Adenine uptake cells in the rat kidney with remarks on the expression of Ia antigen

The patterns of [14C] adenine ([14C]A) incorporation into DNA by proliferative cells in the kidney were studied by the autoradiographic technique. It was revealed that, after 3 daily injections of [14C]A (1 microCi/g body weight each), a portion of the glomerular cells and a few fibroblastoid cells in the cortical intexstitium incorporated [14C]A into DNA to a remarkable extent. Such cells also incorporated [3H]thymidine, but to a lesser extent. The cells which incorporate [14C]A to a particularly great extent (adenine uptake cells) also occur in other tissues. Such cells are confined to a few cell types of either the macrophage or fibroblast or reticulum cell lines. This fact suggests that the adenine uptake cells observed in the glomerulus are also of a similar cell line and most likely mesangial cells. By immunohistochemical examination for Ia antigen, adenine uptake cells are divided into Ia-positive and Ia-negative types. The present examination showed that the major portion of adenine uptake cells in the glomerulus are Ia-negative, and it is suggested that these cells are analogous to the Ia-negative macrophages in the lung. This suggestion is supported by the fact that, in the glomerulus, colloidal carbon uptake cells (macrophage-like cells) are present in fairly large numbers. The Ia-positive cells seem to be of the same cell line as the adenine uptake cells that express Ia antigen in other tissues, such as septal fibroblasts in the lung.     

The histiocytic origin of the multinucleated giant cells in myeloma kidney.

A recent case of myeloma kidney disease was studied to determine the cellular origin of the syncytial multinucleated giant cell. Light, immunofluorescence, and electron microscopy of the giant cells revealed features characteristic of histiocytes. This finding contradicts the generally accepted conclusion that the giant cell represents a syncytial mass of degenerating or reactive tubular epithelial cells. We conclude that the giant cells arise in the interstitial tissues, migrate through breaks in the tubular basement membrane, and engulf and surround intratubular protein casts.     

The origin of marginal zone B cells in the rat.

The marginal zone is a unique compartment that is only found in the spleen. Rat marginal zone B cells (MZ-B) can be distinguished from other B cells, e.g. recirculating follicular B cells (RF-B), by several phenotypic characteristics. Typically MZ-B cells are surface (s)IgMhi, sIgDlo and CD45R(B220)lo, whereas RF-B cells are sIgMlo, sIgDhi and CD45Rhi. In addition, MZ-B cells stain strongly with HIS57, a newly developed monoclonal antibody. The developmental pathway and origin of MZ-B cells are not exactly known. However, previous studies indicate that recirculating (i. e. thoracic duct) B cells can give rise to MZ-B cells. Here the origin of (naive) MZ-B cells was studied using adriamycin (doxorubicin)-induced B cell depletion. Using three-color flow cytometry and immunohistology we show that 2 days after a single i.v. injection of the anti-tumor drug adriamycin only RF-B cells can be detected, while all other B cell subpopulations are depleted, including all bone marrow precursor B cells. By studying the sequential reappearance of various B cell subsets and their precursors after adriamycin administration we show that MZ-B cells and the splenic marginal zone can be detected at a time point at which newly generated B cells (immature B cells) are not yet present. Given the observation that only RF-B cells were present at this time, we conclude that RF-B cells are the immediate MZ-B precursor cells.     

Immune induction of Ia antigens in activated T cells and in kidney epithelial cells in mice.

A rat monoclonal antibody against mouse Ia antigens was used to investigate the induction of Ia expression by activated T cells and renal epithelial cells during immune responses. The induction of low levels of surface Ia on activated helper and cytotoxic/suppressor T-cell phenotypes was directly demonstrated by differential fluorescence staining. Renal epithelial cells were observed to become strongly Ia-positive after primary immunization with alum-precipitated protein antigen, or after secondary challenge with the soluble antigen used for priming.     

The bivalence of Juxtaglomerular cells in the maturing rat kidney

Summary A comparative immunofluorescence and light microscopical study of the three cell types of the Juxtaglomerular apparatus (pure muscle cells, pure granular cells and mixed cells) was performed on the growing and maturing kidney of the rat. Mixed cells, containing contractile protein and secretory granules, are detectable on the first postnatal day in about one third of the JGAs. From the third week, the number of bivalent cells increases, while the proportion of pure muscle or pure granular cells decreases. Morphological and functional maturation, achieved by 3 to 4 weeks, is associated with increasing numbers of bivalent cells and a shift in the main site of renin production from the inner to the outer cortical zone.The divergent internal structure of JGA cell types expresses the range of varied differentiation expressed by one cell line. Pure muscle or granular cells are at the extremes of the range and mixed cells take up an intermediate position.     

