consuming diets containing n-6 fatty acids only, at all time-points studied. Macrophages from mice consuming the n-3 PUFA showed an earlier increase in cell-associated and secreted TNF compared with macrophages from mice consuming n-6 fatty acids only. Kinetics of maximum TNF production was not affected by the diets and dietary n-3 PUFA did not cause a prolonged increase in TNF secretion. Macrophages from mice consuming the n-3 PUFA produced less PG than macrophages from mice consuming the n-6 fatty acids only. PG secretion increased following appearance of cell-associated TNF but when PG had accumulated in the medium there was no further increase in TNF production.     

Mycobacterium bovis-infected cervine alveolar macrophages secrete lymphoreactive lipid antigens

Tuberculosis is caused by intracellular bacteria belonging to the genus Mycobacterium, including M. tuberculosis and M. bovis. Alveolar macrophages (AMs) are the primary host cell for inhaled mycobacteria. However, little is known about the mechanisms by which infected AMs can process and present mycobacterial antigens to primed lymphocytes and how these responses may affect ensuing protection in the host. In the present study, we sought to determine whether AMs from a naturally susceptible host for Mycobacterium bovis (red deer) could produce and secrete soluble immunoreactive antigens following mycobacterial infection in vitro. Confluent monolayers of deer AMs were infected with either heat-killed or live virulent M. bovis or M. bovis BCG at a multiplicity of infection of 5:1 and cultured for 48 h. Culture supernatants were collected, concentrated, and tested for the presence of mycobacterial antigens in a lymphocyte proliferation assay by using peripheral blood mononuclear cells from M. bovis-sensitized or naive deer. Supernatants derived from macrophages which had been infected with live bacilli stimulated the proliferation of antigen-sensitized, but not naive, lymphocytes. Supernatants derived from uninoculated AMs or AMs inoculated with heat-killed bacilli failed to stimulate lymphocyte proliferation. The lymphoproliferative activity was retained following lipid extraction of the supernatants, which were free of amino groups as determined by thin-layer chromatography. These results demonstrate that mycobacteria which are actively growing within AMs produce lipids which are secreted into the extracellular milieu and that these lipids are recognized by lymphocytes from mycobacterium-primed hosts. We suggest that mycobacterial lipids are released from AMs following aerosol infection in vivo and that they play an important role in the early immune response to tuberculosis.     

Correlation Between Modification of Membrane Phospholipids and Some Biological Activity of Lymphocytes,Neutrophils and Macrophages

Abstract

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. the results are as follows.

a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested.

b) the phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids.

c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.

d) the cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested.     

Genesis of pulmonary foam cells in rats with diet-induced hyper beta-lipoproteinaemia

Hyper beta-lipoproteinaemia in rats was produced by feeding a standard diet to which was added excess cholesterol and cholic acid, with or without olive oil, for 4, 8, and 12 weeks. The beta-lipoprotein percentage in serum lipoprotein electrophoresis and lipid contents in very low density lipoprotein and low density lipoprotein fractions in these rats were significantly higher than in the control rats fed the standard diet only. The percentage of foamy monocytes (FMs) to the total number of blood monocytes (BMs) from mononuclear leucocyte fractions and percentage of pulmonary foam cells (PFCs) to the number of alveolar macrophages (AMs) from bronchopulmonary lavage fluids in the rats increased with the extension of the feeding period and were significantly higher than those in the controls. An increase in the percentage of PFCs was closely correlated with that of FMs in the rats. FMs and PFCs had cytoplasmic fine vacuoles proved to be neutral lipid and cholesterol. Histologically, PFCs made an appearance in the lungs of all the rats as early as 4 weeks after the start of feeding. The degree of the PFCs' development increased as the feeding period lengthened. When latex particles were injected intravenously into rats at feeding week 4, the percentage of latex-ingested AMs to the number of AMs in the rats was significantly higher than that of the controls at 4 and 8 days post-injection. The percentage of latex-ingested PFCs to the number of latex-ingested AMs increased with the lapse of a day after injection and was significantly higher than that of the controls at 2, 4, and 8 days post-injection. The present findings suggest that the foamy transformation of BMs and their migration into the pulmonary alveoli may be a potential mechanism of the PFCs' development in rats with hyper beta-lipoproteinaemia.     

Depletion of Alveolar Macrophages by Treatment with 2-Chloroadenosine Aerosol

Alveolar macrophages (AMs) are localized in the alveoli and alveolar ducts of the lung and are the only macrophages living in an aerobic environment. Recent studies have demonstrated that AMs play a central role in lung diseases, such as pneumonia and acute respiratory distress syndrome. It has become important to find a simple, effective way to eliminate AMs in order to investigate the function of AMs in vivo. 2-Chloroadenosine (2-CA), a purine analog, is reported to be selectively cytotoxic to cultured macrophages, and we hypothesized that it would deplete the number of AMs in the bronchoalveolar lavage fluid (BALF) of mice without any effect on neutrophil or lymphocyte counts. After mice had inhaled 1 mM aerosolized 2-CA for 2 h, AMs were found to be significantly depleted at 0 h [(4.42 ± 0.16) × 10⁴/ml], 24 h [(4.17 ± 0.89) × 10⁴/ml], 48 h [(3.17 ± 0.21) × 10⁴/ml], and 72 h [(5.00 ± 0.64) × 10⁴/ml] compared with concentrations in untreated controls [(12.1 ± 0.21) × 10⁴/ml]. Neutrophil and lymphocyte counts in BALF did not change and histological changes in the lung were not observed after 2-CA treatment. The lung wet-to-dry weight ratio did not change at 0, 24, and 48 h after 2-CA aerosol application. The 2-CA aerosol had no effect on lung vascular permeability, as assessed by the intravenous administration of Evans blue, or on other phagocytes, as assessed by Kupffer cell counts. Our study demonstrates the efficacy of 2-CA in reducing AM numbers in vivo.