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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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The complex IL-15/IL-15R alpha induced by P31-43 on the surface of CaCo-2 cells functions as a growth factor for CTLL2 cells.3H-thymidine incorporation by CTLL2 cells induced to proliferate by CaCo-2 cells untreated or treated with P31-43 or P31-43 and anti-IL-15 or P57-68 was measured. CaCo-2 and CTLL2 cells were co-cultivated overnight. Data are expressed as 3H-TdR (CpM 1×10 6 cells). Columns represent the mean, and bars represent the standard deviation of five independent experiments. *p<0.05 (Student's t-test). For methods, see supplementary material.
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pone-0017039-g008: The complex IL-15/IL-15R alpha induced by P31-43 on the surface of CaCo-2 cells functions as a growth factor for CTLL2 cells.3H-thymidine incorporation by CTLL2 cells induced to proliferate by CaCo-2 cells untreated or treated with P31-43 or P31-43 and anti-IL-15 or P57-68 was measured. CaCo-2 and CTLL2 cells were co-cultivated overnight. Data are expressed as 3H-TdR (CpM 1×10 6 cells). Columns represent the mean, and bars represent the standard deviation of five independent experiments. *p<0.05 (Student's t-test). For methods, see supplementary material.

Mentions: Most of the biological activity of IL-15 is believed to be mediated by the membrane-attached form of the protein [27], [28]. We therefore evaluated the functional activity of IL-15 on the CaCo-2 cell surface by co-culturing irradiated CaCo-2 cells, treated or not with P31-43, with CTLL2, a cell line responsive to the mitogenic effects of both IL-15 and IL-2 [35]. As shown in Fig. 8, the proliferation rate of CTLL2 cells, evaluated as 3H-thymidine incorporation, increased from 24,945 cpm±13,792 of the untreated sample, to 36,431 cpm±13,265 after P31-43 treatment of CaCo-2 cells. As expected, P57-68 treatment of CaCo-2 cells was not able to induce proliferation of CTLL2 (11,952+/−6,108). Furthermore, CTLL2 cells did not proliferate in response to direct treatment with P31-43 alone (not shown). The increase of 3H-thymidine incorporation was dependent on IL-15 because IL-15 blocking antibody treatment prevented CTLL2 proliferation induced by CaCo-2 cells treated with P31-43 (21,129+/−12,648). This finding indicates that IL-15 increased on the cell surface after P31-43 treatment can function as a growth factor. (Methods are described in Text S3)


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)

The complex IL-15/IL-15R alpha induced by P31-43 on the surface of CaCo-2 cells functions as a growth factor for CTLL2 cells.3H-thymidine incorporation by CTLL2 cells induced to proliferate by CaCo-2 cells untreated or treated with P31-43 or P31-43 and anti-IL-15 or P57-68 was measured. CaCo-2 and CTLL2 cells were co-cultivated overnight. Data are expressed as 3H-TdR (CpM 1×10 6 cells). Columns represent the mean, and bars represent the standard deviation of five independent experiments. *p<0.05 (Student's t-test). For methods, see supplementary material.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017039-g008: The complex IL-15/IL-15R alpha induced by P31-43 on the surface of CaCo-2 cells functions as a growth factor for CTLL2 cells.3H-thymidine incorporation by CTLL2 cells induced to proliferate by CaCo-2 cells untreated or treated with P31-43 or P31-43 and anti-IL-15 or P57-68 was measured. CaCo-2 and CTLL2 cells were co-cultivated overnight. Data are expressed as 3H-TdR (CpM 1×10 6 cells). Columns represent the mean, and bars represent the standard deviation of five independent experiments. *p<0.05 (Student's t-test). For methods, see supplementary material.
Mentions: Most of the biological activity of IL-15 is believed to be mediated by the membrane-attached form of the protein [27], [28]. We therefore evaluated the functional activity of IL-15 on the CaCo-2 cell surface by co-culturing irradiated CaCo-2 cells, treated or not with P31-43, with CTLL2, a cell line responsive to the mitogenic effects of both IL-15 and IL-2 [35]. As shown in Fig. 8, the proliferation rate of CTLL2 cells, evaluated as 3H-thymidine incorporation, increased from 24,945 cpm±13,792 of the untreated sample, to 36,431 cpm±13,265 after P31-43 treatment of CaCo-2 cells. As expected, P57-68 treatment of CaCo-2 cells was not able to induce proliferation of CTLL2 (11,952+/−6,108). Furthermore, CTLL2 cells did not proliferate in response to direct treatment with P31-43 alone (not shown). The increase of 3H-thymidine incorporation was dependent on IL-15 because IL-15 blocking antibody treatment prevented CTLL2 proliferation induced by CaCo-2 cells treated with P31-43 (21,129+/−12,648). This finding indicates that IL-15 increased on the cell surface after P31-43 treatment can function as a growth factor. (Methods are described in Text S3)

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