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Thymosin beta 4 expression in normal skin, colon mucosa and in tumor infiltrating mast cells

Mast cells (MCs) are metachromatic cells that originate from multipotential hemopoietic stem cells in the bone marrow. Two distinct populations of MCs have been characterized: mucosal MCs are tryptase-positive while mast cells in skin contain tryptase and chymase. We now show that a sub-population of MCs is highly immunoreactive for thymosin β4, as revealed by immunohistochemical analyses of normal skin, normal colon mucosa and salivary gland tumors. Four consecutive serial sections from each case were immunostained for thymosin β4 (Tβ4), chymase, tryptase and stained for toluidine blue. In skin biopsies, MCs showed a comparable immunoreactivity for Tβ4, chymase and tryptase. In normal colon mucosa the vast majority of mucosal MCs expressed a strong cytoplasmic immunoreactivity for tryptase and for Tβ4, in the absence of chymase reactivity. A robust expression of Tβ4 was detected in tumor-infiltrating and peritumoral mast cells in salivary gland tumors and breast ductal infiltrating carcinomas. Tumorinfiltrating MCs also showed a strong immunoreactivity for chymase and tryptase. In this paper, we first demonstrate that normal dermal and mucosal mast cells exhibit strong expression of thymosin β4, which could be considered a new marker for the identification of mast cells in skin biopsies as well as in human tumors. The possible relationship between the degree of Tβ4 expression in tumor-infiltrating mast cells and tumor behaviour warrants further consideration in future investigations.


Introduction
Mast cells (MCs) are metachromatic cells that originate from multipotential hemopoietic stem cells in the bone marrow, and reside in vascularized tissues where they play an important role in the innate immune system. 1 The developmental pathways of mast cells within different tissues appear to be different. 2,3 The observation of two distinct populations of mast cells, i.e. mucosal mast cells characterized by reactivity for chondroitin sulfate E proteoglycan 4 and serosal or connective tissue-type mast cells, characterized by the ability to sinthesize heparin 5 led to the concept of mast cell heterogeneity. Heterogeneity of mast cells has been reported in different tissues both in animals6 and in men. 7 It has been demonstrated that mucosal mast cells differ from skin and peritoneal mast cells, even in the expression of two classes of proteases, one with trypsin-like activity, called tryptase, 8 and one with a chimotrypsin-like activity, called chymase, 9 which are among the major components of mast cell granules. In humans, these proteinases are differentially expressed in mast cell subtypes: mucosal MC are tryptase-positive, while mast cells in skin contain tryptase and chymase. 10 These MCs subtypes are designated MCt and MCtc, respectively. The specialization of mast cells in different tissue sites has been hypothesized to be related to their involvement in physiology and pathology, which is yet poorly understood. 11 MCs can also be stained with antibodies against c-kit. 12 Histochemically, mast cells may be easily revealed by toluidine blue 13 and by alcian blue-safranin 14 stains. The latter method is able to detect a higher number of mast cells than toluidine blue staining. 15 The fixation mode of tissue samples also influences the number of detected mast cells. It is commonly accepted that Carnoy's solution has the best capacity to fix mast cell granules; 16 formalin is also good and allows appropriate histochemistry and immunohistochemistry for MCs. The aim of this study is to propose the introduction of thymosin β 4 (Tβ 4 ) as a new marker for serosal, mucosal and tumor infiltrating mast cells.

Materials and Methods
In order to test Tβ 4 immunoreactivity in the different subtypes of human MCs, we examined 10 surgical specimens of normal skin containing tryptase and chymase-positive serosal MCs and 10 normal colon biopsies containing tryptase-positive mucosal mast cells. Tβ 4 expression in tumor-infiltrating MCs was evaluated in 4 acinic cell carcinomas of salivary glands and in 10 ductal infiltrating breast carcinomas. All 34 specimens were selected for this study by the preliminary demonstration through toluidine-blue stain of metachromatic granules-containing MCs. Positive controls included tonsil and reactive lymph nodes for chymase and tryptase and for Tβ 4 , respectively. All samples were fixed in 10% formalin, paraffin-embedded and routinely processed. The immunohistochemical technique was performed in a DAKO autostainer. Paraffin sections from each case were immunostained with the policlonal antibodies against thymosin β 4 (Bachem-Peninsula Lab, San Carlos, CA, USA; diluted 1:500), chymase (Novocastra, Benton Lane, Newcastle, UK clone CC1, diluted 1:200) and tryptase (Novocastra, Benton Lane, Newcastle, UK clone 10D11, diluted 1:300). A LSAB2 system-HRP kit (DAKO) was used and the signal was visualized by the AEC (3-amino-9-ethylcarbazole) substrate. All samples were also stained with the toluidine-blue method.

