Limits...
Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis.

Foey A, Green P, Foxwell B, Feldmann M, Brennan F - Arthritis Res. (2001)

Bottom Line: PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B.Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population.IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, Hammersmith, London, W6 8LH, UK. a.foey@ic.ac.uk

ABSTRACT
IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.

Show MeSH

Related in: MedlinePlus

T cells from synovial membranes in rheumatoid arthritis (RA-Ts) induce macrophage TNF-α production. RA-Ts were co-cultured with M-CSF- primed macrophages and incubated for 24 hours at 37°C/5% CO2, after which supernatants were harvested and stored at -20°C until ELISA. Bars show mean TNF-α in triplicate culture supernatants ± SD, for a representative of N = 3 replicate experiments. Macros = macrophages; M-CSF = macrophage-colony-stimulating factor; T:M = ratio of RA-T to M-CSF-primed macrophages; TNF = tumour necrosis factor. *P < 0.05, **P < 0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC64854&req=5

Figure 8: T cells from synovial membranes in rheumatoid arthritis (RA-Ts) induce macrophage TNF-α production. RA-Ts were co-cultured with M-CSF- primed macrophages and incubated for 24 hours at 37°C/5% CO2, after which supernatants were harvested and stored at -20°C until ELISA. Bars show mean TNF-α in triplicate culture supernatants ± SD, for a representative of N = 3 replicate experiments. Macros = macrophages; M-CSF = macrophage-colony-stimulating factor; T:M = ratio of RA-T to M-CSF-primed macrophages; TNF = tumour necrosis factor. *P < 0.05, **P < 0.01.

Mentions: After demonstrating that Tck could induce IL-10 production in M-CSF-primed monocytes, we investigated whether RA-Ts and without any further activation also could induce IL-10. Neither fixed RA-Ts nor freshly elutriated peripheral blood monocytes spontaneously produce IL-10 secreted into tissue culture supernatant. When these cell types were co-cultured together, however, monocyte IL-10 was produced (126 ± 35 pg/ml at a T:macrophage ratio of 5:1 [P = 0.0305] and 146 ± 26 pg/ml at a ratio of 7:1 [P = 0.0128]) (Fig. 3a). This IL-10 production is a consequence of physical interaction between these cells, as separation by a semipermeable membrane insert abrogated this production. The ability of monocytes to produce IL-10 was shown using lipopolysaccharide at 1 ng/ml as a positive control; IL-10 was routinely produced at levels greater than 200 pg/ml. In addition, RA-T cells also induced IL-10 production upon physical interaction with M-CSF-primed macrophages, which produced similar or slightly higher concentrations of IL-10 in co-culture (189 ± 23 pg/ml at a T:macrophage ratio of 3:1 [P = 0.0047] rising to 243 ± 28 pg/ml at a T:macrophage ratio of 7:1 [P = 0.009]) (Fig. 3b). RA-Ts also induced macrophage TNF-α production (81 ± 21 pg/ml at 3:1 [P = 0.0293], rising to 150 ± 26 pg/ml at 7:1 [P = 0.0047]) (Supplementary Fig. 2). These CD3+ RA-T cells were predominantly CD4+CD45RO+. In addition, the T-cell activation markers HLA-DR and CD69 were expressed, suggesting that RA-Ts were of an activated, memory phenotype closely resembling that of Tck [6].


Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis.

Foey A, Green P, Foxwell B, Feldmann M, Brennan F - Arthritis Res. (2001)

T cells from synovial membranes in rheumatoid arthritis (RA-Ts) induce macrophage TNF-α production. RA-Ts were co-cultured with M-CSF- primed macrophages and incubated for 24 hours at 37°C/5% CO2, after which supernatants were harvested and stored at -20°C until ELISA. Bars show mean TNF-α in triplicate culture supernatants ± SD, for a representative of N = 3 replicate experiments. Macros = macrophages; M-CSF = macrophage-colony-stimulating factor; T:M = ratio of RA-T to M-CSF-primed macrophages; TNF = tumour necrosis factor. *P < 0.05, **P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64854&req=5

Figure 8: T cells from synovial membranes in rheumatoid arthritis (RA-Ts) induce macrophage TNF-α production. RA-Ts were co-cultured with M-CSF- primed macrophages and incubated for 24 hours at 37°C/5% CO2, after which supernatants were harvested and stored at -20°C until ELISA. Bars show mean TNF-α in triplicate culture supernatants ± SD, for a representative of N = 3 replicate experiments. Macros = macrophages; M-CSF = macrophage-colony-stimulating factor; T:M = ratio of RA-T to M-CSF-primed macrophages; TNF = tumour necrosis factor. *P < 0.05, **P < 0.01.
Mentions: After demonstrating that Tck could induce IL-10 production in M-CSF-primed monocytes, we investigated whether RA-Ts and without any further activation also could induce IL-10. Neither fixed RA-Ts nor freshly elutriated peripheral blood monocytes spontaneously produce IL-10 secreted into tissue culture supernatant. When these cell types were co-cultured together, however, monocyte IL-10 was produced (126 ± 35 pg/ml at a T:macrophage ratio of 5:1 [P = 0.0305] and 146 ± 26 pg/ml at a ratio of 7:1 [P = 0.0128]) (Fig. 3a). This IL-10 production is a consequence of physical interaction between these cells, as separation by a semipermeable membrane insert abrogated this production. The ability of monocytes to produce IL-10 was shown using lipopolysaccharide at 1 ng/ml as a positive control; IL-10 was routinely produced at levels greater than 200 pg/ml. In addition, RA-T cells also induced IL-10 production upon physical interaction with M-CSF-primed macrophages, which produced similar or slightly higher concentrations of IL-10 in co-culture (189 ± 23 pg/ml at a T:macrophage ratio of 3:1 [P = 0.0047] rising to 243 ± 28 pg/ml at a T:macrophage ratio of 7:1 [P = 0.009]) (Fig. 3b). RA-Ts also induced macrophage TNF-α production (81 ± 21 pg/ml at 3:1 [P = 0.0293], rising to 150 ± 26 pg/ml at 7:1 [P = 0.0047]) (Supplementary Fig. 2). These CD3+ RA-T cells were predominantly CD4+CD45RO+. In addition, the T-cell activation markers HLA-DR and CD69 were expressed, suggesting that RA-Ts were of an activated, memory phenotype closely resembling that of Tck [6].

Bottom Line: PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B.Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population.IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, Hammersmith, London, W6 8LH, UK. a.foey@ic.ac.uk

ABSTRACT
IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.

Show MeSH
Related in: MedlinePlus