Antigen-induced mucosal damage and restitution in the small intestine of the immunized rat

Intestinal mucosal damage and restitution were examined following antigen-induced systemic anaphylaxis in Nippostrongylus brasiliensis immunized rats. The rats were injected intravenously with N. brasiliensis antigen or saline. At 60 min, morphological and biochemical parameters were determined in jejunum and ileum, and the epithelial permeability was assessed by measuring recovery of 51Cr-ethylenediaminetetraacetic acid in the blood after injecting it into a ligated segment. Antigen challenge resulted in significant abnormalities: (1) villus damage with sloughing of enterocytes; (2) decreased activities of brush border enzymes; (3) decreased levels of mucosal histamine and rat mast cell protease II (mast cell mediators), and (4) increased uptake of 51Cr-ethylenediaminetetraacetic acid. Progression of the injury was examined by taking consecutive biopsies at 15-min intervals for 60 min and then at 5 h. At 15 min, an abnormality was present in all sections which ranged from minor oedema and enterocyte detachment at villus tips to virtual complete destruction of the apical region. Restitution occurred by villus contraction with migration of the epithelium over the damaged regions. At 5 h, the epithelium had resealed, but the villi were significantly reduced in height.     

Lymphokine secretion and proliferation of intraepithelial lymphocytes from murine small intestine.

Compared to other peripheral lymphocytes, intestinal intraepithelial lymphocytes (IEL) have previously been shown to have low proliferative capabilities. However, there are two main populations of IEL in the small intestine of mice. First, there is the thymus-dependent CD3+,Thy-1+ population, most of which expresses the alpha beta T-cell receptor (TcR), and second there is the thymus-independent CD3+,Thy-1- population, most of which expresses the gamma delta TcR. In this study Thy-1-enriched and Thy-1-depleted lymphocytes from murine intestinal epithelium were studied separately for their ability to proliferate and secrete lymphokines in vitro after mitogenic stimulation, after stimulation via the TcR-CD3 complex and after stimulation with the superantigen Staphylococcus aureus B (SEB). Here we show that Thy-1-enriched IEL are not an immunocompromised population of cells but are functionally competent T cells that are capable of proliferation and lymphokine secretion after stimulation with concanavalin A (Con A), phorbol myristate acetate (PMA) and anti-CD3 monoclonal antibody (mAb). Furthermore, Thy-1-enriched IEL proliferate and secrete lymphokines after 'superantigenic' stimulation with SEB. In contrast, the majority of Thy-1-depleted IEL do not proliferate, and secrete only minimal levels of lymphokine to any of the stimuli tested in this study.     

The histamine-diamine oxidase system and mucosal proliferation under the influence of aminoguanidine and seventy percent resection of the rat small intestine

We have suggested previously that the histamine-diamine oxidase system is involved in cell proliferation. Therefore, the diamine oxidase activity and the histamine content were studied during mucosal proliferation induced by 70% resection of the small intestine of the rat with and without aminoguanidine (AG), a specific inhibitor of diamine oxidase (DAO). The DAO activity underwent a marked alteration during the period of mucosal proliferation, probably as an expression of an antiproliferative regulation mechanism. The histamine content decreased in the proliferating mucosa. No influence of AG on mucosal proliferation could be found, which might be due to a compensatory activity of the interconversion pathway.     

Influence of starvation and refeeding on mucosal size and epithelial renewal in the rat small intestine

Adult male rats were fasted for 0 (controls), three, five and seven days; a group was refed for one day after six days of starvation. Histological samples were taken from five regions along the length of the small intestine. The sizes of the villi, crypts and mitotic pool were estimated by measuring the number of epithelial cells per villus and crypt section and the number of mitotic figures per crypt section. Additional studies were carried out using colchicine for estimating mitotic time and methotrexate for inhibiting mitosis. All three parameters decreased progressively during starvation; the decrease in villus size was most pronounced in the duodenum and gradually less distalward. Refeeding increased the mitotic pool in every region; crypt size did not increase and villus size increased slightly in duodenum and jejunum only. When refeeding was combined with mitotic inhibition, the cell population of the crypts became depleted by 30–40% without change in villus size; thus, renewal appeared to continue by crypt cells migrating to the villi. Mitosis in the crypts is used for epithelial renewal in the adult intestine. The calculated turnover time of the epithelium was longer than normal and similar in every stage of starvation. Refeeding appeared to stimulate renewal. Since villus size changed somewhat independently from mitotic activity, the involvement of a separate mechanism controlling villus size was indicated.     

