Activation of Alveolar Macrophages Exposed to Lavage-Procured Immunoglobulin G Obtained from Normal Rabbit Lungs

Pulmonary washings from rabbits were freed of cells and added to the monolayers of homologous alveolar macrophages (AM). At 1 h after incubation with the pulmonary washings, many more cells adhered to glass, spread out, and showed enhanced Nitro Blue Tetrazolium reduction. The maximal effect of the pulmonary washings on AM activation was obtained 12 h after incubation. The AM activated by the pulmonary washings showed a higher capacity to inhibit the growth of intracellular BCG, and that capacity was correlated with the intensity of Nitro Blue Tetrazolium reduction by the AM. Gel filtration of the pulmonary washings through Sepharose 4B yielded five fractions. The factor that activated the AM functions was in fraction 4. When the immunoglobulin G in the fraction was removed by an immunoadsorbent column, AM activity was abolished. The effect of the immunoglobulin G was dose dependent, and minimal responses to 10(6) cells per ml were obtained at a protein concentration of 20 mug/ml. Lymphokines had no effect on AM activation with respect to the morphological alterations and Nitro Blue Tetrazolium reduction during the 24-h observation time. In summary, AM from normal rabbits were soon activated markedly by lavage-procured immunoglobulin G, but not by lymphokines.     

Nontypeable Haemophilus influenzae Clearance by Alveolar Macrophages Is Impaired by Exposure to Cigarette Smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-α) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-α secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-α secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.The human respiratory tract is one of the largest body surfaces in contact with the environment and, therefore, is a main entry portal for microorganisms. In healthy humans, the lungs are sterile due to the combined actions of a repertoire of defense mechanisms. The components of lung innate immunity include mechanical barriers such as the mucociliary barrier, humoral elements present in the fluid in contact with the lung epithelium such as surfactants, complement, antimicrobial peptides, lysozyme, and lactoferrin, and resident innate immunity cells such as alveolar macrophages and dendritic cells (32, 37). Alveolar macrophages are professional phagocytes and antigen-presenting cells which patrol the lungs as sentinels and are endowed with, among other things, a collection of pattern recognition receptors used to recognize microorganisms containing pathogen-associated molecular patterns. As professional phagocytes, alveolar macrophages recognize, ingest, and process foreign material using a phagolysosomal pathway and thus play an essential role in the clearance of infections (18).Cigarette smoke is the main risk factor for the development of lung cancer, chronic obstructive pulmonary disease (COPD), and respiratory infections (26). In this context, the so-called “British hypothesis” states that recurrent bronchial infections were the reason, at least partially, that some smokers developed progressive airway obstruction and others did not (12, 13). Exposure to cigarette smoke markedly alters lung immunity by disruption of the mucociliary function, mucus hypersecretion, and disturbance of the mucosal integrity (31). Cigarette smoke also causes oxidative stress which triggers local lung inflammation by activation of epithelial cells, alveolar macrophages, neutrophils, and T lymphocytes (2). These cells secrete inflammatory cytokines, proteases, and reactive oxygen species, causing necrosis, tissue damage, and further amplification of the inflammatory response with enhanced recruitment of neutrophils into the lung. Tissue damage promotes the release of inflammatory mediators and inhibits lung tissue repair functions, further increasing the tissue damage in the lungs of smokers (35, 38, 39). It is generally accepted, although it has not been formally proven, that these alterations could allow access of microorganisms to the otherwise sterile lungs, thereby leading to microbial colonization (28-30). Supporting this hypothesis, mice exposed to cigarette smoke were impaired in the ability to clear a Pseudomonas aeruginosa infection (10). However, there is currently limited information concerning the effect of cigarette smoke at the molecular and cellular levels on the interaction between pathogens and alveolar macrophages.Glucocorticoids are drugs that are widely used to control many inflammatory and immune diseases, including respiratory diseases. Moreover, adjunctive glucocorticoid therapy is currently being used against a variety of bacterial infections, including otitis media, and COPD (7, 21). However, despite their importance in suppressing inflammatory responses, little is known about the effects of glucocorticoids in host defense against pathogens.Nontypeable Haemophilus influenzae (NTHI) is a frequent gram-negative asymptomatic colonizer of the upper respiratory tract in healthy humans, but it is also an opportunistic bacterial pathogen. NTHI causes invasive diseases such as meningitis and acute respiratory infections such as otitis media with effusion, sinusitis, pneumonia, and bronchitis (24). Moreover, NTHI is the pathogen isolated most frequently from lower respiratory tract secretions from patients suffering from chronic respiratory diseases such as COPD and chronic bronchitis (30). Lipooligosaccharide (LOS) is the main glycolipid on the NTHI cell surface and comprises a membrane-anchoring lipid A molecule linked to oligosaccharide chains that extend from the bacterial cell surface (27). Phosphocholine (PCho) is a substituent frequently present in NTHI LOS chain extensions (36). This modification has been shown to be a virulence factor that is involved in NTHI adhesion and invasion of the respiratory epithelium and hence promotes pathogen persistence on the mucosal surface of the respiratory tract (33, 34).The importance of NTHI as a respiratory pathogen has been extensively demonstrated, and alveolar macrophages play an essential role in the clearance of bacterial infections. However, little is known about the interaction between NTHI and alveolar macrophages and about the influence of PCho on this interaction. It is tempting to speculate that NTHI might be able to escape alveolar macrophage-mediated killing and that PCho could play an important role in this process. In addition, given the association between cigarette smoke and respiratory infections caused by NTHI, we hypothesized that cigarette smoke could modify the characteristics of the interaction between NTHI and alveolar macrophages. In the present study, we investigated the features of the interaction between NTHI and alveolar macrophages. Furthermore, we analyzed the impact of cigarette smoke on the ability of alveolar macrophages to engulf and process this respiratory pathogen and whether glucocorticoids affect this interaction.     

