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Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Woerly G, Roger N, Loiseau S, Dombrowicz D, Capron A, Capron M - J. Exp. Med. (1999)

Bottom Line: CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected.Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release.These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie et Biologie Parasitaire, Institut National de la Santé et de la Recherche Médicale U167, Institut Pasteur, and Université de Lille II, 59019 Lille Cedex, France.

ABSTRACT
Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.

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Biological activity of IL-2 and IFN-γ released by CD28-activated eosinophils. (A and B) Detection of the proliferative activity of IL-2 released by eosinophils on an IL-2–dependent cell line. Supernatants collected from eosinophils (2 × 106/ml) stimulated with immobilized anti-CD28 mAb or isotype control Ab for 18 h were able to sustain the IL-2–dependent growth of the mouse cytotoxic T cell line CTLL-2 (American Type Culture Collection). The CTLL-2 cells were cultured in the presence of rhIL-2 (A) or eosinophil supernatants (50% dilution; B) for 24 h. Proliferation was measured by [3H]thymidine incorporation. Each supernatant obtained from individual patients (Table , donors 2, 11, and 16) was assayed in duplicate. Results are expressed as proliferation index ± SD, according to the following formula: proliferation index = dpm (eosinophils stimulated with anti-CD28 mAb)/dpm (eosinophils stimulated with isotype control mAb). An index >3 was considered significant. (C and D) Supernatants collected from eosinophils (2 × 106/ml) stimulated with cross-linked anti-CD28 mAb or isotype control Ab for 18 h were assayed for IFN-γ–induced MHC class II expression on Colo 205 cells. The Colo 205 cells were cultured in the presence of rhIFN-γ (C) or eosinophil supernatants (20% dilution; D). After 48 h, cells were harvested and stained with a PE-conjugated anti–HLA-DR mAb or an isotype-matched Ab and examined by flow cytometry. Specificity of the bioassay was controlled by adding a neutralizing anti–IFN-γ mAb to the culture. Cell fluorescence was measured using a FACSCalibur™ flow cytometer, and data were analyzed using CellQuest software. Data are presented as mean ± SD from three different experiments (Table , donors 2, 8, and 16).
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Figure 5: Biological activity of IL-2 and IFN-γ released by CD28-activated eosinophils. (A and B) Detection of the proliferative activity of IL-2 released by eosinophils on an IL-2–dependent cell line. Supernatants collected from eosinophils (2 × 106/ml) stimulated with immobilized anti-CD28 mAb or isotype control Ab for 18 h were able to sustain the IL-2–dependent growth of the mouse cytotoxic T cell line CTLL-2 (American Type Culture Collection). The CTLL-2 cells were cultured in the presence of rhIL-2 (A) or eosinophil supernatants (50% dilution; B) for 24 h. Proliferation was measured by [3H]thymidine incorporation. Each supernatant obtained from individual patients (Table , donors 2, 11, and 16) was assayed in duplicate. Results are expressed as proliferation index ± SD, according to the following formula: proliferation index = dpm (eosinophils stimulated with anti-CD28 mAb)/dpm (eosinophils stimulated with isotype control mAb). An index >3 was considered significant. (C and D) Supernatants collected from eosinophils (2 × 106/ml) stimulated with cross-linked anti-CD28 mAb or isotype control Ab for 18 h were assayed for IFN-γ–induced MHC class II expression on Colo 205 cells. The Colo 205 cells were cultured in the presence of rhIFN-γ (C) or eosinophil supernatants (20% dilution; D). After 48 h, cells were harvested and stained with a PE-conjugated anti–HLA-DR mAb or an isotype-matched Ab and examined by flow cytometry. Specificity of the bioassay was controlled by adding a neutralizing anti–IFN-γ mAb to the culture. Cell fluorescence was measured using a FACSCalibur™ flow cytometer, and data were analyzed using CellQuest software. Data are presented as mean ± SD from three different experiments (Table , donors 2, 8, and 16).

Mentions: In spite of the numerous studies published on cytokine secretion by eosinophils, very few have evaluated the biological activity of the cytokines released. Therefore, appropriate bioassays were performed in order to evaluate the functional activity of IL-2 and IFN-γ in eosinophil supernatants. The biological activity of IL-2 in the supernatants of CD28-activated eosinophils was evaluated in a proliferation assay using the IL-2–dependent mouse cytotoxic T cell line CTLL-2. The results presented in Fig. 5 indicate that eosinophil supernatants induced the proliferation of CTLL-2 cells, with large variations according to individual eosinophil donors. Comparison with the results obtained for purified rhIL-2 (Fig. 5 A) indicated that between 100 and 250 pg/ml of biologically active IL-2 could be detected in eosinophil supernatants after CD28 ligation (Fig. 5 B).


Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Woerly G, Roger N, Loiseau S, Dombrowicz D, Capron A, Capron M - J. Exp. Med. (1999)

Biological activity of IL-2 and IFN-γ released by CD28-activated eosinophils. (A and B) Detection of the proliferative activity of IL-2 released by eosinophils on an IL-2–dependent cell line. Supernatants collected from eosinophils (2 × 106/ml) stimulated with immobilized anti-CD28 mAb or isotype control Ab for 18 h were able to sustain the IL-2–dependent growth of the mouse cytotoxic T cell line CTLL-2 (American Type Culture Collection). The CTLL-2 cells were cultured in the presence of rhIL-2 (A) or eosinophil supernatants (50% dilution; B) for 24 h. Proliferation was measured by [3H]thymidine incorporation. Each supernatant obtained from individual patients (Table , donors 2, 11, and 16) was assayed in duplicate. Results are expressed as proliferation index ± SD, according to the following formula: proliferation index = dpm (eosinophils stimulated with anti-CD28 mAb)/dpm (eosinophils stimulated with isotype control mAb). An index >3 was considered significant. (C and D) Supernatants collected from eosinophils (2 × 106/ml) stimulated with cross-linked anti-CD28 mAb or isotype control Ab for 18 h were assayed for IFN-γ–induced MHC class II expression on Colo 205 cells. The Colo 205 cells were cultured in the presence of rhIFN-γ (C) or eosinophil supernatants (20% dilution; D). After 48 h, cells were harvested and stained with a PE-conjugated anti–HLA-DR mAb or an isotype-matched Ab and examined by flow cytometry. Specificity of the bioassay was controlled by adding a neutralizing anti–IFN-γ mAb to the culture. Cell fluorescence was measured using a FACSCalibur™ flow cytometer, and data were analyzed using CellQuest software. Data are presented as mean ± SD from three different experiments (Table , donors 2, 8, and 16).
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Related In: Results  -  Collection

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Figure 5: Biological activity of IL-2 and IFN-γ released by CD28-activated eosinophils. (A and B) Detection of the proliferative activity of IL-2 released by eosinophils on an IL-2–dependent cell line. Supernatants collected from eosinophils (2 × 106/ml) stimulated with immobilized anti-CD28 mAb or isotype control Ab for 18 h were able to sustain the IL-2–dependent growth of the mouse cytotoxic T cell line CTLL-2 (American Type Culture Collection). The CTLL-2 cells were cultured in the presence of rhIL-2 (A) or eosinophil supernatants (50% dilution; B) for 24 h. Proliferation was measured by [3H]thymidine incorporation. Each supernatant obtained from individual patients (Table , donors 2, 11, and 16) was assayed in duplicate. Results are expressed as proliferation index ± SD, according to the following formula: proliferation index = dpm (eosinophils stimulated with anti-CD28 mAb)/dpm (eosinophils stimulated with isotype control mAb). An index >3 was considered significant. (C and D) Supernatants collected from eosinophils (2 × 106/ml) stimulated with cross-linked anti-CD28 mAb or isotype control Ab for 18 h were assayed for IFN-γ–induced MHC class II expression on Colo 205 cells. The Colo 205 cells were cultured in the presence of rhIFN-γ (C) or eosinophil supernatants (20% dilution; D). After 48 h, cells were harvested and stained with a PE-conjugated anti–HLA-DR mAb or an isotype-matched Ab and examined by flow cytometry. Specificity of the bioassay was controlled by adding a neutralizing anti–IFN-γ mAb to the culture. Cell fluorescence was measured using a FACSCalibur™ flow cytometer, and data were analyzed using CellQuest software. Data are presented as mean ± SD from three different experiments (Table , donors 2, 8, and 16).
Mentions: In spite of the numerous studies published on cytokine secretion by eosinophils, very few have evaluated the biological activity of the cytokines released. Therefore, appropriate bioassays were performed in order to evaluate the functional activity of IL-2 and IFN-γ in eosinophil supernatants. The biological activity of IL-2 in the supernatants of CD28-activated eosinophils was evaluated in a proliferation assay using the IL-2–dependent mouse cytotoxic T cell line CTLL-2. The results presented in Fig. 5 indicate that eosinophil supernatants induced the proliferation of CTLL-2 cells, with large variations according to individual eosinophil donors. Comparison with the results obtained for purified rhIL-2 (Fig. 5 A) indicated that between 100 and 250 pg/ml of biologically active IL-2 could be detected in eosinophil supernatants after CD28 ligation (Fig. 5 B).

Bottom Line: CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected.Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release.These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie et Biologie Parasitaire, Institut National de la Santé et de la Recherche Médicale U167, Institut Pasteur, and Université de Lille II, 59019 Lille Cedex, France.

ABSTRACT
Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.

Show MeSH
Related in: MedlinePlus