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Pivotal Advance: Pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish.

Loynes CA, Martin JS, Robertson A, Trushell DM, Ingham PW, Whyte MK, Renshaw SA - J. Leukoc. Biol. (2010)

Bottom Line: Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations.The occurrence of apoptosis was observed by time-lapse analysis and confirmed by dual staining for neutrophil-specific mpx activity (TSA staining) and an apoptotic marker (TUNEL).This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom.

ABSTRACT
Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations. Using a transgenic line specifically labeling neutrophils in vivo (mpx:GFP), we studied the effects of a range of pharmacological agents on the resolution of inflammation in vivo. These agents were selected for their ability to modulate neutrophil function and lifespan in human neutrophils in vitro. Agents delaying neutrophil apoptosis (LPS, dbcAMP, and several caspase inhibitors) all lead to a delay in resolution of neutrophilic inflammation. Reciprocally, pyocyanin and roscovitine (inducers of neutrophil apoptosis) lead to reduced neutrophil numbers. The occurrence of apoptosis was observed by time-lapse analysis and confirmed by dual staining for neutrophil-specific mpx activity (TSA staining) and an apoptotic marker (TUNEL). During inflammation, macrophages follow neutrophils into the inflamed site, and TUNEL/TSA dual-positive material can be demonstrated within macrophages, consistent with their uptake of apoptotic neutrophils. This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.

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Macrophages take up apoptotic neutrophils during inflammation resolution in vivo. (A) Three dpf mpx:GFP zebrafish were injured as described previously. Two hours before the time indicated, larvae were stained with neutral red for 2 h. The number of neutral red-positive macrophages and GFP-positive neutrophils was counted for each fish under bright-field illumination and 488 nm fluorescent illumination, respectively. (B and C) Seven dpf wild-type zebrafish were injured and fixed 12 h later in 4% paraformaldehyde at 4°C, followed by staining for mpx activity and L-plastin immunohistochemistry, as described in Materials and Methods. Single macrophages can be seen containing TSA-positive neutrophilic material. (C) A neutrophil is shown alongside for comparison. (D) Cy5-TSA staining (purple) was combined with L-plastin staining (green) and TUNEL staining (red) to show colocalization of apoptotic markers and neutrophil markers within a macrophage. Three-dimensional reconstructions are shown in Supplemental Movies 4 and 5 to confirm location of the Cy-3 TSA-positive material completely within the macrophage. Images were taken using a ×60 Plan Apo Oil immersion NA1.40 (Olympus).
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Figure 5: Macrophages take up apoptotic neutrophils during inflammation resolution in vivo. (A) Three dpf mpx:GFP zebrafish were injured as described previously. Two hours before the time indicated, larvae were stained with neutral red for 2 h. The number of neutral red-positive macrophages and GFP-positive neutrophils was counted for each fish under bright-field illumination and 488 nm fluorescent illumination, respectively. (B and C) Seven dpf wild-type zebrafish were injured and fixed 12 h later in 4% paraformaldehyde at 4°C, followed by staining for mpx activity and L-plastin immunohistochemistry, as described in Materials and Methods. Single macrophages can be seen containing TSA-positive neutrophilic material. (C) A neutrophil is shown alongside for comparison. (D) Cy5-TSA staining (purple) was combined with L-plastin staining (green) and TUNEL staining (red) to show colocalization of apoptotic markers and neutrophil markers within a macrophage. Three-dimensional reconstructions are shown in Supplemental Movies 4 and 5 to confirm location of the Cy-3 TSA-positive material completely within the macrophage. Images were taken using a ×60 Plan Apo Oil immersion NA1.40 (Olympus).

Mentions: To demonstrate the remains of ingested neutrophils within macrophages, zebrafish phagocytes were stained by L-plastin immunofluorescence [29] and the presence of mpx identified using Cy3-TSA staining. Two populations of myeloid cells could be distinguished: neutrophils were L-plastin-positive/Cy3-TSA-positive, and macrophages were L-plastin-positive/Cy3-TSA-negative. In uninjured fish, no Cy3-TSA-positive material was seen within macrophages. However, at 12 h postinjury in 7 dpf fish, macrophages containing small, localized foci of Cy-3-TSA-positive neutrophil material could be identified clearly. This material was always seen as small circular bodies within the macrophages, consistent with macrophage ingestion of apoptotic neutrophils (Fig. 5, B and C, and Supplemental Movies 4 and 5). In a further experiment, using triple staining for neutrophilic material (Cy5-TSA), apoptotic markers (rhodamine TUNEL), and leukocyte markers (L-plastin immunohistochemistry), it was possible to identify rare examples of macrophages containing triple-positive, small rounded inclusions, consistent with ingestion of apoptotic neutrophil material (Fig. 5D). This is consistent with a functional role for neutrophil apoptosis and macrophage clearance in the resolution of inflammation in this model.