Results
In normal skin, dermal-serosal mast cells showed substantially the same immunoreactivity for chymase ( Figure 1c) and tryptase ( Figure 1b). Moreover, the vast majority of MCs showed reactivity for Tβ 4 (Figure 1a). Toluidin blue stain confirmed the same reactivity pattern (data not shown).
In normal colon mucosa, a different immunoreactivity pattern was detected. The In salivary gland tumors, peri-and intratumoral MCs showed a comparable immunoreactivity for tryptase ( Figure 3b) and chymase (Figure 3c). Immunoreactivity for Tβ 4 was found only in a subset of intratumoral mast cells. (Figure 3a).
In breast ductal infiltrating carcinomas, tumor infiltrating and peritumoral MCs showed a strong reactivity for Tβ 4 and tryptase; immunoreactivity for chymase was detected only in a subset of MCs. The same pattern of reactivity was found in MCs surrounding fibrocystic changes (Figure 4 a, b, c).

Discussion
In this paper, we first demonstrate that normal dermal (serosal) and mucosal mast cells exhibit strong expression of thymosin β 4 . Tβ 4 expression was also found in tumor-infiltrating and peritumoral mast cells in salivary gland and in breast tumors. These observations extend the knowledge regarding the immunophenotypic profile of mast cells and provide a useful new marker for the identification of scattered mast cells in skin, colon mucosa as well as in tumors. The co-expression or separate expression of chymase and tryptase is currently used as a specific immunophenotypic profile to distinguish and characterize, on tissue sections, different subtypes of mast cells. 10 According to our results, Tβ 4 could be considered as a marker for MCs in this context, showing a degree of immunoreac-tivity comparable in sensitivity to chymase and tryptase. In this study Tβ 4 demonstrated to stain both serosal and mucosal MCs, paralleling the sensitivity of tryptase in revealing tissue MCs.
In addition, in this study we found a pattern of reactivity for Tβ 4 which could be useful to better characterize, on tissue sections, a new subtype of tumor infiltrating and peritumoral MCs. In salivary gland tumors, Tβ 4 was detected only in a part of tumoral MCs, while reactivity for chymase and tryptase was found in all MCs. A different protein reactivity pattern for Tβ 4 was found in intra-and peri-tumoral MCs in infiltrating ductal carcinoma of the breast. In this contest, MCs showed the highest level of reactivity for Tβ 4 and tryptase, whereas only a minority of MCs were positive for chymase. All together, these findings show a previously unreported heterogeneity of tumor infiltrating MCs, and introduce Tβ 4 as a useful marker, in Original paper   As far as the role of mast cells inside and around malignant neoplasms is concerned, the demonstration of strong Tβ 4 expression in intratumoral MCs can contribute to explain their function as effectors of the innate immune systems in the surveillance of tumor progression. A number of experimental, biochemical and microarray studies have already documented the presence of a high level of expression of Tβ 4 in experimental and human tumors. 17,18 In particular, the role of Tβ 4 has been analyzed in relation to tumor metastasis, [19][20][21] in human lung cancer, 22 and in human colorectal cancer. 23 Recently, an immunohistochemical study on Tβ 4 expression in human breast cancer revealed a low reactivity for the peptide in cancer cells, while intense reactivity for Tβ 4 was detected in other cells of the tumor microenvironment. 24 On the basis of our data, we may hypothesize that Tβ 4 -positive peritumoral cells observed in that study could be identified as MCs.
These data taken together, one may pose the question whether the levels of Tβ 4 recorded in extracts of tumor samples in previous studies reflected levels in tumor cells or in the tumour microenvironment. Our data give an answer to this question, and clearly indicate mast cells as partly responsible for the connection between Tβ 4 and tumour progression. The finding of a strong reactivity for Tβ 4 in tumour-infiltrating mast cells lays stress on the hypothesis that Tβ 4 may constitute a local paracrine mediator which could be relevant to our understanding of cellular cross-talk within the tumor microenvironment.24 Tβ 4 has been shown to exert anti-inflammatory and cytoprotective functions, by suppressing secretion of the proinflammatory cytokine, IL8 and by protecting cells against apoptosis induced by tumor necrosis factor-a. 25 Moreover, in a murine corneal model, Tβ 4 potently inhibited neutrophil infiltration and significantly decreased the expression of pro-inflammatory cytokines. 26 These data taken together, we may speculate that, because of its multifunctional roles in protecting cells against apoptosis 25 and in stimulating neoangiogenesis, [27][28][29] Tβ 4 released by mast cells in the tumor microenvironment could significantly contribute to cancer cell survival. As a consequence, Tβ 4 -producing MCs could represent a new molecular target for antitumoral strategies 30 in different human tumors.
Finally, the expression of Tβ 4 in the cytoplasm of tumor-infiltrating mast cells reported in this study extends our knowledge regarding the immunophenotypic profile of mast cells and can significantly contribute to our understanding of cross-talk between cells of the immune system and tumour cells. The possible relationship between the degree of mast cell infiltration and tumor behaviour warrants further consideration in future investigations.