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Neuronal nicotinic alpha7 receptors modulate early neutrophil infiltration to sites of skin inflammation.

Gahring LC, Osborne AV, Reed M, Rogers SW - J Neuroinflammation (2010)

Bottom Line: RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance.In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes.Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes. nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education and Clinical Center, Salt Lake City VA Medical Center, USA. Lorise.Gahring@hsc.utah.edu

ABSTRACT

Background: A major site of initiation of inflammatory responses upon physical perturbation(s) and infection by invading organisms is the skin. Control of responses in this organ is, in part, modulated by the neuronal nicotinic acetylcholine receptor (nAChR) alpha7.

Methods: To further investigate the role of alpha7 in skin inflammatory responses, a local inflammatory response was induced by topical application of croton oil to the ear skin of wild-type (alpha7WT) and alpha7 knock-out (alpha7KO) mice. Cells infiltrating the inflamed tissue were characterized by flow cytometry and RNA analysis.

Results: Six hours following croton oil application, analysis of infiltrating cells showed that the alpha7KO mice exhibited a significantly enhanced number of cells, and specifically, of Ly6G positive neutrophils. Macrophage and lymphocyte infiltration was equivalent in the alpha7KO and alpha7WT mice. RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance. In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes. Both resident and infiltrating cell populations from alpha7KO mice did show elevated message levels for the adhesion protein ICAM1. Measurement of chemokines revealed enhanced expression of the skin-related CCL27 by resident cells in alpha7KO mice. Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes.

Conclusion: nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.

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Related in: MedlinePlus

The local inflammatory response to croton oil is enhanced in the α7KO mouse. Ears of WT and α7KO mice (5 animals/experiment) were treated with croton oil. Six hours post-treatment ears were removed, dorsal and ventral halves separated before floating them dermis side down on media overnight. A) Total number of cells isolated per treatment group was then measured (99% viability, not shown). The light grey bars represent WT mice and dark grey bars the α7KO. Error bars reflect ± of the standard error of the mean of all experiments (3-5 experiments per group); * p < 0.05 and **, p < 0.01 (two-tailed Student's T-test). B) Expression of key pro-inflammatory cytokine transcripts from infiltrating cells using qPCR (Methods). The average fold increase normalized to a value of 1 for IL-1β, IL-6 and TNFα from α7WT mice (light grey) and α7KO (grey bars) are compared. Error bars reflect the standard error of the mean from 3 independent experiments with 5-6 mice per experimental group. C) Analysis of transcripts from ear skin after removal of infiltrating cells from untreated mice, or mice treated with croton oil. The expression of these cytokines does not differ between α7WT and α7KO. Light gray bars represent ear skin from WT mice and dark gray for α7KO mice. Non-shaded bars represent ear skin from untreated WT mice. Ear skin RNA amounts recovered did not differ between genotypes (not shown).
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Figure 1: The local inflammatory response to croton oil is enhanced in the α7KO mouse. Ears of WT and α7KO mice (5 animals/experiment) were treated with croton oil. Six hours post-treatment ears were removed, dorsal and ventral halves separated before floating them dermis side down on media overnight. A) Total number of cells isolated per treatment group was then measured (99% viability, not shown). The light grey bars represent WT mice and dark grey bars the α7KO. Error bars reflect ± of the standard error of the mean of all experiments (3-5 experiments per group); * p < 0.05 and **, p < 0.01 (two-tailed Student's T-test). B) Expression of key pro-inflammatory cytokine transcripts from infiltrating cells using qPCR (Methods). The average fold increase normalized to a value of 1 for IL-1β, IL-6 and TNFα from α7WT mice (light grey) and α7KO (grey bars) are compared. Error bars reflect the standard error of the mean from 3 independent experiments with 5-6 mice per experimental group. C) Analysis of transcripts from ear skin after removal of infiltrating cells from untreated mice, or mice treated with croton oil. The expression of these cytokines does not differ between α7WT and α7KO. Light gray bars represent ear skin from WT mice and dark gray for α7KO mice. Non-shaded bars represent ear skin from untreated WT mice. Ear skin RNA amounts recovered did not differ between genotypes (not shown).

