ANTICANCER RESEARCH 27: 391-394 (2007)

Mast Cell and Eosinophil Interaction in Gastric Carcinomas: Ultrastructural Observations

R.A. CARUSO, F. FEDELE, V. ZUCCALÀ, M.G. FRACASSI and A. VENUTI

Department of Human Pathology, Policlinico Universitario, Messina, Italy

Abstract. Background: An increase in the number of around tumours and may either promote or inhibit tumour mastocytes has been described in some human neoplasms, growth depending on the local stromal conditions (10). mainly in gastric and colorectal cancer. Materials and Methods: This report provides morphological evidence of active A case of diffuse-type gastric carcinoma, mainly infiltrated by interaction between mast cells and eosinophils in the tumour eosinophils and mast cells, was studied using light and electron stroma of gastric carcinoma. microscopy. Results: Using light microscopy, cell clusters containing one mast cell and one to three eosinophils were found Case Report in the tumour stroma. Electron microscopy of this unusual stromal element revealed that mast cells established areas of A 57-year-old man was admitted to our hospital with junctions with eosinophils. Moreover, focal, polarized exocytosis hematemesis. The history was notable for epigastric discomfort of mast cell granules was found in the areas of junctions with for 3 weeks before admission. Upper gastrointestinal eosinophils. Eosinophils in contact with mast cells showed signs endoscopy revealed a large ulcerated Borrmann type III of important in situ activation, such as alterations in the size and tumour on the lesser curvature of the gastric antrum. Biopsies number of granules, cytoplasmic vacuoles, and scattered of the tumour demonstrated a diffuse-type gastric carcinoma. extracellular granules. Conclusion: Our ultrastructural study Preoperative chest radiography and whole-body computerized provides morphological evidence of cross-talk between activated tomography showed no metastatic lesions. Partial gastrectomy mast cells and eosinophils that may play an important role in the with regional lymph node dissection was performed. The enhancement of host immunity against cancer cells. patient died 12 months after the presentation of his symptoms. No autopsy was performed. Mast cells and eosinophils are bone marrow-derived cells that play a key role in the early and late stages of IgE-mediated Materials and Methods allergic inflammation (1,2). It has been suggested that mast cells interact with, and are activated by, eosinophil mediators In our Department, gastric tumours are routinely processed for both (3, 4). These interactions can take place during the late and light and electron microscopic observations (6, 7). Briefly, the chronic phases of the allergic inflammation, as well as in other fragments of fresh tumour tissue were divided into two portions with a sharp razor blade. The first member of the pair was processed for inflammatory conditions in which mast cells and eosinophils routine paraffin-embedding together with additional tissue samples can be found in close proximity (3, 4). taken from the tumour as well as from the surgical borders of the An increase in the number of mastocytes was described in specimens. These sections were stained with haematoxylin and eosin some human neoplasms, mainly in gastric and colorectal (H&E). The second piece of the paired samples was minced into cancer, where these cells were dispersed or formed islets (5-9). smaller pieces and destined for electron microscopy. This material was Profound knowledge of the morphology and function of mast fixed in 3% glutaraldeyde in phosphate-buffered solution, postfixed in cells may create new paths for pharmacological therapies. 1% osmium tetroxide, and subsequently dehydrated in graded ethanol and embedded in Araldite. Semi-thin Giemsa-stained sections were Mounting evidence indicates that mast cells accumulate reviewed. Areas selected for study by electron microscopy included those with mast cell and eosinophil infiltration in the tumour. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (Zeiss EM 109). Correspondence to: Prof. R.A. Caruso, Dipartimento di Patologia Umana – Policlinico Universitario pad D, 1 via Consolare Valeria, Pathological Findings I-98125 Messina, Italy. Tel: +39 090 312291, Fax: +39 090 2212523, e-mail: [email protected] Surgical pathology revealed a 3.0 x 5.0 cm ulcerated Borrmann Key Words: Gastric carcinoma, eosinophils, mast cells, electron type III tumour on the lesser curvature of the gastric antrum. microscopy. Microscopically, the tumour consisted of a population of