Pivotal Advance: Pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish.

Loynes CA, Martin JS, Robertson A, Trushell DM, Ingham PW, Whyte MK, Renshaw SA - J. Leukoc. Biol. (2010)

Macrophages take up apoptotic neutrophils during inflammation resolution in vivo. (A) Three dpf mpx:GFP zebrafish were injured as described previously. Two hours before the time indicated, larvae were stained with neutral red for 2 h. The number of neutral red-positive macrophages and GFP-positive neutrophils was counted for each fish under bright-field illumination and 488 nm fluorescent illumination, respectively. (B and C) Seven dpf wild-type zebrafish were injured and fixed 12 h later in 4% paraformaldehyde at 4°C, followed by staining for mpx activity and L-plastin immunohistochemistry, as described in Materials and Methods. Single macrophages can be seen containing TSA-positive neutrophilic material. (C) A neutrophil is shown alongside for comparison. (D) Cy5-TSA staining (purple) was combined with L-plastin staining (green) and TUNEL staining (red) to show colocalization of apoptotic markers and neutrophil markers within a macrophage. Three-dimensional reconstructions are shown in Supplemental Movies 4 and 5 to confirm location of the Cy-3 TSA-positive material completely within the macrophage. Images were taken using a ×60 Plan Apo Oil immersion NA1.40 (Olympus).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Macrophages take up apoptotic neutrophils during inflammation resolution in vivo. (A) Three dpf mpx:GFP zebrafish were injured as described previously. Two hours before the time indicated, larvae were stained with neutral red for 2 h. The number of neutral red-positive macrophages and GFP-positive neutrophils was counted for each fish under bright-field illumination and 488 nm fluorescent illumination, respectively. (B and C) Seven dpf wild-type zebrafish were injured and fixed 12 h later in 4% paraformaldehyde at 4°C, followed by staining for mpx activity and L-plastin immunohistochemistry, as described in Materials and Methods. Single macrophages can be seen containing TSA-positive neutrophilic material. (C) A neutrophil is shown alongside for comparison. (D) Cy5-TSA staining (purple) was combined with L-plastin staining (green) and TUNEL staining (red) to show colocalization of apoptotic markers and neutrophil markers within a macrophage. Three-dimensional reconstructions are shown in Supplemental Movies 4 and 5 to confirm location of the Cy-3 TSA-positive material completely within the macrophage. Images were taken using a ×60 Plan Apo Oil immersion NA1.40 (Olympus).
Mentions: To demonstrate the remains of ingested neutrophils within macrophages, zebrafish phagocytes were stained by L-plastin immunofluorescence [29] and the presence of mpx identified using Cy3-TSA staining. Two populations of myeloid cells could be distinguished: neutrophils were L-plastin-positive/Cy3-TSA-positive, and macrophages were L-plastin-positive/Cy3-TSA-negative. In uninjured fish, no Cy3-TSA-positive material was seen within macrophages. However, at 12 h postinjury in 7 dpf fish, macrophages containing small, localized foci of Cy-3-TSA-positive neutrophil material could be identified clearly. This material was always seen as small circular bodies within the macrophages, consistent with macrophage ingestion of apoptotic neutrophils (Fig. 5, B and C, and Supplemental Movies 4 and 5). In a further experiment, using triple staining for neutrophilic material (Cy5-TSA), apoptotic markers (rhodamine TUNEL), and leukocyte markers (L-plastin immunohistochemistry), it was possible to identify rare examples of macrophages containing triple-positive, small rounded inclusions, consistent with ingestion of apoptotic neutrophil material (Fig. 5D). This is consistent with a functional role for neutrophil apoptosis and macrophage clearance in the resolution of inflammation in this model.

Bottom Line: Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations.The occurrence of apoptosis was observed by time-lapse analysis and confirmed by dual staining for neutrophil-specific mpx activity (TSA staining) and an apoptotic marker (TUNEL).This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom.

ABSTRACT
Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations. Using a transgenic line specifically labeling neutrophils in vivo (mpx:GFP), we studied the effects of a range of pharmacological agents on the resolution of inflammation in vivo. These agents were selected for their ability to modulate neutrophil function and lifespan in human neutrophils in vitro. Agents delaying neutrophil apoptosis (LPS, dbcAMP, and several caspase inhibitors) all lead to a delay in resolution of neutrophilic inflammation. Reciprocally, pyocyanin and roscovitine (inducers of neutrophil apoptosis) lead to reduced neutrophil numbers. The occurrence of apoptosis was observed by time-lapse analysis and confirmed by dual staining for neutrophil-specific mpx activity (TSA staining) and an apoptotic marker (TUNEL). During inflammation, macrophages follow neutrophils into the inflamed site, and TUNEL/TSA dual-positive material can be demonstrated within macrophages, consistent with their uptake of apoptotic neutrophils. This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.

Show MeSH
Related in: MedlinePlus