Morphometry of postnatal development in the porcine lung

Alveolar regions of normal pig lungs (newborn to 60-day-old) were characterized morphometrically to provide a basis for comparison in future investigations of porcine respiratory diseases. Endotracheal installation of fixative was done to expand the lungs uniformly at total capacity. Differential effects of lobar variations were determined by stratified random sampling of lung lobes. A stereologic study was done by point and intersection counts on electron micrographs. At birth, the lungs were remarkably well developed. Relative alveolar and capillary surface densities and air-blood tissue barrier thicknesses were at adult levels. In allometric regressions, volumes and surfaces of lung components regressed directly to lung volume, but monoexponentially (to the 3/4 power) with body weight. In the first postnatal week, however, relative volume densities of cellular interstitium in septal tissue and of capillary lumina in parenchyma increased at statistically significant levels. Composition of lung parenchyma and septa was changed, although without statistically significant direct impact on parameters related to gas exchange. Type II pneumocytes had increased nuclear to cytoplasmic volume ratios in 7- to 14-day-old pigs, probably reflecting cell activation and increased surfactant production. Age (postnatal lung growth) created the most substantial variance of results; interanimal variation in pigs of the same age was less important and no consistent lobar variations were seen.     

Presence of pulmonary intravascular macrophages in the equine lung: Some structuro-functional properties

The pulmonary intravascular macrophages (PIMs) have been described in several species of animals. This study demonstrates for the first time that the equine lung has PIMs as resident phagocytes in its microvasculature. Their salient features such as globular surface coat, structures of the endocytic pathway, and related cell organelles closely resemble those of the calf, goat, and sheep. The exquisite organization of the coat globules in the form of a linear chain was structurally similar to the lipolytic lipase and the heparin-sensitive globular coat from PIMs of calf, goat, and sheep. Monastral blue (MB) when employed as a tracer to assess the phagocytic properties of equine PIMs induced similar modification of the globules of the coat into lipid droplets, reminiscent of neutral lipids. Lipid droplets (modified coat globules) were delivered into acid phosphatase-positive endosomes and lysosomes. Concurrently, the unaltered globules of the coat, probably internalized via fluid-phase constitutive pinocytoses, followed a different endocytic pathway. Large-scale platelet uptake by the PIMs was observed with thrombocytopenia in MB-treated ponies. The possible significance of hypothetical LDL-coat and the endocytic organelles as equivalents of synthetic apparatus of vasoactive lipids in the PIMs of horse needs to be assessed in future studies.© Willey-Liss, Inc.     

Presence of pulmonary intravascular macrophages in the equine lung: some structuro-functional properties.

The pulmonary intravascular macrophages (PIMs) have been described in several species of animals. This study demonstrates for the first time that the equine lung has PIMs as resident phagocytes in its microvasculature. Their salient features such as globular surface coat, structures of the endocytic pathway, and related cell organelles closely resemble those of the calf, goat, and sheep. The exquisite organization of the coat globules in the form of a linear chain was structurally similar to the lipolytic lipase and the heparin-sensitive globular coat from PIMs of calf, goat, and sheep. Monastral blue (MB) when employed as a tracer to assess the phagocytic properties of equine PIMs induced similar modification of the globules of the coat into lipid droplets, reminiscent of neutral lipids. Lipids droplets (modified coat globules) were delivered into acid phosphatase-positive endosomes and lysosomes. Concurrently, the unaltered globules of the coat, probably internalized via fluid-phase constitutive pinocytoses, followed a different endocytic pathway. Large-scale platelet uptake by the PIMs was observed with thrombocytopenia in MB-treated ponies. The possible significance of hypothetical LDL-coat and the endocytic organelles as equivalents of synthetic apparatus of vasoactive lipids in the PIMs of horse needs to be assessed in future studies.     

Heterogeneity of swine lung macrophages inoculated by porcine reproductive and respiratory syndrome virus

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susceptibility to PRRSV.     

