Activation Of Human Alveolar Macrophages Research Articles

Reduced phagocytic activity of human alveolar macrophages infected with Mycobacterium avium complex

IntroductionCo-infection of nontuberculous mycobacteria (NTM) with other bacteria is associated with increased frequency of hospitalization and reduced quality of life. However, the clinical significance of co-infection with NTM and other bacteria remains unclear. Here, we investigated the distribution of alveolar macrophage populations, characterized their phagocytic function in bronchoalveolar lavage fluid (BALF), and assessed the bactericidal function of macrophages infected with NTM using cell lines. MethodsBALF samples were prospectively obtained from 30 patients with suspected NTM lung disease to evaluate phagocytic activities of macrophages using immunostaining. Bactericidal activities of Staphylococcus aureus (S. aureus) and Mycobacterium intracellulare (M. intracellulare)-infected or -non-infected macrophages were evaluated using macrophage cell lines. ResultsEleven patients with Mycobacterium avium complex (MAC) infection and 19 patients with chronic lower respiratory tract infections except for NTM infection (controls) were enrolled. The percentage of non-polarized (HLA-DR+, CD40−, and CD163−) macrophages in patients infected with MAC was significantly higher than that in controls; non-polarized macrophages demonstrated an impaired ability to phagocytose S. aureus. In vitro experiments revealed higher intracellular S. aureus colony-forming unit counts and proinflammatory cytokine levels in M. intracellulare-infected macrophages than in non-NTM-infected macrophages. Electron microscopy showed morphologically damaged macrophages and M. intracellulare and S. aureus growing in the same phagosome. ConclusionThe proportion of alveolar macrophages (HLA-DR+, CD40−, and CD163−) with impaired phagocytosis increased in MAC-infected individuals. M. intracellulare-infected macrophages reduced bactericidal activity in vitro. Dysfunction of alveolar macrophages may contribute to persistent infection by other bacteria, leading to MAC lung disease progression.

Adenosine A2A receptors are up-regulated and control the activation of human alveolar macrophages

Chronic inflammatory lung diseases remain a health concern and new anti-inflammatory treatments are needed. Targeting adenosine A2A receptors (A2AR) affords robust anti-inflammatory effects in animal models, but the translation of this promising strategy to humans has been challenging, possibly due to interspecies differences in receptor distribution and effects. Thus, we now assessed the efficiency of a selective A2AR agonist to control the activation of fresh human alveolar inflammatory cells. We collected bronchoalveolar lavage fluid from patients with interstitial lung disease and loaded alveolar cells with the intracellular free calcium probe FURA-2/AM. Calcium transients were then recorded in response to superfusion with a proinflammatory peptide (N-formylmethionyl-leucyl-phenylalanine - FMLP), in the absence or presence of the selective A2AR agonist CGS21680. In a second experiment, cells were continuously exposed to FMLP and A2AR density was assessed by immunocytochemistry. Sixteen patients were included, nine for analysis of calcium transients, and seven for immunocytochemistry. When alveolar macrophages were exposed to 100 nM FMLP for 120 s, a peak elevation of intracellular free calcium levels (97.0% over baseline) was recorded; CGS21680 (100 and 300 mM) significantly reduced this peak to 89.5% and 81.5%, respectively. The immunofluorescence analysis revealed a time-dependent increase of A2AR density in alveolar macrophage upon exposure to 1 μM FMLP, up to 148% of control at 6 h. These results show that pro-inflammatory stimuli up-regulate A2AR and their activation dampens the impact of pro-inflammatory stimuli. This supports that targeting A2AR is a promising therapy for human lung inflammatory diseases, especially for diseases with a strong inflammatory component.