0250-7005/2007 $2.00+.40 391 ANTICANCER RESEARCH 27: 391-394 (2007) large, poorly cohesive cells with abundant cytoplasm, invading the mucosal, submucosal and muscular layers of the stomach. The surgical findings were pT2, N2, M0, Stage IIIa according to the TNM system. Neoplastic cells were characterized by large vesicular nuclei with single large nucleoli (Figure 1). The inflammatory reaction mainly comprised eosinophils. Mast cells were found in clusters containing one mast cell and one to three eosinophils (Figure 1). Electron microscopy revealed areas of intimate contact between mast cells and eosinophils (Figure 2). These cells delimited an intercellular space, where focal degranulation of mast cells could take place (Figures 2-4). This process was evident from the loss of several subjacent granules with residual cytoplasmic lacunae (Figures 2-4). There was formation of small secretory channels and granule opening to the cell exterior (Figures 3-4). Eosinophils in contact with mast cells showed ultrastructural signs of activation such as a reduced number of granules and cytoplasmic vacuolization. A few apoptotic eosinophils were also seen and exhibited condensed chromatin, cytoplasmic vacuolization, and peripheral translocation of granules (Figures 4-5). Clusters of three or more extracellular membrane-bound secondary granules were adjacent to apoptotic eosinophils (Figure 6).

Discussion Figure 1. Diffuse-type gastric carcinoma. Cluster containing one mast cell Occasionally, an ordinary gastric adenocarcinoma exhibits an and two eosinophils (arrow) is present in the tumour stroma. Giemsa; unusual stromal response, including a massive infiltration of semi-thin section x400. eosinophils, neutrophils or lymphocytes (11). In our case, clusters containing one mast cell and eosinophils constituted piecemeal degranulation, because there is continuity an unusual stromal reaction. Electron microscopy showed that between the mast cell secretory events and these two basic mast cells established areas of junctions with eosinophils. We degranulation pathways (12). ruled out the possibility that this association was casual, Experimental studies have shown that mast cells enhance because a focal exocytosis of mast cell granules appeared to the cytotoxic activation of mainly peritumoral macrophages be polarized toward the area of junction with the and eosinophils and may indirectly exert a cytotoxic effect on eosinophils. These ultrastructural observations suggest the cancer cells (10). Tumour stroma mast cells and/or eosinophils presence of a cross-talk between mast cells and eosinophils have generally been associated with improved prognosis in a in the tumour stroma. wide variety of epithelial neoplasms (5, 14, 15). In our case, Focal exocytosis was characterized by the opening of eosinophils in contact with mast cells showed ultrastructural single granules to the cell exterior and/or fusion of a few signs of activation, including alterations in the size and granules into small secreting channels (12). Two basic number of granules, cytoplasmic vacuoles and numerous free patterns of mast cell degranulation have been identified: granules in the extra-cellular space. Ultrastructural evidence anaphylactic and piecemeal (13). Anaphylactic degranulation for release of major basic protein-containing crystalline cores is characterized by extensive fusion of granules with each of eosinophil granules suggests cytotoxic potential in tumour other and with the plasma membrane, giving rise to open tissue (16). From these results, we suggest that mast cells may secretory channels which allow the release of granule play an important role in the enhancement of local stromal contents into the local extracellular environment (13). reaction against cancer cells through an indirect mechanism Piecemeal degranulation is characterized by the loss of dense involving adjacent eosinophils. granule materials and the retention of granule containers in the cytoplasm, without membrane fusion events and granule Acknowledgements opening to the cell exterior (13). Focal exocytosis may be considered an intermediate pattern of mast cell The authors are grateful to Mariannina Stancampiano (Bibilioteca; degranulation between anaphylactic degranulation and Università degli Studi di Roma - Tor Vergata) for bibliographic help.

392 Caruso et al: Mast Cells and Eosinophils in Gastric Carcinoma

Figure 2. Mast cell (M), eosinophil (E), tumour cell (T) and a long cytoplasmic process of fibroblast-like cell (F) delimit an intercellular space, where focal exocytosis of single mast cell granules is noted. x10,000.

Figure 4. Mast cell in contact with eosinophil shows evidence of focal Figure 3. Extensive interdigitating plasmalemmal folds were observed on exocytosis. Intracellular channels, containing altered cytoplasmic granules, the surface of mast cells in contact with eosinophils (arrow) x20,000. are present. x12,000.