The neonatal porcine lung: ultrastructural morphology and postnatal development of the terminal airways and alveolar region

Morphology and postnatal development of the porcine lung are described in animals ranging in age from newborn through 60 days. Standardized fixation was accomplished by intratracheal instillation of glutaraldehyde under constant pressure. Light microscopic, scanning, and transmission electron microscopic investigations revealed that the porcine lung follows the common architecture of mammalian lungs, but has certain peculiarities as well: intravascular macrophages, ultrastructurally similar to Kupffer cells, are attached to endothelial cells in pulmonary capillaries and are involved in erythrophagocytosis during the first postnatal weeks. Type II pneumocytes of newborn pigs exhibit signs of cell activation, mainly complex nuclear bodies in the cell nuclei. At the same time high levels of 17-hydroxycorticosteroids are observed in the newborn blood plasma. Terminal airways of the porcine lung are nonalveolarized and are, therefore, of purely conductive function. At birth the porcine lung exhibits a high degree of maturity, and thick-walled primary saccules, as described in newborn rodents, are not seen. Septa appear straight and smooth, owing to rare ramification. Septal buds are discernible, and two capillary networks visible on both sides of septal cross sections are seen. Further subdivision of the airspaces occurs in the first two postnatal weeks. Precociousness and fast postnatal growth of the porcine species are assumed to be the reason of this advanced degree of lung maturity at birth and the following rapid pulmonary development.     

Isolation and preliminary in vitro characterization of the porcine pulmonary intravascular macrophage

Porcine intravascular macrophages were isolated by perfusion of the pulmonary vasculature with 0.1% collagenase solution. The isolated cells formed intercellular adhesion plaques with endothelial cells when incubated with porcine pulmonary artery, aorta, and corneal cups. Intercellular adhesion plaques were focal junctionlike membrane specializations consisting of paired submembranous amorphous densities subjacent to 15-20 nm gaps between parallel apposing cell membranes. The intermembranous space was filled with moderately electron dense, finely granular material. Adhesion plaques formed in 4-8 hours and resembled the adhesion plaques formed between pulmonary intravascular macrophages and endothelium in vivo. Alveolar macrophages and peripheral blood monocytes did not form intercellular adhesion plaques with endothelial cells. Intravascular macrophages had histologic and ultrastructural features of macrophages, were alpha naphthyl butyrate esterase positive, adhered to plastic coverslips after 1 hour of incubation, and were smaller than alveolar macrophages and endothelial cells. The formation of intercellular adhesion plaques in vivo and in vitro by cells with morphologic and histochemical features of macrophages distinguishes intravascular macrophages from monocytes and alveolar macrophages.     

Intravascular macrophages in the sheep lung

The presence of a resident population of intravascular macrophages in the sheep lung was investigated. Injection of fluorescent microspheres into the sheep results in uptake by a population of large phagocytic cells scattered within the alveolar capillaries of the lung. By contrast, the rat lung has no uptake of these fluorescent microspheres into the alveolar walls. The cells are large, extremely irregular in shape, often exceed 30 micron in length, and lie within the vascular lumen, apposed to the underlying endothelium. They have several features characteristic of mature macrophages including numerous pseudopodia, a partially preserved cell coat, wormlike bodies ("micropinocytosis vermiformis"), and numerous phagosomes that often contain erythrocytes. Unlike other species such as the rat, in which the mononuclear phagocyte system is distributed primarily in the liver and spleen, the sheep lung has a significant clearance function. Moreover, the presence of these cells in the lung may have important implications for studies of the inflammatory response to pulmonary vascular injury in the sheep.     

Ultrastructural response of pulmonary intravascular macrophages to exogenous oestrogen in the bovine lung: translocation of the surface-coat and enhanced cell membrane plasticity and angiogenesis

The pulmonary intravascular macrophages (PIMs) of domestic ungulates are recognised by their specific surface coat, consisting of linearly arranged globules along the external leaf of the plasma membrane. The coat is sensitive to in vitro digestion with lipolytic lipase (LPL), intravenous heparin and clinical exposure to halothane anaesthesia. The sensitivity to these experimental manipulations suggests that the globules of the coat are predominantly composed of lipoproteins (LDL). The present administration of oestradiol proprionate in castrated male calves potentiated the translocation of the surface coat into the endocytotic pathway of the PIMs. Concurrently with mobilisation of the coat, the plasma membrane was thrown into prominent arrays of lamellipodial extensions. The sprawling macrophages made extensive adhesive contacts with the lining endothelium of the capillaries. Consequently, the endothelial cells were highly attenuated and precariously maintained the integrity of the vascular wall. At some focal points, the vascular wall was penetrated by the filopodial processes of PIMs, which protruded into the perivascular space. Furthermore, there were signs of neovascularisation in the form of overt mitotic changes, sprouting and precursor capillary formation. It is conceivable that the evolving profile of angiogenesis is due to the vascular endothelial growth factor (VEGF) paracrine function of PIMs. Endothelial cell specificity has been considered an important advantage of VEGF for neovascularisation. It allows pleotrophic response of endothelial cells to proliferate and to assemble into endothelial tubes.     

