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SP-D counteracts GM-CSF-mediated increase of granuloma formation by alveolar macrophages in lysinuric protein intolerance.

Douda DN, Farmakovski N, Dell S, Grasemann H, Palaniyar N - Orphanet J Rare Dis (2009)

Bottom Line: At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain.Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.We propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lung Innate Immunity Research, Program in Physiology and Experimental Medicine, Research Institute, The Hospital For Sick Children, Toronto, Ontario, M5G 1X8, Canada. david.douda@utoronto.ca

ABSTRACT

Background: Pulmonary alveolar proteinosis (PAP) is a syndrome with multiple etiologies and is often deadly in lysinuric protein intolerance (LPI). At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain. We hypothesized that GM-CSF and surfactant protein D (SP-D) would enhance the clearance of proteins and dying cells that are typically present in the airways of PAP lungs.

Methods: Cells and cell-free supernatant of therapeutic bronchoalveolar lavage fluid (BALF) of a two-year-old patient with LPI were isolated on multiple occasions. Diagnostic BALF samples from an age-matched patient with bronchitis or adult PAP patients were used as controls. SP-D and total protein content of the supernatants were determined by BCA assays and Western blots, respectively. Cholesterol content was determined by a calorimetic assay or Oil Red O staining of cytospin preparations. The cells and surfactant lipids were also analyzed by transmission electron microscopy. Uptake of Alexa-647 conjugated BSA and DiI-labelled apoptotic Jurkat T-cells by BAL cells were studied separately in the presence or absence of SP-D (1 microg/ml) and/or GM-CSF (10 ng/ml), ex vivo. Specimens were analyzed by light and fluorescence microscopy.

Results: Here we show that large amounts of cholesterol, and large numbers of cholesterol crystals, dying cells, and lipid-laden foamy alveolar macrophages were present in the airways of the LPI patient. Although SP-D is present, its bioavailability is low in the airways. SP-D was partially degraded and entrapped in the unusual surfactant lipid tubules with circular lattice, in vivo. We also show that supplementing SP-D and GM-CSF increases the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo. Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.

Conclusions: We propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI. To improve the lung condition of LPI patients with PAP, it would be useful to explore alternative therapies for increasing dead cell clearance while decreasing cholesterol content in the airways.

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SP-D and GM-CSF affect the shape and size of the cells. (A-D) Adherent viable cells were grown in the absence or presence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml) for 24 h. (A) Cells without any added proteins have relatively the same size and shape. (B) In the presence of SP-D, more cells became elongated in shape (arrows). (C) In the presence of GM-CSF, more cells became small, round or oval (arrowheads). (D) In the presence of both SP-D and GM-CSF, both populations (arrowheads, oval; arrow, elongated) are present. (E) Average cell size shown in the above conditions. Presence of GM-CSF significantly reduced cell size, reflecting the formation of small and spherical/oval cells. The sizes of at least 50 cells were measured in each category. Data are presented as means ± SEM; n = two independent experiments. The means were compared with respective controls using Dunnett's multiple mean comparison procedure (* p < 0.05). (F) Over one week time, many BAL cells changed into elongated and stopped internalizing foreign materials (labeled BSA). Number of cells that actively internalize the foreign material was quantified from the randomly selected images obtained in uptake assays. The increase in cell size was associated with decrease in % cells that internalize BSA. For percent cells that actively internalize, data are presented as the proportion of cells that were able to take up Alexa fluor 647-conjugated BSA in total number of cells present. The mean values were compared with Day1 value using Dunnett's multiple mean comparison procedure (* p < 0.05).
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Figure 4: SP-D and GM-CSF affect the shape and size of the cells. (A-D) Adherent viable cells were grown in the absence or presence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml) for 24 h. (A) Cells without any added proteins have relatively the same size and shape. (B) In the presence of SP-D, more cells became elongated in shape (arrows). (C) In the presence of GM-CSF, more cells became small, round or oval (arrowheads). (D) In the presence of both SP-D and GM-CSF, both populations (arrowheads, oval; arrow, elongated) are present. (E) Average cell size shown in the above conditions. Presence of GM-CSF significantly reduced cell size, reflecting the formation of small and spherical/oval cells. The sizes of at least 50 cells were measured in each category. Data are presented as means ± SEM; n = two independent experiments. The means were compared with respective controls using Dunnett's multiple mean comparison procedure (* p < 0.05). (F) Over one week time, many BAL cells changed into elongated and stopped internalizing foreign materials (labeled BSA). Number of cells that actively internalize the foreign material was quantified from the randomly selected images obtained in uptake assays. The increase in cell size was associated with decrease in % cells that internalize BSA. For percent cells that actively internalize, data are presented as the proportion of cells that were able to take up Alexa fluor 647-conjugated BSA in total number of cells present. The mean values were compared with Day1 value using Dunnett's multiple mean comparison procedure (* p < 0.05).

Mentions: We next asked whether SP-D and GM-CSF alter the cells isolated from BALF. Analysis of randomly selected images obtained from the feeding experiments showed that SP-D and GM-CSF affect the shape and size (as defined by the area occupied by each cell) of the cells (Figure 4). While control cells consisted of relatively uniform shapes and sizes (Figure 4A), the presence of SP-D promoted cell spreading (Figure 4B) and GM-CSF made cells more spherical/oval in shape (Figure 4C). Presence of both SP-D and GM-CSF generated mixed populations of both elongated and spherical cells (Figure 4D). Measurement of cell size also showed that GM-CSF, both in the presence and absence of SP-D, caused a significant decrease in cell size (p < 0.05; Figure 4E). Thus, these results show that SP-D may increase cell spreading, whereas GM-CSF maintains cells in a spherical shape.


