Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.     

Differential effects of vasoactive intestinal peptide, substance P, and somatostatin on immunoglobulin synthesis and proliferations by lymphocytes from Peyer's patches, mesenteric lymph nodes, and spleen

We examined the effect of vasoactive intestinal peptide, substance P, and somatostatin on concanavalin A (1 microgram/ml)-induced lymphocyte proliferation and immunoglobulin (IgA, IgM, and IgG) synthesis by cells from spleens, Peyer's patches, and mesenteric lymph nodes. These neuropeptides (10(-7) to 10(-12) M) modulated immune responses in a dose-dependent manner. For a comparative study, neuropeptides were used at 10(-8) M concentration. Both vasoactive intestinal peptide and somatostatin significantly decreased DNA synthesis (30 to 50%), whereas substance P increased synthesis (40%) in lymphocytes from all organs tested. IgA synthesis was significantly altered by all of the neuropeptides tested, whereas IgM synthesis was less affected and IgG synthesis was virtually unchanged. Somatostatin inhibited IgA (20 to 50%) and IgM (10 to 30%) synthesis in lymphocytes from all three organs. Substance P increased IgA synthesis in mesenteric lymph nodes (50%), spleens (70%), and Peyer's patches (300%). It also increased IgM synthesis in Peyer's patches (20%) and spleens (30%), but was without effect on IgM synthesis in mesenteric lymph nodes. Vasoactive intestinal peptide increased the IgA response in mesenteric lymph nodes (20%) and spleens (30%), but inhibited IgA synthesis in lymphocytes from Peyer's patches (60%). Interestingly, in Peyer's patches, IgM synthesis was increased by vasoactive intestinal peptide (80%), whereas it was unchanged in mesenteric lymph nodes and spleen. Thus, not only did these neuropeptides have different effects on the production of different immunoglobulin isotypes, but their effect was also organ-specific. Because neuropeptides which are abundant in the intestine can modulate IgA and other immunoglobulin synthesis in vitro, they may play a significant regulatory role in mucosal immune responses in vivo.     

Immunomodulatory effect of (--)-matairesinol in vivo and ex vivo

Matairesinol is one of the lignan compounds found in a variety of plant foodstuffs. We investigated the immunomodulatory effects of (-)-matairesinol in vivo and ex vivo by using mice. Although we found no significant differences in the IgG, IgA and IgM levels in the serum, the IgE level was strongly suppressed by the uptake of (-)-matairesinol in both intact and ovalbumin-immunized mice. The immunoglobulin produced by lymphocytes from the spleen was not activated by the intake of (-)-matairesinol. However, lymphocytes in such gut-associated lymphatic tissues as Peyer's patches and mesenteric lymph nodes were activated by the administration of (-)-matairesinol.     

Early response to rotavirus infection involves massive B cell activation

Rotavirus is an acute enteric pathogen which induces severe diarrhea in infants and children. To determine the immune response to rotavirus in vivo, we used a mouse model of rotavirus infection. We observed dramatic increases in the sizes of both Peyer's patches and mesenteric lymph nodes, but not spleen, between 1 and 6 days after infection with a homologous strain of murine rotavirus, EC wild type. Histological analysis showed large increases in the numbers of lymphocytes in these same tissues in rotavirus-infected mice. Flow cytometric analysis confirmed the increase in numbers of lymphocytes and revealed a large increase in the percentage of activated B, but not T, lymphocytes in both Peyer's patches and mesenteric lymph nodes of rotavirus-infected mice compared with control mice. Fragment cultures from these tissues established at 3-4 days postinfection contain rotavirus-specific IgM but not IgA Ab. A similar degree of lymphoid hyperplasia and percentage of activated B cells were observed in rotavirus-infected TCR knockout mice. Taken together, our findings show that rotavirus infection, in the context of a normal immune response, induces a large increase in the percentages of activated B cells in the absence of any detectable increase in the percentage of activated T cells, implicating a T cell-independent B cell response as the primary mechanism for initial rotavirus clearance.     

Physiology of B cells in mice with X-linked immunodeficiency. I. Size, migratory properties, and turnover of the B cell pool

In terms of certain immune functions and density of surface IgM, B cells from xid mice are often viewed as the equivalent of the immature (Lyb-5-) B cell subset of normal adult mice. In this paper we examine xid B cells with regard to certain physiologic functions, including homing to the lymphoid tissues, recirculation, and turnover. Xid mice were found to possess about one-third of the total number of B cells found in normal mice. This applied irrespective of whether one examined the spleen, lymph nodes, or outputs of B cells in thoracic duct lymph. In terms of migration to spleen, lymph nodes, and Peyer's patches, capacity to recirculate from blood to thoracic duct lymph, and turnover, xid B cells proved to be indistinguishable from normal spleen or thoracic duct B cells. Within these parameters, most xid B cells closely resemble the normal mature long-lived population of B cells residing in the recirculating pool of normal mice. Because xid B cells are functionally quite different from normal mature B cells, it seems reasonable to view xid B cells as an abnormal population not represented in normal mice.     

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.     

Detection and quantification of IgM(+) lymphocytes in fetal lamb spleen, liver and lymph nodes by flow cytometry

The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.     

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses

Peyer's patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA(+) and IgG2a(+) B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.     

