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Interleukin (IL)-4 induces leukocyte infiltration in vivo by an indirect mechanism.

Ratthé C, Ennaciri J, Garcês Gonçalves DM, Chiasson S, Girard D - Mediators Inflamm. (2009)

Bottom Line: The IL-4-induced expression of CCL-2 was confirmed by ELISA.Air pouch resident lining cells were harvested and were found to express IL-4Ralpha.CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Université du Québec, Québec, Canada.

ABSTRACT
Interleukin (IL)-4 is a cytokine known mainly for its anti-inflammatory activity. Using the in vivo murine air pouch model, we found that IL-4 significantly increased the number of leukocytes after 9 hours of treatment, consisting mainly of neutrophil (60%) and monocytic (40%) cell populations. Using an antibody array, we found that the expression of several analytes (predominantly CCL2) was increased by IL-4 before the arrival of leukocytes. The IL-4-induced expression of CCL-2 was confirmed by ELISA. Air pouch resident lining cells were harvested and were found to express IL-4Ralpha. CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions. We conclude that IL-4 can attract leukocytes in vivo by an indirect mechanism involving the production of several analytes by, at least, resident cells.

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Expression of CCL2 mRNA in different cell populations. (a) air pouches were created and IL-4 or buffer was administered for 6 hours. Cells were harvested, pooled (n = 6) and CCL2 (MCP-1) mRNA were detected as described in Materials and Methods. (b) Murine air pouches were created and the lining cells and skin cells were harvested as described in Materials and Methods. Lining cells, skin cells, murine macrophages (RAW) and epithelial Mode-K cells were stimulated for 6 hours with buffer (Ctrl) or 250 ng/ml IL-4 and the expression of CCL2 mRNA was performed by RT-PCR. Results are from one representative experiment out of at least 3. Number between parentheses represents the ratio of the signal intensity of (IL-4/18S)/Ctrl/18S) obtained after densitometry analysis using a Fluor-S multi-imager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program.
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Related In: Results  -  Collection


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fig5: Expression of CCL2 mRNA in different cell populations. (a) air pouches were created and IL-4 or buffer was administered for 6 hours. Cells were harvested, pooled (n = 6) and CCL2 (MCP-1) mRNA were detected as described in Materials and Methods. (b) Murine air pouches were created and the lining cells and skin cells were harvested as described in Materials and Methods. Lining cells, skin cells, murine macrophages (RAW) and epithelial Mode-K cells were stimulated for 6 hours with buffer (Ctrl) or 250 ng/ml IL-4 and the expression of CCL2 mRNA was performed by RT-PCR. Results are from one representative experiment out of at least 3. Number between parentheses represents the ratio of the signal intensity of (IL-4/18S)/Ctrl/18S) obtained after densitometry analysis using a Fluor-S multi-imager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program.

Mentions: Because several analytes detected in the exudates of IL-4-induced air pouch were present before the emigration of leukocytes, and since most of them are not necessarily typical neutrophil or monocyte chemoattractants, it is highly plausible that the lining or resident cells from the pouch are the first to interact with IL-4. Therefore, we isolated the lining cells from air pouches and investigated the expression of IL-4R. As illustrated in Figure 4 the lining cells expressed both CD132 (IL-2Rγ) and IL-4Rα, as assessed by RT-PCR (Figure 4(a)). We confirmed the expression of IL-4Rα at the cell surface of lining cells by flow cytometry (Figure 4(b)). Of note, expression of this component alone is sufficient to induce a signal in response to IL-4. Knowing that the harvested lining cells express the IL-4R, we next stimulated them in vitro (pool of n = 9) with IL-4 for 6 hours and investigated the expression of the same analytes. As illustrated in Table 2, 20 of 39 analytes were detected with a ratio greater than 1. Among these, 5 (Eotaxin-2, G-CSF, MIG, SDF-1 and sTNFR-I), exhibited a ratio ≥ 1.2. Of note, CCL2 was only detected at a ratio of 1.12. This suggests that the high level expression of several analytes observed in vivo in IL-4-induced air pouch necessitates other factors and/or players. Thus, the profile of analytes detected after 6 hours in response to IL-4 differs in terms of expression level and in terms of specificity in vivo and in vitro, suggesting that other cells are involved and that the responses are context specific. Because CCL2 is the predominant analyte detected in the exudates in response to IL-4, and in order to better elucidate the participation of different cell populations for such production, we first verified the possibility that the attracted leukocytes themselves participate in the increased production of CCL2. As illustrated in Figure 5(a), leukocytes attracted by IL-4 can produce higher levels of CCL2 mRNA when compared to controls. We next collected the lining cells and the skin cells from the inside part of the air pouch treated with buffer or IL-4 and monitored the gene expression of CCL2. In addition, we investigated the gene expression of CCL2 in murine RAW 264.7 macrophages and epithelial Mode-K cells. As illustrated in Figure 5(b), IL-4 increased the CCL2 mRNA expression in all conditions when compared to controls. Thus, according to the results obtained for CCL2, the production of the different analytes induced by IL-4 could originate from resident cells expressing IL-4R, as well as from leukocytes attracted by IL-4.


