Patch Lymphocytes Research Articles

Elevated membrane IgA + and CD4 + (T helper) populations in murine peyer's patch and splenic lymphocytes during dietary administration of the trichothecene vomitoxin (deoxynivalenol)

Recent investigations indicate that dietary exposure to the trichothecene vomitoxin increases total and antigen-specific serum immunoglobulin A (IgA) and glomerular IgA accumulation in mice. In this study, the effects of 25 ppm dietary vomitoxin on the histological and lymphocytic profile of component immune organs in the mucosal lymphocyte migratory pathway were evaluated in the B6C3F 1 mouse. Vomitoxin administration resulted in marked stimulation of the size and frequency of germinal centres in Peyer's patches, mesenteric lymph nodes and the spleen. A slight increase in the percentage of B cells in the Peyer's patch was observed, although vomitoxin treatment had no effect on the percentage of B cells in the spleen. The percentage of IgA + cells in Peyer's patches and spleen were approximately twice that of controls at 4, 8 and 12 wk of vomitoxin exposure whereas the percentage of IgG + cells decreased in these two organs. Exposure to vomitoxin increased the percentage of T cells in Peyer's patches and the spleen. The percentage of CD4 + cells (T helper subset) increased slightly in Peyer's patches and more markedly (30–50%) in the spleen following vomitoxin treatment. Contrastingly, there was only a slight increase in the percentage of CD8 + cells (T cytotoxic/suppressor subset) in the spleens of vomitoxin-treated mice in comparison with controls, and no effect in Peyer's patches. The relative effects of vomitoxin on these two T cell populations was also reflected in increased CD4 +:CD8 + ratios in Peyer's patches and spleen. These results are consistent with the hypothesis that dietary vomitoxin modulates normal regulation of the IgA response at the Peyer's patch level and that this is manifested in an altered lymphocyte distribution pattern in both the mucosal and systemic compartment. Notably increased levels of IgA + and CD4 + cells are indicative of IgA-producing progenitors and T helper subsets, respectively, that in tandem could favour IgA hyperproduction and elevated IgA in serum.

In vitro adhesion of K88ac +Escherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac + enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88 + enterotoxigenic E. coli in the porcine small intestine.

Rotavirus-specific cytotoxic T lymphocytes appear at the intestinal mucosal surface after rotavirus infection

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria and viruses. The first line of defense of the host against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. Little is known about the function, memory, or trafficking of IELs after intestinal infection. We found that IELs obtained 6 days after oral inoculation of mice with the intestinal pathogen rotavirus (simian strain RRV) lysed rotavirus-infected target cells; cytotoxic T lymphocytes (CTLs) were responsible for rotavirus-specific cytotoxic activity. Rotavirus-specific cytotoxic activity by IELs was (i) eliminated by treatment with Thy 1.2-specific immunoglobulin M plus complement, (ii) restricted by proteins encoded at the major histocompatibility complex, and (iii) absent in mock-infected animals. Oral inoculation of mice with RRV also induced rotavirus-specific CTLs in splenic and intestinal lymphocytes (mesenteric lymph nodes, Peyer's patch). Parenteral inoculation induced rotavirus-specific CTLs in splenic, intestinal (IELs, mesenteric lymph nodes, Peyer's patch), and nonintestinal lymphocytes (inguinal nodes). Therefore, presentation of rotavirus to the intestinal mucosal surface was not necessary to induce IELs with virus-specific cytotoxic activity. At 4 weeks after oral or parenteral inoculation of mice with RRV, rotavirus-specific CTL precursors appeared among splenic, Peyer's patch, inguinal, and mesenteric node lymphocytes, but not among IELs. IELs with rotavirus-specific cytotoxic activity may be generated from precursors at a site other than the intestinal mucosal surface. Part of the response of the host to enteric infection may include surveillance and lysis of virus-infected villus epithelial cells by IELs.

Neuroendocrine regulation of mucosal immunity.