Enhanced IA Expression by Alveolar Macrophages Following Intratracheal Administration of Bleomycin to Rats

Bleomycin is an important anticancer drug that causes severe, and sometimes life-threatening, pulmonary toxicity. Initially, there is an acute inflammation followed by an irreversible pulmonary fibrosis. Our studies have focussed on the effects of the acute pulmonary inflammation on the state of alveolar macrophage activation. To study this, we administered a single dose of 3.6 mg bleomycin/kg body weight intratracheally to rats and obtained alveolar macrophages at selected times thereafter. Ia expression was determined by fluorescent microscopy of cells labelled with a fluorochrome-tagged antibody against rat Ia molecules. We report that: 1.) alveolar macrophages have elevated Ia expression shortly after receiving intratracheally administered bleomycin; 2.) Ia expression is not limited to a specific subpopulation of alveolar macrophages; 3.) Ia expression is transient in nature returning to control levels 7-14 days after bleomycin administration; and, 4.) the degree of upregulation of Ia expression is directly related to the dose of bleomycin administered.     

The Effect of Marijuana Smoke Exposure on Murine Sarcoma 180 Survival in Fisher Rats

Fisher rats were treated for 28 or 60 days to multiple exposures to the smoke of marijuana or marijuana placebo cigarettes. Primary, secondary and in some instances tertiary tumor implants were performed. Murine sarcoma 180 tumor cells (7.5 × 10⁷) were implanted subcutaneously on day 1, 14 and 28 following initiation of smoke exposure (28 day studies) or on day 1, 14 after cessation of smoke exposure (60 day studies). Tumor areas were measured on alternate days beginning on the second or third day after implantation for 13 or 14 days. Exposure to both marijuana and placebo smoke for 28 days (6, 9 and 18 cigarettes per day) resulted in suppressed growth of secondary and tertiary implants. Administration of △⁹ tetrahydrocannabinol (50 mg/kg, i.p., 20 days) failed to suppress the growth of primary and secondary tumors. This suggests that noncannabinoid constituents of the smoke may contribute to the suppression of tumor growth. Exposure of rats to 9, but not 4 or 6, marijuana or placebo cigarettes per day for 60 days suppressed the growth of primary but not secondary tumors. Thus, the effects of smoke exposure appear to be lost by two weeks after cessation of treatment. The possible existence of a non-cannabinoid immunostimulant in the smoke is discussed.     

The Effect of Marijuana Smoke Exposure on Murine Sarcoma 180 Survival in Fisher Rats

Abstract

Fisher rats were treated for 28 or 60 days to multiple exposures to the smoke of marijuana or marijuana placebo cigarettes. Primary, secondary and in some instances tertiary tumor implants were performed. Murine sarcoma 180 tumor cells (7.5 × 10⁷) were implanted subcutaneously on day 1, 14 and 28 following initiation of smoke exposure (28 day studies) or on day 1, 14 after cessation of smoke exposure (60 day studies). Tumor areas were measured on alternate days beginning on the second or third day after implantation for 13 or 14 days. Exposure to both marijuana and placebo smoke for 28 days (6, 9 and 18 cigarettes per day) resulted in suppressed growth of secondary and tertiary implants. Administration of △⁹ tetrahydrocannabinol (50 mg/kg, i.p., 20 days) failed to suppress the growth of primary and secondary tumors. This suggests that noncannabinoid constituents of the smoke may contribute to the suppression of tumor growth. Exposure of rats to 9, but not 4 or 6, marijuana or placebo cigarettes per day for 60 days suppressed the growth of primary but not secondary tumors. Thus, the effects of smoke exposure appear to be lost by two weeks after cessation of treatment. The possible existence of a non-cannabinoid immunostimulant in the smoke is discussed.     

