Different Thymosin Beta 4 Immunoreactivity in Foetal and Adult Gastrointestinal Tract

Background Thymosin beta 4 (Tβ4) is a member of beta-thymosins, a family of peptides that play essential roles in many cellular functions. A recent study from our group suggested a role for Tβ4 in the development of human salivary glands. The aim of this study was to analyze the expression of Tβ4 in the human gut during development, and in the adult. Methodology/Principal Findings Immunolocalization of Tβ4 was studied in autoptic samples of tongue, oesophagus, stomach, ileum, colon, liver and pancreas obtained from two human foetuses and two adults. Tβ4 appeared unevenly distributed, with marked differences between foetuses and adults. In the stomach, superficial epithelium was positive in foetuses and negative in adults. Ileal enterocytes were strongly positive in the adult and weakly positive in the foetuses. An increase in reactivity for Tβ4 was observed in superficial colon epithelium of adults as compared with the foetuses. Striking differences were found between foetal and adult liver: the former showed a very low reactivity for Tβ4 while in the adult we observed a strong reactivity in the vast majority of the hepatocytes. A peculiar pattern was found in the pancreas, with the strongest reactivity observed in foetal and adult islet cells. Significance Our data show a strong expression of Tβ4 in the human gut and in endocrine pancreas during development. The observed differential expression of Tβ4 suggests specific roles of the peptide in the gut of foetuses and adults. The observed heterogeneity of Tβ4 expression in the foetal life, ranging from a very rare detection in liver cells up to a diffuse reactivity in endocrine pancreas, should be taken into account when the role of Tβ4 in the development of human embryo is assessed. Future studies are needed to shed light on the link between Tβ4 and organogenesis.


Introduction
Beta-thymosins (Tbs) constitute a highly conserved family of actin-binding polypeptides [1], presenting a well conserved fouraminoacid motif, corresponding to the sequence LKKT, which interacts with actin, promoting or inhibiting actin assembly [2]. Thymosin beta-4 (Tb 4 ) is the archetypal member of the betathymosins family: it is a 43-aminoacid peptide, isolated from human blood platelets [3] which forms a 1:1 complex with actin, inhibits its polymerization [4], and acts as an extremely effective actin-monomer sequestering peptide [5]. Tb 4 has multiple functions: it moonlights to repair injured tissues [6], has antiinflammatory efficacy in monocyte/macrophages [7], promotes wound healing [8] and mediates angiogenesis [9]. Tb 4 has been also shown to play a relevant role during the development of different neural cell types in the rat brain [10]. In particular, Tb 4 plays a neurotrophic and antiapoptotic role during the development of the nervous system [11]. In the embryo, reduced myocardial Tb 4 levels have been reported to cause a disrupted coronary vasculogenesis and a number of cardiac defects [12]. During coronary vessels development, Tb 4 should act on cardiac stem cells, also known as epicardially derived cells (EPDCs) [13] inducing their migration into the myocardium, where they differentiate into either endothelial or smooth muscle cells [9]. The theory on the putative role of Tb 4 in the physiological development of embryos, as well as in vascularization and tissue recovery in acute and chronic ischemia, has been reinforced by the discovery that Tb 4 is one of the most abundant factors secreted by embryonic endothelial progenitor cells [14]. Recently a study from our group evidenced a strong reactivity for Tb 4 in developing foetal salivary glands, with a switch from the acinar component to the ductal cells in the adult [15]. Tb 4 has also been identified as a predominant transcript in intraepithelial lymphocytes (IEL) in the murine gut [16], in which it could exert a relevant antiinflammatory effect by inhibiting neutrophilic infiltration [17].
On the basis of these data, suggesting a relevant role for Tb 4 during the development of the foetus and the embryo, it seemed of some interest to investigate the expression of the peptide in the gastrointestinal tract of human fetuses and in adults, with the aim to gain insights into the expression of Tb 4 in the human gastrointestinal tract during development.

Materials and Methods
Two human fetuses of 20 weeks (male) and 21 weeks (female) of gestational age, and two adults 65 and 74 year old respectively were the object of the present study. In each subject, we obtained, at autopsy, multiple samples from the following segments of the gut: tongue, oesophagus, stomach, ileum, colon. Samples were also obtained from liver and pancreas. Tissue samples were fixed in 10% formalin, routinely processed and paraffin-embedded. Immunohistochemistry was performed on 5 mm-thick sections, using the labeled streptavidin-biotin complex system (LSAB2, Dako) in a Dako Autostainer (DakoCytomation, Carpintera, CA, USA). Heat-induced antigen retrieval was carried out by steaming unstained sections in Target Retrieval Solution (Dako TRS pH 6.1) for 30 min. Tissue sections were incubated (30 min at room temperature) with the monoclonal anti-thymosin beta 4 antibody (Bachem, Bubendorf, Switzerland). Sections of a reactive human adult lymph node with activated macrophages were used as positive controls. As a negative control, we utilized sections of foetal oesophagus: immunoistochemistry was performed using isotype antibody (Fig. 1). The immunostaining was interpreted as positive when at least 10% of cells expressed the antigen. The positive expression was further categorized into focal (10-33%) and diffuse (.33%).
All cases were independently reanalyzed by two pathologists specialized in gastrointestinal pathology (GF, SN).