Immunity, gut microbiota and infection

Recurrent infections are very common in children and a major challenge for pediatricians. They affect the children’s quality of life, cause absences from school and lost parental working days, and repeated medical examinations, hospital admissions as well as antibiotic therapies lead to high costs for society. Given their prevalence and clinical importance, various prevention strategies have been developed. Some of these strategies have considered that innate and adaptive immunity are strictly related with gut microbiota and interact in the modulation of host’s reactivity to infections. Variations in gut microbiota related to age, nutrition or underlying disease may influence immune system defenses against viruses and bacteria. Furthermore, disturbed gut colonization patterns are proposed to be associated with the development of allergic disease. This explains why probiotics represent an important option in the prophylaxis and the management of recurrent infectious diseases as well as allergic diseases. However, the incomplete understanding of what constitutes a healthy gut microbiota that promotes tolerance, remains a challenge. Further understanding of gut microbial functions may pave the way for more effective prevention and treatment strategies. Another approach used for prevention of recurrent infections is represented by the administration of immunostimulants: i.e. molecules of bacterial or synthetic origin that interact with immunological mechanisms in vitro and in vivo. Pidotimod (3-L-pyroglutamil-L-thiaziolidine-4-carboxylic acid) is a synthetic dipeptide which stimulates the phagocytic activity of polymorphonuclear leukocytes, enhances the killing activity of human alveolar macrophages, increases the cytotoxic activity of natural killer cells, and stimulates cytokine production. It also influences the maturation and activity of dendritic cells by increasing the expression of the key surface markers that help to trigger T cell activation and increased IL-12 production. The majority of studies have shown that the number of infections decreases after pidotimod treatment, but they are affected by some methodological weaknesses. Further studies are urgently needed to confirm the pediatric population that should have the greatest benefit with the administration of immunostimulants. More studies are needed to identify the most promising probiotic strains and study populations, and to evaluate the mechanisms behind the possible effects of probiotics on OM.

Activation of Human Alveolar Macrophages via P2 Receptors: Coupling to Intracellular Ca2+ Increases and Cytokine Secretion

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X(1), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(13), and P2Y(14). We also showed that extracellular ATP induced Ca(2+) transients and increased IL-1beta secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-alpha, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.

Mechanism of Intracellular Candidacidal Activity Mediated by Calcium Ionophore in Human Alveolar Macrophages

In the present study, we investigated the effect of in vitro treatment with calcium ionophore (A23187) on candidacidal activity of human alveolar macrophages (AM) from normal subjects. In vitro incubation of AM with A23187 results in a significant dose-dependent enhancement of candidacidal activity. The availability of Ca2+ and Mg2+ ions in the culture medium is crucial for phagocytosis and killing to occur, but appears irrelevant for the binding between Candida albicans and AM. Enhancement of the killing effect mediated by A23187 does not correlate with increased phagocytic activity; in fact, the availability of ions is required for the phagocytic event, but an increase of cations does not correlate with enhancement of this activity. On the contrary, the augmentation of killing activity correlates with increased production of superoxide anion. Moreover, soluble material endowed with candidacidal activity has been extracted from cytoplasmic granules of AM both unstimulated and following A23187 treatment in vitro. Indeed, the granules extracted contain cationic proteases and, when isolated from stimulated cells, appear to be significantly more cytotoxic for C. albicans with respect to those obtained from unstimulated AM. In conclusion, the results reported here show that the phagocytic and killing events are ion dependent and the enhancement of intracellular candidacidal activity mediated by A23187 in AM is correlated with an augmented anti-Candida activity of cation-activated proteases.

Characterization of Suppressor Function of Human Alveolar Macrophages for T Lymphocyte Responses to Phytohemagglutinin: Cellular Selectivity, Reversibility, and Early Events in T Cell Activation

The predominant immunoregulatory activity of alveolar macrophages (AM) on T lymphocytes is to suppress their responses to antigenic and mitogenic stimuli. The suppressive activity of human AM for T cell responses to phytohemagglutinin (PHA) was further characterized. At ratios of AM to T lymphocytes of 0.4:1 to 1.6:1, AM inhibited the blastogenic response (3H-thymidine uptake into DNA) to PHA by 26 to 87%, respectively. Blood monocytes precultured in vitro for 5 to 7 days inhibited responses to PHA similarly. Freshly isolated blood monocytes, peritoneal macrophages, and A-549 epithelial and CCD-18Lu fibroblast cell lines failed to inhibit T lymphocyte responses. AM were capable of suppressing PHA-induced blastogenesis of purified CD4 cells without the addition of other cells. Cell contact was required for suppression of CD4 cells, as demonstrated using dual chambers. T cells precultured with AM with or without PHA retained the ability to respond to PHA compared with control T cells not precultured with AM. Kinetic experiments showed that AM needed to be added at the initiation of a 3-day culture period for suppression to occur. Analysis of the T cell DNA cycle revealed that AM decreased the percentage of cells entering the synthesis phase of DNA production. Flow cytometry also was used to assess the effect of AM on early markers of T cell activation. AM inhibited the percentage of T cells expressing the interleukin-2 receptor 46 to 83% and the transferrin receptor 58 to 78% at 24 to 48 h after stimulation with PHA. There was no effect of AM on expression of HLA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