Encapsulated Bifidobacterium bifidum potentiates intestinal IgA production

We asked whether Bifidobacterium bifidum regulates the synthesis of IgA by mucosal lymphoid cells. B. bifidum alone, but not Clostridium perfringens, significantly induced total IgA and IgM synthesis by both mesenteric lymph nodes (MLN) and Peyer's patch (PP) cells. We, further, investigated the mucosal antibody production following peroral administration of B. bifidum to mice. Ingested B. bifidum significantly increased the number of Ig (IgM, IgG, and IgA) secreting cells in the culture of both MLN and spleen cells. Nonetheless, B. bifidum itself does not induce the own specific antibody responses, implying that B. bifidum does not provoke unnecessary immune reaction. Subsequently, it was found that encapsulation of B. bifidum further augments the total IgA production in the culture of both MLN and spleen cells. Finally, we found that the immuno-stimulating activity of B. bifidum is due to its cellular components but not due to any actively secreting component(s) from bacteria.     

Immunoglobulin isotypes in plasma cells of normal and athymic mice.

The distribution of plasma cells was determined in various lymphoid tissues and exocrine glands of athymic (nude) mice. Compared to values for normal mice, the total number of plasma cells in organs of athymic mice showed a variable decrease as follows: 0%, small intestine; 29%, respiratory tree; 33%, spleen; 50%, lymph nodes; 75%, lactating mammary gland; 85%, Peyer's patches; and 90%, parotid gland. Plasma cells containing IgG₁ or IgA showed the greatest decrease, whereas IgM-containing plasma cells were actually increased by 100% or more in most organs. In exocrine glands the absolute deficit of IgA-containing plasma cells was most marked in the parotid and lactating mammary gland, and least in the small intestine. All lymphoid tissues had a striking deficit in the absolute numbers of IgA as well as IgG, plasma cells. Total plasma cell numbers and their isotype distribution were similar for BALB/c +/+ (homozygous) and +/nu (heterozygous) mice.     

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.     

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.     

Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

In vitro tapeworm extract-induced proliferative responses of gut-associated lymphoid cells from Hymenolepis diminuta infected mice

The development of lymphoid cells reactive to tapeworm-associated antigens during the course of Hymenolepis diminuta rejection from mice was studied using an in vitro tapeworm extract (TWE)-induced cell proliferation culture system. Mice infected with three cysticercoids on day 0 developed three adult worms by day 7 but worms were rejected by day 21 post-infection. Concomitant with worm rejection was the development of TWE-sensitized lymphoid cells which responded by proliferation when stimulated in vitro with TWE. Sensitized cells were detected in gut-associated mesenteric lymph nodes but were not detected in spleen, axillary lymph nodes, or Peyer's patches of infected mice, or in lymphoid organs of non-infected mice. These studies suggest that rejection of H. diminuta from mice is associated with the activities of gut-associated, tapeworm antigen-sensitized immune cells localized in the mesenteric lymph nodes.     

Active synthesis of hemagglutinin-specific immunoglobulin A by lung cells of mice that were immunized intragastrically with inactivated influenza virus vaccine

Intragastric inoculation with whole-virion vaccine of inactivated influenza virus resulted in production of hemagglutinin (HA)-specific immunoglobulin A (IgA) and IgG both in lung lavage fluids and in serum samples of mice. HA-specific IgA was the predominant isotypic antibody secreted in the lung lavage fluids (average IgA/IgG ratio, 13:1), whereas HA-specific IgG was the major antibody class in serum (average IgA/IgG ratio, 0.3:1). These responses were similar to the antibody responses stimulated by intranasal infection with live influenza virus. In vitro cultures of lymphoid cells from lungs and Peyer's patches, but not from spleens, in the presence of homologous antigen, from mice vaccinated intragastrically synthesized mostly HA-specific IgA. Mice immunized parenterally with inactivated influenza virus produced only IgG in lung lavage fluids and sera. Cultures of lymphoid cells from their spleens, but not their lungs, synthesized HA-specific IgG upon antigenic stimulation in vitro; neither synthesized IgA. These in vitro cell culture results, as well as the inverse relationship of IgA/IgG ratios in lung lavage fluids and sera, demonstrated that the IgA antibody in lung lavage fluids was actively synthesized locally in the lungs of intragastrically immunized mice. This finding was consistent with the migratory distribution of antigen-primed lymphoid cells from Peyer's patches to distant lymphoid tissue such as lung. Intragastric vaccination conferred protection against intranasal challenge with a lethal dose of virulent virus.     

Changes in antigen distribution and lymphocyte migration pattern after peroral immunization

The migration pattern of lymphoid cells in long-term p.o. immunized and control mice using the transfer of 51Cr-labelled cells from spleen, Peyer's patches and mesenteric or peripheral lymph nodes was studied. There are no differences between the homing activity of spleen of PLN cells to different organs of recipient animals. Peyer's patch cells from SRBC-fed mice home significantly more to the gut of antigen-fed mice; also the MLN cells from these mice exhibit a higher localization in the gut of SRBC-fed mice. There were no differences between the localization of antigen (SRBC) in different organs of SRBC-fed and control mice. The clearance of this antigen was higher in SRBC-fed animals.     

Organogenesis of lymphoid tissues

The development of lymphoid organs depends on the correct expression of several molecules within a defined timeframe during ontogeny. Although this is an extremely complex process, with each secondary lymphoid tissue requiring subtly different signals, a common framework for lymphoid development is beginning to emerge. Drawing on studies of lymph nodes, Peyer's patches and nasal-associated lymphoid tissue, an integrative model of lymphoid-tissue development, involving adhesion molecules, cytokines and chemokines, which emphasizes the role of interactions between CD3-CD4+CD45+ 'inducer' cells and VCAM1+ICAM1+ stromal 'organizer' cells is presented.     

The localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.