The distribution,ontogeny and origin in the rat of Ia-positive cells with dendritic morphology and of Ia antigen in epithelia,with special reference to the intestine

Ia antigens were localized in cryostat sections of rat intestine and other tissues by an indirect immunoperoxidase technique using monoclonal antibodies that recognize the rat antigens homologous to the gene products of the I-A and I-E subregions of the mouse major histocompatibility complex (MHC). Two categories of Ia⁺ cells were characterized, namely epithelial cells and bone marrow-derived cells with dendritic morphology. In the small intestine Ia antigen was present in the distal 2/3 of the absorptive epithelium but absent from the bases of the villi, the crypts and the epithelium covering the Peyer's patches. The distribution in nude rats was similar, indicating that T lymphocytes are not obligatory for its expression. In ontogeny Ia antigen was absent in the epithelium of neonatal gut, appearing at about 4 weeks of age and reaching adult levels at about 6 weeks. Different rat strains showed large differences in the amount of Ia antigen expressed by villus epithelium and the traits for the level of expression were shown to map outside the MHC. The levels of expression of Ia antigen in the proximal tubules of the kidney followed that of the gut epithelium in the different strains and in both tissues was mostly intracellular. Studies with chimeras showed that the Ia antigen in epithelial cells was not acquired from bone marrow-derived cells. The second category of cell studied had a characteristic dendritic morphology and was present in large numbers in the lamina propria of the villi and in the crypts. In the Peyer's patches these cells were present both in the subepithelial dome region and within the epithelium itself. These Ia⁺ dendritic cells were present in nude rat jejunum and appeared in normal fetal gut by 18 days gestation and were also shown to migrate into antigen-free grafts of fetal gut. This suggests that they do not require stimulation from antigens, bacterial products or T lymphocytes in order to localize in the gut or to express Ia antigen. Studies with other cell surface markers suggest that the Ia⁺ cells with dendritic morphology represent a range of cell types, some with similarities to macrophages and others to nonphagocytic dendritic cells.     

Ultrastructural identification of Ia positive dendritic cells in the lactating rat mammary gland

Summary Dendritic cells which express Ia antigen have been demonstrated for the first time in the lactating rat mammary gland. Ultrastructurally, the dendritic cells appear as electron-lucent pale cells interspersed among the epithelial cells of the alveoli, forming a cell population distinct from classical macrophages. They show morphological resemblance to the dendritic cells of lymphoid organs as well as the Langerhans cells of skin. The Ia antigen has been localised by electron microscopic immunocytochemistry on the cell membrane and endocytotic vesicles and tubules. Ia positive cells are also seen in the stroma of the mammary gland. It is proposed that the dendritic cells of the mammary gland belong to the lineage of epidermal Langerhans cells and lymphoid dendritic cells, subserving an immunological role in the lactating breast.     

The macrophagic origin of multinucleated giant cells in myeloma kidney: an immunohistologic study

The cast-engulfing giant cells in a case of kappa light chain nephropathy/Bence Jones cast nephropathy were characterized with monoclonal antibodies Uro-5 (renal epithelium: Henle's loop, distal tubule, collecting duct) and Leu-M5 (macrophage/monocyte/dendritic cell specific). The giant cells were Leu-M5 positive and Uro-5 negative. There was an increased concentration of Leu-M5-positive cells surrounding cast-containing tubules, and occasional Leu-M5-positive cells were found within the tubular epithelial layer. Although there were no Uro-5-positive giant cells, an occasional Uro-5-positive cell was found within casts. The majority of casts were found within Uro-5-positive tubules. These results confirm earlier electron microscopic reports that the cell of origin of the cast-engulfing multinucleated giant cell is the macrophage (Leu-M5 positive, Uro-5 negative). These giant cells may originate from macrophages that have migrated from the interstitium through the tubular epithelium.     

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

Expression of Ia determinants on immunocompetent cells.

The expression of Ia (immune response region-associated) antigens on the surface of lymphocyte subpopulations with defined immunological function has been investigated by negative selection of subpopulations with anti-Ia sera and complement. Ia determinants were found on both unprimed (IgM) and on primed (IgG) antibody-forming precursor cells. No Ia antigens were detected on the surface of helper T cells. In contrast, suppressor T cells were sensitive to treatment with anti-Ia sera and complement demonstrating the presence of Ia determinants on this T cell subpopulation.     

The intracellular chloride activity of rat kidney proximal tubular cells

The intracellular Cl^? activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl^? sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measureV _Cl and using — in separate experiments — conventional KCl-filled microelectrodes to measure the membrane potential,V _m. After correction for interference from other anions onV _Cl the intracellular Cl^? activity averaged 13.1 mmol·l^?1 SD±4.5 mmol·l^?1 (n=96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl^? activity of 90–103 mmol·l^?1 andV _m of ?71.2 mV, SD±4.9 mV (n=23) to amount to 6.3 to 6.7 mmol·l^?1. Since both cell membranes are permeable for Cl^? ions, as concluded from luminal and/or peritubular Cl^? substitution experiments, we conclude that the cellular Cl^? accumulation above equilibrium results from transcellular active Cl^? transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl^? concentration after substitution of luminal Cl^? by gluconate, however, we deduce that transcellular Cl^? absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl^? flux is passive and paracellular.     