Requirement for Cell-to-Cell Contact for the Immunosuppressive Activity of Human Alveolar Macrophages

The ability of alveolar macrophages (AM) obtained by bronchoalveolar lavage of healthy volunteers to suppress T lymphocyte responses to the mitogen phytohemagglutinin (PHA) in vitro was investigated. AM but not monocytes (MN) inhibited responses of peripheral blood mononuclear cells (PBMC) to PHA as measured by incorporation of [3H]thymidine [( 3H]TdR) and interleukin-2 (IL-2) expression. Supernatants of AM generated for various periods and with various concentrations of cells did not, however, inhibit PBMC responses to PHA. To examine the role of cell contact in the inhibitory activity of AM, AM or MN were added to PBMC in 6-well plates either directly (in co-culture) or separated by a 0.45-micron filter. MN did not inhibit PBMC blastogenic responses under either condition. AM at a 1:2 ratio with PBMC inhibited blastogenesis by 75 +/- 11% (mean +/- SD, n = 3, P less than 0.01) when cultured directly with PBMC but had no inhibitory effect on blastogenesis when physically separated from target PBMC. AM in co-culture with PBMC also inhibited PHA-stimulated IL-2 production by 70% but did not inhibit IL-2 production when AM were separated from PBMC in dual chambers. To assess the role of the cell surface in the inhibitory activity of AM, AM and MN were fixed with 2% paraformaldehyde. Neither fixed nor unfixed MN inhibited PBMC blastogenic responses, but both fixed and unfixed AM inhibited responses similarly (77 to 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Short-Chain Fatty Acids Produced by Anaerobic Bacteria Inhibit Phagocytosis by Human Lung Phagocytes

The effect of short-chain fatty acids on the phagocytic activity of human alveolar macrophages and neutrophils was investigated. These acids, butyric, propionic, and succinic, are produced by anaerobic bacteria. The results indicate that phagocytosis of Staphylococcus aureus by human lung phagocytes is strongly inhibited by the end products of anaerobic catabolism and support the hypothesis that the antiphagocytic activity present in the supernatants of anaerobic cultures may be dependent on the presence of short-chain fatty acids.

Modulation of anti-Candida Activity of Human Alveolar Macrophages by Interferon-gamma or Interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.

Nedocromil Sodium Inhibition of IgE-Mediated Activation of Human Mononuclear Phagocytes and Platelets from Asthmatics1

The demonstration of a specific receptor for IgE on non-mast cell or basophil leucocytes, such as mononuclear phagocytes, eosinophils and platelets, suggests that these cells may participate directly in immunological disorders of allergy. In this study, inhibition by nedocromil sodium of IgE-dependent activation of human alveolar macrophages, blood monocytes and platelets was investigated. Nedocromil sodium produced an inhibition of IgE-mediated generation of cytotoxic molecules from monocytes and platelets together with a concomitant inhibition of their oxidative metabolism, measured by chemiluminescence. In addition, nedocromil sodium reduced the ability of alveolar macrophages to synthesise and release mediators, estimated by beta-glucuronidase activity. Furthermore, nedocromil sodium inhibited the abnormal response to aspirin of platelets from aspirin-sensitive asthmatics at therapeutic concentrations. These studies confirm that nedocromil sodium acts on a cell compartment other than the classical mast cell population in IgE-dependent allergy and asthma.

Measurement of angiotensin-converting enzyme activity in intact human alveolar macrophages and effect of smoking.

Serum angiotensin-converting enzyme (ACE) activity is known to be elevated in various granulomatous conditions such as sarcoidosis, whose characteristic epithelioid cells are thought to belong to the macrophage series. It was recently shown that alveolar macrophages had ACE activity in their sonicated and homogenate forms. We investigated ACE activity of human alveolar macrophages in the intact form using a sensitive radioimmunoassay of generated angiotensin II. We also investigated the effect of smoking on ACE activity of alveolar macrophages using this method. Alveolar macrophages generated angiotensin II (218 +/- 106 pg/20 min/10(5) cells). More angiotensin II was generated in smokers (246 +/- 119 pg/20 min/10(5) cells) than in nonsmokers (160 +/- 50 pg/20 min/10(5) cells) (p less than 0.05). These data showed that ACE activity of macrophages is higher in smokers than nonsmokers.

