Interaction with Monocytes Enhances IL-5 Gene Transcription in Peripheral T Cells of Asthmatic Patients

Abstract

Background: The regulatory mechanisms of IL-5 gene transcription in human peripheral T cells are unclear because the transfection efficiency of plasmid constructs into nontransformed T cells is very low. Methods: Concanavalin A (ConA)-stimulated blastocytes derived from peripheral blood lymphocytes of asthmatic subjects were transiently transfected with the human IL-5 gene promoter/enhancer-luciferase gene construct, pIL-5 (–511)Luc, and cultured with THP-1 cells (human monocytoid cells) and anti-CD3 monoclonal antibody (mAb). IL-5 level in the culture medium was determined by an enzyme-linked immunosorbent assay. Transcriptional activity of the IL-5 gene was measured by luciferase reporter analysis. Results: ConA-blast lymphocytes of asthmatic patients produced a significant amount of IL-5 upon combined stimulation with anti-CD3 mAb and THP-1 cells, but not with anti-CD3 mAb alone. Costimulation with anti-CD28 mAb also enhanced the anti-CD3 mAb- induced IL-5 production. Accordingly, luciferase activity induced by anti-CD3 mAb stimulation in pIL-5(–511)Luc-transfected ConA-blast lymphocytes was increased 1.9- and 3.4-fold by the addition of anti-CD28 mAb and THP-1 cells, respectively. Serial 5′ deletion analysis of the reporter gene demonstrated that the cis-regulatory element located at –119 to –80 is critical for anti-CD3 mAb-induced IL-5 gene transcription. Conclusions: Our present findings provide a useful model reflecting IL-5 gene transcription in human peripheral T cells in vivo, and clearly demonstrate that an interaction with monocytes enhances IL-5 gene transcription.

Eosinophilic inflammation: mechanisms regulating IL‐5 transcription in human T lymphocytes

“…The importance of the Ets1 site and the synergistic interactions with GATA-3 and AP-1 were also demonstrated for the first time with freshly prepared normal human T cells. Our use of a high efficiency transfection method allowed these studies to be done with primary T cells from healthy donors rather than needing to use T cells from asthma patients (25,26). Taken together our findings suggest that Ets1, AP-1 and GATA-3 may be part of a higher order transcriptional complex involved in regulating human IL-5 expression in T cells.…”

“…Earlier work with T lymphocytes from asthmatic patients (25,26) showed a role for the proximal promoter region in regulating IL-5 transcription but no detailed studies were carried out. We have used HSB-2 cells to look in detail at the role of three putative sites in this region.…”

“…In terms of a role for GATA, deletion data from Gruart-Gouilluex et al (23) indicated no involvement of the proximal GATA site in promoting human IL-5 transcription, whereas a selective role for GATA-4 rather than GATA-3 at this site was shown with ATL-16T cells (24). Two studies using T lymphocytes from asthmatic patients have shown a role for the proximal promoter region to )119 in human IL-5 transcription (25,26) but detailed transactivation studies with transcription factors have not been carried out with primary T cells.…”

Resident Alveolar Macrophages Suppress, whereas Recruited Monocytes Promote, Allergic Lung Inflammation in Murine Models of Asthma

“…As noted previously, the fact that BALF TGF-β levels decreased upon monocyte depletion in parallel with those of all other measured cytokines argues against the importance of TGF-β as an inhibitory factor in our asthma model. A pathogenic role of monocytes might be explained by previous observations identifying circulating monocytes from asthmatics as a source of IL-5 [46–48], which is an eosinophil chemoattractant. Finally although our data fail to support that circulating monocytes serve as precursors for AMs, previous studies have shown that blood monocytes can be precursors for lung dendritic cells in both steady-state conditions [49] and in models of allergic asthma [50] and infectious lung disease [51, 52].…”

Human and murine model cell lines for dendritic cell biology evaluated

“…THP-1 cells secrete T cell recruiting and differentiating cytokines, such as IL-6 and IL-8 [56], leading to moderate stimulation of T cells [57,58]. HL-60 cells produce IL-6 [59] and IL-8 [60] and can also stimulate T cells upon activation, albeit moderately [61,62].…”