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The vacA and cagA geno- and phenotypes of two mouse-adapted strains of Helicobacter pylori, SS1 and SPM326, were determined. The SS1 strain, which had the cagA+ and vacA s2-m2 genotype, induced neither vacuole formation in HeLa cells nor interleukin-8 (IL-8) production in KATO III cells. In contrast, H. pylori SPM326, with the cagA+ and vacA s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro. Furthermore, a spontaneous mutant of SPM326, which produced a vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8(-)), was detected. C57Bl/6 and BALB/c mice were infected with these three strains to investigate the colonization pattern and the effect on the immune response in vivo. The SS1 strain colonized the stomachs of all mice in large numbers which remained constant over time. Colonization with the SPM326/IL-8(+) and SPM326/IL-8(-) strains was lesser, or even absent, and decreased over time. At 5 weeks postinoculation all three H. pylori strains induced a mild increase of neutrophil count in the gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both 5 and 12 weeks postinoculation C57Bl/6 mice colonized with SPM326/IL-8(+) showed an increased expression of major histocompatibility complex (MHC) class II antigen in the cardia which was accompanied by an increased number of T cells. C57Bl/6 mice that were infected with SS1 and SPM326/IL-8(-) did not show chronic inflammation. BALB/c mice colonized with SS1 and SPM326/IL-8(-) also showed an increase in neutrophil count at 5 weeks, which normalized again by 12 weeks postinoculation. At this time point SS1-infected mice showed inflammation in the corpus and antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with SS1, their incidence as well as their number was increased at 12 weeks. These results show that inflammation induced by H. pylori depends both on the bacterial strain and the host.

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.

Oral administration of DSS has been reported to induce an acute and chronic colitis in mice. The aim of our study was to evaluate if the chronic phase of DSS-induced colitis was characterized by a Th1/Th2 response and how this would relate to mucosal regeneration. Swiss Webster mice were fed 5% DSS in their drinking water for 7 days, followed by 2-5 weeks consumption of water. Control mice received only water. The animals were killed at 3 and 6 weeks after induction. Their colons were isolated for histology and immunohistochemistry, using specific MoAbs for T and B cells, macrophages, interferon-gamma (IFN-gamma), IL-4 and IL-5. Colons were scored for inflammation, damage and regeneration. Two weeks after stopping DSS the colonic epithelium had only partially healed. Total colitis scores were still increased, especially in the distal colon, which was due to more inflammation, damage and less regeneration. In areas of incomplete colonic healing the basal parts of the lamina propria contained macrophages and CD4+ T cells. These CD4+ T cells showed a focal increase of IFN-gamma and IL-4 staining compared with control animals. These findings were still observed 5 weeks after stopping DSS in some mice, albeit less extensive. Chronic DSS-induced colitis is characterized by focal epithelial regeneration and a Th1 as well as Th2 cytokine profile. We postulate that chronic immune activation mediated by both populations of Th cells can interfere with colonic healing and can play a role in the pathogenesis of chronic colitis.

Glucocorticosteroids (GCS) are effective in treatment of inflammatory bowel disease (IBD), but also have unwanted systemic side effects. Here, we describe the effects of budesonide and dexamethasone on acute experimental colitis and on T cells in thymus and spleen, as well as the effect of budesonide treatment on relapsing colitis. Acute colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol, and a relapse was induced by an intraperitoneal booster of TNBS. GCS were administered intrarectally on days 1, 4, and 6 after induction of acute colitis or a relapse. Inflammatory cells in the colon were studied on day 7, and in acute colitis also on days 13 and 16. Budesonide treatment in acute and relapsing colitis resulted in reduction of macroscopic damage and decreased the numbers of macrophages and neutrophils in the colon. Dexamethasone was less effective. Dexamethasone, but not budesonide, reduced the number of T cells in the thymus. It is concluded that local budesonide is more effective in treatment of acute experimental colitis than dexamethasone and, in contrast to dexamethasone, did not cause a general suppression of T cells. Although budesonide was very effective in the treatment of relapsing colitis, this effect was not accomplished by affecting the number of T cells in the colon.