Inhibition by nedocromil sodium of IgE-mediated activation of human mononuclear phagocytes and platelets in allergy.

The demonstration of a specific receptor for IgE on nonmast cell or basophil leukocytes, such as mononuclear phagocytes, eosinophils, and platelets, suggests that these cells may participate directly in immunological disorders of allergy. Thus, a full understanding of the mode of action of antiallergic or antiasthma drugs must take into account their activity on these nonmast cell leukocytes. Consequently, inhibition by nedocromil sodium of IgE-dependent activation of human alveolar macrophages, blood monocytes and platelets, was investigated. This compound induced an inhibition of the IgE-mediated generation of cytotoxic molecules from monocytes and platelets, together with a concomitant inhibition of their oxidative metabolism, measured by chemiluminescence, and a reduction of the potential ability of alveolar macrophages to synthesize and release mediators, estimated by lysosomal beta-glucuronidase activity. These observations confirm the hypothesis that nedocromil sodium acts on a cell compartment other than the classical mast cell population, in IgE-dependent allergy and, more particularly, in asthma.

Human alveolar macrophage activation: Inhibition by forskolin but not β-adrenoceptor stimulation or phosphodiesterase inhibition

Alveolar macrophages are the most numerous cells within human airways. They release inflammatory mediators following immunological challenge and have been implicated in the pathogenesis of asthma. beta-agonists and phosphodiesterase inhibitors are frequently used in the treatment of asthma and are potent inhibitors of human mast cells. We have examined the role of the beta-agonist, isoprenaline, the phosphodiesterase inhibitor Ro-20 1724, and the adenylate cyclase stimulator forskolin on the activation of human alveolar macrophages. This was assessed by monitoring the release of thromboxane B2 (TXB2), leukotriene B4, N-acetyl-beta-D-glucosaminidase (NAG), and superoxide (SO) following stimulation of the cells by opsonised zymosan or IgE/anti IgE complexes. Neither isoprenaline (1nM-10 microM) nor Ro-20 1724 (0.5-50 microM) alone or in combination had any inhibitory effect on release of these mediators. However, forskolin (0.1-100 microM) significantly inhibited release of both TXB2 and SO but not NAG. This result shows that human alveolar macrophages do not possess functional beta-receptors, although stimulation of adenylate cyclase with forskolin, inhibits some of the elements of macrophage activation.

Procoagulant activity of human alveolar macrophages: Different expression in patients with lung cancer

Procoagulant activity of human alveolar macrophages: Different expression in patients with lung cancer

Procoagulant activity of human alveolar macrophages: different expression in patients with lung cancer.

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of many malignant tissues, might contribute to tumor-associated fibrin deposition through the production of procoagulant activity (PCA). We have studied the PCA of human alveolar macrophages in 28 patients with primary lung cancer and in 9 control subjects. Alveolar macrophages (greater than 97% esterase positive) were isolated form bronchoalveolar lavage fluids by adherence onto plastic. PCA was evaluated by a one-stage clotting assay immediately after isolation (basal PCA) and after incubation (4 hr at 37 degrees C) in the absence and in the presence of endotoxin. Cells from control subjects had low basal PCA (3.9 +/- 1.0 units/5 X 10(4) cells) but, upon exposure to endotoxin, they displayed a 5- to 16-fold increase in PCA. In patients, different patterns of PCA were observed. In the 8 patients in whom lavage had been carried out on the contralateral side to the neoplasm, alveolar macrophages behaved essentially like those from controls. In contrast, in the 20 patients in whom macrophage populations close to the site of the tumor were examined, PCA was abnormal in many respects. In 12 of these, alveolar macrophages had basal PCA comparable to or somewhat lower than control cells, but exhibited a poor procoagulant response when incubated in vitro in the presence of endotoxin. Alveolar macrophages from the remaining 8 patients expressed far higher levels of basal PCA than control cells (25.1 +/- 5.9 units as compared to 3.9 +/- 1.0 units/5 X 10(4) cells). These cells retained their ability to respond to endotoxin in vitro with a 3-fold increase in PCA. In all instances, alveolar macrophage PCA had the characteristics of tissue factor. These data suggest that the presence of primary lung cancer may modulate the expression of PCA in alveolar macrophages close to the tumor site. PCA might be useful to better characterize the functional state of macrophages near the tumor.

Opsonic activity of plasma fibronectin for Staphylococcus aureus by human alveolar macrophages: inefficacy of trypsin-sensitive staphylococcal fibronectin receptor.

