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IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.

Loke P, Nair MG, Parkinson J, Guiliano D, Blaxter M, Allen JE - BMC Immunol. (2002)

Bottom Line: Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes.Differential expression was confirmed by real time RT-PCR analysis.Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK. ploke@uclink.berkeley.edu

ABSTRACT

Background: "Alternatively-activated" macrophages are found in Th2-mediated inflammatory settings such as nematode infection and allergic pulmonary inflammation. Due in part to a lack of markers, these cells have not been well characterized in vivo and their function remains unknown.

Results: We have used murine macrophages elicited by nematode infection (NeM(phi)) as a source of in vivo derived alternatively activated macrophages. Using three distinct yet complementary molecular approaches we have established a gene expression profile of alternatively activated macrophages and identified macrophage genes that are regulated in vivo by IL-4. First, genes abundantly expressed were identified by an expressed sequence tag strategy. Second, an array of 1176 known mouse genes was screened for differential expression between NeM(phi) from wild type or IL-4 deficient mice. Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes. Differential expression was confirmed by real time RT-PCR analysis.

Conclusions: Our data demonstrate that alternatively activated macrophages generated in vivo have a gene expression profile distinct from any macrophage population described to date. Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings. Our data also provide additional in vivo evidence for parallels between the inflammatory processes involved in nematode infection and allergy.

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FIZZ1/RELMα expression is increased in response to parasite implant in C57BL/6 but not IL-4 -/- mice. Semiquantitative RT-PCR of FIZZ1/RELMα and β-actin was performed on total RNA samples from the peritoneal lavages of individual mice (A). Real Time PCR analysis of FIZZ1 expression by total cells (B) or F4/80 purified macrophages (C) obtained from peritoneal lavages of implanted or naïve control mice. For the purified NeMφ, each cDNA sample was obtained from individual mice. For the purified control macrophages, the cDNA samples represent pooled macrophages from groups of 2 or 3 mice for a total of 5 mice per control group. Expression levels were measured as a percentage of β-actin expression. Error bars represent one SD from the mean.
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Figure 3: FIZZ1/RELMα expression is increased in response to parasite implant in C57BL/6 but not IL-4 -/- mice. Semiquantitative RT-PCR of FIZZ1/RELMα and β-actin was performed on total RNA samples from the peritoneal lavages of individual mice (A). Real Time PCR analysis of FIZZ1 expression by total cells (B) or F4/80 purified macrophages (C) obtained from peritoneal lavages of implanted or naïve control mice. For the purified NeMφ, each cDNA sample was obtained from individual mice. For the purified control macrophages, the cDNA samples represent pooled macrophages from groups of 2 or 3 mice for a total of 5 mice per control group. Expression levels were measured as a percentage of β-actin expression. Error bars represent one SD from the mean.

Mentions: To confirm that FIZZ1/RELM-α was regulated by IL-4 in vivo, we compared the expression in PEC recovered from WT or IL-4-/- mice implanted with B. malayi using quantitative real-time PCR. Consistent with the results from the subtractive hybridization, the lack of IL-4 in vivo prevented a significant upregulation of FIZZ1/RELM-α (Figure 3A &3B). Expresssion was further analyzed using F4/80 purified macrophages. A 40-fold induction of gene expression at the level of mRNA, was found in WT NeMφ though expression remained basal in IL-4-/- NeMφ (Figure 3C).


IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.

Loke P, Nair MG, Parkinson J, Guiliano D, Blaxter M, Allen JE - BMC Immunol. (2002)

FIZZ1/RELMα expression is increased in response to parasite implant in C57BL/6 but not IL-4 -/- mice. Semiquantitative RT-PCR of FIZZ1/RELMα and β-actin was performed on total RNA samples from the peritoneal lavages of individual mice (A). Real Time PCR analysis of FIZZ1 expression by total cells (B) or F4/80 purified macrophages (C) obtained from peritoneal lavages of implanted or naïve control mice. For the purified NeMφ, each cDNA sample was obtained from individual mice. For the purified control macrophages, the cDNA samples represent pooled macrophages from groups of 2 or 3 mice for a total of 5 mice per control group. Expression levels were measured as a percentage of β-actin expression. Error bars represent one SD from the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117781&req=5

Figure 3: FIZZ1/RELMα expression is increased in response to parasite implant in C57BL/6 but not IL-4 -/- mice. Semiquantitative RT-PCR of FIZZ1/RELMα and β-actin was performed on total RNA samples from the peritoneal lavages of individual mice (A). Real Time PCR analysis of FIZZ1 expression by total cells (B) or F4/80 purified macrophages (C) obtained from peritoneal lavages of implanted or naïve control mice. For the purified NeMφ, each cDNA sample was obtained from individual mice. For the purified control macrophages, the cDNA samples represent pooled macrophages from groups of 2 or 3 mice for a total of 5 mice per control group. Expression levels were measured as a percentage of β-actin expression. Error bars represent one SD from the mean.
Mentions: To confirm that FIZZ1/RELM-α was regulated by IL-4 in vivo, we compared the expression in PEC recovered from WT or IL-4-/- mice implanted with B. malayi using quantitative real-time PCR. Consistent with the results from the subtractive hybridization, the lack of IL-4 in vivo prevented a significant upregulation of FIZZ1/RELM-α (Figure 3A &3B). Expresssion was further analyzed using F4/80 purified macrophages. A 40-fold induction of gene expression at the level of mRNA, was found in WT NeMφ though expression remained basal in IL-4-/- NeMφ (Figure 3C).

Bottom Line: Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes.Differential expression was confirmed by real time RT-PCR analysis.Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK. ploke@uclink.berkeley.edu

ABSTRACT

Background: "Alternatively-activated" macrophages are found in Th2-mediated inflammatory settings such as nematode infection and allergic pulmonary inflammation. Due in part to a lack of markers, these cells have not been well characterized in vivo and their function remains unknown.

Results: We have used murine macrophages elicited by nematode infection (NeM(phi)) as a source of in vivo derived alternatively activated macrophages. Using three distinct yet complementary molecular approaches we have established a gene expression profile of alternatively activated macrophages and identified macrophage genes that are regulated in vivo by IL-4. First, genes abundantly expressed were identified by an expressed sequence tag strategy. Second, an array of 1176 known mouse genes was screened for differential expression between NeM(phi) from wild type or IL-4 deficient mice. Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes. Differential expression was confirmed by real time RT-PCR analysis.

Conclusions: Our data demonstrate that alternatively activated macrophages generated in vivo have a gene expression profile distinct from any macrophage population described to date. Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings. Our data also provide additional in vivo evidence for parallels between the inflammatory processes involved in nematode infection and allergy.

Show MeSH
Related in: MedlinePlus