In view of the extensive innervation of the gastrointestinal tract, including the mucosa, and the high number of immune effector cells present in this tissue, we have studied the effects of certain neurotransmitters on immune responses. We have concentrated on the effects of the neuropeptides substance P (SP), somatostatin (SOM) and vasoactive intestinal peptide (VIP). We have found that SP causes an increase in the proliferation of Peyer's patch lymphocytes as well as immunoglobulin (especially IgA) synthesis, when compared to splenic cells; and that there is a greater expression of SP receptors on lymphocytes derived from Peyer's patches compared to the spleen, without a significant difference in the expression between subsets of T and B cells. Furthermore, we have shown that intraepithelial leukocytes (IEL) show significantly increased cytotoxic activity following incubation with SP; whereas splenic lymphocytes were not stimulated in the same system. The effects of SOM where bi-directional depending upon the concentration employed but in general, SOM was inhibitory, in terms of proliferation, as was VIP. Although many more experiments are required to prove a physiological significance for the results that we have obtained, and to examine the whole gamut of neurotransmitters, we suggest that neuroendocrine regulation may play an important part in mucosal immunity.

Enhancement of host resistance against Salmonella typhimurium infection by a diet supplemented with yogurt.

The effect of a diet supplemented with yogurt containing live lactobacilli (LAB) - Lactobacillus bulgaricus and Streptococcus thermophilus - on the response of inbred mice to infection with Salmonella typhimurium was elaborated. The results of our experiments were consistent with the hypothesis that modifications of the microflora influence the adherence of S. typhimurium to intestinal mucosa, the natural antibacterial activity of the Peyer's patches lymphocytes, the accumulation of the macrophages in the liver, the proliferative responses of the splenocytes. The relationship between modifications of the immune response following ingestion of yogurt with live LAB and increased defense mechanisms was confirmed by the bacterial counts in livers and spleens and by the reduced mortality to S. typhimurium infection.

The differential effect with time of neuropeptides on the proliferative responses of murine Peyer's patch and splenic lymphocytes

The neuropeptides substance P (SP), somatostatin (SOM), and vasoactive intestinal peptide (VIP) have been shown to modulate lymphocyte DNA, RNA, and immunoglobulin synthesis. We have previously shown that SP enhances while SOM and VIP inhibit proliferation of murine splenic and Peyer's patch lymphocytes when cells were cultured with concanavalin A and neuropeptides for 72 h. Here we show that the effect of neuropeptides, in particular SP, is dependent on the amount of time that lymphocytes are incubated with NP. We found that SOM and VIP always inhibited cell proliferation for incubation times of 2 to 72 h. In contrast when cells were exposed to SP for 24 h or less, there was inhibition of [ 3H]thymidine uptake by both Peyer's patches and splenic lymphocytes. Significant enhancement in DNA synthesis by lymphocytes from both organs was only seen when cells were incubated with SP for the whole 72 h. We believe that our data may explain some of the conflicting reports regarding the effects of neuropeptides on cell proliferation.

Thymus derived (T) lymphocyte subsets restore the immune responsiveness of Peyer's patch lymphocytes from mice fed a diet reduced in protein

The principal immunoglobulin (Ig) that inhibits absorption of intact dietary protein and microbial colonization of the small intestine is secretory IgA produced by lamina propria plasma cells terminally differentiated from B lymphocytes of the Peyer's patches (PP). To determine the effect of reduced dietary protein upon the ability of PP lymphocytes to differentiate into IgA-secreting plasma cells, weanling female BDF mice were fed either a 20% casein (20C) or a 4% casein (4C) diet for 5 to 6 weeks. Mice were then orally primed with sheep erythrocytes (SRBC) four times during a 2-week period to initiate differentiation of PP lymphocytes to anti-SRBC IgA production. Both the PP and splenic lymphocytes of primed mice were incubated with SRBC in vitro to evaluate the capacity to terminally differentiate to anti-SRBC IgA- and IgM-secreting plasma cells upon antigenic stimulation. The in vitro IgA and IgM anti-SRBC immune response of PP lymphocytes from mice fed 4C was significantly lower than the immune response of PP from 20C-fed mice. In contrast, splenic IgM and IgA in vitro immune responses to SRBC of orally primed 4C-fed mice were higher than the response of 20C-fed mice. Addition of Lyt1 + T lymphocytes from SRBC-primed donor mice or Interleukin 2 (IL2) to the in vitro PP lymphocyte cultures partially restored the anti-SRBC IgA and IgM immune responses of mice fed 4C. These results strongly suggest that mice fed a diet low in protein possess a PP T-lymphocyte populations that is unable to support a local IgA or IgM immune response, while at the same time possessing a splenic lymphocyte population that has increased capability to mount a systemic IgA and IgM immune response.