Comparative Densities of Hydrolase-Containing Granules from Normal and BCG-Induced Alveolar Macrophages

Compared to cells from normal rabbit lungs, BCG-induced alveolar macrophages have a marked increase in hydrolase levels and the number of large electron-dense subcellular structures. This study was performed to investigate the possibility that these electron-dense structures were responsible for the increased hydrolase levels of these cells. Using sucrose gradient centrifugation, nuclei-free homogenates of normal and BCG-induced macrophages were analyzed with respect to the subcellular particles they contain. Gradient fractions were assayed for enzymes commonly associated with lysosomes as well as the mitochondrial cytochrome oxidase. Fractions of peak hydrolase activity from the BCG-induced preparations were consistently more dense than those from the normal cell preparations. Ultrastructural studies of the particulate material found in fractions of peak hydrolase activity from BCG-induced preparations revealed the presence of electron-dense, often dumbbell-shaped, granules. The data suggest that these peculiar granules are responsible for the elevated hydrolase levels of BCG-induced alveolar macrophages.     

Synthesis of Complement by Alveolar Macrophages from Patients with Sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown aetiology. Alveolar macrophages (AM) in sarcoidosis release a variety of mediators important to the pathogenesis of the disease. Complement is essential for the inflammatory response and we investigated whether there were any major defects in the potential for sarcoidosis AM to synthesize complement in vitro. AM from 11 patients with active sarcoidosis and three healthy controls were cultured under serum-free conditions. There was a significant binding of polyclonal (anti-C5, -C6, -C7, -C8) and monoclonal anti-complement antibodies (anti-C3c and anti-C9 neoepitope (aE11] to agarose beads incubated with unstimulated AM for 24, 48, or 72 h. A significant and inhibitable production of soluble C3c, C5, C9, and S-protein was found in the harvested medium as detected by enzyme immunoassays. Activated C3 and C9 were also detected based on neoepitope expression. Presence of co-cultured agarose beads reduced the amount of soluble S-protein due to deposition on the agarose. We argue that the C9 neoepitope is an integral part of the terminal complement complex (TCC), both in the fluid and solid phase when bound to the agarose. In the fluid phase, SC5b-9 was generated, whereas the agarose-bound S-protein is assumed not to be associated with TCC on the beads. The results demonstrate for the first time that AM from sarcoidosis patients synthesize the functional alternative and terminal pathway of complement.     

Structural and Functional Analysis of the Spermatogenic Epithelium in Rats Exposed to Titanium Dioxide Nanoparticles

Bulletin of Experimental Biology and Medicine - We studied immunohistochemical and morphometric characteristics of the spermatogenic epithelium in rats against the background of peroral...     

Effects of Afobazole on Cognitive Behavior of the Offspring of Rats Exposed to Tobacco Smoke during Gestation Period

Experiments on the model of foraging behavior formation under conditions of free choice (T-maze) revealed learning failure against the background of reduced motor activity in the offspring of rats exposed to tobacco smoke on gestation days 1–20. Afobazole administered to pregnant rats orally in doses of 1 or 10 mg/kg daily during the whole gestation and/or entering rat pup body with breast milk from mothers receiving 200 mg/kg to day 20 of their life normalized their learning capacity. The formation of short-term and long-term memory in animals receiving afobazole did not differ from the control. Hence, afobazole corrects cognitive disorders in rats exposed to tobacco smoke during prenatal development.     

