Proliferation of a-Smooth Muscle Actin-containing Stromal Cells (Myofibroblasts) in the Lamina Propria Subjacent to Intraepithelial Carcinoma of the Esophagus

Background: The lamina propria of the digestive tract is the space containing vessels, myofibroblasts, and other interstitial components. The present study was undertaken to elucidate the relationships between the proliferation of myofibroblasts within this space and other histological features such as inflammatory cell infiltration and proliferation of blood vessels. Methods:     

Pathogenesis of esophageal squamous cell carcinoma with lymphoid stroma

BACKGROUND/AIMS: Lymphocyte infiltration in esophageal cancer, especially when beneath intraepithelial carcinoma, is frequently seen. However, cases of esophageal cancer with a dense stromal infiltration of lymphocytes are rare and the pathogenesis of such cases has yet to be clearly demonstrated. The objective of this study is to clarify its pathogenesis. METHODOLOGY: Four cases of esophageal squamous cell carcinoma with lymphoid stroma were investigated by immunohistochemical staining for the detection of Epstein-Barr virus, human papillomavirus, human leukocyte antigen-DR, as well as T and B cells in cancer tissue. RESULTS: In these four cases, neither positive staining of Epstein-Barr virus nor human papillomavirus infection was detected. On the other hand, the expression of human leukocyte antigen-DR antigen was evident in all cases with dense T-cell infiltration in the tumor tissue and moderate B-cell infiltration around the tumor. CONCLUSIONS: The expression of human leukocyte antigen-DR antigen without Epstein-Barr virus or human papillomavirus infection could thus be one possible pathogenesis of patients demonstrating esophageal squamous cell carcinoma with a lymphoid stroma.     

Immunohistological studies of surface antigen on colonic lymphoid cells in normal and inflamed mucosa. Comparison of follicular and lamina propria lymphocytes

Mucosal samples from 16 patients with idiopathic inflammatory bowel disease were examined immunohistologically using several monoclonal antibody combinations, and the results were compared with those obtained in other colonic inflammatory disorders and in normal mucosa. Within and around lymphoid follicles, most T cells expressed the restricted common leucocyte antigen (CD45R, displayed by unprimed T cells). Conversely, most lamina propria T cells were negative for CD45R but stained positively with UCHL1 (a monoclonal antibody recognizing an antigen displayed by primed T lymphocytes). The proportions of T-lymphocyte subpopulations in normal and inflamed mucosa were similar, except that CD6-negative, CD7-positive cells were significantly more frequent in inflammatory bowel disease. A characteristic feature of ulcerative colitis and Crohn's colitis was marked infiltration by CD45R+ lymphoid cells that did not coexpress T- or B-cell surface antigens; some stained positively with plasma cell reagents, suggesting that they may be activated B cells. The observations in the colitis sections are consistent with the interpretation that T and B cells alter their surface antigen expression as they emerge from follicles and enter the inflamed lamina propria.     

Anti-CD2 and anti-CD3 induced T cell cytotoxicity of human intraepithelial and lamina propria lymphocytes.

The effector function of immunocompetent cells in the gut mucosa has not yet been defined. The cytotoxic function of these cells might be important in the normal immune response and could be relevant to the mucosal damage seen in inflammatory conditions. The cytotoxic function of isolated intraepithelial and lamina propria mononuclear cells in six and 18 hour assays after the addition of various stimuli that interact with the human leukocyte antigens CD2 and CD3 on the mucosal effector cells was investigated. T cell phenotypes were determined using CD4, CD8, and HML1 to characterise cells of the appropriate compartments. Anti-CD3 and phytohaemagglutinin can induce toxic activity of lamina propria lymphocytes in most individuals after six hours and in all individuals after 18 hours. Anti-CD2, anti-CD3, and phytohaemagglutinin are similarly effective at triggering lamina propria lymphocytes. Intraepithelial lymphocytes contain predominantly CD8 and HML1 positive T cells, differentiating phenotypically intraepithelial lymphocytes from lamina propria lymphocytes. Intraepithelial lymphocytes are not cytotoxic at six hours, but have a toxic function comparable with lamina propria lymphocytes after 18 hours with all three triggers. Intraepithelial lymphocytes from inflamed mucosa (Crohn's disease and diverticulitis) mediate significantly reduced cytotoxicity in vitro compared with normal mucosa, whereas lamina propria lymphocyte toxicity is not different. Reduced numbers of cytotoxic cells and reduced reactivity to the trigger substances used after in vivo activation or cold target inhibition could explain the observed differences between intraepithelial lymphocytes from inflamed and uninflamed mucosa. Changes in cell mediated cytotoxicity of intraepithelial lymphocytes and lamina propria lymphocytes may be involved in the mucosal damage in these inflammatory conditions.     

