Quantitative studies of lymphoid organs, blood and lymph in inbred athymic and euthymic LEW rats under germfree and specified-pathogen-free conditions

Four groups of inbred male LEW rats were examined: A, germfree athymic; B, specified pathogen free (SPF) athymic; C, germfree euthymic; D, SPF euthymic. All animals were killed at 18 weeks and compared with respect to body weight, histological appearance and cell density of the lymphoid organs, haematological values and differential counts of bone marrow, peripheral blood and lymph. Athymic rats had a lower body weight, less densely populated lymphoid organs, and fewer lymphocytes in the blood and lymph compared with euthymic animals. No difference was seen between athymic rats under germfree and SPF conditions, and in general the differences between athymic and euthymic animals were less pronounced under germfree conditions.     

免疫应答期间脑和淋巴器官中去甲肾上腺素含量的变化

应用高效液相色谱－电化学检测法（ＨＰＬＣ－ＥＣＤ）测定大鼠在用绵羊红细胞（ＳＲＢＣ）免疫后第２－７天期间,下丘脑、海马、脑干、胸腺和脾脏中去甲肾上腺素（ＮＡ）含量的变化。实验结果表明,下丘脑内ＮＡ含量在免疫后第４－７天明显增加,其中第７天有回降趋势。海马内ＮＡ含量在第４－５天显著增多。而胸腺和脾脏中ＮＡ水平在第４－５天均明显降低。脑干在免疫应答期间无明显改变。以上结果说明,体液免疫应答可影响脑和淋     

The use of a rat-derived microflora for providing colonization resistance in SPF rats

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb: WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37 degrees C) for 7 days and harvested. This cultured flora was given to germ-free Cpb: SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the 'quality' of the intestinal microflora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-microflora which can be used to start new SPF units.     

Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs

Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.     

Possibilities of using neurotropic agents for preventing the development of aphthous stomatitis

Aphthous lesions of the oral mucosa were simulated in dogs by common bile duct ligation. In one of the experimental groups, the animals were administered the beta-adrenoblocker obsidan 30 min before operation. Two hours after operation the regions of the oral mucosa mostly affected by aphthous lesions were examined for the content of adrenaline and noradrenaline (NA). The data obtained indicate a significant increase in the content of NA and adrenaline in the oral mucosa 2 h following operation. In animals pretreated with obsidan, the content of catecholamines remained at the level seen in the control group. Therefore, blocking the transmission of nerve impulses to the sympathetic nervous system obsidan interferes with the reflex influence from the involved organs of the abdominal cavity, thereby protecting the oral mucosa tissues from the damage-inducing action of high concentrations of NA.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Toxicity and proteolysis in gnotobiotic rats with thermal burns]

Experiments were conducted on germ-free (gnotobiotic) rats. A study was made of the toxic properties of the organs and the activity of proteolytic enzymes in them after thermal burns. Despite the absence of pathogenic microbial flora the germ-free rats developed the state of toxemia with an increase of the level of proteolytic enzymes. The extent of the toxic and catabolic reaction depended on the severity of the burn. Intoxication and an increase in proteolysis in the gnotobiotic rats were similar, and, in a number of cases greater than in usual animals. A conclusion was drawn on the important role of the tissue sources of intoxication in burns. A correlation between the toxemia and proteolysis was established.     

CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.     

Nonsynaptic noradrenaline release in neuro-immune responses

Evidence has recently been obtained that the branches of the autonomic nervous system, mainly, the sympathetic [25], regulate cytokine production. Not only the primary (thymus, bone marrow) and secondary (spleen, tonsils, and lymph nodes) lymphoid organs, but also many other tissues are involved in immune responses and are heavily influenced by noradrenaline (NA) derived from varicose axon terminals of the sympathetic nervous system [25, 100]. Besides NA released from nonsynaptic varicosities of noradrenergic terminals [92], circulating catecholamines (adrenaline, dopamine, NA) are also able to influence immune responses, the production of pro- and anti-inflammatory cytokines by different immune cells. The sympathetic nervous system (catecholamines) and the hypothalamic-pituitary-adrenal (HPA) axis (cortisol) are the major integrative and regulatory components of different immune responses. In our laboratory convincing evidence has been obtained that NA released non-synaptically [90, 92] from sympathetic axon terminals and enhanced in concentration in the close proximity of immune cells is able to inhibit production of proinflammatory (TNF-alpha, IFN-gamma, IL-12, IL-1) and increase antiinflammatory cytokines (IL-10) in response to LPS [25, 91], indicating a fine-tuning control of the production of TNF-alpha and other cytokines by sympathetic innervation under stressful conditions. This effects are mediated via beta2-adrenoceptors expressed on immune cells and coupled to cAMP levels.     