Gastritis was induced in mice by oral administration of acetic acid 5%, a cagA positive Helicobacter pylori strain, or both. The induction of a mild gastritis by acetic acid before inoculation with H. pylori resulted in a slight but not significantly decreased colonisation rate. To study the initial stage of inflammation, the presence of gastric lymphoid and non-lymphoid cells was studied by immunohistochemistry during the first 2 weeks after induction of gastritis. Treatment with acetic acid alone or in combination with H. pylori resulted in an increase in the number of neutrophils in the mucosa and submucosa, without evident epithelial damage. The influx of neutrophils was most prominent in the mice that received a combined treatment of acetic acid and H. pylori. Macrophages were also increased in both acetic acid and acetic acid plus H. pylori-treated groups, although a different kinetic pattern was present in these groups. In mice infected with H. pylori alone, only a slight but not significant increase in neutrophils and macrophages was observed. The early presence of lymphoid aggregates in the gastric mucosa of mice in which colonisation was shown with H. pylori was remarkable. This phenomenon was not seen in control mice, in mice that received acetic acid alone or when colonisation was not shown. These data suggest that gastritis induced by a chemical agent such as acetic acid occurs by a different mechanism than gastritis induced by H. pylori and that the continued presence of H. pylori is required for local lymphocyte activation.

Alveolar macrophages (AM) are important in the regulation of immune responses in the lung, through their role as scavenger cells and through the production of many bioactive factors. Because in early infancy pulmonary infections are a recurrent problem, we studied the postnatal functional maturation of AM in a rat model. AM were isolated from rat lungs by bronchoalveolar lavage at several time intervals after birth and tested for their ability to ingest Escherichia coli in the presence of surfactant protein A (SP-A). Furthermore, their capacity to produce nitric oxide (NO) and interleukin-1 beta (IL-1 beta) after in vitro lipopolysaccharide (LPS) stimulation was analysed, as well as their capacity to downregulate proliferation of T cells from both mature and neonatal rats. SP-A-mediated phagocytosis of E. coli by AM was reduced in 14-day-old neonatal rats, as compared with mature rats (P < or = 0.05). Also the IL-1 beta production by rat AM after LPS stimulation was impaired at 14 days of age, as compared with IL-1 beta production by AM from mature rats (P < or = 0.05). In contrast, the LPS-induced NO production by rat AM as well as the capacity to inhibit T-cell proliferation were well developed at all ages tested. In conclusion, during postnatal development the rat AM is functionally immature, with respect to phagocytosis and secretion of inflammatory mediators. These differences may underly the enhanced susceptibility to pulmonary infections as found in human neonates.

CD4+ T cells play an important role in the aetiology of inflammatory bowel disease (IBD), but it is not clear which factor(s) cause activation of these cells. The aim of this study was to examine the effects of adoptive transfer of splenic (CD4+) T cells from TNBS/ethanol-sensitized donor rats to naive recipients and the migration pattern of transferred T cells. For the transfer experiments, colitis was induced in rats by colonic administration of TNBS/ethanol. Seventeen days later, either total splenic T cells or CD4+, or CD8+ T cells were transferred to naive recipients. At days 1, 2 and 3 after transfer, the recipients were killed and the migration pattern of the transferred T cells was studied, as well as inflammatory cells in several organs, including the colon. To determine cytokine profiles of the T cells, colitis was induced in mice. Therefore, different combinations of 2,4-dinitrobenzene sulfonic acid (DNBS) in ethanol or saline, or ethanol alone were intrarectally administered. At day 9 after induction of colitis, mice were killed and cytokine profiles in the colon were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The results show that CD4+ T cells from donor rats with TNBS/ethanol-induced colitis migrate in particular to the colon upon transfer to naive recipients, and that this process is down-regulated by CD8+ T cells. This migration is probably caused by T cell recognition of the colonic bacterial flora and initiates an inflammatory reaction in the recipient's colon, characterized by an increase of the recipient's own T cells, macrophages, and neutrophils. In the mice experiments we showed that a second administration of DNBS/ethanol or ethanol alone, which presumably causes bacterial translocation, results in increased numbers of T cells into the colon, accompanied by an increase in Th1 cytokines. These data suggest that Th1 cells recognize the colonic bacterial flora.

Genetic diversity of 32 Helicobacter pylori strains isolated from patients with gastritis, gastric or duodenal ulcer, carcinoma, or lymphoma was determined by repetitive sequence element polymerase chain reaction (REP-PCR), and by the new typing method restriction fragment end-labelling (RFEL). Furthermore, these two methods were used to investigate a possible correlation between clinical symptoms and the genetic background of Helicobacter pylori. Both REP-PCR and RFEL revealed 31 different patterns for the 32 strains tested, but the pair of isolates with identical REP-PCR patterns was not the same as the pair of isolates with identical RFEL patterns. Computer-assisted analysis of the DNA fingerprints was used to determine similarity coefficients. This analysis revealed no clustering of disease-specific strains by any of the two methods.