Jejunal biopsy in the diagnosis of malabsorption syndromes

Summary This paper concerns the use of peroral jejunal biopsy in the diagnosis of malabsorption syndromes. Such biopsy reveals characteristic findings in the following diseases which may cause steatorrhea: sprue, amyloidosis, Whipple’s disease, lymphoma, a-beta-lipoproteinemia, and scleroderma. In nontropical sprue (and to a lesser degree in tropical sprue), microscopic examination of the jejunum reveals blunting or absence of the villi, substitution of cuboidal for columnar surface cells, infiltration of the lamina propria with lymphocytes and plasma cells, and increased mitotic activity in the crypts of the unusually deep intestinal glands. In amyloidosis, which affects the intestinal tract in 50% of cases, there is deposition of amyloid in the lamina propria and around blood vessels. In Whipple’s disease, villi of the intestinal mucosa are thickened and have bulbous tips, and the laminae propria contain foamy histiocytes and lipid deposits; also the macrophages contain a carbohydrate-protein complex which is readily demonstrated by the periodic-acid, Schiff staining technic. In lymphoma, mucosal lesions may be identical to those seen in sprue; on the other hand, diagnostic nodules of lymphocytes or unusual cellular infiltrations may be seen in the lamina propria. In a-beta-lipoproteinemia, numerous vacuoles of fat are found in the epithelial cells. In scleroderma, which may involve the intestine, collagen deposition has been demonstrated around Brunner’s glands in the duodenum. Other diseases associated with malabsorption may show nonspecific villous changes and infiltration of the lamina propria with inflammatory cells. In giardiasis,Giardia may be observed between the villi and in the surface mucus. Read at the meeting of the American Proctologic Society, Minneapolis, Minnesota, June 14 to 17, 1965.     

Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease

BACKGROUND: Keratinocyte growth factor (KGF) stimulates gastrointestinal epithelial cells in vivo, and is protective against gastrointestinal injury and colitis. Endogenous KGF is increased in inflammatory bowel disease (IBD), and may be an important mediator of mucosal repair. KGF is expressed by mesenchymal cells and activated intraepithelial lymphocytes (IEL). AIMS: To investigate the relative contributions of these cellular sources of KGF expression in IBD. METHODS: IELs and lamina propria lymphocytes (LPL) were isolated from inflamed and uninflamed IBD tissues. mRNA expression was determined by ribonuclease protection assay. In situ hybridisation was combined with immunohistochemistry to determine whether KGF mRNA was expressed by specific cell types in vivo. RESULTS: Low levels of KGF mRNA expression were detected in three of five IEL samples derived from inflamed tissue, but not in two IEL samples from uninflamed tissue. No KGF expression was detected in LPLs from either inflamed or uninflamed tissue. In contrast, KGF was expressed by primary cultures of human intestinal fibroblasts, and was induced by treatment with interleukin 1. CONCLUSIONS: The major source of KGF expression in IBD was lamina propria cells of non-immune origin, most likely fibroblasts and/or smooth muscle cells. Compared with these cell types, relatively little KGF synthesis was associated with IELs in inflamed IBD tissue.     