The lymphatic system in body homeostasis: physiological conditions

The lymphatic system is an organized network composed of functionally interrelated lymphoid tissue, and transportation pathways of tissue fluid/lymph and lymphoid cells. Its main components are 1. migrating dendritic cells, macrophages and lymphocytes, organized lymphoid tissue such as lymph nodes, thymus, spleen, bone marrow, and lymphoid tissue in gut and lungs, liver lymphoid cells, and the dendritic cell network of nonlymphoid organs; 2. vessels (intercellular space, lymphatics, and perivascular spaces); 3. fluids (tissue fluid and lymph). The lymphatic system can be divided into the following compartments: peripheral (from the interstitial space to and within the nearest lymph node), and central (efferent lymphatics, cysterna chyli, and thoracic duct, all lymphoid organs). Organs and tissues with the most active afferent arm of the lymphatic system are skin, gut, and lungs. These are the body structures exposed to the external environment. All other nonlymphoid bodily tissues are also percolated by tissue fluid/lymph, and contain a network of dendritic cells and macrophages. Data obtained from normal human subjects on lymph composition and flow are presented. Future trends in lymphatic research are outlined.     

Morphology of germ-free piglets

The postnatal ontogeny of primary (thymus) and secondary (spleen, lymph node, lingual tonsil) lymphoid tissues was studied in germ-free colostrum-deprived piglets up to age of 68 days. The thymus, which is morphologically fully developed by the end of gestation, showed no significant differences in the germ-free and conventional state. In germ-free piglets, slow development of periarteriolarly organized lymph follicles occurred in the spleen up to the end of the observation period. As distinct from the conditions in the spleen of conventional animals, the presence of a large number of pyroninophilic cells was not observed in germ-free piglets and no germinal centres were found. A similar situation was seen in the mesenteric lymph nodes, in which, in conventional piglets, cells belonging to the plasmacyte series, as well as the germinal centres, proliferate by the 13th day. Differences were also found in the organization of the follicular lymphoid tissue in the wall of the terminal ileum. In germ-free piglets, the lymph follicles increased only very slowly in size during the observation period and germinal centres were absent, while in conventional piglots germinal centres were present from the 12th day. The view is expressed that the intestinal lymphoid tissue ought rather to be classified as peripheral lymphoid tissue.     

The Gut Microbiota and Developmental Programming of the Testis in Mice

Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.     

Lactational alcohol exposure elicits long-term immune deficits and increased noradrenergic synaptic transmission in lymphoid organs

Increasing evidence suggests that the sympathetic nervous system plays an important role in immunomodulation. While chronic alcohol consumption has been associated with immune deficits, the effects of exposure to alcohol during early postnatal life on subsequent immunocompetence and activity of sympathetic neurons in lymphoid organs are not known. This study examined the long-term effects of lactational alcohol consumption on cellular immune responses and noradrenergic synaptic transmission in lymphoid and other organs of the young adult C57BL/6 mouse. The data show that exposure to alcohol via the mother's milk was associated with long-term deficits in cellular immunity, including suppression of the local graft vs host and contact hypersensitivity responses. The animals also displayed enhanced noradrenergic synaptic transmission and decreased beta-adrenoceptor density selectively in lymphoid organs. These neuroimmune changes are particularly striking since body weight-gain of the suckling pups was normal and their blood alcohol concentration was considerably lower than that of the alcohol-consuming dam. This suggests an increased sensitivity of the nascent immune and nervous systems during the critical period of early postnatal development.     

The specific anti-parasite immune responses of germ-free and conventional rats infected with Trypanosoma lewisi

To test the hypothesis that the rapid immune response of rats to Trypanosoma lewisi is elicited by prior exposure to cross-reacting environmental antigens, the early immune response to infection with this nonpathogenic protozoan was studied in germ-free and conventional rats. In germ-free rats, initial levels of both IgG and IgM were significantly lower than those of conventional rats. After infection, the germ-free rats made more immunoglobulins of both classes, and made them more quickly, than did conventional rats. Trypanosome-specific antibodies appeared earlier and in higher titers in the germ-free rats. Because they lacked intestinal microflora, it is unlikely that the germ-free rats' responses had been primed; thus, these observations indicated that the conventional rats' responses to some trypanosome antigens had been down-regulated by their prior exposure to environmental antigens. However, protective antibodies that inhibited parasite reproduction (ablastin) may have been primed, because these appeared in sera 2 days earlier in conventional rats. Despite much lower rates of production of trypanosome-specific antibodies, the conventional rats had the same peak parasitemias and times to crisis as germ-free rats. Thus it is apparent that protective immunity to this nonpathogenic parasite is not down-regulated by prior exposure to environmental antigens, as would be predicted.     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

Glycoconjugate expression in follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in specific pathogen-free and conventional rats.