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.     

Differences in the effects of age on intestinal proliferation, crypt fission and apoptosis on the small intestine and the colon of the rat

The increase in gastrointestinal epithelial tissue mass and the development of the gut can occur through three main mechanisms, namely elevated cell production from the intestinal crypts, by raised crypt number, which occurs through the process of crypt fission or by altered apoptosis. The small bowel and the colon have various rates of these, which were studied in rats of various ages. Wistar rats were fed ad libitum, and were killed at 3, 4, 6, 9, 12, 18, 26 and 48 weeks of age. Tissue was later stained and microdissected and the number of native mitoses and apoptotic figures per crypt and the percentage of crypts in fission were determined. There was an almost linear increase in body weight from 3 to 9 weeks, followed by a more gradual rise until 18 weeks. The weight of the stomach and the small intestine reached maximum values at 9 weeks, whereas the caecum and the colon approached this at 12 weeks. Mitotic activity per crypt in the small intestine increased from 3.8 +/- 0.1 at 3 weeks to 7.8 +/- 0.4 mitoses per crypt (P < 0.001) at 9 weeks and then decreased slightly; crypt fission increased from 4.6% +/- 0.8 at 3 weeks to 8.4 +/- 0.9% at 6 weeks and then decreased gradually reaching a value of 1.5 +/- 0.4% at 48 weeks. Apoptosis also peaked at 6 weeks and was then very low. In the colon, the proliferation decreased from 4.2 +/- 0.2 mitoses per crypt in the young (3 weeks) rat and reached a plateau by 9 weeks (2.5 +/- 0.1 mitoses per crypt, P < 0.001). Crypt fission also declined rapidly in the first 9 weeks (from 67.6 +/- 4.2 to 23.1 +/- 4.6%, P < 0.01) and then continued to decline, although at a lower rate. The crypt fission index at 48 weeks was 9.8 +/- 1.0. Apoptosis in the colon persisted throughout the duration of the study, 0.19 +/- 0.06 apoptotic bodies per crypt were seen at week 48. The development of the small intestine is more dependent on cell proliferation, whereas in the colon crypt fission is far more predominant, with the colon having fission indices approximately six times greater than those of the small intestine. Proliferative activity in the colon was approximately half that of the small intestine.     

Mucosal lymphocytes in the rat small intestine: phenotypical characterization in situ.

Lymphocyte subpopulations in the intestinal mucosa of the rat were quantified in situ and compared with data obtained by other authors using enzymic or mechanical methods (Selby et al., 1984; Gibson et al., 1985) in order to assess any selective loss of cell types or contamination with lamina propria lymphocytes after these enzymic or mechanical isolation procedures. Intraepithelial lymphocytes (IEL) were predominantly of the suppressor/cytotoxic phenotype (MRC OX-8) and nearly all cells bore the pan T marker W3/13. About 10% of the IEL phenotypically belonged to the T-helper (W3/25) lineage. MRC OX-19, the rat equivalent of the mouse Lyt-1 antigen, was present on about 15% of the IEL. The main subpopulation of lamina propria T lymphocytes (T-LPL) showed a T-helper phenotype and a smaller subpopulation a suppressor/cytotoxic phenotype. Practically all T-LPL expressed the pan T marker W3/13, and half of these T cells were MRC OX-19+. The results proved to be in agreement with the data obtained after enzymic or mechanical isolation procedures and indicate that proportional contamination with LPL is not likely to occur with these methods.     

Stimulatory effects of bestatin on human B-cell colony formation

Bestatin, (2S, 3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucine, is a small molecular immunomodifier. Effects of this compound on human immune function were studied, in vitro, using the human B-cell colony formation technique. B-cell colonies were obtained from enriched B-cell populations placed in conditioned methylcellulose medium containing stimulators and irradiated T-cells as feeders. Addition to the culture of Bestatin at concentrations of 0.1 μg/ml and 1 μg/ml led to a significant increase (P < 0.05) in the number of B-cell colonies and this effect was abolished when irradiated T-cells were not added to the culture. Bestatin increased soluble factor production induced by phytohemagglutinin (PHA)-stimulated T-cells. Such findings suggest that T-cells probably mediate this stimulatory effect of Bestatin on B-cell colony formation.     