Interleukin (IL)-4 induces leukocyte infiltration in vivo by an indirect mechanism.

Ratthé C, Ennaciri J, Garcês Gonçalves DM, Chiasson S, Girard D - Mediators Inflamm. (2009)

Expression of CCL2 mRNA in different cell populations. (a) air pouches were created and IL-4 or buffer was administered for 6 hours. Cells were harvested, pooled (n = 6) and CCL2 (MCP-1) mRNA were detected as described in Materials and Methods. (b) Murine air pouches were created and the lining cells and skin cells were harvested as described in Materials and Methods. Lining cells, skin cells, murine macrophages (RAW) and epithelial Mode-K cells were stimulated for 6 hours with buffer (Ctrl) or 250 ng/ml IL-4 and the expression of CCL2 mRNA was performed by RT-PCR. Results are from one representative experiment out of at least 3. Number between parentheses represents the ratio of the signal intensity of (IL-4/18S)/Ctrl/18S) obtained after densitometry analysis using a Fluor-S multi-imager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742652&req=5

fig5: Expression of CCL2 mRNA in different cell populations. (a) air pouches were created and IL-4 or buffer was administered for 6 hours. Cells were harvested, pooled (n = 6) and CCL2 (MCP-1) mRNA were detected as described in Materials and Methods. (b) Murine air pouches were created and the lining cells and skin cells were harvested as described in Materials and Methods. Lining cells, skin cells, murine macrophages (RAW) and epithelial Mode-K cells were stimulated for 6 hours with buffer (Ctrl) or 250 ng/ml IL-4 and the expression of CCL2 mRNA was performed by RT-PCR. Results are from one representative experiment out of at least 3. Number between parentheses represents the ratio of the signal intensity of (IL-4/18S)/Ctrl/18S) obtained after densitometry analysis using a Fluor-S multi-imager (Bio-Rad, Hercules, CA) and the Multi-Analyst version 1.1 program.
Mentions: Because several analytes detected in the exudates of IL-4-induced air pouch were present before the emigration of leukocytes, and since most of them are not necessarily typical neutrophil or monocyte chemoattractants, it is highly plausible that the lining or resident cells from the pouch are the first to interact with IL-4. Therefore, we isolated the lining cells from air pouches and investigated the expression of IL-4R. As illustrated in Figure 4 the lining cells expressed both CD132 (IL-2Rγ) and IL-4Rα, as assessed by RT-PCR (Figure 4(a)). We confirmed the expression of IL-4Rα at the cell surface of lining cells by flow cytometry (Figure 4(b)). Of note, expression of this component alone is sufficient to induce a signal in response to IL-4. Knowing that the harvested lining cells express the IL-4R, we next stimulated them in vitro (pool of n = 9) with IL-4 for 6 hours and investigated the expression of the same analytes. As illustrated in Table 2, 20 of 39 analytes were detected with a ratio greater than 1. Among these, 5 (Eotaxin-2, G-CSF, MIG, SDF-1 and sTNFR-I), exhibited a ratio ≥ 1.2. Of note, CCL2 was only detected at a ratio of 1.12. This suggests that the high level expression of several analytes observed in vivo in IL-4-induced air pouch necessitates other factors and/or players. Thus, the profile of analytes detected after 6 hours in response to IL-4 differs in terms of expression level and in terms of specificity in vivo and in vitro, suggesting that other cells are involved and that the responses are context specific. Because CCL2 is the predominant analyte detected in the exudates in response to IL-4, and in order to better elucidate the participation of different cell populations for such production, we first verified the possibility that the attracted leukocytes themselves participate in the increased production of CCL2. As illustrated in Figure 5(a), leukocytes attracted by IL-4 can produce higher levels of CCL2 mRNA when compared to controls. We next collected the lining cells and the skin cells from the inside part of the air pouch treated with buffer or IL-4 and monitored the gene expression of CCL2. In addition, we investigated the gene expression of CCL2 in murine RAW 264.7 macrophages and epithelial Mode-K cells. As illustrated in Figure 5(b), IL-4 increased the CCL2 mRNA expression in all conditions when compared to controls. Thus, according to the results obtained for CCL2, the production of the different analytes induced by IL-4 could originate from resident cells expressing IL-4R, as well as from leukocytes attracted by IL-4.

Bottom Line: The IL-4-induced expression of CCL-2 was confirmed by ELISA.Air pouch resident lining cells were harvested and were found to express IL-4Ralpha.CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Université du Québec, Québec, Canada.

ABSTRACT
Interleukin (IL)-4 is a cytokine known mainly for its anti-inflammatory activity. Using the in vivo murine air pouch model, we found that IL-4 significantly increased the number of leukocytes after 9 hours of treatment, consisting mainly of neutrophil (60%) and monocytic (40%) cell populations. Using an antibody array, we found that the expression of several analytes (predominantly CCL2) was increased by IL-4 before the arrival of leukocytes. The IL-4-induced expression of CCL-2 was confirmed by ELISA. Air pouch resident lining cells were harvested and were found to express IL-4Ralpha. CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions. We conclude that IL-4 can attract leukocytes in vivo by an indirect mechanism involving the production of several analytes by, at least, resident cells.

Show MeSH