Early tissue transglutaminase–mediated response underlies K562(S)-cell gliadin-dependent agglutination

Articles nature publishing group Basic Science Investigation Early tissue transglutaminase–mediated response underlies K562(S)-cell gliadin-dependent agglutination Marco Silano1, Olimpia Vincentini1, Alessandro Luciani2, Cristina Felli1, Sergio Caserta3, Speranza Esposito2, Valeria Rachela Villella3, Massimo Pettoello-Mantovani2, Stefano Guido3 and Luigi Maiuri2 INTRODUCTION: K562(S) agglutination has been used as a rapid and economic tool for the in vitro screening of the toxicity of cereal fractions and prolamins in celiac disease (CD). A strict correlation has been reported between the toxicity of cereals and cereal fractions for celiac patients and their ability to agglutinate K562(S) cells. Whether this specificity of K562(S)-cell agglutination is caused by the activation of the same pathogenic events triggered by toxic cereal fractions in CD intestine or simply represents a bystander event of gluten toxicity is, however, unknown. METHODS: K562(S) cells were incubated in vitro with the peptic-tryptic digest of wheat gliadin. RESULTS: The agglutination of K562(S) cells by wheat gliadin peptides is orchestrated by a cascade of very early events occurring at the K562(S)-cell surface similar to those occurring at the intestinal epithelial surface. They involve a rapid increase in intracellular calcium levels that activate tissue transglutaminase (TG2), leading to a rapid actin reorganization that is pivotal in driving cell agglutination. These specific effects of toxic cereals are phenocopied by the gliadin-derived peptide p31–43, which orchestrates the activation of innate response to gliadin in CD. DISCUSSION: Our study provides the rationale for the extensive use of K562(S)-cell agglutination as a valuable tool for screening cereal toxicity. C eliac disease (CD) is a permanent intolerance to gluten, an alcohol-soluble protein fraction present in some cereals such as wheat, rye, and barley, occurring in genetically predisposed individuals (1,2). During the past few years, a series of seminal papers have provided a clearer picture of the pathogenic mechanisms of CD with a specific activation by gluten/gliadin-derived peptides of HLA (histocompatibility leukocyte antigen)-DQ 2/8 restricted CD4+ T cells at the small-intestinal level (3). Despite this unassailable evidence, a series of studies have indicated that T cells, although essential for the full CD manifestation, cannot explain all the facets of CD (4,5). These studies have suggested that different portion(s) of gliadin, ostensibly not the ones recognized by T cells, modulate an innate activation of celiac small intestine that sets the tone and intensity of the adaptive immune response induced by the immunodominant gliadin epitopes (6). Surprisingly, gliadin and prolamin preparations also show biological activity on celiac-unrelated in vitro systems, including intestinal epithelial cells lines, fetal rat jejunum, and K562(S) cells, a highly undifferentiated cell line isolated from an outgrowth of a patient with chronic myelogenous leukemia (7–9). K562(S) cells undergo agglutination within few minutes after contact with gluten digests. A strict correlation has been reported between the toxicity of cereals and cereal fractions for celiac patients and their biological activity in these unrelated in vitro systems (10). Therefore, the ability to agglutinate K562(S) cells has been used for screening of toxicity of gluten fractions belonging to different cultivars (11). The mechanisms underlying such a puzzling behavior of toxic cereal fractions are unknown. In particular, this evidence raised a series of issues; the most pressing is the clarification of where the specificity for gliadin of these unrelated models lies. A common feature of gliadin/prolamin response between gluten/gliadin-responsive intestinal epithelial cell lines, such as T84, and the K562(S) model is the rapidity of response. Indeed, K562(S) cell agglutination is a very rapid event (11), and gliadin digests are able to upregulate tissue transglutaminase (TG2) protein and activity as well as induce cytoskeleton reorganization (12) very early upon gluten/gliadin exposure in sensitive epithelial cells. Moreover, neutralizing such precocious events is effective in controlling the downstream gliadin-triggered epithelial activation (13). Whether these mechanisms also underlie the agglutination of K562(S) cells is, however, still unknown. Here, we demonstrate how the agglutination of K562(S) cells by gliadin preparation or gliadin-derived peptides is induced by a cascade of very precocious events occurring at the cell surface upon contact with toxic gluten fractions and involves surface mechanisms similar to those induced in intestinal e pithelial cells. Results PT Gliadin Digest Induces Very Early TG2 Activation in K562(S) Cells Upon Gliadin Exposure TG2 is a Ca2+-dependent ubiquitous enzyme with multifaceted functions that mainly catalyzes deamidation or cross-linking of substrate proteins via ε-(γ-glutamyl)lysine isopeptide bonds (14,15). TG2 is also involved in cytoskeleton rearrangement (14). Because TG2 plays a key role in the most precocious Unit of Human Nutrition and Health, Istituto Superiore di Sanità, Rome, Italy; 2Institute of Pediatrics, University of Foggia, Foggia, Italy; 3Department of Chemical Engineering, University of Napoli Federico II, Naples, Italy. Correspondence: Marco Silano () 1 Received 06 July 2011; accepted 13 December 2011; advance online publication 7 March 2012. doi:10.1038/pr.2012.4 532 Pediatric Research      Volume 71 | Number 5 | May 2012 Copyright © 2012 International Pediatric Research Foundation, Inc. Gliadin and K562(S)-cell agglutination b 1,200 K562(S) TG2 Actin OD (arbitrary units) a T84 1,000 * 800 600 digest could increase intracellular Ca2+ levels in K562(S) agglutinated cells. We found that PT gliadin digest induced a dramatic increase in the intracellular Ca2+ levels within 5 min of incubation, whereas PT zein digest or phosphate-buffered saline (PBS) did not (Figure 3b,c). L,2-bis(aminophenoxy) ethane-N,N,N,N′-tetraacetic acid (BAPTA)-AM (5 mmol/l), a Ca2+ chelator also prevented both PT gliadin-induced TG2 activation (data not shown) and agglutination in K562(S) cells (Figure 3d). Furthermore, ionomycin (5 mmol/l) induced K562(S) agglutination (data not shown). Together, these findings demonstrate that PT digests from toxic cereals agglutinate K562(S) cells by inducing an early increase in intracellular Ca2+ levels, leading to TG2 activation. Next, we addressed whether such an early Ca2+-dependent TG2 activation, occurring in K562(S) cells upon gliadin exposure, could also regulate the rearrangement of cytoskeleton that is pivotal in cell agglutination (23,24). Blocking TG2 activation, a central player of these cell surface events (14), by means of the Ca2+ chelator BAPTA-AM or the anti-TG2 antibody CUB 7402 prevented the early rearrangement of the actin fibers occurring after a few (...truncated)