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Alveolar epithelial type II cells activate alveolar macrophages and mitigate P. Aeruginosa infection.

Kannan S, Huang H, Seeger D, Audet A, Chen Y, Huang C, Gao H, Li S, Wu M - PLoS ONE (2009)

Bottom Line: The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies.Mechanistically, we found that Lyn mediated NFkappaB activation led to increased gene expression and secretion of MCP-1.Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota, USA.

ABSTRACT
Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1(-/-) mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1(-/-) mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1(-/-) mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFkappaB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn(-/-) mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.

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Conditioned medium from AECII by PAO1 infection activated AM (12 h).(A) Increase of AM migration as determined by chemotaxic chamber methods (see Material and Methods). Migration index was calculated based on conditioned medium or control medium. (B) Increase in phagocytosis of AM as detected by CFU (colony forming units). (C) Increase in superoxide production of AM analyzed by H2DCF fluorescence (Molecular Probes). (D) The morphology of the activated AM is shown typically with lamllipodium and filopodium by confocal microscopy. (E) The morphology of the activated AM is further evidenced by high resolution and magnification Scanning Electron Microscopy (SEM). Statistical analysis was done by comparing mean individual values versus controls using student's t test, *p<0.05 (95% Confidence Interval, CI). The results are representative of three experiments.
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pone-0004891-g001: Conditioned medium from AECII by PAO1 infection activated AM (12 h).(A) Increase of AM migration as determined by chemotaxic chamber methods (see Material and Methods). Migration index was calculated based on conditioned medium or control medium. (B) Increase in phagocytosis of AM as detected by CFU (colony forming units). (C) Increase in superoxide production of AM analyzed by H2DCF fluorescence (Molecular Probes). (D) The morphology of the activated AM is shown typically with lamllipodium and filopodium by confocal microscopy. (E) The morphology of the activated AM is further evidenced by high resolution and magnification Scanning Electron Microscopy (SEM). Statistical analysis was done by comparing mean individual values versus controls using student's t test, *p<0.05 (95% Confidence Interval, CI). The results are representative of three experiments.

Mentions: Our objective was to test whether AECII in a culture system can boost the immune function of AM. Through co-culturing macrophages with lung epithelial cells, macrophages were significantly activated, demonstrating multiple functional activities including enhanced migration and actin reorganization (Figure S1A, B). To investigate whether secreted (soluble) substances are major mediators, we collected a conditioned medium from primary mouse AECII following PAO1 infection for 1 h and added to primary AM culture. After 24 h, AM activity was detected by three measurements: migration (Figure 1A), phagocytosis (Figure 1B) and superoxide production (Figure 1C). Our data indicates that the three AM function parameters were significantly increased by the AECII conditioned medium compared to the control medium from AECII without infection (P<0.05, Student's t test). The activated AM also showed more significant morphological changes with membrane projections such as lamellipodia and filopodia by confocal microscopy (Figure 1D). Moreover, we demonstrated apparent morphology alterations by scanning electron microscopy (SEM) (Figure 1E). Although we found that AECII have a role in activating AM, it is possible that AM themselves can be activated by P. aeruginosa, which enhances the function of resting AM through an autocrine loop. Thus, we evaluated whether the activated AM can bolster immunity by stimulating other naïve AM. We co-incubated resting AM with a conditioned medium that was derived from AM following P. aeruginosa time course infection. As expected, we have found that the AECII conditioned medium indeed showed greater increases in AM migration and superoxide production compared to the AM-conditioned medium, particularly at earlier times (6 h, Figure S1C). However, AM also secreted comparable MCP-1 possibly through autocrine activation at 24 h.


Alveolar epithelial type II cells activate alveolar macrophages and mitigate P. Aeruginosa infection.

Kannan S, Huang H, Seeger D, Audet A, Chen Y, Huang C, Gao H, Li S, Wu M - PLoS ONE (2009)

Conditioned medium from AECII by PAO1 infection activated AM (12 h).(A) Increase of AM migration as determined by chemotaxic chamber methods (see Material and Methods). Migration index was calculated based on conditioned medium or control medium. (B) Increase in phagocytosis of AM as detected by CFU (colony forming units). (C) Increase in superoxide production of AM analyzed by H2DCF fluorescence (Molecular Probes). (D) The morphology of the activated AM is shown typically with lamllipodium and filopodium by confocal microscopy. (E) The morphology of the activated AM is further evidenced by high resolution and magnification Scanning Electron Microscopy (SEM). Statistical analysis was done by comparing mean individual values versus controls using student's t test, *p<0.05 (95% Confidence Interval, CI). The results are representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654511&req=5

pone-0004891-g001: Conditioned medium from AECII by PAO1 infection activated AM (12 h).(A) Increase of AM migration as determined by chemotaxic chamber methods (see Material and Methods). Migration index was calculated based on conditioned medium or control medium. (B) Increase in phagocytosis of AM as detected by CFU (colony forming units). (C) Increase in superoxide production of AM analyzed by H2DCF fluorescence (Molecular Probes). (D) The morphology of the activated AM is shown typically with lamllipodium and filopodium by confocal microscopy. (E) The morphology of the activated AM is further evidenced by high resolution and magnification Scanning Electron Microscopy (SEM). Statistical analysis was done by comparing mean individual values versus controls using student's t test, *p<0.05 (95% Confidence Interval, CI). The results are representative of three experiments.
Mentions: Our objective was to test whether AECII in a culture system can boost the immune function of AM. Through co-culturing macrophages with lung epithelial cells, macrophages were significantly activated, demonstrating multiple functional activities including enhanced migration and actin reorganization (Figure S1A, B). To investigate whether secreted (soluble) substances are major mediators, we collected a conditioned medium from primary mouse AECII following PAO1 infection for 1 h and added to primary AM culture. After 24 h, AM activity was detected by three measurements: migration (Figure 1A), phagocytosis (Figure 1B) and superoxide production (Figure 1C). Our data indicates that the three AM function parameters were significantly increased by the AECII conditioned medium compared to the control medium from AECII without infection (P<0.05, Student's t test). The activated AM also showed more significant morphological changes with membrane projections such as lamellipodia and filopodia by confocal microscopy (Figure 1D). Moreover, we demonstrated apparent morphology alterations by scanning electron microscopy (SEM) (Figure 1E). Although we found that AECII have a role in activating AM, it is possible that AM themselves can be activated by P. aeruginosa, which enhances the function of resting AM through an autocrine loop. Thus, we evaluated whether the activated AM can bolster immunity by stimulating other naïve AM. We co-incubated resting AM with a conditioned medium that was derived from AM following P. aeruginosa time course infection. As expected, we have found that the AECII conditioned medium indeed showed greater increases in AM migration and superoxide production compared to the AM-conditioned medium, particularly at earlier times (6 h, Figure S1C). However, AM also secreted comparable MCP-1 possibly through autocrine activation at 24 h.

Bottom Line: The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies.Mechanistically, we found that Lyn mediated NFkappaB activation led to increased gene expression and secretion of MCP-1.Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota, USA.

ABSTRACT
Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1(-/-) mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1(-/-) mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1(-/-) mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFkappaB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn(-/-) mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.

Show MeSH
Related in: MedlinePlus