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Objective- Macrophages play key roles in inflammation and diabetic vascular complications. Emerging evidence implicates long noncoding RNAs in inflammation, but their role in macrophage dysfunction associated with inflammatory diabetic complications is unclear and was therefore investigated in this study. Approach and Results- RNA-sequencing and real-time quantitative PCR demonstrated that a long noncoding RNA Dnm3os (dynamin 3 opposite strand) is upregulated in bone marrow-derived macrophages from type 2 diabetic db/db mice, diet-induced insulin-resistant mice, and diabetic ApoE -/- mice, as well as in monocytes from type 2 diabetic patients relative to controls. Diabetic conditions (high glucose and palmitic acid) induced Dnm3os in mouse and human macrophages. Promoter reporter analysis and chromatin immunoprecipitation assays demonstrated that diabetic conditions induce Dnm3os via NF-κB activation. RNA fluorescence in situ hybridization and real-time quantitative PCRs of subcellular fractions demonstrated nuclear localization and chromatin enrichment of Dnm3os in macrophages. Stable overexpression of Dnm3os in macrophages altered global histone modifications and upregulated inflammation and immune response genes and phagocytosis. Conversely, RNAi-mediated knockdown of Dnm3os attenuated these responses. RNA pull-down assays with macrophage nuclear lysates identified nucleolin and ILF-2 (interleukin enhancer-binding factor 2) as protein binding partners of Dnm3os, which was further confirmed by RNA fluorescence in situ hybridization immunofluorescence. Furthermore, nucleolin levels were decreased in diabetic conditions, and its knockdown enhanced Dnm3os-induced inflammatory gene expression and histone H3K9-acetylation at their promoters. Conclusions- These results demonstrate novel mechanisms involving upregulation of long noncoding RNA Dnm3os, disruption of its interaction with nucleolin, and epigenetic modifications at target genes that promote macrophage inflammatory phenotype in diabetes mellitus. The data could lead to long noncoding RNA-based therapies for inflammatory diabetes mellitus complications.

Cell mediated immunity in the respiratory tract, operating somewhat independently of systemic cell mediated immunity CMI, has been recognized only rather recently. Local CMI has been shown to be more consistently and more potently stimulated following local administration of immunogen. In addition, in the few studies that have been done relative to protection, local immunization has resulted in greater resistance to organisms usually considered protected against by CMI. Thus, like the humoral immune system, CMI appears to operate locally in the respiratory tract, at least partially independent of systemic CMI. This paper deals with the evidence relative to local CMI, as well as a discussion of the role of the pulmonary macrophage, the mechanisms whereby local CMI may operate in protection, and the evidence for the presence of T-cell memory in the respiratory tract.

This study was initiated to determine whether purified slime polysaccharide(PSP) from P aeruginosa inhibits the ingestion of heat killed Saccharomyces cerevisiae particles in macrophage cultures. Relative to controls, direct phagocytosis assays revealed that the percentages of phagocytes and the numbers of ingested yeast particles per phagocyte decreased in a dose-dependent manner in PSP-treated cultures. Thus, PSP may act as a virulence factor in vivo by impairing the phagocytic capacity of macrophages.

Groups of young adult and senescent guinea pigs were fed normal and vitamin C deficient diets for 4 weeks and tested for their peritoneal macrophage functions. Serum levels of vitamin C in deficient animals indicated a progressive state of ascorbic acid deficiency and correlated well with the clinical signs and symptoms of scurvy. Fewer macrophages were obtained from the peritoneal cavities of deficient animals and morphologically they were smaller in size. Adverse effects of vitamin C deficiency were enhanced in aged animals. Significantly greater number of aged animals died by 4 weeks of deficiency. Deficient senescent animals had greater decline in macrophage random migration and bactericidal capacity. Following phagocytic stimuli, superoxide anion generation also significantly decreased. Data suggest that vitamin C deficiency might affect macrophage functions in the aged more profoundly and could compromise parameters of host defenses effective against microbial infections.

Guinea pigs were fed a vitamin C-deficient diet and at various time periods thereafter their peritoneal cells were tested for biological activity. The serum levels of vitamin C in the deficient animals indicated a progressive state of ascorbic acid deficiency with time and this correlated well with clinical signs and symptoms of scurvy. Fewer macrophages were obtained from the peritoneal cavities of deficient animals and in structural appearance under the phase contrast and light microscope they were smaller in size. They showed no significant impairment in phagocytosis of bacterial cells. The macrophages, however, exhibited significantly reduced migration on glass as compared to the normal cells. In vitro addition of vitamin C partially reversed this reduced migration.