Differential expression of estrogen-regulated CD4 and Ia positive cells in the immature rat uterus

Estradiol treatment of sexually immature Sprague-Dawley, Cr:NIH-nu/+ and Cr:NIH-nu/nu rats for 2 days results in a significant increase in the number of uterine eosinophils and OX-42 positive macrophages. However, only Sprague-Dawley rats exhibit an estrogen regulated increase in the number of cells expressing immunoreactive CD4 and Ia, suggesting that the differential responsiveness to the effects of estradiol between the two strains of rats may be genetically controlled. Nevertheless, Cr:NIH-nu/nu rats, which are deficient in T-cells, possess virtually identical numbers of CD4 positive staining uterine cells as compared to their nu/+ littermate controls, indicating that the expression of immunoreactive CD4 is associated with a resident non-T-cell population. In addition, a message for rat CD4 was observed in Northern blots of uterine mRNA, suggesting that the immunoreactive CD4 expressed by uterine cells is probably a fully functional cell surface antigen/receptor rather than a truncated form which only expresses the antigenic determinant recognized by the W3/25 monoclonal antibody.     

The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract.

Extracts of muscle and mucosal layers of the rat stomach contained material cross-reacting in radioimmunoassays for the amphibian skin peptide bombesin. In the intestine, immunoreactive bom-besin was confined to the muscle layers. Two molecular forms of immunoreactive bombesin were identified; one of these components was eluted in a similar position to tetradecapeptide bombesin on gel filtration and accounted for about 90% of the immunoreactivity in intestinal extracts, compared with about 40% in stomach. The two components were distinguishable from synthetic bombesin, and from the structurally related peptide substance P, on the basis of their pattern of immunochemical properties with three different antisera. Immunohistochemical studies using the same antisera revealed a rich distribution of nerve fibres in the mucosa of the rat stomach, but few fibres were seen in the intestinal mucosa. Abundant fibres with bombesin-like immunoreactivity were found surrounding nerve cell bodies in the myenteric plexus throughout the gut. Immunoreactivc nerve cell bodies were not identified, neither was convincing evidence obtained to indicate the presence of bombesin in mucosal endocrine cells.The results support the possibilities that bombesin-like peptides are neurotransmitters in the gut and that they could play a role in the modulation of gastrointestinal motility and in the release of gastrin.     

Effect of HgCl2 on rat kidney cells in primary culture.

Single cell suspensions, obtained from rat kidneys, were cultured with various concentrations of HgCl2 for different times to examine the effects on cellular synthetic activity. Unexpectedly, the RNA synthesis of kidney cells was stimulated with low doses of HgCl2, whereas it was inhibited with higher doses. Even at a higher dose, RNA synthesis was stimulated during early incubation but was inhibited during longer incubations. Thus, the effect of mercury showed a biphasic pattern both in dose-response and time-response relationships. Moreover, once the cells were exposed to HgCl2 for a certain period, RNA syntehsis was irreversibly inhibited. Since RNA synthesis was inhibited earlier than DNA or a protein synthesis, the RNA synthesizing system appeared most susceptible. Without HgCl2, by the time DNA synthesis reached its maximum, protein and RNA synthesis had already declined. In contrast, RNA synthesis of HgCl2-treated cells remained at its maximal level and showed no decline when DNA synthesis reached its peak.     

Regulation of antigen-expressing dendritic cells by double negative regulatory T cells

TCRαβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) double negative (DN) Tregs comprise 1-3% of peripheral T lymphocytes in mice and humans. It has been demonstrated that DN Tregs can suppress allo-, xeno- and auto-immune responses in an Ag-specific fashion. However, the mechanisms by which DN Tregs regulate immune responses remain elusive. Whether DN Tregs can regulate DCs has not been investigated previously. In this study, we demonstrate that DN Tregs express a high level of CTLA4 and are able to down-regulate costimulatory molecules CD80 and CD86 expressed on Ag-expressing mature DCs (mDCs). DN Tregs from CTLA4 KO mice were not able to downregulate CD80 and CD86 expression, indicating that CTLA4 is critical for DN Treg-mediated downregulation of costimulatory molecule expression on Ag-expressing mature DCs. Furthermore, DN Tregs could kill both immature and mature allogeneic DCs, as well as Ag-loaded syngeneic DCs, in an Ag-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. These data demonstrate, for the first time, that DN Tregs are potent regulators of DCs and may have the potential to be developed as a novel immune suppression treatment.