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Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Huff KR, Akhtar LN, Fox AL, Cannon JA, Smith PD, Smythies LE - Mucosal Immunol (2011)

Bottom Line: In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells.Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)).These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (Gastroenterology), University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)). Antibody neutralization studies showed that TGF-β in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1β in Crohn's S-CM promoted T-cell proliferation, and IL-1β alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

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TGF-β promotes down-regulation of CD4+ T-cell responses(A) Proliferation of CD3/CD28-stimulated T-cells cultured in the presence of rhTGF-β alone for 4 days in a representative experiment (p ≤ 0.05 at 50, 250 and 500 pg/mL for n=4). (B) Percent inhibition of CD3/CD28-stimulated T-cell proliferation by rhTGF-β alone (n=4) at 50 pg/mL or 500 pg/mL compared to normal S-CM (250 µg/mL) (n=7). (C) CD3/CD28-stimulated IFN-γ release by T-cells cultured in the presence rhTGF-β (n=3). For B and C, error bars indicate the standard error of the mean. * p ≤ 0.05 and ** p ≤ 0.01.
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Figure 3: TGF-β promotes down-regulation of CD4+ T-cell responses(A) Proliferation of CD3/CD28-stimulated T-cells cultured in the presence of rhTGF-β alone for 4 days in a representative experiment (p ≤ 0.05 at 50, 250 and 500 pg/mL for n=4). (B) Percent inhibition of CD3/CD28-stimulated T-cell proliferation by rhTGF-β alone (n=4) at 50 pg/mL or 500 pg/mL compared to normal S-CM (250 µg/mL) (n=7). (C) CD3/CD28-stimulated IFN-γ release by T-cells cultured in the presence rhTGF-β (n=3). For B and C, error bars indicate the standard error of the mean. * p ≤ 0.05 and ** p ≤ 0.01.

Mentions: We have shown that stromal TGF-β mediates the differentiation of pro-inflammatory monocytes into cells with the phenotype and functional profile of non-inflammatory intestinal macrophages 2–4. Others have shown that blockade of TGF-β in mucosal biopsies leads to increased mucosal Th1 responses 6. Since TGF-β associates with the extracellular matrix 18 and is released into S-CM 2, 4, we assessed the contribution of stromal TGF-β to the down-regulation of T-cell function mediated by normal S-CM. Neutralization of TGF-β in normal S-CM significantly inhibited S-CM down-regulation of inducible T-cell proliferation (Figure 2A, B and Supplemental Figure 4A) and IFN-γ production (Figure 2C and Supplemental Figure 4B) in a dose-dependent manner, implicating a critical role for TGF-β in normal S-CM down-regulation of T-cell function. In parallel experiments, equivalent amounts of irrelevant isotype control antibody did not affect T-cell proliferation or cytokine release (Supplemental Figure 5A–C), and neutralization of TGF-β in complete media with human serum containing low levels of TGF-β also had no effect on T-cell proliferation and IFN-γ release. We next evaluated whether the level of TGF-β in S-CM was sufficient to down-regulate T-cell proliferation and cytokine production. rhTGF-β alone at 50–500 pg/mL induced dose-dependent down-regulation of T-cell proliferation (Figure 3A and Supplemental Figure 4C). However, rhTGF-β alone at 50 pg/mL and 500 pg/mL was less effective at down-regulating T-cell proliferation than normal S-CM (250 µg/mL) (p < 0.01 for each concentration) (Figure 3B). (Note: 50 pg/mL rhTGF-β is equivalent to the level of TGF-β in normal S-CM at a concentration of 250 µg protein/mL (Supplemental Table 1)). rhTGF-β at 50 – 500 pg/mL also inhibited T-cell IFN-γ release (p < 0.05 – 0.01) (Figure 3C and Supplemental Figure 4D). These findings suggest that TGF-β is necessary for T-cell down-regulation in the mucosa, although alone it is not sufficient to achieve the full down-regulation of T-cell responses induced by normal S-CM.


Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Huff KR, Akhtar LN, Fox AL, Cannon JA, Smith PD, Smythies LE - Mucosal Immunol (2011)

TGF-β promotes down-regulation of CD4+ T-cell responses(A) Proliferation of CD3/CD28-stimulated T-cells cultured in the presence of rhTGF-β alone for 4 days in a representative experiment (p ≤ 0.05 at 50, 250 and 500 pg/mL for n=4). (B) Percent inhibition of CD3/CD28-stimulated T-cell proliferation by rhTGF-β alone (n=4) at 50 pg/mL or 500 pg/mL compared to normal S-CM (250 µg/mL) (n=7). (C) CD3/CD28-stimulated IFN-γ release by T-cells cultured in the presence rhTGF-β (n=3). For B and C, error bars indicate the standard error of the mean. * p ≤ 0.05 and ** p ≤ 0.01.
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Figure 3: TGF-β promotes down-regulation of CD4+ T-cell responses(A) Proliferation of CD3/CD28-stimulated T-cells cultured in the presence of rhTGF-β alone for 4 days in a representative experiment (p ≤ 0.05 at 50, 250 and 500 pg/mL for n=4). (B) Percent inhibition of CD3/CD28-stimulated T-cell proliferation by rhTGF-β alone (n=4) at 50 pg/mL or 500 pg/mL compared to normal S-CM (250 µg/mL) (n=7). (C) CD3/CD28-stimulated IFN-γ release by T-cells cultured in the presence rhTGF-β (n=3). For B and C, error bars indicate the standard error of the mean. * p ≤ 0.05 and ** p ≤ 0.01.
Mentions: We have shown that stromal TGF-β mediates the differentiation of pro-inflammatory monocytes into cells with the phenotype and functional profile of non-inflammatory intestinal macrophages 2–4. Others have shown that blockade of TGF-β in mucosal biopsies leads to increased mucosal Th1 responses 6. Since TGF-β associates with the extracellular matrix 18 and is released into S-CM 2, 4, we assessed the contribution of stromal TGF-β to the down-regulation of T-cell function mediated by normal S-CM. Neutralization of TGF-β in normal S-CM significantly inhibited S-CM down-regulation of inducible T-cell proliferation (Figure 2A, B and Supplemental Figure 4A) and IFN-γ production (Figure 2C and Supplemental Figure 4B) in a dose-dependent manner, implicating a critical role for TGF-β in normal S-CM down-regulation of T-cell function. In parallel experiments, equivalent amounts of irrelevant isotype control antibody did not affect T-cell proliferation or cytokine release (Supplemental Figure 5A–C), and neutralization of TGF-β in complete media with human serum containing low levels of TGF-β also had no effect on T-cell proliferation and IFN-γ release. We next evaluated whether the level of TGF-β in S-CM was sufficient to down-regulate T-cell proliferation and cytokine production. rhTGF-β alone at 50–500 pg/mL induced dose-dependent down-regulation of T-cell proliferation (Figure 3A and Supplemental Figure 4C). However, rhTGF-β alone at 50 pg/mL and 500 pg/mL was less effective at down-regulating T-cell proliferation than normal S-CM (250 µg/mL) (p < 0.01 for each concentration) (Figure 3B). (Note: 50 pg/mL rhTGF-β is equivalent to the level of TGF-β in normal S-CM at a concentration of 250 µg protein/mL (Supplemental Table 1)). rhTGF-β at 50 – 500 pg/mL also inhibited T-cell IFN-γ release (p < 0.05 – 0.01) (Figure 3C and Supplemental Figure 4D). These findings suggest that TGF-β is necessary for T-cell down-regulation in the mucosa, although alone it is not sufficient to achieve the full down-regulation of T-cell responses induced by normal S-CM.

Bottom Line: In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells.Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)).These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (Gastroenterology), University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)). Antibody neutralization studies showed that TGF-β in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1β in Crohn's S-CM promoted T-cell proliferation, and IL-1β alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

Show MeSH
Related in: MedlinePlus