SP-D counteracts GM-CSF-mediated increase of granuloma formation by alveolar macrophages in lysinuric protein intolerance.

Douda DN, Farmakovski N, Dell S, Grasemann H, Palaniyar N - Orphanet J Rare Dis (2009)

SP-D and GM-CSF affect the shape and size of the cells. (A-D) Adherent viable cells were grown in the absence or presence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml) for 24 h. (A) Cells without any added proteins have relatively the same size and shape. (B) In the presence of SP-D, more cells became elongated in shape (arrows). (C) In the presence of GM-CSF, more cells became small, round or oval (arrowheads). (D) In the presence of both SP-D and GM-CSF, both populations (arrowheads, oval; arrow, elongated) are present. (E) Average cell size shown in the above conditions. Presence of GM-CSF significantly reduced cell size, reflecting the formation of small and spherical/oval cells. The sizes of at least 50 cells were measured in each category. Data are presented as means ± SEM; n = two independent experiments. The means were compared with respective controls using Dunnett's multiple mean comparison procedure (* p < 0.05). (F) Over one week time, many BAL cells changed into elongated and stopped internalizing foreign materials (labeled BSA). Number of cells that actively internalize the foreign material was quantified from the randomly selected images obtained in uptake assays. The increase in cell size was associated with decrease in % cells that internalize BSA. For percent cells that actively internalize, data are presented as the proportion of cells that were able to take up Alexa fluor 647-conjugated BSA in total number of cells present. The mean values were compared with Day1 value using Dunnett's multiple mean comparison procedure (* p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807424&req=5

Figure 4: SP-D and GM-CSF affect the shape and size of the cells. (A-D) Adherent viable cells were grown in the absence or presence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml) for 24 h. (A) Cells without any added proteins have relatively the same size and shape. (B) In the presence of SP-D, more cells became elongated in shape (arrows). (C) In the presence of GM-CSF, more cells became small, round or oval (arrowheads). (D) In the presence of both SP-D and GM-CSF, both populations (arrowheads, oval; arrow, elongated) are present. (E) Average cell size shown in the above conditions. Presence of GM-CSF significantly reduced cell size, reflecting the formation of small and spherical/oval cells. The sizes of at least 50 cells were measured in each category. Data are presented as means ± SEM; n = two independent experiments. The means were compared with respective controls using Dunnett's multiple mean comparison procedure (* p < 0.05). (F) Over one week time, many BAL cells changed into elongated and stopped internalizing foreign materials (labeled BSA). Number of cells that actively internalize the foreign material was quantified from the randomly selected images obtained in uptake assays. The increase in cell size was associated with decrease in % cells that internalize BSA. For percent cells that actively internalize, data are presented as the proportion of cells that were able to take up Alexa fluor 647-conjugated BSA in total number of cells present. The mean values were compared with Day1 value using Dunnett's multiple mean comparison procedure (* p < 0.05).
Mentions: We next asked whether SP-D and GM-CSF alter the cells isolated from BALF. Analysis of randomly selected images obtained from the feeding experiments showed that SP-D and GM-CSF affect the shape and size (as defined by the area occupied by each cell) of the cells (Figure 4). While control cells consisted of relatively uniform shapes and sizes (Figure 4A), the presence of SP-D promoted cell spreading (Figure 4B) and GM-CSF made cells more spherical/oval in shape (Figure 4C). Presence of both SP-D and GM-CSF generated mixed populations of both elongated and spherical cells (Figure 4D). Measurement of cell size also showed that GM-CSF, both in the presence and absence of SP-D, caused a significant decrease in cell size (p < 0.05; Figure 4E). Thus, these results show that SP-D may increase cell spreading, whereas GM-CSF maintains cells in a spherical shape.

Bottom Line: At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain.Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.We propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lung Innate Immunity Research, Program in Physiology and Experimental Medicine, Research Institute, The Hospital For Sick Children, Toronto, Ontario, M5G 1X8, Canada. david.douda@utoronto.ca

ABSTRACT

Background: Pulmonary alveolar proteinosis (PAP) is a syndrome with multiple etiologies and is often deadly in lysinuric protein intolerance (LPI). At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain. We hypothesized that GM-CSF and surfactant protein D (SP-D) would enhance the clearance of proteins and dying cells that are typically present in the airways of PAP lungs.

Methods: Cells and cell-free supernatant of therapeutic bronchoalveolar lavage fluid (BALF) of a two-year-old patient with LPI were isolated on multiple occasions. Diagnostic BALF samples from an age-matched patient with bronchitis or adult PAP patients were used as controls. SP-D and total protein content of the supernatants were determined by BCA assays and Western blots, respectively. Cholesterol content was determined by a calorimetic assay or Oil Red O staining of cytospin preparations. The cells and surfactant lipids were also analyzed by transmission electron microscopy. Uptake of Alexa-647 conjugated BSA and DiI-labelled apoptotic Jurkat T-cells by BAL cells were studied separately in the presence or absence of SP-D (1 microg/ml) and/or GM-CSF (10 ng/ml), ex vivo. Specimens were analyzed by light and fluorescence microscopy.

Results: Here we show that large amounts of cholesterol, and large numbers of cholesterol crystals, dying cells, and lipid-laden foamy alveolar macrophages were present in the airways of the LPI patient. Although SP-D is present, its bioavailability is low in the airways. SP-D was partially degraded and entrapped in the unusual surfactant lipid tubules with circular lattice, in vivo. We also show that supplementing SP-D and GM-CSF increases the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo. Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.

Conclusions: We propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI. To improve the lung condition of LPI patients with PAP, it would be useful to explore alternative therapies for increasing dead cell clearance while decreasing cholesterol content in the airways.

Show MeSH
Related in: MedlinePlus