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.     

Proliferative kinetics of large and small intraepithelial lymphocytes in the small intestine of the mouse

The proliferative kinetics of the intraepithelial lymphocytes (IL) of the mouse intestine have been evaluated. By inducing mitotic arrest it was found that large IL — constituting about 50% of the IL — showed a mitotic rate of 2.3. Autoradiographic results obtained after two different schedules of ³H-thymidine injections showed that 30% of the large IL were in DNA synthesis, and that the large IL were renewed at a rate comparable to that of blast cells from Peyer's patches, mesenteric lymph nodes and thoracic duct lymph. The small IL were renewed very rapidly compared to small lymphocytes of peripheral lymphoid tissues, although small lymphocytes with lifespans of several weeks were also present in the epithelial sheet. By the use of intestinal perfusion, in vivo, it was estimated that the loss of lymphocytes from intestinal villi into the lumen of the gut was negligible, and it is concluded that the most probable kinetic model for the majority of IL is: B and T lymphoblasts invade the epithelium and undergo mitosis. B lymphoblasts give rise predominantly to plasma cells, and T lymphoblasts give rise to small lymphocytes — probably long-lived — which reenter the circulation.     

Differential tolerance of thymus-independent and thymus-dependent antibody responses.

The effect of pre-immunization with soluble haptenated serum proteins or haptenated carbohydrates on the subsequent responses to the hapten in thymus-dependent or thymus-independent form was investigated. In certain cases both thymus-dependent and -independent responses were suppressed whilst in others a differential suppression of thymus-independent responsiveness was observed. These results are discussed in terms of B-cell subpopulations and mechanisms of tolerance induction.     

Effect of adenovirus and influenza virus on the thymus-dependent and thymus-independent immune response

Experiments in mice showed influenza virus and adenovirus to inhibit the secondary and the primary immune response to thymus-dependent antigens and not to influence production of antibodies to thymus-independent antigens.     

The effect of pertussis adjuvant on antibody production: the need for thymus-dependent lymphocytes

The response to human serum albumin in the BALB/c mouse immunoglobulin classes has been examined both with and without B. pertussis (BP) organisms as adjuvant. Without BP, IgG1 was found to be the only thymus-dependent class. The presence of thymus-dependent lymphocytes was necessary for BP to have its full adjuvant effect, although some activity was observed in neonatally thymectomized mice. In thymectomized mice IgG1 antibody was stimulated significantly by BP, but only to half the level seen in intact mice. Although IgG2a and IgG2b both rose in these mice after BP treatment, the difference was not significant. IgA and IgM were both unchanged. Thus, as well as being needed for increased stimulation of IgG1, the thymus was particularly required for BP to increase the response in the classes of immunoglobulin not usually found to be thymus-dependent for soluble antigens.     

Phenotypic heterogeneity of intraepithelial T lymphocytes from mouse small intestine.

We have used two-colour flow cytometry to examine the heterogeneity of intraepithelial lymphocytes (IEL) from mouse small intestine. We have confirmed the predominance of CD3+ Thy 1- CD8+ IEL and show that a substantial but variable proportion of CD8+ IEL does not express the alpha beta T-cell receptor (TcR) for antigen. Simultaneous analysis of the co-expression of the alpha and beta chains of the CD8 heterodimer and of the alpha beta TcR revealed three populations of CD8+IEL. The first of these expressed both CD8 alpha and beta chains and had normal expression of V beta families and so represented conventional CD8+ alpha beta TcR+ T cells. The second population comprised alpha beta TcR- T cells (presumed gamma delta TcR+) which expressed only the alpha chain of the CD8 molecule. Finally, we identified a second, unique population of alpha beta TcR+ CD8+ IEL which were also CD8 beta-. Gamma delta + IEL predominated in mice aged less than 8 weeks, but there was a rapid increase in both populations of alpha beta TcR+ CD8+ IEL in older mice. CD8+ IEL were similar to peripheral CD8+ T cells in having high expression of the CD45RB molecule, but CD4+ IEL had generally lower expression of CD45RB than their peripheral counterparts, despite having normal expression of TcR. These findings emphasize the heterogeneity of IEL and underline the need to study phenotypically defined populations.     

