Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat

One of the diabetes susceptibility genes of the BB rat is a mutation at the lyp locus that decreases the thymic output of T cells and the life span of most recent thymic emigrants (RTE). Consequently, there is a 10-fold reduction in the number of CD4+ and CD8+ T cells in secondary lymphoid organs. Results presented in this work demonstrate that the BB rat lyp mutation is associated with an accelerated apoptotic death in vitro of mature CD4+ 8- and CD4- 8+ thymocytes and peripheral T cells. The stability of the pool of recirculating T cells (PRL) of BB rats over time results from a > 10-fold increase in the mitotic activity of T cells as assessed in vivo by bromodeoxyuridine incorporation. This increased mitotic activity is not observed when BB T cells develop in the context of a normal sized PRL. MHC haploidentical WF and BB rats differ at minor histocompatibility loci. Intravenous injection of (WF x BB)F1 T cells into euthymic BB rats led to the rejection of donor T cells within 3 wk by unprimed recipients and within 1 wk by primed recipients. This secondary immune response was unaffected by postpriming thymectomy. F1 T cells were not rejected, but rather expanded after their injection into thymectomized BB rats that had been primed as early as 48 h after thymectomy. These results strongly suggest that the BB rat PRL is devoid of long-lived naive T cells and that rescue of recent thymic emigrants from programmed cell death is initiated by Ags, exclusively.     

T cell receptor gene deletion circles identify recent thymic emigrants in the peripheral T cell pool

Progenitor cells undergo T cell receptor (TCR) gene rearrangements during their intrathymic differentiation to become T cells. Rearrangements of the variable (V), diversity (D), and joining (J) segments of the TCR genes result in deletion of the intervening chromosomal DNA and the formation of circular episomes as a byproduct. Detection of these extrachromosomal excision circles in T cells located in the peripheral lymphoid tissues has been viewed as evidence for the existence of extrathymic T cell generation. Because all of the T cells in chickens apparently are generated in the thymus, we have employed this avian model to determine the fate of the V(D)J deletion circles. In normal animals we identified TCR Vgamma-Jgamma and Vbeta-Dbeta deletion circles in the blood, spleen, and intestines, as well as in the thymus. Thymectomy resulted in the gradual loss of these DNA deletion circles in all of the peripheral lymphoid tissues. A quantitative PCR analysis of Vgamma1-Jgamma1 and Vbeta1-Dbeta deletion circles in splenic gamma delta and Vbeta1(+) alphabeta T cells indicated that their numbers progressively decline after thymectomy with a half-life of approximately 2 weeks. Although TCR deletion circles therefore cannot be regarded as reliable indicators of in situ V(D)J rearrangement, measuring their levels in peripheral T cell samples can provide a valuable index of newly generated T cells entering the T cell pool.     

Phenotypic analysis of resident lymphoid cells in the conjunctiva and adnexal tissues of rat

The conjunctival associated lymphoid tissue is considered to be an integral part of the mucosal immune system. Under normal circumstances immune mechanisms in mucosal associated lymphoid tissue of the gut and bronchus can selectively suppress, rather than enhance, immune responsiveness to encountered antigens, inducing a state of tolerance. It is possible that conjunctival associated lymphoid tissue can also induce a state of tolerance to encountered antigens. Such a response may be exploited to modulate immune mediated ocular disease. Enhanced tolerance may protect the host against foreign antigen. Alternatively, under certain circumstances when the normal immune system is altered or disrupted the mucosal tissue may act to induce sensitisation and trigger immune mediated disease. The rat is frequently used as an animal model of immune mediated eye disease, but the normal profile of immune cells in the rat conjunctiva has not been studied. This information is essential for meaningful interpretation in the experimental situation. In this study we examined the immunophenotype of lymphoid tissue associated with the conjunctiva, lacrimal gland and Harderian gland of the Lewis rat. CD4+, Ia+ and the monocyte/macrophage population of cells were found predominantly in the substantia propria of the conjuctiva and interstitial connective tissue of the glands. CD8+ cells were distributed mainly in relation to the conjunctival and glandular epithelium. Goblet cells stained strongly with the monoclonal antibody (MAb) MRC OX-39, which is a marker for IL-2 receptors. The overall pattern of distribution of immunocompetent cells in the rat was found to be similar to that reported in humans.     