Distribution of substance P receptors on murine spleen and Peyer's patch T and B cells.

Previous studies have demonstrated that the sensory neuropeptide substance P (SP) can modulate immune responses in vitro. Work from this laboratory has shown that SP enhances immunoglobulin synthesis by murine splenic and Peyer's patch lymphocytes stimulated with concanavalin A. One mechanism underlying these effects is the binding of SP to specific receptors on lymphocytes. We examined the distribution of SP receptors on murine T and B lymphocytes and their subsets by one and two color fluorescence-activated cell sorter analysis. The specificity and nature of binding was examined using radiolabeled SP, and competitive inhibition experiments were performed with cold SP. In cytofluorimetry experiments, both T and B lymphocytes from Peyer's patches and spleen were bound to SP, with those from Peyer's patches having a higher proportion than lymphocytes from the spleen. The majority of T cells from both organs bound SP with binding being evenly distributed between Lyt-1+ and Lyt-2+ cells. Similarly, the majority of B lymphocytes from spleen and Peyer's patches showed SP binding. There were no significant isotype-specific differences within any organ. Studies using 125I-labeled SP showed specific binding to all lymphocyte subpopulations examined. SP receptors were fewer in number on cells isolated from spleen than on cells from Peyer's patches although the dissociation constants were similar for all populations examined. These studies demonstrated that SP receptors are present both on murine T and B lymphocytes from Peyer's patches and spleen. There is no evidence for differential SP receptor expression on distinct lymphocyte subsets in spleen or Peyer's patches.

Cyclic adenosine 3′,5′-monophosphate response to dopamine in mouse lymphoid cells and cell lines

Cyclic adenosine 3′,5′-monophosphate responses to dopamine and isoproterenol were studied in mouse and rat spleen, thymus, lymph nodes and Peyer's patches lymphocytes and in 7 mouse cell lines of T- and B-lymphoid derivation. The responses of normal cells to dopamine were moderate, of the same extent, but selective to spleen and thymus in mouse, and to spleen and lymph nodes in rat. The YAC-1 T lymphoma cell line was sensitive to dopamine with a higher magnitude than normal lymphoid cells. Dopamine was less potent than isoproterenol in all cells, and whereas dopamine-sensitive and isoproterenol-sensitive cells, or dopamine-insensitive and isoproterenol-insensitive cells were found, no cell type was dopamine-sensitive and isoproterenol-insensitive. Altogether, these results suggest that only a small subset of lymphocytes is susceptible to the cAMP-elevating action of dopamine.

Distribution of somatostatin receptors on murine spleen and Peyer's patch T and B lymphocytes

Recent evidence suggests that the neuropeptide somatostatin (SOM) plays an immunoregulatory role. We demonstrated previously that SOM inhibits concanavalin A-induced cell proliferation and immunoglobulin synthesis by murine Peyer's patch and splenic lymphocytes. Available data suggest that these effects are in part mediated by specific SOM receptors expressed by lymphocytes, but as yet these receptors have not been characterized. Using cytofluorimetry we investigated the distribution and specificity of binding of fluorescent SOM (SOM∗) to murine Peyer's patch and splenic T- and B-lymphocyte subpopulations. The specificity of binding was confirmed by radioassay. T and B cells from both organs showed specific binding of SOM∗. In Peyer's patches, approximately 50% of all cell populations studied (whole, T- and B-cell-enriched) bound SOM specifically and this was significantly higher than the corresponding splenic lymphocyte populations. Eighty to eighty-four percent of Peyer's patch Thy1.2 +, Lyt1 +, or L3T4 + cells and 94% of Lyt2 + cells bound SOM. Greater than 80% of B cells from this organ bound SOM (sIgA + = sIgM + > sIgG + cells). In spleen, approximately 30% of Thy1.2 +, Lyt1 +, or L3T4 + cells bound SOM and this was significantly less than the proportion of Lyt2 + cells (53%) which did so. More sIgA + (89%) than sIgG + (66%) than sIgM + (55%) B cells bound SOM∗. Although we have previously shown that the effect of SOM on immunoglobulin synthesis was relatively isotype-specific (IgA synthesis was predominantly affected, especially in Peyer's patches) this cannot be explained solely on the basis of preferential expression of SOM receptors by distinct lymphocyte subsets. Instead, it is probably the result of the specific immunological microenvironment in which the lymphocytes reside.