Specific priming of mouse thymus-dependent lymphocytes to allogeneic cells in vitro

Blast cells from one-way mixed lymphocyte cultures (MLC) revert to lymphocytes in vitro and in vivo. The blast-derived lymphocytes (BDL) respond promptly in second MLC to the original and H-2 cross-reactive stimulator cells. The peak response by primed cells takes place on the 2nd – 3rd culture day, as compared to 6th – 7th day by nonprimed cells. BDL do not respond to the mitogens, phytohemagglutinin or concanavalin A.     

A new surface antigen on intraepithelial lymphocytes in the intestine

A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.     

Interactions between the immunological responses of a thymus-independent antigen (Salmonella adelaide O antigen) with a thymus-dependent antigen (sheep erythrocytes) in the adult bird.

The bird's antibody response to a thymus-dependent antigen (sheep erythrocytes) (SRBC) and a thymus-independent antigen (SALMONELLA ADELAIDE O antigen) were characterized: whereas the former proceeded through a brief 19S response to a declining 7S response, the latter failed to switch from 19S TO 7S for several weeks and consisted in repeated excursions of 19S antibodies. When injected intravenously and simultaneously an injection of S. adelaide-killed organisms and SRBC interact, so that the response to the latter fails to switch from 19S TO 7S and consists of repeated excursions of 19S antibodies. The changed character of the SRBC response is interpreted to be due to the relative lack of 7S antibody: passive 7S antibody to S. adelaide O antigen or 7S anti-SRBC produces a negative feedback inhibition of their respective responses, so that only one excursion of 19S antibody is observed. The effect is not, however, symmetrical; the thymus-independent antigen is dominant. Thus, whereas 7S antibody to S. adelaide produces the same negative feedback inhibition on the response to S. adelaide and the response to SRBC (when injected with adlaide), 7S antibody to SRBC inhibits only the response to SRBC and not the response to S. adelaide. These results are discussed relation to current hypotheses of antibody biosynthesis and mechanisms of adjuvant action. They are also discussed in relation to the function of the germinal centres of the spleen which may function to mediate the negative feedback of 7S antibody on the antibody response.     

Subpopulations of rat and mouse thoracic duct small lymphocytes in the Salmonella flagellar antigen system

The purpose of the present experiments was to determine whether populations of lymphocytes from mouse or rat thoracic duct lymph deprived of the larger, dividing cells (large and medium lymphocytes) could still transfer adequate primary or secondary adoptive immune responses using syngeneic irradiated hosts. The transfer system used involved the antigen polymerized flagellin from Salmonella adelaide and assay of anti-H antibody levels in the recipients. Two methods of preparation of `small lymphocytes' were used, namely the glass bead column filtration method of Shortman and the agitated culture method of Gowans and Uhr (1966). Both of these yield lymphocyte fractions essentially free of dividing cells.

Unfractionated thoracic duct lymphocytes gave satisfactory adoptive immune responses in both species, primed cells being more effective than normal cells.

In both species and for both primary and secondary immune responses, the Shortman-column fractionated small lymphocytes (col-SL) were grossly impaired in their capacity to transfer adoptive responses. In contrast, the Gowans-type post-incubation small lymphocytes (inc-SL) gave rather better adoptive immune responses. Gowans and Uhr (1966) had found some enhancement of reactivity of inc-SL in comparison with normal lymph cells in another transfer system. We, however, found a mild impairment with unstimulated mouse and rat inc-SL and primed rat inc-SL and a slightly more marked impairment with primed mouse inc-SL.

We have confirmed the finding of Gowans and Uhr (1966) that lymphocyte populations free of dividing cells can mount an adoptive antibody response, showing that a proportion, at least, of the antigen-reactive cells can be described as `small lymphocytes'. The study also revealed the functional heterogeneity of morphologically similar lymphocytes, in that col-SL, morphologically the same as inc-SL, are functionally quite different; they appear to contain very few antigen-reactive cells. In the Salmonella system, therefore, the antigen-reactive thoracic duct cells can be described as lymphocytes which are fairly resistant to agitated culture but which fail to pass through Shortman columns.