Mentions: The mouse ear pinna provides a unique site to study cellular infiltration following skin inflammation. This is due to the distinct dorsal and ventral sides, each with an epidermal and dermal layer, that are separated by auricular cartilage. Upon application of an inflammatory agent, cells collect between the dorsal and ventral ear skin and infiltrate the tissue. The infiltrating cells are recovered from the media after mechanical separation of the ear into the respective halves, and overnight incubation of the ear halves in media allows cells to be released into the media. This procedure results in the isolation of recruited cells without using more disruptive interventions that can inconsistently impact upon cells more sensitive to harsh isolation conditions. To examine an inflammatory response in the skin, we used the well-characterized method of topical application of croton oil, an organic phorbol compound derived from the seeds of the Croton tiglium tree. A particular advantage of this method is the relatively mild inflammatory response which could facilitate detection of α7 modulatory components. Mouse ears were treated with topically applied 3.5% croton oil to induce local inflammation. At six hours post croton oil application, the mice (groups of 5 α7WT or α7KO) were sacrificed and the ears removed. The dorsal and ventral sides of the ear were separated and incubated overnight in media (see Methods). The results presented in Figure 1a show that the total number of cells isolated from α7KO mice is significantly (p < 0.01) enhanced relative to wild-type mice. Cell numbers reflect the total number of cells obtained from 5 mice in each group. The standard error of the mean is derived by averaging the number of cells obtained from each of these groups (WT or KO) from at least 5 separated experiments. In the absence of an inflammatory stimulus such as croton oil, there are too few cells to isolate and characterize. Further, we have found that the overnight incubation of the ear halves is required to obtain the infiltrating cells suggesting that the cells are not just in the edematous fluid of the ear.


Neuronal nicotinic alpha7 receptors modulate early neutrophil infiltration to sites of skin inflammation.

Gahring LC, Osborne AV, Reed M, Rogers SW - J Neuroinflammation (2010)