Modulation of murine Peyer's patch immunoglobulin A response by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) may have profound effects on the capacity of gut-associated lymphoid tissue to mount a secretory immune response because of the potential ability of heat-stable toxin or heat-labile toxin to modulate the immune response. To examine the effects of ETEC or its purified enterotoxins upon the humoral immune response of murine small intestinal Peyer's patch lymphocytes, BDF1 (lipopolysaccharide-responder) and C3H/HeJ (lipopolysaccharide-nonresponder) mice were orally primed with sheep erythrocytes (SRBC) four times during a 2-week period to initiate differentiation of Peyer's patch B lymphocytes to cells committed to anti-SRBC immunoglobulin A (IgA) production. Halfway through the oral priming regimen the mice were gastrically intubated with 10(8) ETEC, 10(8) non-ETEC, or saline. ETEC persisted in the small intestine for at least 7 days at a level of 10(3) to 10(4) bacteria per mouse. Seven days after the last oral dosing with SRBC, Peyer's patch lymphocytes were removed from infected or saline-treated mice and incubated in vitro with SRBC. The ETEC infection had a small effect on the anti-SRBC IgM plaque-forming cell response of SRBC-primed mice but inhibited significantly the anti-SRBC IgA plaque-forming cell response in both BDF1 and C3H/HeJ mice as compared with uninfected controls. The non-ETEC, an isolate from normal mouse small intestine, had no significant effect on either IgM or IgA anti-SRBC plaque-forming cell response. Purified heat-labile toxin, not heat-stable toxin, alone in a dose-dependent manner significantly inhibited both the IgA and IgM plaque-forming cell response of Peyer's patch lymphocytes from primed mice. These data suggest that ETEC can inhibit the development of the gut-associated lymphoid tissue IgA immune response through the immunopharmacological effect of an enterotoxin, the heat-labile toxin.

A call for software

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4–5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minima1 changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-l-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

Influence of ofloxacin on murine Peyer's patch lymphocytes

Several factors must be considered before the clinical impact of antimicrobials on the immune function can be determined. No data are available as yet on the influence of ofloxacin, a new fluoro-quinolone, on the lymphocytes of Peyer's patches. The present study offers evidence that murine Peyer's patches are influenced by ofloxacin administered by the oral route for seven days (15 mg/kg). No modifications of the percentage of the Thy 1,2 and SIg positive splenocytes and of the PHA and LPS-induced proliferative responses were observed studying cell suspensions from the spleen of the same animals. An immunotoxic effect of the compound on the cells of Peyer's patches or an impairment of their homing tendency has been postulated.

Adoptive Transfer of Small Bowel Intraepithelial and Peyer's Patch Lymphocytes from Orally Immunised Animals

Intraepithelial (IEL) and Peyer's patch lymphocytes (PPL) were isolated from the small bowel mucosa of mice in which specific systemic antibody responses to ovalbumin (OVA) had been suppressed by oral exposure to that antigen. These lymphocytes were then adoptively transferred into syngeneic animals. The PPL transferred 24 h after oral OVA showed a tendency to depress secondary serum antibody responses to intraperitoneal OVA in the recipients. Conversely, IEL transferred 1–5 days after oral OVA significantly enhanced specific serum antibody responses.

Adoptive transfer of small bowel intraepithelial and Peyer's patch lymphocytes from orally immunised animals: effects on the antibody response to parenteral antigen.