Lymphocytic infiltration and expression of inducible nitric oxide synthase in human duodenal and colonic mucosa is a characteristic feature of ankylosing spondylitis

OBJECTIVE: In patients with ankylosing spondylitis (AS), inflammatory processes have been detected in the ileal and colonic mucosa. The inducible isoform of nitric oxide synthase (iNOS) may be expressed early in the inflammatory process. We investigated iNOS activity and lymphocytic infiltration in the duodenum and colon in patients with AS and ulcerative colitis compared with controls. METHODS: Gastroscopy with duodenal biopsies and/or colonoscopy with biopsies were conducted in 42 patients with AS treated or not treated with nonsteroidal antiinflammatory drugs (NSAID), in 15 with ulcerative colitis, and in 46 controls. Lymphocytic infiltration in the lamina propria and intraepithelial infiltration were quantified by histological score. iNOS expression was assessed by immunohistochemistry with monoclonal antibodies, and iNOS activity was determined by radiochemical assay. RESULTS: Endoscopic examination of the gastroduodenal or colonic mucosa did not reveal macroscopic lesions in the AS patients. In the duodenum, mucosal lymphocytic infiltration was found in 83.3% of the AS group compared to 48.6% of controls (p = 0.02), and was independent of the NSAID intake. Intraepithelial lymphocyte infiltration was increased in both duodenum and colon in AS patients compared to controls. iNOS activity in duodenum and colon and expression of iNOS protein in lamina propria inflammatory cells was increased in AS patients compared to controls. CONCLUSION: Lymphocytic infiltration and iNOS expression and activity were detected in duodenal and colonic mucosa from patients with AS. Such findings may indicate an inflammatory process in the small intestine and colon of patients with AS.     

Lymphoid and non-lymphoid cells in the epithelium and lamina propria of intestinal mucosa of pigs.

The jejunum and ileum of 5 day old and adult normal pigs and of 45 day old germ free pigs were used to study the lymphocyte pools in the epithelium and lamina propria by sequential treatments with EDTA, four hours, and 12 hours of collagenase treatment. In adult animals the incubation of the jejunal wall with EDTA resulted in mean (SD) 26.8 (10.9) x 10(6) intraepithelial lymphocytes per g of tissue. The ileal wall gave lower cell yields. After complete digestion of the lamina propria by collagenase a further yield of 35.2 (10.2) x 10(6)/g lymphocytes was achieved. The separation of the gut wall from 5 day old pigs resulted in a 10-fold lower total lymphocyte yield, and the tissue was totally digested after four hours of collagenase treatment. Many eosinophils and mast cells were found in the suspensions from adult animal tissues after the collagenase treatment; 4.7 x 10(6)/g and 4.8 x 10(6)/g, respectively. The suspensions after 12 hour collagenase incubation contained up to 30% plasma cells. Almost all cells isolated by EDTA incubation were CD8+ T cells. After collagenase incubation CD4+ and CD8+ T lymphocytes were found in all animal groups, and in adult animals up to 20% surface Ig+ cells were harvested. When the incorporation of the thymidine analogue bromodesoxyuridine was used to study the lymphocyte production in vivo 3 to 7% lymphocytes in the epithelium were labelled 24 hours later (lamina propria T lymphocytes about 1%). In this study lymphoid as well as non-lymphoid cells have been analysed in mucosal cell suspensions. The absolute cell yield per gram of mucosal tissue is a basis to estimate the pool sizes of intraepithelial and lamina propria lymphocytes.     