Exposure to synthetic glucocorticoids (GCs) or other stimuli around birth may affect neuroendocrine and immune responsiveness in the offspring. Experiments were conducted to investigate whether maternal manipulation with saline or with GCs alters the corticosterone (CORT) response to a mild stressor in the offspring, and whether maternal manipulation results in long-term altered in vivo humoral and cellular immune responsiveness in the offspring. Pregnant rats were given dexamethasone (DEX, 1.2 mg/kg body weight, i.p.) or saline (SAL) at day 17 and 19 of gestation. A third group of pregnant rats was left undisturbed (UNTR-group). After maternal DEX treatment, no altered CORT response was seen to a novel environment at 20 days of age, as compared to both the SAL-treated group and the UNTR-group. However, saline administration to pregnant rats caused an increased CORT response in female offspring, but not male offspring, as compared to the UNTR-group (P < or = 0.01). Furthermore, no effects of maternal DEX exposure were seen on IgG2a production after immunization with a conjugated pneumococcal polysaccharide (PPS-14-CRM197) at 6 weeks of age. However, maternal SAL treatment enhanced anti-PPS-14 IgG2a antibody levels in female offspring, but not in male offspring, as compared to the UNTR-group (P < or = 0.05). Cellular immune responses were measured by an oxazolone-induced contact hypersensitivity response (CHS-response), at 8 weeks of age. Maternal SAL treatment increased the CHS response in adult male rats, but not in female rats, as compared to both the UNTR-group and the DEX-group (P < or = 0.005). These data suggest that manipulations during late pregnancy not only affect endocrine responsiveness, but also influence immune responsiveness in the rat offspring. Furthermore, these effects may be long-term and gender-specific.

One of the major advances in the understanding of inflammatory bowel disease has been the observation that mice with immunoregulatory defects, such as interleukin-2 knockout (IL-2 -/-) mice, develop spontaneous gut inflammation. Here we have characterized the immune response in the ileum, caecum and colon of these mice before and after the onset of colitis by examining the cellular infiltrate, the cytokines produced by these cells and the mucosal vascular addressin MAdCAM-1. IL-2 -/- mice developed colitis after 35 days of age and before this the mice were apparently healthy. IL-2 -/- mice aged over 35 days with colitis had large numbers of CD4+, CD8+, alpha beta T-cell receptor (TCR)+ and gamma delta TCR+ T cells, macrophages, dendritic cells and MAdCAM-1+ endothelial cells in the caecum and colon. This was associated with an increase in the number of interferon-gamma (IFN-gamma), IL-1 and tumour necrosis factor-alpha (TNF-alpha) transcripts and a decrease in IL-4 and IL-10 transcripts. Treatment of IL-2 -/- mice with cyclosporin A significantly delayed mortality. Interestingly, IL-2 -/- mice under 35 days, although healthy, did show some subtle immunological signs of preclinical disease. There was a significant increase in the number of macrophages and dendritic cells in the colonic lamina propria and increased mRNA for IL-1 and TNF-alpha. There were also increased numbers of MAdCAM-1+ endothelial cells, but IFN-gamma transcripts were not elevated. These results suggest that T-cell-mediated colitis in IL-2 -/- mice may be secondary to an initial non-specific inflammation.

Synthetic glucocorticoids (GC) are widely used drugs, also in the prevention of diseases that occur in the preterm newborn. Previously we have found that GC treatment of pregnant rats resulted in a persistent increase in the ratio of AVP over CRH in the mediobasal hypothalamus, and in an increased CD4/CD8 ratio in the thymus of the newborn. The objective of the present study was to investigate whether such effects were also seen after neonatal GC exposure, given in clinically-relevant doses. Dexamethasone-21-phosphate (DEX; 1.2 microg/g BW, i.p.) was given at day 5 and day 7 after birth. At day 18, 33, and 48 after birth effects of GC on the HPA-axis, and on CD4+ and CD8+ T cells in thymus and spleen were examined. Neonatal DEX treatment temporarily increased (p < 0.01) AVP stores in the external zone of the median eminence (ZEME) on day 18 after birth, and did not affect CRH stores. Resting plasma levels of ACTH and corticosterone remained unchanged after neonatal DEX treatment at any time interval studied. In the thymus and spleen, neonatal DEX treatment decreased (p < 0.0001) T cell numbers on day 18 after birth. Furthermore, neonatal DEX treatment increased (p < 0.01) the ratio of mature CD4-CD8+ over CD4-CD8+ thymocytes on day 18 after birth, but not on day 33 and day 48 after birth. In conclusion, neonatal DEX treatment has temporary effects on peptide expression in hypothalamic CRH neurons, and on thymocyte maturation. Apparently, neonatal exposure to GC affects potentially sensitive targets within the endocrine system and immune system but these alterations are reversible and readjusted during development.