Intraepithelial (IEL) and Peyer's patch lymphocytes (PPL) were isolated from the small bowel mucosa of mice in which specific systemic antibody responses to ovalbumin (OVA) had been suppressed by oral exposure to that antigen. These lymphocytes were then adoptively transferred into syngeneic animals. The PPL transferred 24 h after oral OVA showed a tendency to depress secondary serum antibody responses to intraperitoneal OVA in the recipients. Conversely, IEL transferred 1-5 days after oral OVA significantly enhanced specific serum antibody responses.

Modulation of in vitro natural cell-mediated activity against enteropathogenic bacteria by simple sugars.

Lymphoid cells from mouse Peyer's patches and spleens were tested in a 2-h in vitro assay for their natural activity against the enteropathogenic bacteria Salmonella typhimurium, Salmonella enteritidis, Salmonella tel aviv, and Shigella sp. X16. The antibacterial activity expressed by normal cells was detected against all the bacterial strains tested with the exception of Peyer's patch lymphocytes against S. tel aviv and splenocytes against Shigella sp. X16. To determine whether the different expression of natural antibacterial activity might be due to lectin-like proteins interacting with the saccharidic moieties of the bacterial wall, 11 simple sugars were preincubated with the effector cells before the in vitro assays. We found that some of them could block the natural antibacterial activity as well as induce antibacterial activity when this was not spontaneously expressed. Interestingly, a different panel of sugars among those employed was observed to affect the antibacterial activities for each of the above-mentioned bacterial targets and each effector cell. However, the same panel of sugars was able to block or stimulate the lymphocyte activity when bacteria with the same somatic antigens as two substrains of S. typhimurium and one strain of Salmonella schottmuelleri were employed. To further investigate the interaction between effector cells and bacteria, effector cells or Shigella sp. X16 targets were treated with proteolytic, glycolytic, and lipolytic enzymes before the in vitro assays. Furthermore, EDTA was used to analyze the role of divalent cations in this experimental system. The results obtained suggest that lectin-like proteins playing a role in this interaction are present not only on lymphocytes but also on bacteria and that divalent cations are essential for the expression of in vitro antibacterial activity.

Effect of Sublethal Ionizing Radiation on Rat Peyer's Patch Lymphocytes

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.

Selective emigration of suppressor T cells from Peyer's patches

The emigration of Peyer's patch lymphocytes to mesenteric lymph nodes was studied by injecting fluorescein isothiocyanate (FITC) directly into Peyer's patches. Using double immunofluorescence it was demonstrated that at 2 and 4 hr after FITC injection 70% of the labeled cells that migrated to mesenteric lymph nodes were T lymphocytes, although rat Peyer's patches contain only 15–20% T lymphocytes. At later time points after FITC injection this percentage of T cells derived from Peyer's patches gradually declined, most likely caused by selective interaction and/or retention inside the mesenteric lymph node. Determination of helper and suppressor T-cell subsets within this emigrating population showed an increased number of T suppressor cells migrating into mesenteric lymph nodes. The putative role of suppressor T cells in inducing systemic tolerance after oral antigen administration was discussed.

Modulation of antibody responses to Vibrio cholerae in mice by adoptive transfer of Peyer's patch lymphocytes from orally immunised donors.

Peyer's patch lymphocytes from mice orally primed with V. cholerae injected into recipient mice together with V. cholerae led to a considerable suppression of IgM antibody-forming cells in the spleen by comparison with control mice receiving the same schedule but with Peyer's patch lymphocytes from unprimed mice. The effect on the splenic IgA responses of recipients was variable. Incomplete separation of the Peyer's patch cells showed that both T and B cell enriched fractions were active in this suppressive effect.

A cell ELISA technique. The direct detection and semi-quantitation of immunoglobulin positive cells in 7 day lymphocyte cultures using the microtitre culture plates as the solid phase

A sensitive ELISA is described which allows the detection and quantitation of immunoglobulin positive Peyer's patch and spleen lymphocytes in tissue culture microtitre plates. A methanol fixation step is used to fix the cells to the microtitre plate. The effects of various mitogens and an antigen on the number of immunoglobulin positive cells present after a 7 day culture were studied. Results were expressed either in optical densities or as percentages of the unstimulated control. The method proves a useful addition to existing techniques for studying the effects of mitogens and other agents on in vitro immunoglobulin synthesis by lymphocytes, and reflects immunologbulin levels in the culture supernatants.