     

The immune response in NZB mice of different ages to thymus-dependent and thymus-independent phosphorylcholine antigens.

This study was designed to examine aberrations of immune responses in autoimmune NZB strain mice during ageing, at the level of individual B cell clones. The response to phosphorylcholine (PC) was chosen because murine responses to PC are restricted to a few B cell clones and can be characterized with idiotypic markers. Responses to both thymus-dependent (TD) and thymus-independently (TI) PC-containing antigens were measured in mice ranging from 1 to 62 weeks of age. We found that: (1) TD responses to phosphorylcholine keyhole limpet haemocyanin (PC-KLH) decreased markedly (about 17-fold) between 28 and 62 weeks of age. TI responses to the R36a strain pneumococcus decreased only slightly during the same period. (2) The PFC responses to both antigens became markedly prolonged in mice older than 24 weeks. (3) The NZB response to either antigen is essentially monoclonal, as measured by inhibition of PFC with specific anti-idiotype serum and PC hapten. No age-related alteration in avidity or idiotype expression was observed. Our results demonstrate that no aberrant PC-reactive B cell clones appear in old NZB, and lend support to the notion that the abnormalities observed are due to defective regulatory mechanisms.     

Role of the striatum in the humoral immune response to thymus-independent and thymus-dependent antigens in rats

Since the role of striatal GABAergic medium-sized spiny (MSP) neurons in the modulation of the immune responses is largely unknown, we evaluated the humoral immune response in rats with bilateral lesion of the striatum caused by quinolinic acid, which destroys MSP neurons. Sham-operated rats and those with striatal lesions were immunized either with TNP-LPS, a T-independent antigen type 1, or one of several T-dependent antigens: ovoalbumin, bovine serum albumin, lysozyme, sheep red blood cells (SRBC) or outer membrane proteins (OMP) of Salmonella enterica serovar Typhimurium. The specific levels of serum IgM and IgG, as well as intestinal IgA antibodies were determined either by enzyme-linked immunosorbent assay (ELISA) or a haemagglutination assay 5 or 7 days after immunization. Our results show that the lesion of striatal MSP neurons attenuated the primary antibody response to the T-independent antigen type 1 (TNP-LPS), but increased the antibody response to T-dependent antigens (proteins, SRBC and OMP), indicating that the striatal neurons modulate the humoral immune response in rats. The mechanisms involved are probably related to a reduction in both the number of B cells and the expression of caveolin-1 in the spleen, as well as an increase in the number of CD4(+) T cells and in corticosterone levels of the serum.     

Age-related changes in the regional variations in the number and subsets of intraepithelial lymphocytes in mouse small intestine

Previously, we reported regional variations in the number and subsets of the small intestinal IELs of mice. In this study, we examined the age-related changes in the regional variations of IELs in mice from 2 to 11 weeks old. IELs were isolated from the proximal, middle and distal parts of the small intestine and analysed by flow cytometry. The total number of IELs gradually increased with age and reached a plateau at 8 weeks old. As to IEL subsets, the percentage of alpha beta T cells was higher in the distal part at and after 2 weeks of age (before weaning). The percentage of the alpha beta T cell subset of extrathymic origin was higher in the proximal part while the percentages of alpha beta T cell subsets of thymic origin were higher in the distal part at and after 3 weeks (just after weaning). It appears that regional variations in IELs may be formed before the weaning period in mice.     

Ontogeny and immunohistochemical localization of thymus-dependent and thymus-independent RT6+ cells in the rat.