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Figure 5. Mature mast cell (arrowhead) with a full complement of Figure 6. Focal exocytosis of a mast cell adjacent to an eosinophil is granules in close contact with two eosinophils showing early apoptotic characterized by granule extrusion to the cell exterior (arrowhead). Late changes (arrows) such as cytoplasmic vacuolization and condensed apoptotic eosinophils show peripheral localization of granules, condensed chromatin. A tumour cell (T) is near the mast cell. x6,000. chromatin and cytoplasmic vacuolization. A few scattered extracellular eosinophil granules (arrows) are also seen. x10,000. References p185, estrogen receptor, and proliferating cell nuclear antigen in 1 Bochner BS and Schleimer RP: Mast cells, basophils, and gastric carcinoma. World J Gastroenterol 8: 1005-1008, 2002. eosinophils: distinct but overlapping pathways for recruitment. 10 Theoharides TC and Conti P: Mast cells: the Jekyll and Hyde of Immunol Rev 179: 5-15, 2001. tumor growth. Trends Immunol 25: 235-241, 2004. 2 Galli SJ, Nakae S and Tsai M: Mast cells in the development of 11 Rosai J: Gastrointestinal tract. In: Rosai and Ackerman’s Surgical adaptive immune responses. Nat Immunol 6: 135-142, 2005. Pathology. Rosai J (ed.). Edinburgh, Mosby, pp. 648-711, 2004. 3 Levi-Schaffer F: Cross-talk between mast cells and eosinophils. 12 Crivellato E, Candussio L, Mallardi F and Ribatti D: Allergy 54(Suppl 58): 36-38, 1999. Recombinant human alpha-2a interferon promotes an atypical 4 Piliponsky AM, Gleich GJ, Nagler A, Bar I and Levi-Schaffer F: process of mast cell secretion with ultrastructural features Non-IgE-dependent activation of human lung- and cord blood- suggestive for piecemeal degranulation. J Anat 201: 507-512, 2002. derived mast cells is induced by eosinophil major basic protein 13 Dvorak AM and Kissell S: Granule changes of human skin mast cell and modulated by the membrane form of stem cell factor. Blood characteristic of piecemeal degranulation and associated with 101: 1898-1904, 2003. recovery during wound healing in situ. J Leuk Biol 49: 197-210, 1991. 5 Fisher RE, Paik SM, Rockette H, Jones J, Caplan R and Fisher 14 Fernandez-Acenero MJ, Galindo-Gallego M, Sanz J and Aljama B: Prognostic significance of eosinophils and mast cells in rectal A: Prognostic influence of tumor-associated eosinophilic infiltrate cancer: Findings from the National Surgical Adjuvant Breast and in colorectal carcinoma. Cancer 88: 1544-1548, 2000. Bowel project (Protocol R-01). Human Pathol 20: 159-163, 1989. 15 Dabiri S, Huntsman D, Makretsov N, Cheang M, Gilks B, Bajdik 6 Caruso RA, Fedele F, Rigoli L and Inferrera C: Mast cell C, Gelmon K, Chia S and Hayes M: The presence of stromal mast interaction with tumor cells in small early gastric cancer: cells identifies a subset of invasive breast cancers with a favorable Ultrastructural observations. Ultrastruct Pathol 21: 173-181, 1997. prognosis. Mod Pathol 17: 690-695, 2004. 7 Caruso RA, Ieni A, Fabiano V, Basile G and Inferrera C: 16 Dvorak AM, Letourneau L, Weller PF and Ackerman SJ: Perivascular mast cells in advanced gastric adenocarcinomas. An Ultrastructural localization of Charcot-Leyden crystal protein electron microscopic study. Anticancer Res 24: 2257-2264, 2004. (lysophospholipase) to intracytoplasmic crystals in tumor cells of 8 Kondo K, Muramatsu M, Okamoto Y, Jin D, Takai S, Tanigawa primary solid and papillary epithelial neoplasm of the pancreas. N and Miyazaki M: Expression of chymase-positive cells in gastric Lab Invest 62: 608-615, 1990. cancer and its correlation with angiogenesis. J Surg Oncol 93: 36- 42, 2006. Received September 22, 2006 9 Jiang YA, Zhang YY, Luo HS and Xing SF: Mast cell density and Revised November 27, 2006 the context of clinicopathological parameters and expression of Accepted November 29, 2006

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