The etiology of chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis is still unknown. Recent studies including animal models of intestinal inflammation have identified interactions between the mucosal immune system, host genetic susceptibility and environmental factors, including normal intestinal microflora. Although the ideal animal model of IBD has not been found, each model can study pathogenetic factors such as acute intestinal injury and healing, acute and chronic inflammation, regulation by key cytokines, T-lymphocyte mediation, the role of luminal bacteria, immunoregulatory factors and genetic susceptibility. The relevance of these studies for the treatment of IBD is also discussed.

Emigration of leukocytes from the circulation into tissue by transendothelial migration, is mediated subsequently by adhesion molecules such as selectins, chemokines and integrins. This multistep paradigm, with multiple molecular choices at each step, provides a diversity in signals. The influx of neutrophils, monocytes and lymphocytes into inflamed tissue is important in the pathogenesis of chronic inflammatory bowel disease. The importance of each of these groups of adhesion molecules in chronic inflammatory bowel disease, either in human disease or in animal models, will be discussed below. Furthermore, the possibilities of blocking these different steps in the process of leukocyte extravasation in an attempt to prevent further tissue damage, will be taken into account.

The inhibitory effect of steroids such as dexamethasone on the immune response of adults has been widely documented. Little is known, however, about the effect of these agents on the developing immune system in newborns. The present study describes the ontogeny of the sensitivity of peripheral blood T cells to dexamethasone during the first year of life. Cord blood T cells of preterm or term infants appear to be extremely sensitive to inhibition of the proliferative response. This high sensitivity of cells to dexamethasone can still be observed in the first 2 weeks after birth. Subsequently, the sensitivity to dexamethasone inhibition of T-cell proliferation gradually decreases. At 1 year of age, the adult response pattern has been acquired.

The effects of glucocorticoid (GC) treatment on the mature immune and neuroendocrine system are known to be reversible. However, prenatal GC exposure may have irreversible consequences on the development of the newborn. In this study, possible long-lasting effects of short-term prenatal GC treatment were examined on the developing thymus, spleen and hypothalamo-pituitary adrenal axis (HPA axis). Female rats were given dexamethasone (DEX, 400 micrograms, i.p.) on day 17 and 19 of pregnancy and offspring was studied at several time intervals (1-20 days) after birth, for examination of thymus, spleen, hypothalamus and blood plasma. Examination of thymus and spleen revealed that prenatal exposure to DEX resulted in decreased T cell numbers in thymus and spleen on day 1 after birth. Thymus regeneration after DEX exposure both during pregnancy and in adult life was completed after 24 days. However, the kinetics of regeneration of the thymi after prenatal DEX exposure were different from that seen after DEX in adult life. Whereas DEX treatment during pregnancy resulted in an increased ratio of CD4+/CD8- thymocytes over CD4-/CD8+ thymocytes compared to control groups on day 7 and day 20 after birth (time X treatment interaction; P < 0.05), DEX treatment in adult life did not change this ratio. T cell numbers in the spleen were significantly decreased at all neonatal ages studied. Regarding the hypothalamus, prenatal exposure to DEX altered the pattern of neonatal changes in peptide expression in corticotropin-releasing hormone neurons, with a selective reduction in CRH storage in the median eminence (7 and 9 days after birth) and an increase in AVP storage (9 and 20 days after birth). The ratio of AVP over CRH was significantly increased at all developmental ages studied. No effects were seen on basal ACTH and corticosterone levels in plasma. In conclusion, the kinetics of thymus regeneration after DEX exposure during pregnancy were different from that seen after DEX exposure in adult life. Prenatal DEX exposure also seemed to delay the migration of T cells into the spleen. Furthermore, prenatal DEX treatment exerted major effects on hypothalamic CRH neurons that maintained for at least 20 days after birth, which points towards an enhanced stress responsiveness of the HPA axis in later life.

