pubmed.ncbi.nlm.nih.gov

Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by reciprocal secretion of adenosine and IL-6 - PubMed

Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by reciprocal secretion of adenosine and IL-6

S V Sitaraman et al. J Clin Invest. 2001 Apr.

Abstract

Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca(++)] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.

PubMed Disclaimer

Figures

Figure 1
Figure 1

IL-6 induction by adenosine. T84 monolayers were prewashed in HBSS and equilibrated at 37°C for 20 minutes. Adenosine (Ado; 100 μM) was added to the apical (Ap) or basolateral (Bs) compartment. After an incubation period of 5.5–6 hours at 37°C, IL-6 was measured in the apical or basolateral compartment as described in Methods. Values are expressed as pg/ml. Data represent the responses observed in three separate experiments plotted as mean ± SD, n = 3 samples per treatment group. Footnotes represent values significantly different from monolayers treated with vehicle alone, AP < 0.001, BP < 0.05. (a) Monolayers treated with either vehicle or adenosine. (b) Monolayers treated with either adenosine (100 μM) or adenosine plus 8-SPT (100 μM). Ap, apical; Bs, basolateral.

Figure 2
Figure 2

Time course of IL-6 secretion. T84 monolayers were prewashed in HBSS. After equilibration at 37°C for 20 minutes, cells were stimulated with adenosine (apical or basolateral; 100 μM), HBSS was collected for 1, 2, 3, or 6 hours, and IL-6 was measured as described in Methods. Values are expressed as pg/ml IL-6. Data represent the responses observed in three separate experiments plotted as mean ± SD, n = 2 samples per treatment group. Footnotes represent values significantly different from the respective 0 time point, AP < 0.001, BP < 0.05.

Figure 3
Figure 3

Effect of various proinflammatory agents on IL-6 secretion. T84 cells were washed with HBSS. After equilibration of 20 minutes at 37°C, forskolin (10 μM), TNF-α (100 ng/ml), and carbachol (1 μM) were added basolaterally. Apical and basolateral media were collected for 5.5–6 hours after stimulation. S. typhimurium was added apically for 1 hour for surface colonization (30), after which the monolayers were rinsed thoroughly with HBSS and incubated for an additional 5 hours, and IL-6 assays were performed. Data represent the responses observed in two separate experiments plotted as mean ± SD, n = 2 per treatment group. ASignificantly different from the unstimulated control, P < 0.001. BSignificantly different from unstimulated control, P < 0.05.

Figure 4
Figure 4

Effect of adenosine on IL-6 promoter activity and analysis of adenosine-mediated regulation of IL-6 promoter mutants. (a) Most elements of the IL-6 promoter that have been characterized lie within the 300 bp proximal to the start site (+1) of transcription. These elements include multiple AP-1–binding sites, an IRF-1–binding site, three glucocorticoid response elements (GRE), two second-messenger and cytokine-responsive elements (MREs), the first of which contains ATF/CREB binding sites and the second a binding site for C/EBPβ or NF-IL-6–binding site, and a NF-κB–binding element. The promoter also contains two transcription start sites, one major and one minor. Mutants that were generated in the ATF/CRE, NF–IL-6, and NF-κB sites are indicated in Methods. (b) COS-7 cells were transiently transfected with wild-type (WT) or various mutant IL-6 CAT constructs. Cells were stimulated with adenosine 66 hours after transfection, and a CAT assay was performed 4 hours after stimulation. Data represent fold increase over transfected and unstimulated control (expressed as nanograms CAT/μg protein) observed in two separate experiments plotted as mean ± SD from duplicate determination of two samples per group, n = 2.

Figure 5
Figure 5

Expression of phosphorylated ATF/CREB in response to adenosine. T84 cells were washed with HBSS, equilibrated for 20 minutes at 37°C, and stimulated with adenosine (100 μM, apical or basolateral) for the various times indicated. Whole-cell detergent lysates (approximately 15 μg protein/lane) were resolved by SDS-PAGE and immunoblotted for phosphorylated ATF/CREB (a) and total CREB (b). Scanning densitometry of the blot is shown in b depicting a four- and eightfold increase in ATF-1 and CREB, respectively compared with 0 time. The induction was maximal at 60 minutes and declined at 180 and 360 minutes after stimulation. Data represent the responses observed in two separate experiments with two filters per time point.

Figure 6
Figure 6

Confocal microscopy of phospho-CREB. T84 monolayers were incubated with apical or basolateral adenosine (100 μM) for 5 minutes. Nuclear staining was determined by immunofluorescence labeling and confocal microscopy. The en face images document the presence of activated phospho-CREB staining in cells treated with apical or basolateral adenosine (b and c, respectively). Control cells double-stained with phospho-CREB and rhodamine-phalloidin are shown in a. Similar results were obtained at 30 and 60 minutes after adenosine stimulation.

Figure 7
Figure 7

Effect of IL-6 on neutrophil calcium response. Neutrophils (106) were loaded with the [Ca++] indicator Indo-1 and placed in a thermostated spectrofluorometer, and intracellular [Ca++] was measured as described in Methods. Stimuli were added at 60 seconds. Intracellular [Ca++] was measured in response to fMLP (10–6M), IL-6 in the indicated doses, and monoclonal anti–IL-6 added along with IL-6 (100 pg/ml) (IL-6 + IL-6 Ab).

Figure 8
Figure 8

Schematic representation of epithelial-neutrophil interaction in a crypt abscess. An epithelial monolayer with neutrophils transmigrating to the luminal compartment is shown at the left. Inset shows that adenosine is derived from the enzymatic conversion (mediated by epithelial ectonucleotidase, CD 73) of 5′ AMP released in the lumen by the neutrophils. Adenosine thus released interacts with the adenosine A2b receptor, a G protein–coupled receptor, resulting in an increase in intracellular cAMP that may be involved in the transcriptional activation of IL-6 secretion. IL-6 is preferentially released in the apical compartment and induces intracellular [Ca++] flux in neutrophils, which may be involved in the release of oxygen radicals, elastase, etc., from the neutrophils.

Similar articles

Cited by

References

    1. Nash S, Parkos C, Nusrat A, Delp C, Madara JL. In vitro model of intestinal crypt abscess: a novel neutrophil-derived secretagogue (NDS) activity. J Clin Invest. 1991;87:1474–1477. - PMC - PubMed
    1. Palmer TM, Stiles GL. Adenosine receptors. Neuropharmacology. 1995;34:683–694. - PubMed
    1. Roman RM, Fitz JG. Emerging roles of purinergic signalling in gastrointestinal epithelial secretion and hepatobiliary function. Gastroenterology. 1999;116:964–979. - PubMed
    1. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998;50:413–492. - PubMed
    1. Van Belle H, Gossens F, Wynants J. Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia. Am J Physiol. 1987;252:H886. - PubMed

Publication types

MeSH terms

Substances