Pseudolymphoma of the lung: lymphoid subsets in the lung mass and in peripheral blood

Studies of the lymphocyte markers in a case of pseudolymphoma of the lung indicate a non-neoplastic nature of the lymphoid infiltrate. The relative proportions of T and B cells and of the markers for the various subsets of both populations reflect the morphologically mixed character of the cellular infiltrate of the lung mass. Moreover, an imbalance of T cell subsets was observed in the peripheral blood: the numbers of T cells with receptors for IgM (TM) were persistently decreased while an increase of the values of T lymphocytes with receptors for IgG (TG) was noted. In addition an altered immunologic status of the patient was indicated by the in vivo impairment of cellular immunity as demonstrated by the failure to respond to common antigens and to become sensitized to 1-chloro-2,4-dinitrobenzene (DNBC).     

Evidence for a distinct gap-junctional phenotype in ventricular conduction tissues of the developing and mature avian heart

The gap-junctional proteins connexin43 and connexin42 have been shown to be expressed in the developing and mature avian heart, but their respective spatiotemporal distributions are unknown. In the present study, we have immunolocalized connexin42 in the conduction tissues of the adult avian heart (nonbranching bundle, bundle branches, and Purkinje fibers) and vascular endothelial cells. Connexin43 immunolabeling was confined to vascular smooth muscle. A novel microwave-based method was used to label connexin42 and connexin43 in the same tissue section. Neither connexin42 nor connexin43 was immunolocalized in working myocardium, atrioventricular node, and atrioventricular ring tissue of the bird heart. Although connexin42 first appeared in periarterial conduction myocytes and vascular endothelium at 9-10 embryonic days, the central conduction tissues, including the nonbranching bundle and proximal branches, remained immunonegative for connexin42 up until hatching (approximately 20 embryonic days). During the early postnatal period (1-14 days), connexin42 immunolabeling progressively spread up the bundle branches toward the nonbranching bundle. Connexin42 appeared uniformly distributed along the left bundle branch by 14 postnatal days. The distribution and spread of connexin42 immunoreactivity suggest that the emergence of specialized junctional contacts along ventricular fascicles occurs relatively late in heart development and coincides with the emergence of the chick from incubation within the egg.     

Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver

Fas, a type I membrane protein that transduces an apoptotic signal, is expressed in lymphocytes as well as in various tissues such as the liver, lung and heart. The mouse lymphoproliferation (lpr) mutation is a leaky mutation in Fas. By means of gene targeting, we generated a mouse strain which is completely deficient in Fas. In addition to the massive production of lymphocytes, the Fas-null mice showed substantial liver hyperplasia, which was accompanied by the enlargement of nuclei in hepatocytes. The Fas system seems to play a role in the apoptotic process to maintain homeostasis of the liver as well as the peripheral lymphoid organs.     

The multiple untranslated first exons and promoters system of the oestrogen receptor gene in the brain and peripheral tissues of the rat and monkey and the developing rat cerebral cortex