The local inflammatory response to croton oil is enhanced in the α7KO mouse. Ears of WT and α7KO mice (5 animals/experiment) were treated with croton oil. Six hours post-treatment ears were removed, dorsal and ventral halves separated before floating them dermis side down on media overnight. A) Total number of cells isolated per treatment group was then measured (99% viability, not shown). The light grey bars represent WT mice and dark grey bars the α7KO. Error bars reflect ± of the standard error of the mean of all experiments (3-5 experiments per group); * p < 0.05 and **, p < 0.01 (two-tailed Student's T-test). B) Expression of key pro-inflammatory cytokine transcripts from infiltrating cells using qPCR (Methods). The average fold increase normalized to a value of 1 for IL-1β, IL-6 and TNFα from α7WT mice (light grey) and α7KO (grey bars) are compared. Error bars reflect the standard error of the mean from 3 independent experiments with 5-6 mice per experimental group. C) Analysis of transcripts from ear skin after removal of infiltrating cells from untreated mice, or mice treated with croton oil. The expression of these cytokines does not differ between α7WT and α7KO. Light gray bars represent ear skin from WT mice and dark gray for α7KO mice. Non-shaded bars represent ear skin from untreated WT mice. Ear skin RNA amounts recovered did not differ between genotypes (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: The local inflammatory response to croton oil is enhanced in the α7KO mouse. Ears of WT and α7KO mice (5 animals/experiment) were treated with croton oil. Six hours post-treatment ears were removed, dorsal and ventral halves separated before floating them dermis side down on media overnight. A) Total number of cells isolated per treatment group was then measured (99% viability, not shown). The light grey bars represent WT mice and dark grey bars the α7KO. Error bars reflect ± of the standard error of the mean of all experiments (3-5 experiments per group); * p < 0.05 and **, p < 0.01 (two-tailed Student's T-test). B) Expression of key pro-inflammatory cytokine transcripts from infiltrating cells using qPCR (Methods). The average fold increase normalized to a value of 1 for IL-1β, IL-6 and TNFα from α7WT mice (light grey) and α7KO (grey bars) are compared. Error bars reflect the standard error of the mean from 3 independent experiments with 5-6 mice per experimental group. C) Analysis of transcripts from ear skin after removal of infiltrating cells from untreated mice, or mice treated with croton oil. The expression of these cytokines does not differ between α7WT and α7KO. Light gray bars represent ear skin from WT mice and dark gray for α7KO mice. Non-shaded bars represent ear skin from untreated WT mice. Ear skin RNA amounts recovered did not differ between genotypes (not shown).
Mentions: The mouse ear pinna provides a unique site to study cellular infiltration following skin inflammation. This is due to the distinct dorsal and ventral sides, each with an epidermal and dermal layer, that are separated by auricular cartilage. Upon application of an inflammatory agent, cells collect between the dorsal and ventral ear skin and infiltrate the tissue. The infiltrating cells are recovered from the media after mechanical separation of the ear into the respective halves, and overnight incubation of the ear halves in media allows cells to be released into the media. This procedure results in the isolation of recruited cells without using more disruptive interventions that can inconsistently impact upon cells more sensitive to harsh isolation conditions. To examine an inflammatory response in the skin, we used the well-characterized method of topical application of croton oil, an organic phorbol compound derived from the seeds of the Croton tiglium tree. A particular advantage of this method is the relatively mild inflammatory response which could facilitate detection of α7 modulatory components. Mouse ears were treated with topically applied 3.5% croton oil to induce local inflammation. At six hours post croton oil application, the mice (groups of 5 α7WT or α7KO) were sacrificed and the ears removed. The dorsal and ventral sides of the ear were separated and incubated overnight in media (see Methods). The results presented in Figure 1a show that the total number of cells isolated from α7KO mice is significantly (p < 0.01) enhanced relative to wild-type mice. Cell numbers reflect the total number of cells obtained from 5 mice in each group. The standard error of the mean is derived by averaging the number of cells obtained from each of these groups (WT or KO) from at least 5 separated experiments. In the absence of an inflammatory stimulus such as croton oil, there are too few cells to isolate and characterize. Further, we have found that the overnight incubation of the ear halves is required to obtain the infiltrating cells suggesting that the cells are not just in the edematous fluid of the ear.

Bottom Line: RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance.In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes.Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes. nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education and Clinical Center, Salt Lake City VA Medical Center, USA. Lorise.Gahring@hsc.utah.edu

ABSTRACT

Background: A major site of initiation of inflammatory responses upon physical perturbation(s) and infection by invading organisms is the skin. Control of responses in this organ is, in part, modulated by the neuronal nicotinic acetylcholine receptor (nAChR) alpha7.

Methods: To further investigate the role of alpha7 in skin inflammatory responses, a local inflammatory response was induced by topical application of croton oil to the ear skin of wild-type (alpha7WT) and alpha7 knock-out (alpha7KO) mice. Cells infiltrating the inflamed tissue were characterized by flow cytometry and RNA analysis.

Results: Six hours following croton oil application, analysis of infiltrating cells showed that the alpha7KO mice exhibited a significantly enhanced number of cells, and specifically, of Ly6G positive neutrophils. Macrophage and lymphocyte infiltration was equivalent in the alpha7KO and alpha7WT mice. RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance. In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes. Both resident and infiltrating cell populations from alpha7KO mice did show elevated message levels for the adhesion protein ICAM1. Measurement of chemokines revealed enhanced expression of the skin-related CCL27 by resident cells in alpha7KO mice. Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes.

Conclusion: nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.

Show MeSH
Related in: MedlinePlus