We examined lectin-histochemically the glycoconjugate expression in the follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in the rat under specific pathogen-free (SPF) and conventional (CV) conditions and compared the results for SPF and CV rats as well as for membranous (M) cells and adjacent ciliated respiratory epithelial (CRE) cells in FAE. N-acetylgalactosamine-specific lectins, Dolichos biflorus (DBA), Helix pomatia (HPA), Glycine max (SBA) and Vicia villosa (VVA), and alpha-L-fucose-specific lectin, Ulex europaeus (UEA-I), preferentially bound to M cells mainly in the luminal surface compared with CRE cells in SPF rats, whereas DBA and UEA-I showed signs of preferential binding to the apical and basolateral cytoplasm as well as to the luminal surface of M cells in CV rats. In addition, HPA, SBA and VVA more frequently and extensively labeled M cells than CRE cells in CV rats with the same subcellular staining pattern as DBA and UEA-I. On the whole, the changes in lectin binding frequency and strength were more prominent in M cells than in CRE cells in both SPF and CV rats. The present results indicate that DBA and UEA-I are useful as markers of M cells in NALT. Furthermore, the pattern of expression of carbohydrate residues recognized by such lectins in SPF and CV rats suggests that M cells are highly sensitive to environmental changes.     

The role of noradrenergic nerves in the development of the lymphoproliferative disease in Fas-deficient, lpr/lpr mice

Lpr/lpr mice develop a lymphoproliferative, autoimmune, lupus-like disease. These mice lack functional Fas (CD95) expression and are resistant to Fas ligand (CD178)-mediated apoptosis, a critical mechanism for the maintenance of peripheral tolerance. In this study, we show that noradrenaline (NA), the main sympathetic neurotransmitter, can induce apoptosis of lymphoid cells independently of functional Fas. Based on this finding, we used lpr/lpr mice as model to study the role of noradrenergic nerves in the expression of a lymphoproliferative disease. Early in ontogeny, the concentration of NA was significantly increased in the spleen of lpr/lpr mice, compared with normal littermates. However, splenic sympathetic innervation gradually declined as the disease progressed, and IgM blood levels and splenic NA concentration inversely correlated when the disease was overtly manifested. When the loss of noradrenergic fibers that occurred naturally during adult life in lpr/lpr mice was experimentally advanced by neonatal sympathectomy, the concentration of IgM and IgG2a in blood was markedly higher than that of control lpr/lpr mice, and the appearance of lymphadenopathy was accelerated. Furthermore, although neonatal denervation did not affect the life span of normal animals, it shortened significantly the survival time of lpr/lpr mice. These data show that, in addition to defects in the Fas pathway, an altered sympathetic innervation in lpr/lpr mice also contributes to the pathogenesis of the autoimmune disease, and strongly support the hypothesis that the sympathetic nervous system can modulate the expression of lymphoproliferative diseases.     

Effects of homozygosity of the nude (rnu) gene in an inbred strain of rats: studies of lymphoid and non--lymphoid organs in different age groups of nude rats of LEW background at a stage in the gene transfer

Several age groups of nude homozygous rnu/rnu and heterozygous rnu/+ rats of the same genetic background at an early stage of back-crossing (LEW/Mol) were compared as to body and organ weights, histological appearance and cell density of lymphoid organs, haematological values and differential counts of bone marrow and peripheral blood. No thymic tissue was found in the nude animals. 7-week-old nudes were smaller than control animals and had relatively larger non-lymphoid organs and cell-depleted peripheral lymphoid organs. Other age groups showed little difference. Peripheral blood of nude rats showed no signs of lymphopaenia in contrast with the findings in nude mice. The number of thoracic duct lymphocytes was, however, significantly smaller in all age groups of the nude rats, and the bone marrow tended to contain fewer lymphocytes.     

Intestinal sphingolipid excretion associated with feeding of phytohemagglutinin lectin (Phaseolus vulgaris) to germ-free and conventional rats

Intestinal sphingolipids of feces of germ-free and conventional rats were analyzed during the pair feeding of a complete defined diet containing phytohemagglutinin lectin (PHA) from red kidney beans (Phaseolus vulgaris) as 1% dietary protein in comparison to casein fed controls. Phytohemagglutinin in the diet increased the total fecal excretion of sphingomyelins (18-fold for germ-free and 20-fold for conventional rats), of non-acid glycosphingolipids (3.5-fold for germ-free and 9-fold for conventional rats) and also of the gangliosides (2.5-fold) for the germ-free rats compared to controls. For germ-free rats the increase of non-acid glycolipids was ascribed to an effect of the lectin strictly on the small intestinal mucosa, while for conventional rats an effect was seen also on the large intestinal mucosa. Increase of fecal gangliosides of germ-free rats was due mainly to an increased excretion ofN-acetylneuraminosyl-lactosylceramide, a ganglioside species restricted to epithelial cells of duodenum, of upper jejunum and of large intestines. The effects on glycolipid excretion observed in germ-free rats and the rather similar effects seen in conventional animals suggested that the influence of dietary PHA was due directly to effects elicited by PHA binding to the enterocyte brush border membrane and not to secondary effects induced by increase in the luminal microflora.