Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses.

The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.

Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

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-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha 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-alpha 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.

Characterization of Actinobacillus pleuropneumoniae antiviral effect against porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages.

Le syndrome reproducteur et respiratoire porcin (SRRP) est la maladie infectieuse la plus économiquement importante de l’industrie porcine. Une étude récente a démontré que le surnageant de culture d’Actinobacillus pleuropneumoniae (App) inhibe l’infection du virus SRRP (VSRRP) in vitro dans des cellules de singe. L’objectif de cette étude est de démontrer l’effet antiviral d’App contre le VSRRP dans les cellules cibles du virus in vivo: les macrophages alvéolaires porcins (MAPs) et d’étudier les mécanismes spécifiques impliqués lors de l’inhibition virale. Les MAPs ont été traités avec App, avant et après l’infection avec le VSRRP. À différents temps post-infection, la réplication et la transcription du génome viral ont été quantifiées. L’expression des interférons (IFN) type I et II, ainsi que le profil protéomique en présence ou absence d’App ont été évalués. L’expression de certaines protéines a été confirmée par immunobuvardage et immunofluorescence (IF). Les résultats ont démontré que l’effet antiviral d’App n’est pas via l’induction des IFN type I et II. App inhibe l’infection virale dans MAPs avant la réplication et la transcription du génome viral, ce qui indique qu’App inhibe précocement le cycle réplicatif viral. Le profil protéomique a révélé qu’App augmentait l’expression de la cofiline, une protéine qui provoque la dépolymérisation de l’actine. De plus, ce phénomène de dépolymérisation a été confirmé par IF. Le traitement des MAPs avec la cytochalasin D (un composé qui provoque la fragmentation des microfilments) a démontré que comme pour App, cette drogue inhibe la réplication virale. Les résultats obtenus suggèrent que l’effet antiviral d’App est via l'activation de la cofiline et dépolymérisation de l’actine, affectant probablement l’endocytose du VSRRP.

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Insulin inhibits LPS-induced signaling pathways in alveolar macrophages

The systemic inflammatory response syndrome ( SIRS) is triggered by lipopolysaccharide (LPS) from Gram-negative bacteria. Insulin was shown to have a protective role in SIRS related to sepsis. Lungs are particularly affected in this condition and provide a second wave of mediators/cytokines which amplifies SIRS. The aim of the present study was to investigate the effect of insulin on the signaling pathways elicited by LPS in alveolar macrophages (AMs) and its consequence in cellular response to LPS measured as production of tumor necrosis factor (TNF). To this purpose, resident AMs from male Wistar rats were obtained by lung lavage and stimulated by LPS ( 100 ng/mL). Insulin ( 1 mU/mL) was added 10 min before LPS. Activation ( phosphorylation) of signaling molecules by LPS was analyzed by western blot, 30 min after LPS stimulation. TNF was measured in the AMs culture supernatants by bioassay using L-929 tumor cells. Relative to controls, LPS induced a significant increase in the activation of ERK (3.6-fold), p38 (4.4-fold), Tyr-326 Akt (4.7-fold), Ser-473 Akt (6.9-fold), PKCa (4.7-fold) and PKCd (2.3-fold). Treatment of AMs with insulin before LPS stimulation, significantly reduced the activation of ERK (54%), p38 (48%), Tyr-326 Akt (64%), Ser-473 Akt (41%), PKCa (62%) and PKCd (39%). LPS induced TNF production in AMs which was also inhibited by insulin (60%). These results show that insulin down-regulates MAPK, PI3K and PKCs and inhibits a downstream effect of LPS, TNF production, in rat AMs stimulated with LPS and suggest that the protective effect of insulin in sepsis could be through modulation of signal transduction pathways elicited by LPS in lung macrophages. Copyright (c) 2008 S. Karger AG, Basel.

Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance Fc gamma R-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated Fc gamma R-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50: 1) or IgG-opsonized K. pneumoniae (30: 1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via Fc gamma R (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via Fc gamma R. Copyright (C) 2010 S. Karger AG, Basel

Differential kinase requirement for enhancement of Fc gamma R-mediated phagocytosis in alveolar macrophages by leukotriene B(4) vs. D(4)

In alveolar macrophages, leukotriene (IT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gamma R)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gamma R-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gamma R engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gamma R-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gamma R-mediated phagocytosis reflect their differential activation of specific kinase programs. (C) 2008 Elsevier Ltd. All rights reserved.

Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection

Alveolar macrophages ( AM) are the first host cells to interact with Paracoccidioides brasiliensis (Pb), a primary human pathogen that causes severe pulmonary infections in Latin America. To better understand innate immunity in pulmonary paracoccidioidomycosis, we decided to study the fungicidal and secretory abilities of AM from resistant (A/J) and susceptible (B10.A) mice to infection. Untreated, IFN-gamma and IL-12 primed AM from B10. A and A/J mice were challenged with P. brasiliensis yeasts and cocultured for 72 h. B10. A macrophages presented an efficient fungicidal ability, were easily activated by both cytokines, produced high levels of nitric oxide ( NO), IL-12, and MCP-1 associated with low amounts of IL-10 and GM-CSF. In contrast, A/J AM showed impaired cytokine activation and fungal killing, secreted high levels of IL- 10 and GM-CSF but low concentrations of NO, IL- 12, and MCP-1. The fungicidal ability of B10. A but not of A/J macrophages was diminished by aminoguanidine treatment, although only the neutralization of TGF-beta restored the fungicidal activity of A/J cells. This pattern of macrophage activation resulted in high expression of MHC class II antigens by A/J cells, while B10. A macrophages expressed elevated levels of CD40. Unexpectedly, our results demonstrated that susceptibility to a fungal pathogen can be associated with an efficient innate immunity, while a deficient innate response can ultimately favor the development of a resistant pattern to infection. Moreover, our data suggest that different pathogen recognition receptors are used by resistant and susceptible hosts to interact with P. brasiliensis yeasts, resulting in divergent antigen presentation, acquired immunity, and disease outcomes.

Impaired phagocytosis by alveolar macrophages from diabetic rats is related to the deficient coupling of LTs to the Fe gamma R signaling cascade

Diabetic individuals are more susceptible to infections and this seems to be related to impaired phagocyte function. Alveolar macrophages (AMs) are the first barrier to prevent respiratory infections Leukotrienes (LTs) increase AM phagocytic activity via Fc gamma R. In this study, we compared AMs from diabetic and nondiabetic rats for phagocytosis via Fc gamma R and the roles of LTs and insulin Diabetes was induced in male Wistar rats by alloxan (42 mg/kg, i.v); macrophages were obtained by bronchoalveolar lavage and IgG-opsonised sheep red blood cells (IgG-SRBC) were used as targets. LTs were added to the AMs 5 min before the addition of IgG-SRBC. AMs were treated with a LT synthesis inhibitor (zileuton, 10 mu M), or antagonists of the LTB(4) receptor (CP105 696, 10 mu M) cys-LT receptor (MK571, 10 mu M), 30 or 20 min before the addition of IgG-SRBC, respectively. We found that the phagocytosis of IgG-SRBC by AMs from diabetic rats is impaired compared with non-diabetic rats. Treatment with the LT inhibitor/antagonists significantly reduced AM phagocytosis in non-diabetic but not diabetic rats. During the phagocytosis of IgG-SRBC LTB(4) and LTC(4) were produced by AMs from both groups. The addition of exogenous LTB(4) or LTD(4) potentiated phagocytosis similarly in both groups Phagocytosis was followed by the phosphorylation of PKC-delta. ERK and Akt This was reduced by zileuton treatment in AMs from non-diabetic but not diabetic rats The addition of insulin to AMs further increased the phagocytosis by increasing PKC-delta phosphorylation These results suggest that the impaired phagocytosis found in AMs from diabetic rats is related to a deficient coupling of LTs to the Fc gamma R signaling cascade and that insulin has a key role in this coupling An essential role for insulin in Innate immunity is suggested (C) 2010 Elsevier Ltd. All rights reserved.

Experimental murine mycobacteriosis: evaluation of the functional activity of alveolar macrophages in thalidomide- treated mice

Thalidomide is a selective inhibitor of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in mycobacterial death mechanisms. We investigated the role of this drug in the functional activity of alveolar macrophages in the presence of infection induced by intranasal inoculation of Mycobacterium avium in thalidomide-treated and untreated adult Swiss mice. Sixty animals were inoculated with 5 x 10(6) M. avium by the respiratory route. Thirty animals received daily thalidomide (30 mg/kg mouse) and 30 received water by gavage up to sacrifice. Ten non-inoculated mice were used as a control group. Lots of animals from each group were evaluated until 6 weeks after inoculation. Infection resulted in an increased total number of inflammatory cells as well as increased activity of pulmonary macrophages. Histologically, intranasal inoculation of bacilli resulted in small mononuclear infiltrates located at the periphery of the organ. Culture of lung fragments revealed the presence of bacilli only at the beginning and at the end of the experimental period. Thalidomide administration did not affect the microbiological or histological features of the infection. Thalidomide-treated and untreated animals showed the same amount of M. avium colonies 3 weeks after infection. Although it did not affect bacillary clearance, thalidomide administration resulted in a decreased percent of spread cells and release of hydrogen peroxide, suggesting that factors other than TNF-alpha play a role in the killing of mycobacteria by alveolar macrophages. Thalidomide administration also reduced the number of spread cells among resident macrophages, suggesting a direct effect of the drug on this phenomenon.