Ethics Statements
The study protocol and written consent forms were approved by the Ethics Human Studies Committee of University Medical Centre of Cagliari (according to the instructions of the declaration of Helsinki). Full written consent forms were obtained from the parents of the newborns and all rules were respected. For the specimens from adults, we obtained written consent to revearch use by their next of kin.

Results
The immunostaining for Tb 4 appeared granular or homogeneously diffuse, always restricted to the cytoplasm of positive cells; no nuclear reactivity was observed in this study. Reactivity for the peptide was also observed, in some cases, in the interstitial spaces, as fine granules, mainly located around the epithelial structures. No significant differences were found in the immunoistochemical pattern for Tb 4 between the two foetuses and the two adults analyzed.

Tongue
Foetus (20 weeks of gestation). The epithelium covering the tongue appeared constantly negative in its deeper layers. The superficial epithelial cells showed a diffuse immunoreactivity for Tb 4 , localized in the cytoplasm (Fig. 2a). The underlying corion was negative. A weak positivity was also observed in the muscular cells.
Adult. The immunoistochemical pattern paralleled that detected in the foetuses. The superficial layers of the stratified

Oesophagus
Foetus (21 weeks of gestation). The epithelium covering the oesophageal lumen appeared negative. The oesophageal lumen appeared coated with a thin Tb 4 -immunoreactive layer, which resulted diffuse to the entire oesophageal lumen (Fig. 3a). A weak reactivity for the peptide was observed in the muscular cells.
Adult. Immunoreactivity for Tb 4 was restricted to epithelial cells of the superficial layers covering the esophageal lumen. No reactivity for Tb 4 was found in the intermediated and deep layers (Fig. 3b).

Stomach
Foetus (21 weeks of gestation). The gastric epithelium showed the presence of Tb 4 -immunoreactive granules in the cytoplasm of the majority of columnar cells. Immunoreactive deposits were mainly found aggregated in the perinuclear regions. Abundant deposits were also found dispersed throughout the mucous covering the gastric surface (Fig. 4a).
Adult. Gastric mucosa was covered by a thin superficial layer intensely reactive for Tb 4 extending to gastric foveolae. Immunoreactivity for Tb 4 in gastric glands was restricted to chief and oxyntic cells (Fig. 4b).
Ileum Foetus (21 weeks of gestation). A granular reactivity for Tb 4 was detected in the epithelium covering the ileal villi, more evident in the cytoplasm of mucous cells (Fig. 5a). A reactivity for the peptide was also observed in the mucous occupying the intestinal lumen.
Adult. A mild but diffuse reactivity for Tb 4 was present in the cytoplasm of enterocytes covering villi (Fig. 5b). Fine granular deposits were also observed in the cytoplasm of mucous cells.

Colon
Foetus (21 weeks of gestation). Coarse granules immunoreactive for Tb 4 were observed in the cytoplasm of superficial colon epithelial cells. No reactivity was found in the  crypts (Fig. 6a). The muscular layer did not show any significant reactivity for the peptide. A weak positivity was detected in the nervous cells.
Adult. An intense and diffuse reactivity with coarse sovranuclear granules immunoreactive for Tb 4 were present in the superficial and crypt epithelium. (Fig. 6b) Liver Foetus (21 weeks of gestation). No significant reactivity for Tb 4 was found in hepatocytes and in biliary ducts nor in biliary epithelial cells of the ductal plate in the foetal liver examined in this study. A weak reactivity was frequently observed in the red blood cells in the dilated sinusoids. Only occasionally, scattered large cells in immature portal tracts showed a strong Tb 4 immunoreactivity (Fig. 7a).
Adult. The expression pattern for Tb 4 changed completely in immunostained adult livers. A strong granular immunoreactivity was found in the cytoplasm of the majority of hepatocytes in all acinar zones and in activated Kupffer cells (Fig. 7b). Portal tracts, including bile ducts were constantly negative.

Pancreas
Foetus (20 weeks of gestation). Immunoreactivity for Tb 4 appeared focal, mainly localized inside the islets of Langerhans. A part of the endocrine cells showed the entire cytoplasm strongly immunoreactive for the peptide. Few Tb 4 -reactive cells were also present in the exocrine pancreas, inside the tubular structures, intermingled among the tubular epithelium. A weak granular reactivity for the peptide was also found inside the cytoplasm of the tubular cells, a pattern suggestive for a secretion of Tb 4 in the pancreatic juice (Fig. 8a).
Adult. The distribution of Tb 4 immunoreactivity paralleled that observed in the foetus: the highest reactivity was observed in the islet cells which showed diffuse fine cytoplasmic granular deposits in the majority of endocrine cells. Only rare acinar cells contained cytoplasmic vacuoles reactive for Tb 4 (Fig. 8b).