The influx of monocytes and neutrophils into the inflamed tissue could be an important aspect in the pathogenesis of inflammatory bowel disease (IBD). A membrane protein involved in the monocyte/neutrophil adherence to endothelium is CD11b/CD18 or alpha M beta 2 (complement receptor type 3 = CR3). In the present study the role of CD11b/CD18 in experimental IBD was studied by treatment with ED7 and OX42, two MoAbs against CD11b/CD18. Colitis was induced in rats by a single, rectal administration of 30 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol 30%. Two hours before and 3 days after induction of colitis, the animals were given an i.v. dose of 0.5 mg of either ED7 or OX42 in 1 ml PBS. Controls received PBS or an irrelevant MoAb. Four days after the last treatment with the antibodies, the rats were killed, and macroscopic damage scores of the colon were determined. Macrophages and granulocytes were studied by immunohistochemistry and quantified by Interaktives Bild Analysen System (IBAS), and myeloperoxidase (MPO) activity in colonic tissue was measured. After treatment with ED7 and OX42 the mean damage score of the colon was reduced from 4.2 in IBD animals to 1.0 and 1.3, respectively. Smaller areas of ulcerations and a decrease in the number of ulcerations were observed compared with PBS-treated rats. Furthermore, the amount of infiltrating monocytes and leucocytes in the submucosa was enormously reduced, as well as MPO activity in the colonic tissue. These results show that treatment with MoAbs against CD11b/CD18 reduces clinical signs of experimental IBD in rats by a partial blockade of infiltrating macrophages and granulocytes.

Glucocorticoids (GC) are known to inhibit the proliferative response of leukocytes after mitogenic activation. Until now, the effects of GC on the immune system have been studied predominantly in adults. However, GC are frequently administered to human fetuses and newborns for the prevention and treatment of respiratory distress syndrome. The immune system of human newborns is still a functionally immature system. Therefore, we wondered whether the immaturity is also reflected by altered responses to hormonal signals such as glucocorticoids. We studied the effects of the GC dexamethasone (DEX) on the proliferation of peripheral blood mononuclear cells and T cells in vitro after stimulation with phytohemagglutinin, anti-CD3, anti-CD3/anti-CD28 or anti-CD2/anti-CD28. Our data demonstrate that neonatal cells are much more sensitive to inhibition of the proliferative response by DEX than adult cells (ED50 1 +/- 0.8 nM vs. 221 +/- 135 nM). This difference in sensitivity is not related to differences in affinity and capacity of binding of [3H] DEX. Moreover, we show that the mechanisms of GC inhibition differ between adult and neonatal cells. In adult cells, addition of interleukin (IL)-2 does not restore DEX inhibition of the proliferative response. In contrast, the proliferative response of neonatal cells can be restored completely by the addition of IL-2. These data suggest that the primary target of GC in neonatal cells is inhibition of IL-2 production. In adult cells, other mechanisms are responsible for inhibition of T cell proliferation.

Systemic vaccination does not effectively induce a protective immune response to Streptococcus pneumoniae infections in infants, elderly people and immunosuppressed patients, who are highly susceptible to this pathogen. As mucosal immune responses develop early in life and still function well in the elderly, mucosal vaccination (that is, by oral, intratracheal or intranasal routes) may be an alternative approach to current strategies.

The immune response to pneumococcal polysaccharide type 14 (PPS-14), induced by native PPS-14 was compared with the response induced by PPS-14 conjugated with CRM197 (PPS-14-CRM197). In our animal model, immunization with PPS-14-CRM197 gave a significant enhancement of anti-PPS-14 serum titres for IgM and IgG, but not for IgA. Also an increase in total number of anti-PPS-14 antibody-secreting cells was found. Using immunohistochemical techniques, a different distribution pattern of specific antibody-containing cells in spleen section after immunization with PPS-14-CRM197 was observed. Furthermore, a higher number of IFN-gamma producing cells was found after immunization with PPS-14-CRM197, as compared with immunization with PPS-14. This enhanced IFN-gamma production may be the cause for enhanced IgG response observed after immunization with PPS-14-CRM197. The specific immune response was less affected by splenectomy in animals immunized with PPS-14-CRM197 than with PPS-14. However, an age-related response to the native as well as the conjugated form of the PPS-14 was observed, since no effect of conjugation with CRM197 was seen in the onset of the immune response to PPS-14 in young animals. In conclusion, our results affirm the hypothesis that conjugation of polysaccharides changes the characteristics of the antigen towards a thymus-dependent antigen.

