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. 2000 Feb;99(2):279-86.
doi: 10.1046/j.1365-2567.2000.00948.x.

CD40-CD40 ligand (CD154) engagement is required but may not be sufficient for human T helper 1 cell induction of interleukin-2- or interleukin-15-driven, contact-dependent, interleukin-1beta production by monocytes

C Ribbens  1 , J M Dayer, C Chizzolini
Affiliations

CD40-CD40 ligand (CD154) engagement is required but may not be sufficient for human T helper 1 cell induction of interleukin-2- or interleukin-15-driven, contact-dependent, interleukin-1beta production by monocytes

C Ribbens et al. Immunology. 2000 Feb.

Abstract

To investigate whether antigen-independent, interleukin-2 (IL-2) or IL-15 activation of polarized T helper (Th) cells would result in contact-dependent activation of monocytes, living Th1 and Th2 cell clones were co-cultured with THP-1 cells or fresh peripheral blood monocytes. Under these conditions IL-1beta production was induced almost exclusively by Th1 cells and was dependent on the presence and dose of IL-2 or IL-15, and on cell-cell contact, as demonstrated by double-chamber cultures. Low levels of IL-1 receptor antagonist (IL-1Ra) were induced by Th1 and higher levels by Th2 cells. IL-10 production was similar in Th1/monocyte and Th2/monocyte co-cultures, thus arguing against preferential down-regulation of IL-1beta production by anti-inflammatory IL-10 in Th2 co-cultures. In addition, IL-4 and IL-10 neutralization did not result in enhanced IL-1beta production in Th2/monocyte co-cultures. Preferential expression on Th1 cells of CD11b correlated with their capacity to induce IL-1beta production by THP-1 cells in the presence of IL-2 or IL-15, but anti-CD11b monoclonal antibody could not inhibit this activity. Blockade of the CD40-CD40 ligand interaction resulted in inhibition of IL-1beta-inducing capacity while IL-1Ra induction was unaffected, a result previously unknown. This differential effect indicates the selective relevance of CD40-CD40 ligand engagement in inflammatory monocyte responses upon activation by T cells. CD40 ligand expression levels did not differ in Th1 and Th2 cell clones, thus indicating that additional, unidentified molecule(s) preferentially expressed by Th1 cells are involved in their IL-1beta induction capacity.

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Figures

Figure 1
Figure 1
IL-1β production in co-cultures of THP-1 cells with Th1 and Th2 clones in the presence of IL-2 and IL-15; (a) Th1 cell clones, (b) Th2 cell clones. T-cell clones harvested 13 days after previous stimulation were co-cultured (5, 10, 20, or 40 × 104 cells) with THP-1 cells (5 × 104 cells) in medium alone (▪), in the presence of IL-2 (20 U/ml) (▴), or IL-15 (100 ng/ml) (•). After 48 hr of culture, supernatants were harvested and analysed for IL-1β content.
Figure 2
Figure 2
IL-1Ra, IFN-γ and IL-4 production in T/THP-1 cell co-cultures and IFN-γ/IL-4 production by Th1 and Th2 clones. (a) IL-1Ra produced in co-cultures of Th1 or Th2 with THP-1 cells; (b) IFN-γ and IL-4 production by Th1 clones; (c) IFN-γ and IL-4 production by Th2 clones. Co-culture conditions were the same as reported in Figure 1, and IL-1Ra, IFN-γ and IL-4 were measured in 48 hr supernatants at the highest T:THP-1 ratio. T-cell clones were also cultured alone (5 × 104 cells/well) and activated by CD3-cross-linking. IFN-γ and IL-4 were measured in 24 hr supernatants. Data are means±SEM of five Th1 and five Th2.
Figure 3
Figure 3
Cell–cell contact is required for IL-1β and IL-1Ra production in Th1/THP-1 co-cultures. T cells (2·5, 5, or 10 × 105 cells) and THP-1 cells (2·5 × 105 cells) were co-cultured in a double-chamber 24-well culture plate either together (filled symbols) or separated by a semi-permeable membrane (open symbols). IL-1β and IL-1Ra production was measured in the supernatants after 48 hr of culture. IL-15 (100 ng/ml), IL-2 (20 IU/ml), medium. Similar data were obtained in two distinct experiments.
Figure 4
Figure 4
IL-1β, IFN-γ and IL-4 production in T/monocyte co-cultures and IFN-γ/IL-4 production by Th1 and Th2 clones. (a) IL-1β produced in co-cultures of Th1 or Th2 with monocytes; (b) IFN-γ and IL-4 production by Th1 clones; (c) IFN-γ and IL-4 production by Th2 clones. Culture conditions were specified in legends of Figs 1 and 2 except that freshly isolated peripheral blood monocytes were used instead of THP-1 cells. Data are means±SEM obtained with three Th1 and three Th2 clones.
Figure 5
Figure 5
IL-1β and IL-10 production in T/monocyte co-cultures. Freshly isolated monocytes (5 × 104 cells/well) were cultured with T cells (4 × 105 cells/well) in the presence of IL-2 (20 U/ml) for 48 hr. Data are results from single clones obtained in four distinct experiments (a–d) where Th1 and Th2 cell clones were cultured in parallel. Letters in brackets indicate distinct monocyte donors. In experiments (b) and (c) monocytes and T cells were autologous.
Figure 6
Figure 6
Effect of various mAb on IL-1β production in Th1/monocyte co-cultures. (a) IL-1β production in Th/monocyte co-cultures. (b) IL-1Ra production in Th1/monocyte or Th2/monocyte co-cultures. Freshly isolated monocytes were cultured with Th1 cells as described in Figure 5. The mAb or sCD40-Fc were added at the beginning of the culture. All the mAb were IgG1 and used at 10 µg/ml. The sCD40 was used at 1 µg/ml. The number of individual clones tested is given in parentheses. IL-1β production in the presence of irrelevant IgG1 was 153·5 ± 35·4 in different experiments. Bars indicate SEM.
Figure 7
Figure 7
Representative CD40L expression on Th1 and Th2 clones as function of time. Flow cytometry was performed on Th1 and Th2 cells simultaneously 40 hr (upper panel) or 10 days (lower panel) after subculture. Thick line: CD40L, dotted line: isotype control.
Figure 8
Figure 8
Schematic representation of Th1/monocyte and Th2/monocyte co-culture outcomes. x, x-L, y, y-L denote hypothetical receptors and receptor ligands.
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