Toxicity of Haemophilus pleuropneumoniae for porcine lung macrophages, peripheral blood monocytes, and testicular cells.

Strain CM5 of Haemophilus pleuropneumoniae was toxic for porcine lung macrophages in minimum doses of 10(6) colony-forming units per ml and for peripheral blood monocytes at 10(7) colony-forming units per ml. The organism was not toxic for porcine testicular cells in a concentration of 10(8) colony-forming units per ml. Filtered sterile culture supernatant diluted 10(0), 10(-1), and 10(-2) was toxic for porcine pulmonary lavage cells. In dilutions of 10(0) and 10(-1), culture supernatant was toxic for peripheral blood monocytes, and at 10(0) it was toxic for testicular cells. Toxicity associated with bacterial cells was sensitive to heating (60 degrees, 60 min), whereas that of the culture supernatant was heat stable (100 degrees C, 15 min). Swine convalescent serum neutralized supernatant toxicity in dilutions of greater than or equal to 1:320. The findings promote the understanding of the pathogenesis of pleuropneumonia in swine caused by H. pleuropneumoniae.     

Factors affecting the permissiveness of porcine alveolar macrophages for porcine reproductive and respiratory syndrome virus

Alveolar macrophages from PRRSV-infected and naïve pigs were placed into culture and infected with PRRSV laboratory strain SD-23983. Permissiveness increased with time in culture, and macrophages from infected pigs could be superinfected. Addition of actinomycin D, an inhibitor of mRNA synthesis, blocked infection. Interferon-γ reduced infection in cultures, while the addition of tumor necrosis factor-α or interleukin (IL)-10 did not affect permissiveness. IL-4 produced a marginal increase in the percentage of infected cells, but without a detectable increase in virus yield. These results suggest that the PRRSV-permissive population of cells in culture arises from a non-permissive precursor population and depends on new mRNA synthesis.     

Airway intra-luminal macrophages: evidence of origin and comparisons to alveolar macrophages

Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals. In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo monastral blue-induced lamellar-bodies in lysosomes of pulmonary intravascular macrophages (PIMs) of bovine lung: Implications of the surface coat

We previously reported that the pulmonary intravascular macrophages (PIMs) of sheep, goat, and calf lung contained a heparin and a lipolytic lipase sensitive surface coat by using tannic acid as a component of paraformaldehyde-glutaraldehyde-based fixative. The implication of this sensitivity was that the surface coat was predominantly comprised of lipoprotein-like substance. In this study we report that monastral blue (MB) used as a vascular tracer interacted with the coat globules and lost its original particulate appearance. Its precise localization in the PIMs was in combination with altered macromolecules of the surface coat in the form of lipid droplets, which conformed to the conventional view of neutral lipids. In contrast, pigment particles examined in their native state resembled metalic particles as electron-dense eliptical rods. The lipid droplets were subsequently internalized through endocytic route and found their access into the lysosomal compartments of PIMs at the electron microscopic level. Lamellar bodies (LLBs) arose from the lysosomal matrix after the entry of lipid droplets in the secondary lysosomes. Acid phosphatase activity was located in secondary lysosomes as well as in endosomes. These observations suggest that coat granules of the PIMs acted as a carrier of exogenous MB particles to deliver the complex to the lysosomal compartment where partial digestion lead to the formation of lamellar bodies. The implications of MB (cationic dye) as a vascular tracer for studying phagocytic index of PIMs in the light of their coat and the rapid development of LLBs are discussed. It is proposed that MB by initially combining with the surface coat provokes mobilization of intracellular lipid pools. In this way metabolism of vasoactive lipid in the PIMs is stimulated to influence the dynamics of pulmonary circulation in the calves.© Willey-Liss, Inc.     

The origin and function of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have a complex relationship with the neoplastic cells of the tumor. On the one hand, the two cell types produce reciprocal growth factors and may be considered to have a symbiotic relationship. On the other hand, TAM can be activated to inhibit tumor growth and destroy neoplastic cells. Here, Alberto Mantovani and colleagues describe this delicate balance and the prospects for its therapeutic manipulation.