In rodents, intracolonic administration of ethanol 30% induces an acute colitis, while administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS) in ethanol induces a longer lasting colitis. In the acute and chronic stages of experimental colitis, lymphoid and non-lymphoid cells were studied in the colon by immunohistochemistry. During the acute inflammation a high damage score of the colon was observed, which was related to an increase in the number of macrophages and granulocytes. Also a change in distributional patterns of macrophage subpopulations was found. The chronic stage of TNBS-ethanol-induced colitis was characterized by an increase in the number of lymphocytes, especially T cells. These data suggest that macrophages and granulocytes are important in the acute phase of experimental colitis, while lymphocytes play a pivotal role in the chronic stage. As most inflammatory bowel disease (IBD) patients have relapses during the chronic disease, we attempted to induce a relapse during experimental colitis by giving a second i.p. or s.c. dose of TNBS. This resulted in increased damage scores of the colon, new areas of ulceration and a further increase in macrophage numbers. No effect on the number of granulocytes was seen. These results indicate that it is possible to mimic relapses in experimental colitis by a second administration of TNBS, and suggest that the rats had been sensitized by the first dose of TNBS, given into the colon.

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10-ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.

The role of the spleen in the rat mucosal immune response was investigated to three structural different pneumococcal polysaccharides, type 3, 4, and 14. Following immunization with pneumococcal polysaccharides, a larger amount of free antigen was found in several lymphoid tissues and an increased trapping of immune complexes was seen in follicles of splenectomized animals, as compared to control animals. Thus, clearance of the polysaccharides seems to be less effective after splenectomy. An increase in specific IgA antibody-containing cells (ACC) was found in mesenteric lymph nodes, villi and Peyer's patches in splenectomized rats. Apparently, splenectomy and subsequent decreased clearance of the antigen causes a prolonged stay of the antigen in the system and therefore specific ACC can be induced in different lymphoid tissues. After splenectomy the specific IgM and IgG antibody titers in serum decreased significantly for pneumococcal polysaccharides types 4 and 14, but not for type 3. Furthermore, the serum IgA antibody titers against the three types of polysaccharides under study were not affected. After elimination of macrophages in the spleen by treatment with dichloromethylene diphosphonate liposomes no ACC against type 14 were evoked in the marginal zone of the spleen, and again, an increase was observed in specific IgA ACC in mucosa-associated lymphoid tissues. The IgA antibody titers were also enhanced. In conclusion, IgA responses against pneumococcal polysaccharides can be elicited in absence of the spleen, i.e. at mucosal sites or in the draining lymph nodes. Furthermore, polysaccharide-specific IgA responses are enhanced after reduction of splenic phagocytic functions.

Gut mucosal immune responses to bacterial polysaccharide antigen in rats were investigated in vivo. Rats were immunized with pneumococcal polysaccharide type 3 (PPS-3) via different routes, i.e. in the Peyer's patch (iPP), in the colon (ic), in the peritoneal cavity (ip), and intravenously (iv). The development of specific antibody-forming cells (AFC) and their isotypes in the intestinal mucosa, gut-associated lymphoid tissue (GALT), mesenteric lymph nodes (MLN) and spleen were studied by immunohistochemistry. Furthermore, the serum antibody levels were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that iPP immunization evoked high numbers of anti-PPS-3 AFC of the IgA isotype in the mucosa of the small intestine and in the PP. On the contrary, the ic route did not elicit a mucosal response, though a few AFC were found in the MLN and spleen. Following ip priming, a specific IgA response was found, especially in MLN and spleen, and a low response was detected in the villi. A high response was found in the parathymic lymph nodes (PTLN). Iv immunization gave rise to the development of AFC in the spleen, particularly of the IgM isotype. We failed to induce mucosal responses to PPS-3 antigen in the colon, irrespective of the route of immunization.