Discussion
Tb 4 , the most abundant member in human cells of the thymosin family, has been initially embraced as the ideal actin monomer-  sequestering peptide [18], and its function was restricted to regulate actin polymerization of non-muscle cells [19]. Further data, suggesting a role of Tb 4 in modulating stem cell migration [9], activation [20] and inhibition [21], as well a relation between Tb 4 and integrin signalling [22] induced some authors to speak about ''the b-thymosin enigma'' [9]. The oxidized form of Tb 4 , thymosin beta 4 sulfoxide, has been demonstrated to have an important anti-inflammatory activity inhibiting neutrophil chemotaxis in vitro [23] and in vivo [7].
This study represents the first comprehensive analysis of Tb 4 immunoreactivity in the human gastrointestinal tract, pancreas and liver of human foetus in comparison to adult. Interesting differences were found between foetus and adult Tb 4 immunoreactivity: in some organs, such as liver, the immunoreactivity of Tb 4 was higher in the adult, while in others, such as endocrine pancreas, it was lower. Differences in Tb 4 expression between the foetus and the adult have been previously reported by our group in human salivary glands [15]. In that study developing major and minor salivary glands showed a marked reactivity for Tb 4 , which contrasted with the low levels of immunoreactivity observed in the adult glands. In this study liver showed an opposite pattern, characterized by low reactivity for the peptide in the intrauterine life and much higher levels in the adult life. All together, these data seem to indicate the existence of an exquisite cell type-and differentiation stage-specific regulation and expression pattern of Tb 4 in the human gut and annexed glands during development. As a consequence, Tb 4 could play different roles in different organs during the organogenesis, and these roles should change during adult life.
Immunoreactivity for Tb 4 was detected in all different intestinal segments and in all glands examined, but with striking differences among different sites: pancreas and liver of adults showed the highest levels of reactivity for Tb 4 , while the lowest reactivity was observed in the developing liver. A remarkable heterogeneity of Tb 4 immunoreactivity within the developing gastrointestinal tract was observed, ranging from diffuse immunoreactivity in pancreas and enterocytes, to peptide absence in the foetal hepatocytes. It should be outlined that some differences observed in Tb 4 immunoreactivity between fetuses and adult organs may be  related to the different degree of cell differentiation. For example, chief and oxyntic cells, the site of Tb 4 storage in the adult stomach, are not well differentiated yet in the foetal stomach glands. Noticeable interindividual differences during gut development, regarding the intensity of the positivity for Tb 4 and its subcellular localization, were also observed (data not reported).
On the whole, the high amounts of Tb 4 here reported in the human gut during development are in agreement with previous experimental studies on the role of Tb 4 in organogenesis. High levels of Tb 4 mRNA had been indeed reported in early mouse postimplantation embryos [24] and in the ventricular myocardium of mice at embryonic day 10 [25]. The essential role of Tb 4 in heart development had been demonstrated by the generation of mice with RNAi-mediated cardiac specific knockdown of Tb 4 . Tb 4 -mutant hearts showed severe impairment in the mobilization of cardiac progenitor cells, resulting in impaired cardiac development and survival [26]. Our data clearly indicate a major expression of Tb 4 during the development of the human gastrointestinal tract. Such a strong expression should be related to relevant roles in gut development. The well known function of Tb 4 in the regulation of the equilibrium betwen globular and filamentous actin [27] could partly explain the strong Tb 4 immunoreactivity found in the cytoplasm of enterocytes in this study. Tb 4 has been hypothesized to also function as a sentinel of the cell oxidative stress, its oxidation reflecting an oxidizing environment that often correlates with cell damage [7]. Lymphoid Tb 4 , a splice variant of Tb 4 produced by intraepithelial lymphocytes normally present in the cytoplasm of enterocytes, has been shown to have a great capacity to reduce oxidative stress [17]. Following this hypothesis, a strong immunohistochemical reaction for Tb 4 could be interpreted as a cell's response to stress, also considering that antibodies utilized in this study cannot discriminate the oxidized Tb 4 from the non-oxidized one. Recently, Tb 4 has been shown to be involved in regulating phagocytosis of apoptotic cells mediated by stabilin-2 [28]. Given the fundamental role of apoptosis in embryology, our finding of a strong reactivity for Tb 4 in the developing gut as well as in developing salivary glands [15] reinforces the hypothesis of an important role of Tb 4 in organ development during embryogenesis. Future studies will be necessary to shed light on the link between Tb 4 and organogenesis, with particular emphasis on the role plaied by the peptide in cell response to oxidative stress, in apoptosis, in actin metabolism in the different phases of fetal, embryo and adult life. The heterogeneity of Tb 4 immunoreactivity detected in different organs of gastrointestinal tract and the differences observed between intrauterine and adult life should be taken into account when the role of Tb 4 in human physiology is assessed.