Recent studies on the human oestrogen receptor (ER) gene have revealed the complex system with the multiple untranslated first exons and promoters in the ER gene expression. Little information is however available on the system in the ER gene of the rat or nonhuman primate. The rat genomic library was first screened by the rat ER cDNA (0-1) probe. One of the four positive clones (lambda rEgE1) was subcloned and sequenced. The nucleotide sequence was found to contain the exon 0, the intron 0, and the exon 1 with its 3'-ends. The novel untranslated first exons, the exon ON and the exon OS, were further identified. These results indicated the presence of at least four subtypes of the rat ER mRNAs; the messages transcribed from promoter P-0 (ER mRNA (0-1)), putative promoter P-1 (ER mRNA (1-1)), promoter P-ON (ER mRNA (ON-1)) and promoter P-OS (ER mRNA (OS-1)). The P-O- or P-1 driven message (0-1) or (1-1) appeared to be expressed most strongly in major oestrogen central- (anterior pituitary, AP, hypothalamus-preoptic area, HPOA, and amygdala, AMG) and peripheral targets (uterus and ovary). The message (ON-1) was strongly expressed in the liver and kidney, but not in the HPOA, AMG, cerebral cortex, CC, and cerebellum, Ce. The OS-1 message was expressed variably but generally in the tissues examined except for the CC and Ce. Thus, the region- and tissue specific expression of the rat ER gene is likely to be regulated by the multiple untranslated exons and promoters system. Furthermore, when the ER mRNA subtypes were examined in the rat neonatal CC where the ER protein level rose transiently, considered as a model for the development of the ER or progestin receptor A and B isoforms, the expression of the ER mRNAs seemed to be differential postnatally, implicating some stage dependent usage of the promoters in the development. In the monkey, we identified the untranslated first exon OS, the homologue of the rat exon OS. Interestingly, the exon C was found to consist of two different exons, the exon OK and the exon OG. By the alternative usage of the promoters and the alternative splicing, at least six ER mRNA subtypes, that is, ER mRNAs (0-1), (1-1), (OS-1), (OS-OG-1), (OK-1) and (OK-OG-1) were identified in the monkey tissues. These messages were also differentially distributed in the monkey brain and other tissues. It was noteworthy that the P-OK driven messages were expressed almost exclusively in the monkey liver. These results have suggested that the systems of the multiple untranslated first exons and promoters and the alternative splicing are involved in the regulation of the region- and tissue specific expression of the ER gene in the brain and peripheral tissues of the rat and monkey. Stage-related usage of the promoters was also suggested in the ER gene expression in the CC of the postnatal rat in development.     

Fetal cells in the peripheral blood of pregnant women express thymidine kinase: a new marker for detection

Fetal cells occur in maternal blood in a substantial proportion of normal pregnancies. Several different approaches have been used to detect and enrich these cells for non-invasive prenatal diagnosis. However, before these fetal cells can routinely be used for prenatal diagnosis, perfectly reproducible procedures for detection and enrichment need to be established. We found that these fetal cells express high intracellular levels of the DNA precursor pathway enzyme thymidine kinase. Since normal adult peripheral blood cells do not exhibit any thymidine kinase activity, this enzyme is a potent new marker to detect and enrich fetal cells from maternal blood. We further describe the first successful application of a cytofluorometric thymidine kinase assay to detect fetal cells in the maternal circulation by virtue of their high thymidine kinase activity.     

Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory

Progenitor cells in the early developing cerebral cortex produce neurons destined for discrete functional areas in response to specific inductive signals. Using lineage analysis, we show that cortical progenitor cells at different fetal ages retain the memory of an area-specific inductive signal received in vivo, even though they may pass through as many as two cell cycles in the absence of the signal in culture. When exposed to inductive signals in vitro, only those progenitors that progress through at least one complete cell cycle alter their areal phenotype. Our findings suggest that induction of an areal phenotype is linealy inherited, with the phenotype specified prior to the final cell cycle.     

Functional and morphologic maturation of superficial and juxtamedullary nephrons in the rat

The development of single nephron glomerular filtration rate (SNGFER) was studied in both superficial and juxtamedullary nephrons in rats in relation to concomitant morphologic maturation. These experiments were carried out in rats between 23 and 91 days of age (between 36 nad 275 g body weight) with the [14C]ferrocyanide infusion technique. 2. SNGFR of the superficial and juxtamedullary nephrons increased with body weight, glomerular volume and proximal tubular length. 3. The ratio SNGFR of the superficial (S) nephrons/SNGFR of the juxtamedullary (JM) nephrons rose from 0-60 in the 40-60 g rats to 0-84 in the adult rats, demonstrating the centrifugal functional maturation of the nephrons. 4. The S/JM ratio for both glomerular volume and tubular length was constant and averaged 0-72+/--0-12 and 0-81+/-0-05, respectively, indicating that while the increase in SNGFR was greater for S than for JM nephrons, this was not accompanied by concomitant disproportionate increases of glomerular volume and/or proximal tubular length between these nephron categories during development in the rat.     

The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool

The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a "full" peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.     

NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Pleurobranchaea californica was studied histochemically via NADPH-diaphorase (NADPH-d) reduction of Nitro Blue Tetrazolium (NTB). Whole mounts and cryostat sections were prepared from the central nervous system and peripheral organs, including the buccal muscles, esophagus, salivary glands, foot, mantle, and gills. NADPH-d-positive neurons were localized predominantly to the buccal and pedal ganglia as well as to distinct areas of the cerebropleural and visceral ganglia. A variety of identified neurons were positive for NADPH-diaphorase in various central ganglia, including the metacerebral cells of the cerebropleural ganglion, putative locomotor neurons of the pedal ganglia, and buccal motoneurons. Specific staining was observed only in somata of central neurons, whereas neuropil areas remained unstained. However, NADPH-d-reactive axons were dense in buccal ganglion nerves, whereas peripheral nerves and connectives of other ganglia had few or no NADPH-d positive terminals. In the periphery, NADPH-d activity was detected only in a few neurons of the rhinophore and tentacle ganglia. NADPH-d staining was marked in the salivary glands and gills, but there was no or very little staining in the esophagus, buccal mass, and foot. Histochemical stain production required the presence of both beta-NADPH and NBT; alpha-NADPH could not substitute for beta-NADPH. The inhibitor of NOS, 2,6-dichlorophenol-indophenol, at 10(-3) M, totally abolished NADPH-d-positive staining. The apparent high activity of central NADPH-d contrasts with much lower activity in the ganglia of the related gastropod Tritonia. These data suggest a role for nitric oxide as a signal molecule in the central nervous system of Pleurobranchaea.     

Localization of corticotropin-releasing factor in primary and secondary lymphoid organs of the rat

Cells of the immune system produce a variety of neuropeptides or peptide hormones, either constitutively or upon induction, and possess specific neuropeptide receptors that display ligand-receptor interactions similar to those described in the central nervous system (CNS). These findings suggest that specific subsets of lymphoid cells can produce and respond to peptides previously thought to be principally neural mediators. Recently, corticotropin releasing factor (CRF) mRNA was detected in the rat thymus and spleen, although the cells that synthesize CRF were not identified. We examined the localization of CRF and its mRNA in the rat spleen, thymus, and mesenteric lymph nodes using immunocytochemistry (ICC) and in situ hybridization (ISH), respectively. Immunoreactive CRF was present in cells in the marginal zone and red pulp of the spleen, in connective tissue septa and the subcapsular region of the thymus, and in the medullary cords and sinuses of the mesenteric lymph nodes. Dual ICC/ISH for CRF and its mRNA, respectively, demonstrated CRF mRNA over CRF-immunoreactive cells, suggesting CRF synthesis. Double-label ICC for CRF and markers for specific immunocyte subsets suggest that CRF+ cells in the spleen and thymus are macrophages. CRF+ cells in primary and secondary lymphoid organs reside in compartments that are innervated by sympathetic nerves, and some cells appears to be contacted by noradrenergic sympathetic nerve fibers, suggesting that CRF release may be influenced by the sympathetic nervous system, as it is in the hypothalamo-pituitary-adrenal axis. The presence of CRF in organs of the immune system suggests that this neuropeptide may modulate immune functions after paracrine release.     

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

Corticosterone has independent effects on tissue maturation and growth in the suckling rat

Glucocorticoids are associated with reduced weight gain when used to improve pulmonary function in premature infants. However, tissue maturation is stimulated during normal development by an increase in serum glucocorticoids. We evaluated the effects of glucocorticoid treatment on tissue weight gain and the activity of specific enzymes in the suckling rat, with the hypothesis that these processes are independently regulated. Before the ontogenic surge in corticosterone, 6-d-old rat pups were implanted with a pellet to release corticosterone continuously at 0 (placebo), 48, 120, 240, or 360 micro g/d. We killed the pups at 7, 9, or 12 d of age and measured tissue weights and activities of sucrase and glutamine synthetase. Serum corticosterone concentrations were elevated with dose. Tissue weight gain was proportional to ln(e) serum corticosterone at all ages. In contrast, enzyme indices of tissue maturation did not respond to corticosterone until d9. Also, intestinal tissue was more sensitive than muscle to the effects of corticosterone on weight but less sensitive to its effects on maturation. We conclude that the immediate response, in terms of weight versus the delayed response of the enzymes and their reciprocal sensitivity in muscle and gut, indicates that these processes are independently regulated.     

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2-/-recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas-T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas-) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas-T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.