Some effects of ouabain and potassium on transport and metabolism in rat small intestine

1. Ouabain (10(-3)M) inhibited the transfer of glucose, proline, methionine and fluid but not of galactose and 3-O-methyl glucose by sacs of rat everted intestine.2. Ouabain (10(-3)M) inhibited both glucose and mannose metabolism. Hexose metabolism was also reduced by varying the [K] from 4.8 m-equiv/l.3. Ouabain (10(-3)M) or absence of K reduced the stimulation of amino acid, hexose and fluid transfer by mannose. Although presence of ouabain or absence of K did not inhibit endogenous galactose transfer, these conditions prevented mannose from stimulating this transfer.4. Ouabain (10(-3)M) exerted greater effects on hexose and amino acid transfer when K was omitted from the saline and its inhibitory effects were partially overcome by increased serosal [K].5. Ouabain (10(-2)M) caused further inhibition of amino acid and glucose transfer and also reduced the transfer of galactose and 3-O-methyl glucose.6. The implications of these results are discussed with respect to the relationships between cations and hexose and amino acid transfer.     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

An immunocytochemical double staining technique applied to intraepithelial lymphocytes in the small intestine of the rat

To study lymphocyte antigens in cryostat sections of small intestine of the rat, an indirect double labelling technique was developed, avoiding alkaline phosphatase for the 2nd label, in view of the high endogenous activity of this enzyme in the enterocytes. 2 times a peroxidase was used, but in the 1st sequence an antibody conjugated peroxidase with a reddish brown AEC product and in the 2nd sequence an avidin conjugated peroxidase with a blue colour product via 4CN. 2 antigens on the same cell in the same section can be detected by this method. Double labelled cells were coloured violet. The method is particularly suitable for loosely situated cells.     

Stimulatory and inhibitory effects of cyclic AMP on lymphocytes from atopic children.

The effect of cholera toxin and dibutyryl cAMP on mitogen-activated lymphocytes from atopic and non-atopic individuals was studied. Cholera toxin enhanced stimulation by phytohemagglutinin of cells from small children but not from adults. Dibutyryl cAMP at low concentration (less than 10(-5) M) significantly enhanced the lymphocyte response to mitogens in some, but not all individuals. High concentrations, on the other hand, were consistently inhibitory. In atopic children, the lymphocyte response to T cell mitogen was significantly less stimulated by cholera toxin, and more inhibited by dibutyryl cAMP than the response of cells from non-atopic matched controls. Thus, T cells from atopic individuals appear to have an altered sensitivity to the action of cAMP, possibly resulting in an impaired balance between helper and suppressor T cells. The hypothesis is advanced, that such an altered balance is causally related to hyperproduction of IgE resulting in atopic disease.     

寒冷缺氧降低大鼠脾脏T淋巴细胞的增殖反应

目的观察缺氧和寒冷对大鼠脾脏T淋巴细胞功能的影响及其与腺苷脱胺酶之间的关系,探讨冬季急进高原时机体免疫功能改变的规律和机制。方法大鼠分别在常温常氧、寒冷常氧、常温缺氧以及寒冷缺氧环境中生活3 d,用3H-TdR掺入法检测脾脏T淋巴细胞增殖功能,用比色法测定脾脏T淋巴细胞腺苷脱胺酶活性。结果与常温常压对照组比较,单纯寒冷或单纯缺氧均可引起脾脏T淋巴细胞在刀豆素A刺激下的刺激指数显著降低,但缺氧复合寒冷大鼠脾脏T淋巴细胞的刺激指数并未进一步降低,而是接近单纯缺氧大鼠。单纯缺氧和单纯寒冷并不改变大鼠脾脏T淋巴细胞上的腺苷脱胺酶活性,而缺氧复合寒冷则使其显著降低。结论缺氧和寒冷均可抑制脾脏T淋巴细胞的功能,但两者对脾脏T淋巴细胞功能的影响可能并无叠加效应;腺苷脱氨酶活性的改变可能参与寒冷缺氧大鼠脾脏T淋巴细胞功能的降低。