RT6 is a cell surface alloantigen that identifies a regulatory subset of peripheral T cells in the rat. Diabetes-prone BB rats are deficient in peripheral RT6+ T cells and develop spontaneous autoimmune insulin-dependent diabetes mellitus. Diabetes-resistant BB rats have normal numbers of RT6+ T cells, and insulin-dependent diabetes mellitus can be induced in these animals by in vivo depletion of peripheral RT6+ cells. Athymic rats are also severely deficient in peripheral RT6+ T cells. Although very different with respect to the peripheral RT6+ cell compartment, normal, athymic, and diabetes-prone BB rats all generate RT6+ intestinal epithelial lymphocytes (IELs). The goal of these studies was to analyze the ontogeny of RT6+ IELs and peripheral lymphoid cells by in situ immunohistochemistry. We observed the following. 1) RT6+ IELs appear before alpha(beta) T-cell-receptor- expressing IELs in diabetes-prone BB, diabetes-resistant BB, and athymic WAG rats. 2) In vivo depletion of peripheral RT6+ T cells in diabetes-resistant BB rats using a cytotoxic monoclonal antibody is not accompanied by depletion of RT6+ IELs. 3) A population of RT6+ T-cell-receptor-negative IELs is present in normal, euthymic diabetes-resistant BB rats, constitutes a larger percentage of the euthymic but lymphopenic diabetes-prone BB rat IEL population, and is the predominant IEL phenotype in athymic WAG rats. These results suggest that RT6+ cells are composed of both thymus-dependent and thymus-independent cell subsets that have different developmental characteristics and may differ in function.     

胚胎时期小鼠小肠的组织发生

目的　观察小鼠胚胎各个时期小肠组织的形态结构及杯状细胞在小肠内的分布规律,为小鼠小肠的组织发生提供形态学依据。 方法　采用HE染色和PAS染色,对小鼠胚胎第13.5天（E13.5d）至出生后第1天（P1d）胚胎的石蜡切片染色并行光学显微镜观察。 结果　(1)小鼠肠壁于E13.5 d已分化出现黏膜层、黏膜下层、肌层及浆膜。(2)肠绒毛于E15.5 d分化形成,杯状细胞于E16.5 d逐渐发育分化出现,肠腺于E18.5~P1 d发育分化形成,此时小肠基本结构形成。(3)杯状细胞主要分布于小肠绒毛上皮,其中以回肠末端最多,回肠、空肠、十二指肠顺次递减（P<0.05）。杯状细胞数量随胎龄逐渐增加而逐渐增多,于P1 d最多（P<0.05）。 结论　小肠上皮分化于E15.5 d至E17.5 d最为迅速,胚胎时期小肠基本结构形成,其吸收消化功能基本建立完成。     

A unique surface antigen on intraepithelial lymphocytes in the mouse

This paper reports the discovery in the mouse of a new antigen found almost exclusively on the surface of lymphocytes residing in or immediately adjacent to the gut epithelium. The antigen was expressed by Lyt-2+ and L3T4+ cells but not by B cells or plasma cells and was present on almost all intraepithelial lymphocytes (IELs) in the gut. Only a very small proportion of cells in other lymphoid compartments expressed the antigen. Stimulation of IELs or other lymphocytes in vitro caused a decline in expression. Immunoprecipitation experiments showed the new antigen to be a molecular complex comprising two non-covalently linked chains (175 kDa and 136 kDa) and minor components (27 kDa and 25 kDa). The function of this complex is unknown but its structure has certain features in common with that of cell adhesion molecules and extracellular matrix receptors of the 'integrin' supergene family.     

Protein synthesis in liver and small intestine in protein deprivation and diabetes

Protein synthesis (as a percent of the protein pool synthesized per day) has been measured in liver and small intestine of young male rats from the incorporation of 100 mumol [1-14C]leucine/100 g body wt into protein over 10 min. Dietary protein deprivation for 8 days depressed protein synthesis in liver (30%), jejunal mucosa (20%), and jejunal serosa (25%). In serosa, reduced levels of RNA relative to protein could account for altered synthesis; in liver and mucosa, the amount of protein synthesized per unit RNA was reduced. In liver of streptozotocin-diabetic rats protein synthesis was depressed 45%, whereas it was maintained in jejunal mucosa and serosa. Depressed synthesis in liver was accompanied by both a loss of RNA relative to protein and a reduction in the protein synthesized per RNA.