The binding of 125I-fibronectin (FN) to some bacteria and the phagocytic activity of human alveolar macrophages (AM) by a culture method using 3H-labelled bacteria were examined. FN-binding of Staphylococcus aureus (S. aureus) was higher than that of gram-negative rods and was reduced markedly by trypsin-treatment. FN enhanced the uptake of S. aureus by AM in dose dependent manner. To determine whether FN-binding to S. aureus mediates the promotion of staphylococcal phagocytosis, we compared the uptake of non-treated S. aureus with that of trypsin-treated bacteria by AM. No significant difference was found between the uptake of these bacteria and no effect of FN-binding to S. aureus on the phagocytic activity. It is concluded that FN plays an essential role as a non-immune opsonin for S. aureus in the human lung which is found to be abundant in FN, and that FN-binding to S. aureus is probably not related to this effect.

Degradation of fibrin and elastin by intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation.

Fibrin deposition is prominent in the histopathology of a number of inflammatory lung diseases. Plasmin, activated locally in the lung, can degrade not only this fibrin but potentially structural proteins important to normal lung architecture. Because alveolar macrophages are prominent in inflammatory processes of the lung, we examined the plasminogen activator (PA) activity of human alveolar macrophages. Intact alveolar macrophages from each of 10 healthy subjects expressed PA activity. There was no difference in activity between smoking and nonsmoking individuals. The activator activity was largely cell-associated, but under certain culture conditions, macrophages released a soluble activator into the culture medium. The membrane-bound activator had an apparent molecular mass of 52-55 kD in nonreduced sodium dodecyl sulfate (SDS) gels, and monospecific antibody to urokinase neutralized the enzyme activity. Immunoprecipitation of [35S]methionine-labeled cells showed that human alveolar macrophages actually synthesize the PA in vitro. SDS-gel analysis of the immunoprecipitated material revealed the predominant species of PA to be structurally similar to reduced, active urokinase. We also examined the role of PA in the degradation of both insoluble fibrin and elastin matrices by live macrophages. Cells degraded an insoluble fibrin matrix in the presence of plasminogen whether or not the macrophages contacted the fibrin as long as proteinase inhibitors were not in the culture medium. In the presence of serum proteinase inhibitors, macrophages still degraded a fibrin matrix, but only if they were in contact with the fibrin. Live macrophages also degraded insoluble elastin only when in contact with the elastin but could do so even in the presence of serum proteinase inhibitors. In matrices containing a mixture of fibrin and elastin, cells did not degrade elastin unless plasminogen was added to the medium. These results indicate that normal alveolar macrophages synthesize and express, probably at the cell surface, a PA. The PA is physically and immunochemically similar to urokinase but is membrane bound. The PA is critical to the degradation of fibrin matrices by normal alveolar macrophages. Under tissue conditions where elastin is embedded within other structural proteins, the activator may be rate-limiting in elastin degradation as well. The findings also suggest that live macrophage proteolytic activity is relatively insensitive to the presence of serum proteinase inhibitors, suggesting a mechanism for proteolytic lung injury even in the presence of proteinase-proteinase inhibitor balance in the soluble phase.

Human alveolar macrophages: potentiation of their tumoricidal activity by liposome-encapsulated muramyl dipeptide.

Homogeneous preparations of human alveolar macrophages (AM) were obtained by bronchoalveolar lavage of the lungs of healthy nonsmokers. These AM exhibited a low level of spontaneous cytolytic activity against allogeneic A375 melanoma cells. Tumoricidal activity of AM could be generated and/or augmented by incubation in vitro with either lipopolysaccharides (LPS) or muramyl dipeptide (MDP). MDP derivatives were encapsulated within multilamellar (MLV) liposomes composed of phosphatidylcholine-phosphatidylserine (PC/PS). Dose-response experiments established that liposome-encapsulated MDP augmented the tumoricidal activity of AM at concentrations about 100 times lower than free MDP in the medium. AM activated by MLV containing MDP destroyed allogeneic tumor cell lines, but did not affect cultures of normal, non-neoplastic cells. An inactive derivative, MDP-L, encapsulated in MLV liposomes did not potentiate the tumoricidal activity of AM. We conclude that human AM respond to MDP in vitro and that liposomes containing MDP are far more efficient than unencapsulated, free MDP for enhancing the tumoricidal activity of human AM.