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Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking.

Barone MV, Zanzi D, Maglio M, Nanayakkara M, Santagata S, Lania G, Miele E, Ribecco MT, Maurano F, Auricchio R, Gianfrani C, Ferrini S, Troncone R, Auricchio S - PLoS ONE (2011)

Bottom Line: IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response.The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor.In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. mv.barone@unina.it

ABSTRACT

Background and objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation.

Methods/principal findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor.

Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

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Both IL-15 and IL-15R-alpha expression increase in the isolated membrane fraction after stimulation with P31-43 for 30 min and 3 h.A) Western blot analysis of membrane proteins separated from total cell lysates shows an increase in membrane protein fractions of IL-15 and IL-15R alpha after P31-43 treatment. B and C) densitometric analysis of the Western blot experiment shown in a. EGFR was used to normalise membrane protein measurements. Increments (i) of IL-15 and IL-15R alpha were calculated as follows: iIL-15 =  (IL-15 treated [t]/IL-15 untreated [un])/(EGFR Treated [T]/EGFR Untreated [UN]). iIL-15R  = (IL-15R [t]/Il-15R [un]/(EGFR [T]/EGFR [UN]). The blots shown are representative of three similar independent experiments.
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pone-0017039-g006: Both IL-15 and IL-15R-alpha expression increase in the isolated membrane fraction after stimulation with P31-43 for 30 min and 3 h.A) Western blot analysis of membrane proteins separated from total cell lysates shows an increase in membrane protein fractions of IL-15 and IL-15R alpha after P31-43 treatment. B and C) densitometric analysis of the Western blot experiment shown in a. EGFR was used to normalise membrane protein measurements. Increments (i) of IL-15 and IL-15R alpha were calculated as follows: iIL-15 =  (IL-15 treated [t]/IL-15 untreated [un])/(EGFR Treated [T]/EGFR Untreated [UN]). iIL-15R  = (IL-15R [t]/Il-15R [un]/(EGFR [T]/EGFR [UN]). The blots shown are representative of three similar independent experiments.

Mentions: Duitman et al. demonstrated that membrane-associated IL-15 is directed to the cell surface in complex with IL-15R alpha, which serves as a chaperone for its ligand [30]. We therefore investigated whether cell surface IL-15, which is increased by P31-43, is also attached to the receptor in CaCo-2 cells (Fig. 5–7). PCR analysis confirmed the presence of IL-15R alpha mRNA in CaCo-2 cells (not shown).


Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking.

Barone MV, Zanzi D, Maglio M, Nanayakkara M, Santagata S, Lania G, Miele E, Ribecco MT, Maurano F, Auricchio R, Gianfrani C, Ferrini S, Troncone R, Auricchio S - PLoS ONE (2011)

Both IL-15 and IL-15R-alpha expression increase in the isolated membrane fraction after stimulation with P31-43 for 30 min and 3 h.A) Western blot analysis of membrane proteins separated from total cell lysates shows an increase in membrane protein fractions of IL-15 and IL-15R alpha after P31-43 treatment. B and C) densitometric analysis of the Western blot experiment shown in a. EGFR was used to normalise membrane protein measurements. Increments (i) of IL-15 and IL-15R alpha were calculated as follows: iIL-15 =  (IL-15 treated [t]/IL-15 untreated [un])/(EGFR Treated [T]/EGFR Untreated [UN]). iIL-15R  = (IL-15R [t]/Il-15R [un]/(EGFR [T]/EGFR [UN]). The blots shown are representative of three similar independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045409&req=5

pone-0017039-g006: Both IL-15 and IL-15R-alpha expression increase in the isolated membrane fraction after stimulation with P31-43 for 30 min and 3 h.A) Western blot analysis of membrane proteins separated from total cell lysates shows an increase in membrane protein fractions of IL-15 and IL-15R alpha after P31-43 treatment. B and C) densitometric analysis of the Western blot experiment shown in a. EGFR was used to normalise membrane protein measurements. Increments (i) of IL-15 and IL-15R alpha were calculated as follows: iIL-15 =  (IL-15 treated [t]/IL-15 untreated [un])/(EGFR Treated [T]/EGFR Untreated [UN]). iIL-15R  = (IL-15R [t]/Il-15R [un]/(EGFR [T]/EGFR [UN]). The blots shown are representative of three similar independent experiments.
Mentions: Duitman et al. demonstrated that membrane-associated IL-15 is directed to the cell surface in complex with IL-15R alpha, which serves as a chaperone for its ligand [30]. We therefore investigated whether cell surface IL-15, which is increased by P31-43, is also attached to the receptor in CaCo-2 cells (Fig. 5–7). PCR analysis confirmed the presence of IL-15R alpha mRNA in CaCo-2 cells (not shown).

Bottom Line: IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response.The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor.In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. mv.barone@unina.it

ABSTRACT

Background and objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation.

Methods/principal findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor.

Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

Show MeSH
Related in: MedlinePlus