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Re: The stomach peri-glandular fibroblast sheath: All present and correct

Dear Editor,

We were interested in the paper by Mutoh et al.[1], showing the development of epithelial intestinal metaplasia and mesenchymal proliferation in human stomach resections and Cdx2 transgenic mice. The authors used alpha-smooth muscle actin (alpha-SMA) staining to mark peri-glandular fibroblasts and failed to show any alpha-SMA positive cells surrounding the en-face glands of normal mouse and human stomach mucosa. In metaplastic tissue however, the peri-glandular fibroblast sheath was easily discernible, and the authors concluded that the fibroblast sheath was generated from the intestinal submucosa, possibly through expression of Cdx2.

Mesenchymal cells such as intestinal sub-epithelial myofibroblasts (ISEMF) are widely distributed. They are important coordinating cells that possess significant influence on their environment by virtue of their receptor profile and the signals they produce. Characteristically ISEMF’s form a protective fenestrated sheath around the stem cell compartment, creating the stem cell niche - the optimal microenvironment for stem cells to give rise to differentiated progeny.[2] The stem cell niche is situated in the isthmus/neck region of the gastric gland. ISEMF’s regulate stem cell behaviour via paracrine secretion of growth factors and cytokines, and perform vital functions in the growth, differentiation and development of the embryological stomach. They participate in mucosal wound healing and the response to inflammatory stimuli in the adult gastro-intestinal tract.[3] As these are vital homeostatic roles it seems unlikely that the peri-glandular fibroblast sheath is only generated in abnormal, metaplastic tissue.

We immunostained for ISEMF’s in paraffin embedded normal mouse and human stomach specimens. To identify ISEMF’s we stained for alpha-SMA in 3 different mouse gastric specimens and alpha- SMA and vimentin in 6 different sets of human gastric biopsies. Sections for vimentin staining underwent 10 minutes microwave treatment in citrate buffer for antigen retrieval. Immunostaining was completed using the same antibodies and methods described in detail by Direkze et al.[5] Antibody binding was detected by 1,3-diaminobenzidine (DAB; Sigma). ISEMF’s were identified on the basis of their morphology and positive immunoreactivity for alpha-SMA in mouse tissue, and alpha-SMA and vimentin in human tissue. They were clearly and consistently seen surrounding the stomach glands in normal mouse and human stomach sections, both in the en-face and cross-sectional plane (see figure 1). There was little variation in staining intensity from sample to sample in the 3 mouse and 6 human subjects studied.

Figure 1. The normal stomach periglandular fibroblast sheath (PGFS).
A: Human alpha-SMA control.
B: alpha-SMA immunostaining.
C: Human vimentin control. Normal human gastric glands counterstained with haematoxylin. (40x magnification).
D: Vimentin immunostaining. Normal human gastric glands with surrounding PGFS (brown stain) embracing the epithelial cells of the gastric glands. (40x magnification).
E: Mouse alpha-SMA control. Normal mouse gastric glands counterstained with haematoxylin (40x magnification).
F, G: alpha-SMA immunostaining. Normal mouse gastric glands in en-face (F) and cross sectional (G) orientation showing the close association of the PGFS (brown stain) surrounding and enveloping the normal stomach glands. (60x magnification).

ISEMF’s are involved in the response to damage or disease in the stomach. After epithelial injury, ISEMF contraction limits the exposed area of the wound whilst secreted growth factors such as transforming growth factors alpha and beta (TGF-alpha, TGF-beta) epidermal growth factor (EGF) and fibroblast growth factor (FGF), promote epithelial cell migration and proliferation.[3] In intestinal type gastric cancer myofibroblasts appear not only at the edge of the tumour, contributing to a desmoplastic reaction, but also within the tumour stroma.[4] The presence of increased inter-tubular reticulin, the histological hallmark of increased extra-cellular matrix deposition, is regarded among the first signs of chronic atrophic gastritis, and likely to be caused by elevated numbers or activity of the ISEMFs. The source of these cells is very interesting:- it is more likely that these cells are recruited from circulating precursor cells than being generated by metaplastic mucosa. Direkze et al have shown a large contribution of bone marrow donor-derived myofibroblasts, making up to 64% of the peri-glandular fibroblast sheath in mouse stomach after total body irradiation and bone marrow transplant[5], and Nakayama et al hypothesise that engraftment is responsible for myofibroblast presence in tumours.[4] The mechanisms initiating this engraftment unclear, however it may relate to the release of growth factors such as TGF-beta[6] released from inflammatory cells and the existing peri- glandular fibroblast sheath in response to gastritis induced damage.

We conclude that the peri-glandular myofibroblast sheath in normal stomach is very much a reality and likely to be pivotal in modulating epithelial cell behaviour.

No competing interests declared.

Grant Support – Medical Research Council Clinical Research Fellowship.

References

1. Mutoh H, Sakurai S, Satoh K, Osawa H, Tomiyama T, Kita H, et al. Pericryptal fibroblast sheath in intestinal metaplasia and gastric carcinoma. Gut 2005;54(1):33-9.

2. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001;414(6859):98-104.

3. Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB. Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 1999;277(1 Pt 1):C1-9.

4. Nakayama H, Enzan H, Miyazaki E, Toi M. Alpha smooth muscle actin positive stromal cells in gastric carcinoma. J Clin Pathol 2002;55(10):741 -4.

5. Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, et al. Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. Stem Cells 2003;21(5):514 -20.