Interactions of Thermoactinomyces vulgaris and Mouse Alveolar Macrophages

Interactions of mouse alveolar macrophages from three different inbred strains of mice and Thermoactinomyces vulgaris, a microbe associated with Farmer's lung disease, were studied. Alveolar macrophages were found to abolish the mitogenic activity of T. vulgaris. A prostaglandin synthesis inhibitor indomethacin, could not restore the activity. Alveolar macrophage supernatants generated by T. vulgaris treatment exerted strong suppression in secondary concanavalin A-induced lymphocyte transformation. Indomethacin partly relieved the suppression but a histamine 2 receptor blocker, cimetidine, had no effect. Interleukin 1 activity was practically undetectable by the thymocyte co-stimulation assay unless indomethacin was used. When indomethacin was used, interleukin 1 activity could be detected in all strains of mice tested. Major differences in the abolition of the mitogenic effect, in the suppressive effect, or in the release of interleukin 1 were not detected between inbred strains of mice tested. The results indicate that alveolar macrophages exert suppressive actions in vitro after T. vulgaris treatment but in vivo activities remain to be elucidated.     

CD14 Expression and Binding of Lipopolysaccharide to Alveolar Macrophages and Monocytes

We have studied the expression of the lipopolysaccharide (LPS) receptor CD14 on monocytes (Mo) and alveolar macrophages (AM), including density- and size-defined subpopulations. Bronchoalveolar lavage (BAL) was performed on eleven healthy non-smokers and blood sampled from 5 of them, and the levels of cell CD14 expression was investigated using flow cytometry. The influence of LPS stimulation on the CD14 expression of AM was studied at various intervals during prolonged incubation. Further, the relationship between CD14 expression and LPS binding to Mo and subpopulations of AM was studied by measuring fluorescein isothiocyanate (FITC)-LPS binding (flow cytometry) and binding of radioiodinated LPS (^I25I-LPS). The CD14 expression was 13-fold higher (P < 0.02) on Mo than on unfractionated and high density AM. The CD14 level on the latter was higher than on low density AM, and also higher (P < 0.05) on small AM compared to large (flow cytometrically defined) AM. LPS stimulation had a downregulating effect on AM CD14 level, but after several hours of continuing decreased expression, an increased (P < 0.05) CD14 expression was demonstrated, indicating de novo synthesis. The binding of LPS to subpopulations of AM and isolated Mo was not significantly different, but the binding of FITC-LPS to Mo in whole blood was higher than to AM (P < 0.02). The presented results indicate that AM of different size and maturity have different and variable (activation dependent) CD14 levels. The LPS binding capacity was, however, not proportional to the CD14 expression, indicating that LPS binding mechanisms unrelated to CD14 levels were also operable.     

Histological Changes in Male Accessory Reproductive Organs in Rats Exposed to Cigarette Smoke and the Protective Effect of Honey Supplementation

The effect of cigarette smoke (CS) on histology of male accessory reproductive organs and the possible protective effect of honey supplementation in rats were investigated in this study. Rats received distilled water, honey, CS exposure or honey plus CS exposure. Honey (1.2 g/kg body weight/day) was administered by gavage and CS exposure (3 times per day) was done in a chamber for 13 weeks. CS exposure significantly increased relative weight of epididymis and ventral prostate. There were also significantly increased number of clear cells and epithelial height of cauda epididymis as well as severe interstitial oedema and decreased epithelial height of prostate gland. However, with the supplementation of honey, these histological changes were significantly reversed suggesting the protective effect of honey against the toxic effect of CS on male accessory reproductive organs in rats.     

Ultrastructural Heterogeneity of the Alveolar Macrophages from Tobacco Smokers with Chronic Bronchitis

Alveolar macrophages recovered by bronchoalveolar lavage from 14 heavy smokers with chronic bronchitis were assessed. Ultrastructural examination revealed marked cellular heterogeneity. Three subpopulations of alveolar macrophages were readily identifiable. These have been termed "young," "mature," and "degrading," reflecting their ultrastructural features. In addition, a majority of the cells were found to be positive by TUNEL staining, indicating DNA damage, but a very small percentage tested positive for Caspase-3, suggesting that apoptosis might not account for the DNA damage in at least some of these cells. A small percentage of proliferating cells were noted.     

Human Alveolar Macrophages Synthesize the Functional Alternative Pathway of Complement and Active C5 and C9 in Vitro

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.     

Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages

In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.     

Functional Relationship of Macrophages and Basophils to the Thymus Gland

Human thymus epithelium, depleted of thymocytes and macrophages by means of organ culture, was used in chemotaxis experiments with peripheral blood cells. Such cultured thymus epithelium can attract specifically macrophages and basophils. T-lymphocytes were attracted only by short-term (8 day) cultured thymus tissue which still retains some of the original macrophage population. Thymic macrophages formed rosette structures with thymocytes. In other experiments 'activated' rabbit macrophages had the capability to destroy thymocytes, whether autochthonous or allogeneic. The possible role of macrophages and basophils in thymus function is discussed.