Isolation of intestinal mononuclear cells from colonoscopic biopsies for immunofluorescence analysis by flow cytometry

A method for isolating and characterizing intestinal lymphoid cells from colonoscopic biopsies is presented. Intraepithelial lymphocytes were separated from the lamina propria by incubation in edetic acid (EDTA) and lamina propria lymphoid cells isolated by incubation in collagenase followed by Ficoll-Hypaque density flotation. Quantitation of T lymphocyte helper (OKT4) and suppressor (OKT8) cells was performed using monoclonal antibodies to cell surface markers and analyzed on a flow cytometer. The isolation procedure yielded approximately 400,000 lamina propria cells and 100,000 intraepithelial cells per sample, with better than 90% viability. Surface marker analysis demonstrated significant differences in the ratios of helper to suppressor cells between the intraepithelial lymphocytes and the lamina propria lymphocytes. These demonstrate the feasibility of lymphoid cell isolation from colonoscopic biopsy specimens for surface marker analysis by flow cytofluorimetry. These techniques could prove important in the study of immune mechanisms in inflammatory bowel diseases.     

Intestinal Mesenchymal Cells

The non–white blood cell mesenchymal elements of the intestinal lamina propria are the myofibroblasts, fibroblasts, pericytes, stromal stem cells, muscularis mucosae, and the smooth muscle of the villus core associated with the lymphatic lacteal. We review the functional anatomy of these mesenchymal cells, what is known about their origin in the embryo and their replacement in adults, their putative role in intestinal mucosal morphogenesis, and the intestinal stem cell niche, and we consider new information about myofibroblasts as nonprofessional immune cells. Although our knowledge of the function of mesenchymal cells in intestinal disease is rudimentary, we briefly consider here their roles in cancer and intestinal inflammation.     

Enteropathy associated with the acquired immunodeficiency syndrome

To explore the effect of the acquired immunodeficiency syndrome on gastrointestinal structure and absorption, the cases of 12 homosexual men with the syndrome and 11 homosexual controls were studied. Seven patients had diarrhea with weight loss. Bacterial or parasitic infections were not detected. All patients were malnourished; had significantly fewer T-lymphocyte helper and suppressor cells; and had significantly lower body weights, midarm circumferences, serum albumin concentrations, and iron binding capacities than homosexual controls. D-Xylose malabsorption and steatorrhea were present in patients, especially those with diarrhea. Jejunal and rectal biopsy samples were histologically abnormal in all patients with diarrhea. Jejunal abnormalities included partial villus atrophy with crypt hyperplasia and increased numbers of intraepithelial lymphocytes. Rectal abnormalities included intranuclear viral inclusions, mast cell infiltration in the lamina propria, and focal cell degeneration near the crypt base. The histologic findings suggest that a specific pathologic process occurs in the lamina propria of the small intestine and colon in some patients with the syndrome.     

Human immunodeficiency virus type 1 infection is associated with significant mucosal inflammation characterized by increased expression of CCR5, CXCR4, and beta-chemokines

Mucosal inflammation is characterized by increased expression of proinflammatory cytokines and chemoattractant chemokines, resulting in infiltration of immunocompetent cells. This study compared the degree of mucosal inflammation in human immunodeficiency virus type 1 (HIV-1)-infected gut mucosa with that in tissue samples from subjects with inflammatory bowel disease (IBD) and from healthy seronegative control subjects. Gut mucosal biopsy specimens were immunohistochemically stained and were evaluated by in situ imaging. There was significantly increased expression of HIV-1 coreceptors CCR5 and CXCR4, beta-chemokine RANTES, and macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, as well as increased numbers of T cells in lamina propria of HIV-1-infected patients. The results were similar in patients with IBD and in HIV-1-infected patients, suggesting increased inflammation in the colon of HIV-1-infected patients. To further investigate the effect of inflammation in HIV-1-infected lamina propria, treatments that reduce immune activation in lamina propria must be evaluated.     

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+ T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 <200 cell/mm3) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.     

Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori

BACKGROUND AND AIMS: It is not clear how Helicobacter pylori, an apparently extracellular pathogen colonizing the luminal side of the gastric epithelium, invariably causes an immune-inflammatory response on the stromal side of the mucosa. Penetration of H pylori into epithelial cell lines and its interaction with immune-inflammatory cells have been documented in vitro. Several investigations also showed in vivo bacterial penetration into the epithelium up to the lamina propria; however, the identification as H pylori of the bacteria-like bodies observed in unchanged, metaplastic, or neoplastic mucosa remained sometimes questionable. METHODS: To search for bacteria-like organisms, we used transmission electron microscopy on endoscopic biopsy specimens from 20 dyspeptic subjects and surgical specimens of neoplastic and nonneoplastic mucosa from 20 cancerous stomachs. To ascertain the H pylori nature of the organisms found, we used 6 different antibodies directed against bacterial lysates, purified vacuolating cytotoxin A, or purified cytotoxin-associated antigen A in immunogold tests. The results were compared with those of H pylori strains cultivated in vitro. RESULTS: In nonmetaplastic gastric epithelium, cytochemically proven H pylori were detected, in the majority of cases, inside cytoplasm of epithelial cells, in intraepithelial intercellular spaces, and in underlying lamina propria, often in direct contact with immune-inflammatory cells and sometimes inside small blood vessels. Cytochemically proven H pylori were also observed inside 6 of 8 intestinal metaplasias and 9 of 20 cancers. CONCLUSIONS: H pylori penetrates normal, metaplastic, and neoplastic gastric epithelium in vivo, intracellularly, or interstitially to cause a strong immune-inflammatory response and promote gastric carcinogenesis.     

A regenerative role for bone marrow following experimental colitis: contribution to neovasculogenesis and myofibroblasts

BACKGROUND & AIMS: Bone marrow (BM) cells form differentiated adult lineages within nonhematopoietic tissues, with a heightened propensity with increasing regenerative pressure dictated by disease. We have previously shown that BM cells engraft into the gut and contribute substantially to the subepithelial intestinal myofibroblast population in the lamina propria. To investigate the reparative capacity of BM in inflammatory bowel disease (IBD), a well-established model of experimental colitis was used. METHODS: Lethally irradiated female mice were rescued by a BM transplant from male donors. Colitis was induced 6 weeks posttransplantation by injection of trinitrobenzene sulfonic acid (TNBS), and tissues were analyzed 1-14 days later. Donor-derived cells were detected by in situ hybridization using a Y chromosome-specific probe, and their phenotype was determined by immunohistochemistry. RESULTS: TNBS-induced colitis was manifest as patchy lesions that increased in severity between days 1 and 8, and the mucosa gradually regenerated between days 8 and 14. The contribution of BM to intestinal myofibroblasts was significantly increased in regions of colitis compared with noninflamed regions. Furthermore, BM-derived endothelial cells, pericytes, and vascular smooth muscle cells were frequently interspersed throughout blood vessels, suggesting that these cells facilitate angiogenesis in tissue repair, substantiated by a significant increase in the incidence of BM-derived vascular smooth muscle cells in colitic compared with noninflamed regions. Blood vessels formed entirely from BM-derived cells were also seen, suggesting a role for BM in neovasculogenesis. CONCLUSIONS: Our data show that BM contributes to multiple intestinal cell lineages in colitis, with an important function in tissue regeneration and vasculogenesis after injury.     

Cellular infiltrate of jejunal biopsies in adult coeliac disease in relation to gluten withdrawal

A comparison has been made of inflammatory cell counts in the lamina propria and epithelium of jejunal biopsies in 11 patients with adult coeliac disease with those found in 12 control subjects. In the coeliac patients, there were significant increases in the numbers of total cells, plasma cells, and intraepithelial lymphocytes, but a significant reduction in lamina propria lymphocytes. Following clinical improvement on a strict gluten-free diet, significant changes in cell counts occurred, but with the exception of lymphocytes in the lamina propria, the counts were still abnormal. Analysis of five patients in whom the biopsy improved to near normal morphology and of six in whom there was no such improvement showed that significant falls in plasma cells and rises in lymphocytes in the lamina propria could occur without improvement in other morphological appearances. These results seem relevant to the problem of diagnosing coeliac disease in patients who, on gluten withdrawal, show an unequivocal clinical response, but no gross morphological improvement in the jejunal biopsy. On the basis of the observed changes in cell counts, there seems little justification in questioning the diagnosis of coeliac disease in such patients.     