Immunobiological properties of guinea pig and human peritoneal macrophages were studied at temperatures ranging from 25 degrees C to 37 degrees C. Glass adherence, random migration, response to MIF and killing of Salmonella typhimurium and Saccharomyces cerevesiae by guinea pig macrophages were decreased with temperatures below 37 degrees C. Killing of Sacch. cerevesiae by human macrophages was also reduced at temperatures less than 37 degrees C. Acid phosphatase and beta-N-acetylglucosaminidase (NAG) activity assayed at 37 degrees C did not change when the cells were preincubated for 1 and 5 hours at various temperatures. Impaired macrophage function with subnormal temperatures may contribute to enhanced susceptibility to infection of patients with chronic diseases such as renal failure and cirrhosis.

This study examined the effects of aging on the immunobiological properties of alveolar macrophages in young adult and aged rats. Macrophages, obtained by lung lavage, were enumerated and tested in vitro for yeast phagocytosis and fungicidal activities. Superoxide anion (O2-) generation stimulated by opsonized zymosan was compared between the two groups. Total numbers of alveolar cells and the differential cell distribution were comparable in both the groups. The difference between the groups was not statistically significant, even though the yeast phagocytic capacity in the cells from senescent animals was consistently higher than in the controls. This may be due to the wide degree of variability observed in the aged animals. However, cell fungicidal activity was statistically lower in the aged animals at 90 minutes of incubation which correlated with a significant decline in the O2- production. Aging may adversely affect some of the immunobiologic functions of lung macrophages and thus compromise host defense mechanisms in the respiratory tract.

Storage of intact macrophages would be a useful procedure for laboratories studying the biological functions of these cells. Oil-induced guinea pig peritoneal cells were harvested and frozen at an ultra-low temperature using a cryoprotective media. Studies of the biological properties of the frozen cells upon thawing indicated that the cells retained viability for a fairly long period of time, were metabolically active and comparable to normal cells in morphology. Indications of activation were obtained in enhanced phagocytic activity and significant decrease in migration of the stored cells.

Peritoneal macrophages and peripheral blood monocytes were collected from healthy women undergoing laparoscopic tubal clipping. Monolayers of adherent cells were examined for protein content, intracellular levels of lysosomal enzymes, and antimicrobial capacity. Compared to blood monocytes, peritoneal macrophages were larger and less glass-adherent and were less capable of intracellular killing of Salmonella typhimurium. Acid hydrolase content, however, was increased in peritoneal macrophages. These data suggest that resting human tissue macrophages may be less active in certain respects than their blood precursors. One should then question whether the enhanced functions observed after long-term in vitro culture of blood monocytes-macrophages accurately reflect the in vivo status of their more differentiated descendants.

Peritoneal macrophages are used for immunologic assessment of the lymphokine, migration inhibitory factor (MIF). For this purpose, these cells are induced intraperitoneally in animals with inflammatory agents such as mineral oil (MO) and peptone water (PW). Purpose of the present study was two-fold: To assess the changes in biologic properties of guinea pig macrophages induced peritoneally with MO or PW as compared to control cells after administration of normal saline (NS); and to examine the suitability of induced macrophages to respond to MIF in vitro. Peritoneal exudate cells were harvested from guinea pigs 7 days following intraperitoneal injection of MO, PW or NS. They were enumerated in hemocytometers, their differential counts determined by Wright's stain and morphologic characteristics assessed by scanning electron microscopy. Functional activation of peritoneal macrophages was determined by lysosomal enzyme functions, as well as by yeast phagocytosis in vitro. Random cell migration and responses to migration inhibitory factor (MIF) were determined in Mackaness chambers. Mineral oil injection resulted in significantly higher yield of peritoneal macrophages. Greater than 70% of peritoneal exudate cells were macrophages in all three groups. Spread out structures and ruffled borders were seen in electron micrographs of MO induced cells. Such structures were less evident in PW induced cells and were absent in controls. Acid phosphatase (ACP) and beta-N-acetylglucosaminidase (B-NAG) activities as well as yeast phagocytosis significantly increased in MO and PW induced cells. Random migration and responses to MIF in Mackaness chambers remained comparable in the three experimental groups.