Four immunization routes were investigated to induce an immune response against three structurally different types of pneumoccoccal polysaccharide (PPS) in the rat. In particular, the contribution of the IgA isotype in these immune responses was studied. Six days after administration of PPS type 3, 4 or 14, the localization of specific antibody-containing cells (ACC) in different lymphoid tissues and the antibody titres in serum were studied. All four routes induced anti-PPS ACC in the spleen. After intraduodenal, intravenous and especially intraperitoneal administration of PPS, many IgA-specific anti-PPS ACC were also found in parathymic and mesenteric lymph nodes and in the lamina propria of intestinal tissue. Several anti-PPS ACC were found in Peyer's patches, located peripheral of the B-cell areas. The intratracheal immunization elicited only a local immune response, in bronchus-associated lymphoid tissues and paratracheal lymph nodes. The localization of these anti-PPS ACC was influenced by the route of immunization. After all four investigated routes, specific antibodies were found in serum against PPS. However, some remarkable differences between PPS-3, 4 and 14 were found in the magnitude of the immune response and the distribution of the isotypes. Both route of immunization and structure of the PPS have a profound influence on the immune responses in rats.

The placenta is a unique mixture of histoincompatible cells derived from mother and fetus. The aim of the present study was to obtain information on the development of macrophage subpopulations and reticulum cells during pregnancy in the placenta. Placentas of Wistar rats were removed at several stages of gestation, and were studied by immunohistochemical techniques applying monoclonal antibodies against macrophage subpopulations, lymphoid cells and reticulum cells. The expression of MHC class-II antigens was also studied. Throughout gestation macrophages were demonstrable in large numbers in the endometrium, in the myometrium and in the metrial gland, which is a compartment developing in the myometrium of pregnant rodents. In the labyrinth, a placenta compartment consisting of fetal cells, macrophages (probably of fetal origin) were already found on day 15. In the spongiotrophoblast and decidua basalis, which are layers of the placenta containing both maternal and fetal cells, only a few macrophages were recognized throughout gestation. The monoclonal antibody ED11, raised against reticulum cells, recognized fiber-like structures lining the blood sinuses of the spongiotrophoblast, in which only maternal blood is circulating. As the antigen recognized by ED11 is believed to play a role in the trapping of immune complexes, the spongiotrophoblast may play a role in the protection of the fetus from circulating immune complexes.

Ontogeny of reticulum cells (RC) in the rat intestine in relation to the development of the gut-associated lymphoid tissue (GALT) was studied using a panel of monoclonal antibodies (mab) directed against RC in peripheral lymphoid organs, ED10-ED15. The mab ED10 specific for RC in the spleen T cell area, recognized an epitope on gut RC, which cells seem to be involved in the influx and accumulation of lymphocytes in the lamina propria and in Peyer's patches (PP) and proximal colonic lymphoid tissue (PCLT). The mab ED11 which recognizes RC in the T cell area and B cell follicles of spleen, stained follicular dendritic cells (FEC) in the B cell area of the mesenteric lymph node (MLN), PP and PCLT. The ED11 expression occurs earlier and reveals stronger staining in MLN as compared to those in PP and PCLT, suggesting the prominent role of MLN in the generation and proliferation of B cells in the gut mucosal immune system. The mab ED13 specific for RC in the B cell area of the lymph nodes, stained the basement membrane of the epithelium overlying PP and PCLT, and high endothelial venules (HEV), suggesting that this might be involved in providing the microenvironment for the development and differentiation of follicle-associated epithelium, and facilitating lymphocyte traffic. The mab ED12 specific for RC in the paracortex of peripheral lymph nodes, gave a diffuse nonspecific staining in the gut, whereas mab ED14 and ED15 are markers for common connective tissue cells. We conclude that the gut RC are morphologically and phenotypically heterogenous. ED10+, ED11+, and ED13+ RC are probably involved in the development of the gut lymphoid microenvironment.

Two different methods have been used to study immune responses in the rat to Streptococcus pneumoniae type 3 and type 4 capsular polysaccharides (PPS). First, for simultaneous detection of the specificity and isotype of anti-PPS antibody-containing cells (ACC) in cryostat sections of lymphoid tissue, a double immunocytochemical method was developed. This method is a combination of a three-step immunoperoxidase method to demonstrate specific anti-PPS ACC as bright red cells and a two-step immunophosphatase method to detect the isotype of ACC as blue cells. Double positive cells appear violet. Using this staining procedure, the detection of antigen was also possible. Second, to study the anti-PPS response in serum, an ELISA procedure was modified. In this ELISA, polyvinylchloride microtiter plates are coated directly with type-specific pneumococcal polysaccharide. After intraperitoneal (i.p.) immunization of rats with PPS-3 or PPS-4, both antigen (PPS) and specific ACC could be detected. Specific ACC were found in the spleen and mesenteric lymph nodes. In the spleen, the specific ACC were found in the red pulp, marginal zone, outer PALS, and follicles. Most of these ACC were IgM-positive and to a lesser extent IgG-positive and IgA-positive. However, specific ACC in mesenteric lymph nodes were predominantly of the IgA isotype, with only few IgM or IgG positive cells. The anti-PPS response in serum, as measured by the ELISA, consisted mainly of IgM antibodies with small amounts of IgG and IgA. Both methods were found to be valuable in studies of immune responses against bacterial polysaccharides.