Immunologic characteristics of intestinal lymphoid cells of the guinea pig

Populations of lymphoid cells from the gastrointestinal tract of guinea pigs were compared with splenic cells for both morphologic and functional characteristics. Gastrointestinal lymphoid cells were isolated from mesenteric lymph nodes, Peyer's patches, and also for the epithelium and lamina propria of the small intestinal mucosa. Isolated intraepithelial and lamina propria lymphocyte populations contained (a) T cells, B cells, and macrophages; (b) T cells that proliferated to phytohemagglutinin and concanavalin A; and (c) B cells that proliferated to bacterial lipopolysaccharide. Using inbred guinea pigs, the intraepithelial and lamina propria lymphocytes were shown to contain T cells capable of proliferating to alloantigens in mixed leukocyte culture, and also cells capable of stimulating alloreactive T cells. These studies demonstrate the presence of immunologically reactive T cells and B cells in isolated small intestinal mucosal lymphoid cells from guinea pigs.     

Lymphatics, intraepithelial lymphocytes and endometrial lymphoid tissues in the rabbit uterus: an electron microscopic and immunohistological study

Rabbit uterine intraepithelial lymphocytes, endometrial lymphoid aggregates and lymphatic capillaries were examined electron-microscopically and immunohistochemically at well-defined intervals after the injection of human chorionic gonadotropin (hCG). Lymphatic capillaries originated near the bases of the glands in the uterine cervix and in the border zone between the lamina propria mucosae and myometrium in the uterine body. The lymphatic capillaries were maximally dilated, and their endothelial cells were thinnest 8 hours after hCG injection. Patent junctions between the adjacent endothelial cells of lymphatics in the uterine body were observed in good accordance with the appearance of stromal edema and lymphatic dilatation. The numbers of intraepithelial lymphocytes and macrophages changed cyclically after the induction of ovulation. They were highest 11 hours after hCG injection when lymphocytes were seen occasionally in the lumens of lymphatics located in the lamina propria mucosae of the uterine body. Most of the intraepithelial and interstitial lymphocytes were cells labeled with T cell serum but some were labeled with IgA serum and were occasionally seen beneath the epithelium. Lymphoid aggregates were uniformly present in the stratum basalis and consisted of lymphocytes and macrophages. They had no germinal centers, surrounding lymphatics or high endothelial venules (HEV). The results suggest that lymphatic capillaries are the main route for the removal of edema fluid and for migratory lymphoid cells in the rabbit uterus.     

The cellular infiltrate of the jejunum in adult coeliac disease and dermatitis herpetiformis following the reintroduction of dietary gluten.

The cellular infiltrate of the jejunal mucosa has been studied in patients with both treated and untreated adult coeliac disease and dermatitis herpetiformis and serially in treated patients before and after the reintroduction of gluten to the diet. In adult coeliac disease and dermatitis herpetiformis the jejunal mucosa showed similar abnormalities of the cellular infiltrate which was characterized by an increase in intraepithelial lymphocytes and lamina propria plasma cells and eosinophils, with the greatest numbers of cells occurring in untreated patients. At 24-48 hours following a single 25-g gluten challenge there was an increase in lamina propria plasma cells, lymphocytes and eosinophils and intraepithelial lymphocytes. This rise was sustained after seven days on a gluten-containing diet for all of these cell groups except lamina propria lymphocytes. These responses were essentially similar in both adult coeliac disease and in those dermatitis herpetiformis patients who had jejunal lesions before treatment. In dermatitis herpetiformis patients with normal jejunal morphology on a normal diet there was an upward trend in lamina propria plasma cells and intraepithelial lymphocytes within one to three weeks of taking extra dietary gluten. These results are compatible with the view that more than one immunological mechanism may be responsible for the pathogenesis of the jejunal lesion of coeliac disease and dermatitis herpetiformis.