Early Inhibition of Mycobacterial Growth by Human  Alveolar Macrophages is not Due to Nitric Oxide

Aston, Christopher E.; Rom, William N.; Talbot, Anita; Reibman, Joan

doi:10.1164/ajrccm.157.6.9705028

Cited by 69 publications

(62 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, the bactericiostatic activity of macrophages cultured at high density could not be attributed to the preferential generation of NO by these cells, because treatment with the inducible NO synthase inhibitor L-NIL did not influence their ability to control mycobacterial replication. Consistent with these observations, prior studies have shown that although human macrophages express inducible NO synthase after mycobacterial infection (35,36), the levels of NO produced are low, and the inhibition of NO production by human macrophages does not reproducibly increase mycobacterial growth (36,37). Increased apoptosis of macrophages infected with mycobacteria has been described and in some but not all cases has been associated with mycobacterial killing (38 -41).…”

Section: Figuresupporting

confidence: 52%

Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth

Boéchat

Bouchonnet

Bonay

et al. 2001

The Journal of Immunology

View full text Add to dashboard Cite

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 × 104 cells/well, but increasing the number of cells to 2 × 105 cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 × 104 cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

show abstract

Section: Figuresupporting

confidence: 52%

Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth

Boéchat

Bouchonnet

Bonay

et al. 2001

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…In addition, it has been shown that AM in lung sections from patients with IPF [9], chronic obstructive pulmonary disease (COPD) [23] and sarcoidosis [2] display strong immunoreactivity for NOS2 protein in situ. However, many attempts to induce NOS2 mRNA expression and/or NO production by human AM in vitro failed [7,24,25]. As shown in the present investigation, AM isolated from lung tissue samples exhibited immunoreactive NOS2 protein ex vivo and its level was preserved or slightly decreased during 18-h culture periods with or without stimuli such as pro-inflammatory cytokines.…”

Section: Discussionmentioning

confidence: 47%

“…Although several investigators have shown that human AM can be induced to express NOS2 in vivo, their ability to express NOS2 and to generate NO in response to an in vitro stimulation is still under debate [6,7]. One reason for this controversy may arise from complex inflammatory processes within the alveoli responsible for NOS2 messenger ribonucleic acid (mRNA) expression, enzyme production, and NO generation by human AM.…”

mentioning

confidence: 99%

Human alveolar epithelial cells induce nitric oxide synthase‐2 expression in alveolar macrophages

Pechkovsky¹,

Zissel²,

Stamme³

et al. 2002

Eur Respir J

View full text Add to dashboard Cite

It was hypothesized that cell-to-cell interaction between human alveolar macrophages (AM) and alveolar epithelium, might be an important factor leading to nitric oxide synthase-2 (NOS2) messenger ribonucleic acid (mRNA) and protein expression by constituent cells of the alveolar wall and/or AM.NOS2 mRNA and the protein expression patterns of human AM and alveolar epithelial cells type II (AEC-II) isolated from normal parts of lung resections of patients with pulmonary malignancies were determined. In addition, NOS2 mRNA expression in human AM co-cultured with autologous AEC-II in the presence of pro-inflammatory cytokines interleukin (IL)-1b, tumour necrosis factor (TNF)-a, interferon (IFN)-c or lipopolysaccharide (LPS) was investigated. The effect of human surfactant protein-A (SP-A) on IFN-c-mediated NOS2 mRNA expression in human AM was also studied.Neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated AEC-II. In contrast, freshly isolated AM from bronchoalveolar lavage or lung tissue samples expressed immunoreactivity for NOS2 protein, but no NOS2 mRNA could be detected by reverse transcriptase polymerase chain reaction. All stimuli tested failed to induce NOS2 mRNA expression in human AM in vitro. Only AM-AEC-II co-culture in the presence of IFN-c led to NOS2 mRNA and protein expression. In situ hybridization of NOS2 mRNA on lung tissue explants and immunohistochemical staining of cytospin preparations of AM-AEC-II co-cultures demonstrated that NOS2 is expressed in AM but not in AEC-II. This co-culture effect could not be reproduced by substitution of AEC-II with SP-A.These data give evidence of a regulatory network controlling human nitric oxide synthase-2 expression in the lower respiratory tract.

show abstract

“…Although several reports concerning NOS2 expression in normal human lung tissue have provided evidence that peripheral lung parenchyma, as well as resident lung macrophages, do not express NOS2 mRNA (10) and that no specific immunostaining for NOS2 protein can be observed in alveolar compartments (18), the accumulation of NOS2 protein and NO production have been documented in AM isolated from patients with active pulmonary tuberculosis (22) and acute respiratory distress syndrome (17). However, many attempts to induce NOS2 mRNA expression and/or NO production by human AM in vitro failed (5,7,10). In contrast to data from Guo et al (10) and Kobzik et al (18), but in accordance with our findings, Saleh et al (31) demonstrated a weak expression of nitrotyrosine and NOS2 in the airway epithelium and AM and abundant expression of NOS3 in the pulmonary endothelium and airway epithelium of normal parts of human lungs obtained by pneumectomy performed because of lung cancer.…”

Section: Discussionmentioning

confidence: 99%

Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II

Pechkovsky

Zissel

Goldmann

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-␥, interleukin-1␤, and tumor necrosis factor-␣, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24-or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.nitric oxide synthase; mRNA; RT-PCR; interferon-␥; tumor necrosis factor-␣; interleukin-1␤; type II human alveolar epithelial cells; A549 cell line NITRIC OXIDE SYNTHASES (NOS) constitute a family with at least three distinct isoforms; neuronal (nNOS or NOS1), inducible (iNOS or NOS2) and endothelial (eNOS or NOS3) (20, 21). NOS1 and NOS3 are constitutively active and produce small amounts of nitric oxide (NO), whereas NOS2 is induced by inflammatory cytokines and produces a larger amount of NO. All three types of NOS are expressed in human lung, and NOS mRNA expression and enzyme activity have been described in human bronchial epithelial cells, macrophages, endothelial cells, and vascular smooth muscle cells (18,34). However, relatively little is known regarding the expression of NOS2 and NOS3 in type II human alveolar epithelial cells (AEC II) and how these enzymes are regulated. Although several laboratories have reported that rat AEC II express NOS2 mRNA and produce NO in vitro (12,26) and that human alveolar epithelium exhibits an NOS-like enzymatic activity in vivo (18), there has been neither molecular nor biochemical confirmation of the expression of NOS2 and NOS3 in human primary cultured AEC II. Asano et al. (4) and Kwon and George (19) have demonstrated that the human type II alveolar epithelial cell line (A549) expresses NOS2 and produces NO in response to proinflammatory cytokines such as interferon-␥ (IFN-␥), interleukin-1␤ (IL-1␤), and tumor necrosis factor-␣ (TNF-␣). Therefore, the aims of the present study were 1) to determine whether human AEC II express NOS2 and NOS3 mRNA in primary culture, 2) to characterize the regulation of mRNA expression of NOS isoforms in these cells by proinflammatory cytokines, and 3) to compare these...

show abstract

Early Inhibition of Mycobacterial Growth by Human Alveolar Macrophages is not Due to Nitric Oxide

Cited by 69 publications

References 25 publications

Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth

Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth

Human alveolar epithelial cells induce nitric oxide synthase‐2 expression in alveolar macrophages

Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II

Contact Info

Product

Resources

About