The development of macrophage subpopulations dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gut-associated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.

The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme- and immunohistochemical techniques at the light-microscopical level. The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes. Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla. The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found. Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.

The postnatal development of the various cell populations in the rat popliteal lymph node was investigated applying enzyme-histochemical and immunohistochemical techniques. From birth, T-lymphocytes and interdigitating cells were demonstrable. During the development of the young lymph node, T-lymphocytes of the helper phenotype outnumbered the T-cells with a suppressor phenotype; they account for approximately 70% and 30% of all T-lymphocytes, respectively. At the very first day of postnatal life, post-capillary venules were already present. B-lymphocytes occurred later than T-cells during ontogeny; they were found on the second day after birth, most of them being IgM- or IgG-bearing lymphocytes. The first primary follicles occurred at day 18 and contained principally membrane-stained IgM cells and, to a lesser extent, membrane-stained IgG cells. The appearance of follicular dendritic cells correlated with the formation of primary follicles. With respect to the macrophages, it appeared that the ED1- and ED3-positive subpopulations were present with a similar distributional pattern as seen in adults, but in considerably lower numbers. The expression of ED2, however, showed a sudden increase in the third week of life. Findings of the present study are discussed in relation to those obtained in other investigations dealing with the ontogenetic development of lymphoid organs.

The ontogenetic development of macrophage subpopulations and Ia-positive non-lymphoid cells was studied in gut-associated tissue in fetal and neonatal Wistar rats. A two-step immunoperoxidase method was carried out on cryostat sections, a panel of monoclonal antibodies being applied and aimed specifically at rat macrophages (ED1, ED2 and ED3) and at Ia antigen (Ox4). The first ED1-positive macrophages appeared in the liver on Day 15 (gestational age), and they did not express Ia. In developing mesenteric lymph nodes and in the gut wall, macrophages were found for the first time on Day 17 and 18, respectively, of gestation. These early macrophages were also ED1-positive. Until birth, ED2 recognized few cells in the gut-associated tissue; on the day of birth this subpopulation showed a sudden and considerable increase. The distribution pattern of ED3-positive macrophages appeared to be the same in fetal and in adult rats; it was confined to lymph nodes and Peyer's patches. Ia-positive non-lymphoid cells appeared in the abdomen early in ontogeny. The first Ia-positive cells displayed dendritic features and were found on Day 15 of fetal life in the mesenchymal tissue between intestinal loops. A few days later, many Ia-positive cells with a dendritic appearance were demonstrable in the gut wall and in developing mesenteric lymph nodes. Their number increased rapidly during the following days. Based on these results, the existence of two differentiation lines for dendritic cells and classical macrophages is discussed. The function of early Ia-positive cells in the abdomen is suggested as not being an antigen-presenting one.

To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.

Rats of the BB strain develop diabetes mellitus in a high percentage and display a severe T-cell lymphopenia. In order to investigate the role of micro-environmental factors in the T-cell maturation in BB rats the postnatal development of macrophage subpopulations and T-lymphocyte subsets, in addition to the specific immune response in situ, were studied in thymus, spleen and lymph nodes of BB rats. Wistar rats were used as controls. From the day of birth on, a severe reduction was noticed in the macrophage subpopulations in the thymic cortex of BB rats, but not in spleen and lymph nodes, as compared to Wistar rats. The population of T-suppressor/cytotoxic cells (OX8-positive cells) did not increase any longer from Day 10 after birth in the thymic cortex and from Day 14 in spleen and lymph nodes. This is indicative for an intrathymic maturational defect of the OX8-positive cells in BB rats. No deviations could be observed in the development of the T-helper (ER2-positive) cell population. Young adult BB rats were as capable as Wistars of developing a specific immune response to thymus-independent (TI) antigens, but the response to a thymus-dependent (TD) antigen was delayed and decreased. Also the distribution pattern of the specific antibody-containing cells in a TD response in BB rats differed from that in Wistar rats. The ER2-positive cells, although present in normal numbers, may function insufficiently as T-helper cells in BB rats.